e coli reference strains  (ATCC)


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    ATCC e coli reference strains
    E Coli Reference Strains, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    eleven e coli reference strains  (ATCC)


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    ATCC eleven e coli reference strains
    Eleven E Coli Reference Strains, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cbv3 6 atcc reference strains  (ATCC)


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    ATCC cbv3 6 atcc reference strains
    Cbv3 6 Atcc Reference Strains, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    reference strain e coli atcc 25922  (ATCC)


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    ATCC reference strain e coli atcc 25922
    Reference Strain E Coli Atcc 25922, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    reference strain c parapsilosis atcc 22019  (ATCC)


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    ATCC reference strain c parapsilosis atcc 22019
    Reference Strain C Parapsilosis Atcc 22019, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    reference strain atcc 49226  (ATCC)


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    ATCC reference strain atcc 49226
    a TEM of gonococcal OMV secretion during invasion and exocytosis of HeLa cells. Images are representative of 2 independent experiments. b Enhanced intracellular survival and exocytosis of gonococcal Δ vacJ mutant in gentamicin protection assays with HeLa cells. Data are mean ± s.d. of log-normalized colony forming units (CFU) per well and relative differences in survival between WT and Δ vacJ are provided within the bars; n = 4 independent experiments, two-way ANOVA with posthoc Bonferroni test. c Δ vacJ mutant shows reduced LAMP1-colocalization after a one-hour challenge of HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 53 cells, unpaired two-tailed t -test. d TEM images of purified gonococcal OMVs. Images are representative of 4 (ATCC 49226) and 3 (ZJXSH86) independent experiments. e LC3 Western blots show induction of autophagy flux in HeLa cells stimulated with gonococcal OMVs from strains <t>ATCC</t> <t>49226</t> and ZJXSH86. f Gonococcal OMVs induce accumulation of LC3 puncta in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Vehicle Mock-ZJXSH86 OMVs: P < 10 −15 ). g OMVs from the gonococcal Δ ldcA mutant induce accumulation of LC3 puncta in HeLa cells, but largely lost colocalization with LC3 puncta. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, one-way ANOVA with post-hoc Tukey test for quantification of LC3 puncta (Mock-WT OMVs: P = 3 × 10 −14 ; Mock-Δ ldcA OMVs: P = 6 × 10 −14 ), unpaired two-tailed t -test for quantification of LC3-positive OMVs ( P = 5 × 10 −13 ). h LC3 Western blots of OMV-stimulated HeLa cells show reduced accumulation of LC3 for the Δ ldcA mutant. Cells in c , f , g are from 3 independent experiments. Western blots in e , h are representative of 3 independent experiments. Source data are provided as a Source Data file.
    Reference Strain Atcc 49226, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Gonococcal OMV-delivered PorB induces epithelial cell mitophagy"

    Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

    Journal: Nature Communications

    doi: 10.1038/s41467-024-45961-1

    a TEM of gonococcal OMV secretion during invasion and exocytosis of HeLa cells. Images are representative of 2 independent experiments. b Enhanced intracellular survival and exocytosis of gonococcal Δ vacJ mutant in gentamicin protection assays with HeLa cells. Data are mean ± s.d. of log-normalized colony forming units (CFU) per well and relative differences in survival between WT and Δ vacJ are provided within the bars; n = 4 independent experiments, two-way ANOVA with posthoc Bonferroni test. c Δ vacJ mutant shows reduced LAMP1-colocalization after a one-hour challenge of HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 53 cells, unpaired two-tailed t -test. d TEM images of purified gonococcal OMVs. Images are representative of 4 (ATCC 49226) and 3 (ZJXSH86) independent experiments. e LC3 Western blots show induction of autophagy flux in HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86. f Gonococcal OMVs induce accumulation of LC3 puncta in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Vehicle Mock-ZJXSH86 OMVs: P < 10 −15 ). g OMVs from the gonococcal Δ ldcA mutant induce accumulation of LC3 puncta in HeLa cells, but largely lost colocalization with LC3 puncta. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, one-way ANOVA with post-hoc Tukey test for quantification of LC3 puncta (Mock-WT OMVs: P = 3 × 10 −14 ; Mock-Δ ldcA OMVs: P = 6 × 10 −14 ), unpaired two-tailed t -test for quantification of LC3-positive OMVs ( P = 5 × 10 −13 ). h LC3 Western blots of OMV-stimulated HeLa cells show reduced accumulation of LC3 for the Δ ldcA mutant. Cells in c , f , g are from 3 independent experiments. Western blots in e , h are representative of 3 independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: a TEM of gonococcal OMV secretion during invasion and exocytosis of HeLa cells. Images are representative of 2 independent experiments. b Enhanced intracellular survival and exocytosis of gonococcal Δ vacJ mutant in gentamicin protection assays with HeLa cells. Data are mean ± s.d. of log-normalized colony forming units (CFU) per well and relative differences in survival between WT and Δ vacJ are provided within the bars; n = 4 independent experiments, two-way ANOVA with posthoc Bonferroni test. c Δ vacJ mutant shows reduced LAMP1-colocalization after a one-hour challenge of HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 53 cells, unpaired two-tailed t -test. d TEM images of purified gonococcal OMVs. Images are representative of 4 (ATCC 49226) and 3 (ZJXSH86) independent experiments. e LC3 Western blots show induction of autophagy flux in HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86. f Gonococcal OMVs induce accumulation of LC3 puncta in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Vehicle Mock-ZJXSH86 OMVs: P < 10 −15 ). g OMVs from the gonococcal Δ ldcA mutant induce accumulation of LC3 puncta in HeLa cells, but largely lost colocalization with LC3 puncta. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, one-way ANOVA with post-hoc Tukey test for quantification of LC3 puncta (Mock-WT OMVs: P = 3 × 10 −14 ; Mock-Δ ldcA OMVs: P = 6 × 10 −14 ), unpaired two-tailed t -test for quantification of LC3-positive OMVs ( P = 5 × 10 −13 ). h LC3 Western blots of OMV-stimulated HeLa cells show reduced accumulation of LC3 for the Δ ldcA mutant. Cells in c , f , g are from 3 independent experiments. Western blots in e , h are representative of 3 independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Mutagenesis, Two Tailed Test, Purification, Western Blot

    a Western blots of OMV-stimulated HeLa cells show specific autophagosome/lysosome-dependent degradation of mitochondrial marker proteins (TOM20 and TIM23), but not of Golgi (GM130) or endoplasmic reticulum (PDI). Western blots are representative of 3 independent experiments. b DiO-labeled gonococcal OMVs from strain ATCC 49226 colocalize with MitoBright-labeled mitochondria in HeLa cells. Scale bar, 5 μm. c Live HeLa cell microscopy and 3D image reconstruction shows OMVs remain associated with Cascade Blue-labeled endosomes when delivered to MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. d Gonococcal OMVs from strain ATCC 49226 dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P < 10 −15 . e TEM of HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86 show mitochondrial disruption and capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, one-way ANOVA with post-hoc Tukey test for mitochondrial disruption (Mock-ATCC 49226 OMVs: P = 2 × 10 −11 ; Mock-ZJXSH86 OMVs: P = 2 × 10 −11 ), Kruskal–Wallis with posthoc Dunn test for mitochondria in mitophagy-like structures (Mock-ATCC 49226 OMVs: P = 1 × 10 −6 ; Mock-ZJXSH86 OMVs: P = 7 × 10 −6 ). f Quantitative real-time PCR showing a reduced mitochondrial to genomic DNA ratio in ATCC 49226 OMV-stimulated HeLa cells. Data are mean ± s.d.; n = 3 independent biological replicates, one-way ANOVA with post-hoc Tukey test. g Increased HSP60 and LAMP1 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P = 6 × 10 −9 . h Increased HSP60 and LC3 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test (LC3 puncta Mock-OMVs: P = 7 × 10 −12 ; LC3 colocalized HSP60 Mock-ZJXSH86 OMVs: P = 4 × 10 −15 ). Cells in d , e , g , h are from 3 independent experiments. Images in b , c are representative of 3 independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Western blots of OMV-stimulated HeLa cells show specific autophagosome/lysosome-dependent degradation of mitochondrial marker proteins (TOM20 and TIM23), but not of Golgi (GM130) or endoplasmic reticulum (PDI). Western blots are representative of 3 independent experiments. b DiO-labeled gonococcal OMVs from strain ATCC 49226 colocalize with MitoBright-labeled mitochondria in HeLa cells. Scale bar, 5 μm. c Live HeLa cell microscopy and 3D image reconstruction shows OMVs remain associated with Cascade Blue-labeled endosomes when delivered to MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. d Gonococcal OMVs from strain ATCC 49226 dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P < 10 −15 . e TEM of HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86 show mitochondrial disruption and capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, one-way ANOVA with post-hoc Tukey test for mitochondrial disruption (Mock-ATCC 49226 OMVs: P = 2 × 10 −11 ; Mock-ZJXSH86 OMVs: P = 2 × 10 −11 ), Kruskal–Wallis with posthoc Dunn test for mitochondria in mitophagy-like structures (Mock-ATCC 49226 OMVs: P = 1 × 10 −6 ; Mock-ZJXSH86 OMVs: P = 7 × 10 −6 ). f Quantitative real-time PCR showing a reduced mitochondrial to genomic DNA ratio in ATCC 49226 OMV-stimulated HeLa cells. Data are mean ± s.d.; n = 3 independent biological replicates, one-way ANOVA with post-hoc Tukey test. g Increased HSP60 and LAMP1 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P = 6 × 10 −9 . h Increased HSP60 and LC3 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test (LC3 puncta Mock-OMVs: P = 7 × 10 −12 ; LC3 colocalized HSP60 Mock-ZJXSH86 OMVs: P = 4 × 10 −15 ). Cells in d , e , g , h are from 3 independent experiments. Images in b , c are representative of 3 independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Western Blot, Marker, Labeling, Microscopy, Fluorescence, Membrane, Two Tailed Test, Disruption, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    a TEM of HeLa cells expressing gonococcal PorB from strain ATCC 49226 show mitochondrial capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, two-tailed Mann–Whitney test, P = 2 × 10 −10 . b Western blots showing degradation of mitochondrial proteins TOM20 and TIM23 in HeLa cells expressing gonococcal PorB from strains ATCC 49226 and ZJXSH86, but not for PorB from Neisseria mucosa . c Western blots showing that gonococcal OMVs expressing PorB from N. mucosa lost the ability to induce degradation of TOM20 and TIM23 in HeLa cells. d Gonococcal OMVs expressing PorB from N. mucosa lost the ability to dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 55 cells from 4 independent experiments, Kruskal–Wallis with posthoc Dunn test (Mock-N.g. PorB: P = 8 × 10 −13 ; N.g. PorB-N.m. PorB: P < 10 −15 ). e Flow cytom e try analysis of TMRM fluorescence intensity in HeLa cells stimulated with gonococcal OMVs expressing gonococcal PorB or PorB from N. mucosa . f Gonococcal PorB structure (pdb entry 4AUI) and sequence of PorB from strain ATCC 49226. Lysines are indicated in blue, or in magenta when located within the PorB channel and associated with ATP (orange) binding. g Western blots showing reduced degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q. h Reduced HSP60 and LC3 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 2 × 10 −9 ; WT PorB-PorB K117Q: P = 1 × 10 −5 ). i Reduced HSP60 and LAMP1 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 9 × 10 −8 ). j Western blots of HeLa 4KO cells showing p62 expression restores PorB K117Q-dependent degradation of TOM20 and TIM23, while expression of OPTN or NDP52 restores PorB K5-dependent degradation of TOM20 and TIM23. k Western blots showing impaired degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q, K5 or K null . l Western blots showing that knock-down of PINK1 in HeLa cells inhibits PorB K5-dependent degradation of TOM20 and TIM23, but not for PorB K117Q. Cells in a , h , i are from 3 independent experiments. Western blots in b , c , g , j , k , l are representative of 3 independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: a TEM of HeLa cells expressing gonococcal PorB from strain ATCC 49226 show mitochondrial capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, two-tailed Mann–Whitney test, P = 2 × 10 −10 . b Western blots showing degradation of mitochondrial proteins TOM20 and TIM23 in HeLa cells expressing gonococcal PorB from strains ATCC 49226 and ZJXSH86, but not for PorB from Neisseria mucosa . c Western blots showing that gonococcal OMVs expressing PorB from N. mucosa lost the ability to induce degradation of TOM20 and TIM23 in HeLa cells. d Gonococcal OMVs expressing PorB from N. mucosa lost the ability to dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 55 cells from 4 independent experiments, Kruskal–Wallis with posthoc Dunn test (Mock-N.g. PorB: P = 8 × 10 −13 ; N.g. PorB-N.m. PorB: P < 10 −15 ). e Flow cytom e try analysis of TMRM fluorescence intensity in HeLa cells stimulated with gonococcal OMVs expressing gonococcal PorB or PorB from N. mucosa . f Gonococcal PorB structure (pdb entry 4AUI) and sequence of PorB from strain ATCC 49226. Lysines are indicated in blue, or in magenta when located within the PorB channel and associated with ATP (orange) binding. g Western blots showing reduced degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q. h Reduced HSP60 and LC3 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 2 × 10 −9 ; WT PorB-PorB K117Q: P = 1 × 10 −5 ). i Reduced HSP60 and LAMP1 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 9 × 10 −8 ). j Western blots of HeLa 4KO cells showing p62 expression restores PorB K117Q-dependent degradation of TOM20 and TIM23, while expression of OPTN or NDP52 restores PorB K5-dependent degradation of TOM20 and TIM23. k Western blots showing impaired degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q, K5 or K null . l Western blots showing that knock-down of PINK1 in HeLa cells inhibits PorB K5-dependent degradation of TOM20 and TIM23, but not for PorB K117Q. Cells in a , h , i are from 3 independent experiments. Western blots in b , c , g , j , k , l are representative of 3 independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Expressing, Two Tailed Test, MANN-WHITNEY, Western Blot, Membrane, Fluorescence, Sequencing, Binding Assay

    reference strains b longum subsp longum atcc 15707  (ATCC)


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    ATCC reference strains b longum subsp longum atcc 15707
    Reference Strains B Longum Subsp Longum Atcc 15707, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    reference s typhimurium strain lt2  (ATCC)


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    ATCC reference s typhimurium strain lt2
    Glucose-6-phosphate sensing and transcriptional regulation are impaired in Salmonella Typhi. ( A ) Diagram of the glucose-6-phosphate (G6P) sensing, transcription regulation, and transport in Salmonella enterica . G6P (purple hexagon) is imported into the bacterial cell by the UhpT antiporter, whose expression is strictly controlled by an unconventional two-component regulatory system composed of the membrane-associated UhpC sensor and UhpB kinase/phosphatase, and the cytoplasmic response regulator UhpA. In this system, UhpC directly senses G6P triggering a conformational change in the interacting kinase UhpB, which in turn phosphorylates and activates the response regulator UhpA. Phosphorylation increases the affinity of UhpA for the uhpT promoter (P uhpT ) thus initiating transcription. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . <t>Typhimurium</t> and S . Typhi G6P biosensors after growth in LB medium containing G6P (500 µM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of G6P is shown. ( C ) Investigation of the functionality of UhpA, UhpB, and UhpC regulatory proteins. Each S . Typhi gene was swapped for its S . Typhimurium homolog in the plasmid backbone containing the G6P biosensor as indicated in the diagram. The resulting plasmids were then introduced into the S . Typhi or S. Typhimurium ΔuhpABC mutant backgrounds, and the resulting strains were grown in M9 medium in the presence or absence of G6P (1mM). ( D ) Investigation of the functionality of each of the different uhpA mutations present in S . Typhi in comparison of its functional homolog in S . Typhimurium. Plasmids encoding the G6P biosensor and expressing S . Typhi UhpA mutants in which residues had been individually changed to the amino acid present in its S . Typhimurium homolog as indicated (i. e. UhpA Met77Val , UhpA Glu146Ala , or UhpA Met168Thr ) were introduced into the S. Typhi Δ uhpABC mutant background. The resulting strains were grown into M9 medium in the presence of G6P (1mM). In ( B - D ) the sfGFP fluorescence signal normalized to cell density (sfGFP RFU / OD600) was measured. Values indicate the mean ± SEM of n= 3 or 4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test ***p < 0.001, ( B and D ), or unpaired two-tailed t-test ****p < 0.0001 ( C ). ( E ) Functionality of different UhpA variants expressed in S . Typhi in the context of cultured epithelial cell infection. HeLa cells were infected with wild-type S . Typhi or derivative strains expressing the uhpA allele from S. Typhimurium ( uhpA STm ) or an S . Typhi allele, which had been rendered functional by introducing changes based on the S . Typhimurium sequence (i. e. uhpA Glu146Ala,Met168Thr ). Hela cells were infected with the different S. Typhi strains for 6 hours, fixed, stained with DAPI, and examined under a fluorescence microscope. In all the images, brightness and contrast were optimized for each of the individual color channels to maximize visual clarity. Scale bars, 10 μm. ( F ) Alignment of the modeled atomic structures of S . Typhi UhpA (light blue) and S. Typhimurium UhpA (light pink) showing structural variations in the C-terminal portion at the helix-turn-helix motif (boxed). The models were predicted using AlphaFold2 and aligned with Pymol. The amino acid differences between the two structures (Val77Met, Ala146Glu, and Thr168Met) are shown in green.
    Reference S Typhimurium Strain Lt2, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation"

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    Journal: bioRxiv

    doi: 10.1101/2024.02.14.580360

    Glucose-6-phosphate sensing and transcriptional regulation are impaired in Salmonella Typhi. ( A ) Diagram of the glucose-6-phosphate (G6P) sensing, transcription regulation, and transport in Salmonella enterica . G6P (purple hexagon) is imported into the bacterial cell by the UhpT antiporter, whose expression is strictly controlled by an unconventional two-component regulatory system composed of the membrane-associated UhpC sensor and UhpB kinase/phosphatase, and the cytoplasmic response regulator UhpA. In this system, UhpC directly senses G6P triggering a conformational change in the interacting kinase UhpB, which in turn phosphorylates and activates the response regulator UhpA. Phosphorylation increases the affinity of UhpA for the uhpT promoter (P uhpT ) thus initiating transcription. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . Typhimurium and S . Typhi G6P biosensors after growth in LB medium containing G6P (500 µM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of G6P is shown. ( C ) Investigation of the functionality of UhpA, UhpB, and UhpC regulatory proteins. Each S . Typhi gene was swapped for its S . Typhimurium homolog in the plasmid backbone containing the G6P biosensor as indicated in the diagram. The resulting plasmids were then introduced into the S . Typhi or S. Typhimurium ΔuhpABC mutant backgrounds, and the resulting strains were grown in M9 medium in the presence or absence of G6P (1mM). ( D ) Investigation of the functionality of each of the different uhpA mutations present in S . Typhi in comparison of its functional homolog in S . Typhimurium. Plasmids encoding the G6P biosensor and expressing S . Typhi UhpA mutants in which residues had been individually changed to the amino acid present in its S . Typhimurium homolog as indicated (i. e. UhpA Met77Val , UhpA Glu146Ala , or UhpA Met168Thr ) were introduced into the S. Typhi Δ uhpABC mutant background. The resulting strains were grown into M9 medium in the presence of G6P (1mM). In ( B - D ) the sfGFP fluorescence signal normalized to cell density (sfGFP RFU / OD600) was measured. Values indicate the mean ± SEM of n= 3 or 4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test ***p < 0.001, ( B and D ), or unpaired two-tailed t-test ****p < 0.0001 ( C ). ( E ) Functionality of different UhpA variants expressed in S . Typhi in the context of cultured epithelial cell infection. HeLa cells were infected with wild-type S . Typhi or derivative strains expressing the uhpA allele from S. Typhimurium ( uhpA STm ) or an S . Typhi allele, which had been rendered functional by introducing changes based on the S . Typhimurium sequence (i. e. uhpA Glu146Ala,Met168Thr ). Hela cells were infected with the different S. Typhi strains for 6 hours, fixed, stained with DAPI, and examined under a fluorescence microscope. In all the images, brightness and contrast were optimized for each of the individual color channels to maximize visual clarity. Scale bars, 10 μm. ( F ) Alignment of the modeled atomic structures of S . Typhi UhpA (light blue) and S. Typhimurium UhpA (light pink) showing structural variations in the C-terminal portion at the helix-turn-helix motif (boxed). The models were predicted using AlphaFold2 and aligned with Pymol. The amino acid differences between the two structures (Val77Met, Ala146Glu, and Thr168Met) are shown in green.
    Figure Legend Snippet: Glucose-6-phosphate sensing and transcriptional regulation are impaired in Salmonella Typhi. ( A ) Diagram of the glucose-6-phosphate (G6P) sensing, transcription regulation, and transport in Salmonella enterica . G6P (purple hexagon) is imported into the bacterial cell by the UhpT antiporter, whose expression is strictly controlled by an unconventional two-component regulatory system composed of the membrane-associated UhpC sensor and UhpB kinase/phosphatase, and the cytoplasmic response regulator UhpA. In this system, UhpC directly senses G6P triggering a conformational change in the interacting kinase UhpB, which in turn phosphorylates and activates the response regulator UhpA. Phosphorylation increases the affinity of UhpA for the uhpT promoter (P uhpT ) thus initiating transcription. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . Typhimurium and S . Typhi G6P biosensors after growth in LB medium containing G6P (500 µM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of G6P is shown. ( C ) Investigation of the functionality of UhpA, UhpB, and UhpC regulatory proteins. Each S . Typhi gene was swapped for its S . Typhimurium homolog in the plasmid backbone containing the G6P biosensor as indicated in the diagram. The resulting plasmids were then introduced into the S . Typhi or S. Typhimurium ΔuhpABC mutant backgrounds, and the resulting strains were grown in M9 medium in the presence or absence of G6P (1mM). ( D ) Investigation of the functionality of each of the different uhpA mutations present in S . Typhi in comparison of its functional homolog in S . Typhimurium. Plasmids encoding the G6P biosensor and expressing S . Typhi UhpA mutants in which residues had been individually changed to the amino acid present in its S . Typhimurium homolog as indicated (i. e. UhpA Met77Val , UhpA Glu146Ala , or UhpA Met168Thr ) were introduced into the S. Typhi Δ uhpABC mutant background. The resulting strains were grown into M9 medium in the presence of G6P (1mM). In ( B - D ) the sfGFP fluorescence signal normalized to cell density (sfGFP RFU / OD600) was measured. Values indicate the mean ± SEM of n= 3 or 4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test ***p < 0.001, ( B and D ), or unpaired two-tailed t-test ****p < 0.0001 ( C ). ( E ) Functionality of different UhpA variants expressed in S . Typhi in the context of cultured epithelial cell infection. HeLa cells were infected with wild-type S . Typhi or derivative strains expressing the uhpA allele from S. Typhimurium ( uhpA STm ) or an S . Typhi allele, which had been rendered functional by introducing changes based on the S . Typhimurium sequence (i. e. uhpA Glu146Ala,Met168Thr ). Hela cells were infected with the different S. Typhi strains for 6 hours, fixed, stained with DAPI, and examined under a fluorescence microscope. In all the images, brightness and contrast were optimized for each of the individual color channels to maximize visual clarity. Scale bars, 10 μm. ( F ) Alignment of the modeled atomic structures of S . Typhi UhpA (light blue) and S. Typhimurium UhpA (light pink) showing structural variations in the C-terminal portion at the helix-turn-helix motif (boxed). The models were predicted using AlphaFold2 and aligned with Pymol. The amino acid differences between the two structures (Val77Met, Ala146Glu, and Thr168Met) are shown in green.

    Techniques Used: Expressing, Membrane, Fluorescence, Plasmid Preparation, Mutagenesis, Comparison, Functional Assay, Two Tailed Test, Cell Culture, Infection, Sequencing, Staining, Microscopy

    ( A ) Diagram of the G6P Biosensor for detection of glucose-6-phosphate in S . Typhimurium and S . Typhi. The membrane UhpC sensor detects G6P on the extracellular environment and interacts with the membrane UhpB histidine kinase that phosphorylates the cytosolic UhpA response regulator, which activates the P uhpT promoter. The S. Typhi P uhpT promoter was coupled to a sfGFP reporter gene on a plasmid system and transformed into S . Typhimurium and S . Typhi. In the presence of G6P, the cognate promoter P uhpT STy is activated leading to sfGFP expression. The possible steps where the sensing and regulation could be impaired in S . Typhi are noted. ( B ) Test of the response of the S . Typhimurium and S . Typhi G6P biosensors after growth in media containing increasing concentrations of G6P. The sfGFP fluorescense signal over time for the S . Typhimurium (upper graph) and S . Typhi (lower graph) G6P biosensors are shown. Values indicate the mean ± SEM of n= 3 replicates per condition. ( C ) Functionality of the G6P Biosensor in S. Typhimurium and S. Typhi in the context of cultured epithelial cell infection. Fluorescence microscopy of HeLa cells infected with bacterial strains encoding the G6P biosensors. HeLa cells were infected with wild type S . Typhimurium and S . Typhi strains encoding the G6P biosensors for 20 hs, fixed, stained with DAPI, and examined under a fluorescence microscope. For all images, brightness and contrast were adjusted for each of the individual channels to maximize visual clarity using the same parameters for both strains. Scale bars, 10 μm.
    Figure Legend Snippet: ( A ) Diagram of the G6P Biosensor for detection of glucose-6-phosphate in S . Typhimurium and S . Typhi. The membrane UhpC sensor detects G6P on the extracellular environment and interacts with the membrane UhpB histidine kinase that phosphorylates the cytosolic UhpA response regulator, which activates the P uhpT promoter. The S. Typhi P uhpT promoter was coupled to a sfGFP reporter gene on a plasmid system and transformed into S . Typhimurium and S . Typhi. In the presence of G6P, the cognate promoter P uhpT STy is activated leading to sfGFP expression. The possible steps where the sensing and regulation could be impaired in S . Typhi are noted. ( B ) Test of the response of the S . Typhimurium and S . Typhi G6P biosensors after growth in media containing increasing concentrations of G6P. The sfGFP fluorescense signal over time for the S . Typhimurium (upper graph) and S . Typhi (lower graph) G6P biosensors are shown. Values indicate the mean ± SEM of n= 3 replicates per condition. ( C ) Functionality of the G6P Biosensor in S. Typhimurium and S. Typhi in the context of cultured epithelial cell infection. Fluorescence microscopy of HeLa cells infected with bacterial strains encoding the G6P biosensors. HeLa cells were infected with wild type S . Typhimurium and S . Typhi strains encoding the G6P biosensors for 20 hs, fixed, stained with DAPI, and examined under a fluorescence microscope. For all images, brightness and contrast were adjusted for each of the individual channels to maximize visual clarity using the same parameters for both strains. Scale bars, 10 μm.

    Techniques Used: Membrane, Plasmid Preparation, Transformation Assay, Expressing, Cell Culture, Infection, Fluorescence, Microscopy, Staining

    Investigation of the S . Typhi P uhpABC promoter. ( A ) Wild-type and mutant strains of S . Typhimurium containing the P uhpABC promoter from S . Typhi (P uhpABC STy ), or S . Typhi containing the P uhpABC promoter from S . Typhimurium (P uhpABC STm ), both harboring the G6P biosensor (P uhpT -sfGFP) were grown in presence of G6P for 20 hs and analyzed by flow-cytometry. Histograms show the sfGFP fluorescence intensities of individual bacteria for the indicated concentration of G6P. ( B ) Diagram of the transcriptional reporters in which the P uhpABC STm or the P uhpABC STy (containing an 80 bp insert indicated with a green rectangle) promoters drive the expression of a NanoLuc luciferase (NLuc). ( C ) S . Typhimurium and S . Typhi harboring the P uhpABC STm - NLuc or P uhpABC STy - NLuc reporters were grown for 20 hs, lysed and the luminescence was measured on a microplate reader. The signal in Relative Luminescence (RLU) for the indicated strains is shown. No statistically significative difference was observed between the P uhpABC STm - NLuc or P uhpABC STy - NLuc reporters as determined by Anova with Dunnett’s multiple comparisons test.
    Figure Legend Snippet: Investigation of the S . Typhi P uhpABC promoter. ( A ) Wild-type and mutant strains of S . Typhimurium containing the P uhpABC promoter from S . Typhi (P uhpABC STy ), or S . Typhi containing the P uhpABC promoter from S . Typhimurium (P uhpABC STm ), both harboring the G6P biosensor (P uhpT -sfGFP) were grown in presence of G6P for 20 hs and analyzed by flow-cytometry. Histograms show the sfGFP fluorescence intensities of individual bacteria for the indicated concentration of G6P. ( B ) Diagram of the transcriptional reporters in which the P uhpABC STm or the P uhpABC STy (containing an 80 bp insert indicated with a green rectangle) promoters drive the expression of a NanoLuc luciferase (NLuc). ( C ) S . Typhimurium and S . Typhi harboring the P uhpABC STm - NLuc or P uhpABC STy - NLuc reporters were grown for 20 hs, lysed and the luminescence was measured on a microplate reader. The signal in Relative Luminescence (RLU) for the indicated strains is shown. No statistically significative difference was observed between the P uhpABC STm - NLuc or P uhpABC STy - NLuc reporters as determined by Anova with Dunnett’s multiple comparisons test.

    Techniques Used: Mutagenesis, Flow Cytometry, Fluorescence, Bacteria, Concentration Assay, Expressing, Luciferase

    Alignment of portions of the amino acid sequence of the transcriptional regulator UhpA showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.
    Figure Legend Snippet: Alignment of portions of the amino acid sequence of the transcriptional regulator UhpA showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Techniques Used: Sequencing

    Glucose-6-Phosphate transport is impaired in S . Typhi due to a loss-of-function mutation in UhpT. ( A - C ) Growth kinetics of wild-type (WT) S . Typhimurium and S . Typhi or the indicated mutant strains. Bacteria were grown in M9 medium containing 10 mM G6P as the only carbon source. The OD600 values measured at 30 min intervals are shown. Values indicate the mean ± SEM of n=3 replicates per condition. ( D ) Alignment of the modeled atomic structures of S . Typhi (dark blue) and S . Typhimurium (pink) UhpT predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Asp18Asn, Pro27Ser, Gly61Arg, and Ala395Asp) are shown in green.
    Figure Legend Snippet: Glucose-6-Phosphate transport is impaired in S . Typhi due to a loss-of-function mutation in UhpT. ( A - C ) Growth kinetics of wild-type (WT) S . Typhimurium and S . Typhi or the indicated mutant strains. Bacteria were grown in M9 medium containing 10 mM G6P as the only carbon source. The OD600 values measured at 30 min intervals are shown. Values indicate the mean ± SEM of n=3 replicates per condition. ( D ) Alignment of the modeled atomic structures of S . Typhi (dark blue) and S . Typhimurium (pink) UhpT predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Asp18Asn, Pro27Ser, Gly61Arg, and Ala395Asp) are shown in green.

    Techniques Used: Mutagenesis, Bacteria

    Alignment of portions of the amino acid sequence of the antiporter UhpT showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.
    Figure Legend Snippet: Alignment of portions of the amino acid sequence of the antiporter UhpT showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Techniques Used: Sequencing

    Intracellular growth of different S . Typhimurium or S. Typhi uhp mutants. Cultured HeLa cells were infected with indicated bacterial strains and the CFU 2 and 8 hours after infection were determined by plate dilutions. Values represent the ratio between the CFUs measured at 8 and 2 hours post infection and are the mean ± SEM of n= 7 - 10 replicates per condition. No statistically significative difference was observed as determined using Anova with Dunnett’s multiple comparisons test.
    Figure Legend Snippet: Intracellular growth of different S . Typhimurium or S. Typhi uhp mutants. Cultured HeLa cells were infected with indicated bacterial strains and the CFU 2 and 8 hours after infection were determined by plate dilutions. Values represent the ratio between the CFUs measured at 8 and 2 hours post infection and are the mean ± SEM of n= 7 - 10 replicates per condition. No statistically significative difference was observed as determined using Anova with Dunnett’s multiple comparisons test.

    Techniques Used: Cell Culture, Infection

    ( A ) Diagram of 3PG sensing, transcription regulation, and transport in Salmonella enterica . PgtC senses 3PG (red trapezoid) triggering its interaction with the PgtB histidine kinase/phosphatase that phosphorylates the PgtA response regulator. Phosphorylated PgtA, in turn, activates the P gtpP promoter leading to the expression of the PgtP transporter. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . Typhimurium and S . Typhi strains encoding the 3PG biosensors after growth in LB medium containing 3PG (1 mM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of 3PG is shown. Values indicate the mean ± SEM of n=3 replicates per condition. ( C ) Investigation of the functionality of the S. Typhi PgtA and PgtB regulatory proteins. The indicated S . Typhi genes were swapped for their S . Typhimurium homologs and the resulting mutant strains were tested for their ability to sense 3PG with the P pgtP-sfGFP biosensor. Bacteria were grown in LB medium for 20 h in the presence or absence of 3PG (1mM), and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values in ( B ) and ( C ) are the mean ± SEM of n=3-4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test. ***p < 0.001. ( D ) Alignment of the modeled atomic structures of S . Typhi (blue) and S . Typhimurium (pink) PgtA predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Glu141Asp, Gly173Asp, and His223Tyr) are shown in green. ( E and F ) Growth kinetics of wild type (WT) S . Typhimurium and S . Typhi or the indicated mutant strains. Bacteria were grown in M9 + 0.05% casamino acids medium containing 10 mM 3PG as the only carbon source. The OD600 values measured at 30 min intervals are shown. Values indicate the mean ± SEM of n=3 replicates per condition.
    Figure Legend Snippet: ( A ) Diagram of 3PG sensing, transcription regulation, and transport in Salmonella enterica . PgtC senses 3PG (red trapezoid) triggering its interaction with the PgtB histidine kinase/phosphatase that phosphorylates the PgtA response regulator. Phosphorylated PgtA, in turn, activates the P gtpP promoter leading to the expression of the PgtP transporter. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . Typhimurium and S . Typhi strains encoding the 3PG biosensors after growth in LB medium containing 3PG (1 mM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of 3PG is shown. Values indicate the mean ± SEM of n=3 replicates per condition. ( C ) Investigation of the functionality of the S. Typhi PgtA and PgtB regulatory proteins. The indicated S . Typhi genes were swapped for their S . Typhimurium homologs and the resulting mutant strains were tested for their ability to sense 3PG with the P pgtP-sfGFP biosensor. Bacteria were grown in LB medium for 20 h in the presence or absence of 3PG (1mM), and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values in ( B ) and ( C ) are the mean ± SEM of n=3-4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test. ***p < 0.001. ( D ) Alignment of the modeled atomic structures of S . Typhi (blue) and S . Typhimurium (pink) PgtA predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Glu141Asp, Gly173Asp, and His223Tyr) are shown in green. ( E and F ) Growth kinetics of wild type (WT) S . Typhimurium and S . Typhi or the indicated mutant strains. Bacteria were grown in M9 + 0.05% casamino acids medium containing 10 mM 3PG as the only carbon source. The OD600 values measured at 30 min intervals are shown. Values indicate the mean ± SEM of n=3 replicates per condition.

    Techniques Used: Expressing, Fluorescence, Mutagenesis, Bacteria

    Investigation of the functionality of the PgtA and PgtB regulatory proteins. The S. Typhi pgtA and pgtB genes were swapped for their S . Typhimurium homologs in the 3PG biosensor plasmid. The resulting constructs were introduced into the S . Typhi ΔpgtAB mutant background and tested on the presence or absence of 3PG at 2 mM. The fluorescent gene expression normalized to cell density (sfGFP RFU / OD600) is shown. Values indicate the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001.
    Figure Legend Snippet: Investigation of the functionality of the PgtA and PgtB regulatory proteins. The S. Typhi pgtA and pgtB genes were swapped for their S . Typhimurium homologs in the 3PG biosensor plasmid. The resulting constructs were introduced into the S . Typhi ΔpgtAB mutant background and tested on the presence or absence of 3PG at 2 mM. The fluorescent gene expression normalized to cell density (sfGFP RFU / OD600) is shown. Values indicate the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001.

    Techniques Used: Plasmid Preparation, Construct, Mutagenesis, Expressing

    Alignment of portions of the amino acid sequence of the transcriptional regulator PgtA showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.
    Figure Legend Snippet: Alignment of portions of the amino acid sequence of the transcriptional regulator PgtA showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Techniques Used: Sequencing

    Investigation of the functionality of S . Typhi PgtA amino acid substitutions. ( A ) S . Typhi PgtA amino acids at the indicated positions were substituted for the equivalent S . Typhimurium residues in the plasmid backbone containing the 3PG biosensor. The resulting constructs were introduced into the S . Typhi ΔpgtAB mutant background and the transcriptional response was tested in the presence or absence of 3PG at 2mM. Values represent the fluorescence signal normalized to cell density (sfGFP RFU / OD600) and are the mean ± SEM of n= 4 replicates per condition. ( B ) The S. Typhi pgtA or pgtB were individually swapped for their S. Typhimurium homologues (S. Typhi pgtA Stm and S. Typhi pgtB Stm ). The resulting strains and an S . Typhi strain encoding the pgtA Asp173Gly , all encoding the 3PG biosensor, were tested for their transcriptional response to 3PG at 10 mM. The fluorescence signal normalized to cell density (sfGFP RFU / OD600) is shown. Values indicate the mean ± SEM of n= 3 replicates per condition. For all panels, asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001.
    Figure Legend Snippet: Investigation of the functionality of S . Typhi PgtA amino acid substitutions. ( A ) S . Typhi PgtA amino acids at the indicated positions were substituted for the equivalent S . Typhimurium residues in the plasmid backbone containing the 3PG biosensor. The resulting constructs were introduced into the S . Typhi ΔpgtAB mutant background and the transcriptional response was tested in the presence or absence of 3PG at 2mM. Values represent the fluorescence signal normalized to cell density (sfGFP RFU / OD600) and are the mean ± SEM of n= 4 replicates per condition. ( B ) The S. Typhi pgtA or pgtB were individually swapped for their S. Typhimurium homologues (S. Typhi pgtA Stm and S. Typhi pgtB Stm ). The resulting strains and an S . Typhi strain encoding the pgtA Asp173Gly , all encoding the 3PG biosensor, were tested for their transcriptional response to 3PG at 10 mM. The fluorescence signal normalized to cell density (sfGFP RFU / OD600) is shown. Values indicate the mean ± SEM of n= 3 replicates per condition. For all panels, asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001.

    Techniques Used: Plasmid Preparation, Construct, Mutagenesis, Fluorescence

    ( A ) Diagram of glucarate and galactarate sensing, transcription regulation, transport, and metabolism in Salmonella enterica . Glucarate (green circles) and galactarate (yellow squares) are transported into the bacterial cell by the permease GudT and converted to 5-dehydro-4-deoxy-d-glucarate (orange star) by the dehydratases GudD and GarD, respectively. 5-dehydro-4-deoxy-d-glucarate is further metabolized into D-glycerate (red triangles) by the aldolase GarL and the reductase GarR. The expression of these genes is controlled by the transcription factor CdaR, which senses glucarate, galactarate and D-glycerate in the cytosol and activates the P gudTYD , P garD and P garLRK promoters. The potential steps where the sensing, regulation, transport, or metabolism could be impaired in S . Typhi are noted. ( B and C ) Wild-type S . Typhimurium and S. Typhi or the indicated mutant strains all harboring the PgarL_ sfGFP transcriptional reporter were grown in LB medium containing glucarate, galactarate or glycerate (as indicated) at 10 mM (or water as a negative control) and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values indicate the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences determined using Anova with Tukey’s multiple comparisons test. ***p < 0.001. ( D ) Growth kinetics of wild-type S. Typhimurium and S. Typhi or their isogenic mutants ΔgudT, ΔgudT and gudT as indicated. Growth was monitored in M9 + 0.05% casamino acids medium containing 10 mM glucarate as the only carbon source. The OD600 measured at 30 min intervals are shown and values represent the mean ± SEM of n=3-4 replicates per condition. ( E ) Alignment of the modeled atomic structures of S . Typhi (Teal) and S . Typhimurium (Beige) GudT predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Ser321Phe and Ala357Val) are shown in green. ( F ) Growth kinetics of S. Typhi gudT STm expressing plasmid-borne gudD STm and gudD STy , as indicated. Growth was monitored in M9 medium + 0.05% casamino acids containing 10 mM glucarate as the only carbon source. The OD600 measured at 30 min intervals are shown and values represent the mean ± SEM of n=3-4 replicates per condition.
    Figure Legend Snippet: ( A ) Diagram of glucarate and galactarate sensing, transcription regulation, transport, and metabolism in Salmonella enterica . Glucarate (green circles) and galactarate (yellow squares) are transported into the bacterial cell by the permease GudT and converted to 5-dehydro-4-deoxy-d-glucarate (orange star) by the dehydratases GudD and GarD, respectively. 5-dehydro-4-deoxy-d-glucarate is further metabolized into D-glycerate (red triangles) by the aldolase GarL and the reductase GarR. The expression of these genes is controlled by the transcription factor CdaR, which senses glucarate, galactarate and D-glycerate in the cytosol and activates the P gudTYD , P garD and P garLRK promoters. The potential steps where the sensing, regulation, transport, or metabolism could be impaired in S . Typhi are noted. ( B and C ) Wild-type S . Typhimurium and S. Typhi or the indicated mutant strains all harboring the PgarL_ sfGFP transcriptional reporter were grown in LB medium containing glucarate, galactarate or glycerate (as indicated) at 10 mM (or water as a negative control) and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values indicate the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences determined using Anova with Tukey’s multiple comparisons test. ***p < 0.001. ( D ) Growth kinetics of wild-type S. Typhimurium and S. Typhi or their isogenic mutants ΔgudT, ΔgudT and gudT as indicated. Growth was monitored in M9 + 0.05% casamino acids medium containing 10 mM glucarate as the only carbon source. The OD600 measured at 30 min intervals are shown and values represent the mean ± SEM of n=3-4 replicates per condition. ( E ) Alignment of the modeled atomic structures of S . Typhi (Teal) and S . Typhimurium (Beige) GudT predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Ser321Phe and Ala357Val) are shown in green. ( F ) Growth kinetics of S. Typhi gudT STm expressing plasmid-borne gudD STm and gudD STy , as indicated. Growth was monitored in M9 medium + 0.05% casamino acids containing 10 mM glucarate as the only carbon source. The OD600 measured at 30 min intervals are shown and values represent the mean ± SEM of n=3-4 replicates per condition.

    Techniques Used: Expressing, Mutagenesis, Negative Control, Fluorescence, Plasmid Preparation

    ( A ) ( A ) Diagram of the biosensor for the detection of glucarate, galactarate and glycerate based on the CdaR transcription factor and the cognate promoters P gudTYD , P garD and P garLRK driving expression of sfGFP . ( B ) The indicated wild-type strains of S . Typhimurium (S. Tm) and S . Typhi (S. Ty) harboring the indicated biosensors were tested in LB medium containing glucarate or galactarate (1 mM), as indicated, or water as a negative control, and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. ( C ) The indicated S . Typhimurium and S . Typhi mutant strains all harboring the P garL _ sfGFP transcriptional reporter were grown in LB medium containing galactarate at 10mM, and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values are the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001. ( D ) Growth kinetics of wild-type S. Typhimurium and S. Typhi or their isogenic mutants ΔgudT, ΔgudT and gudT STm as indicated. Growth was monitored in M9 +0.05% casamino acids medium containing 10 mM galactarate as the only carbon source. The OD600 was measured at 30 min intervals and values represent the mean ± SEM of n=3-4 replicates per condition.
    Figure Legend Snippet: ( A ) ( A ) Diagram of the biosensor for the detection of glucarate, galactarate and glycerate based on the CdaR transcription factor and the cognate promoters P gudTYD , P garD and P garLRK driving expression of sfGFP . ( B ) The indicated wild-type strains of S . Typhimurium (S. Tm) and S . Typhi (S. Ty) harboring the indicated biosensors were tested in LB medium containing glucarate or galactarate (1 mM), as indicated, or water as a negative control, and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. ( C ) The indicated S . Typhimurium and S . Typhi mutant strains all harboring the P garL _ sfGFP transcriptional reporter were grown in LB medium containing galactarate at 10mM, and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values are the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001. ( D ) Growth kinetics of wild-type S. Typhimurium and S. Typhi or their isogenic mutants ΔgudT, ΔgudT and gudT STm as indicated. Growth was monitored in M9 +0.05% casamino acids medium containing 10 mM galactarate as the only carbon source. The OD600 was measured at 30 min intervals and values represent the mean ± SEM of n=3-4 replicates per condition.

    Techniques Used: Expressing, Negative Control, Fluorescence, Mutagenesis

    Alignment of portions of the amino acid sequence of the glucarate/galactarate GudT transporter showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.
    Figure Legend Snippet: Alignment of portions of the amino acid sequence of the glucarate/galactarate GudT transporter showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Techniques Used: Sequencing

    Alignment of portions of the amino acid sequences of the GudD (A) and GarD (B) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.
    Figure Legend Snippet: Alignment of portions of the amino acid sequences of the GudD (A) and GarD (B) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Techniques Used:

    Growth kinetics of S . Typhimurium, S . Typhi and S . Paratyphi A in minimal medium containing glucose-6-phosphate, 3-phosphoglyceric acid, glucarate, tartrate, aspartate, or L-histidine as the sole carbon source. Bacteria were grown in M9 medium supplemented with casamino acids (0.05%) and containing 20 mM of the different metabolites and the OD600 was measured at 30 min intervals. Values indicate the mean ± SEM of n= 3-4 replicates per condition.
    Figure Legend Snippet: Growth kinetics of S . Typhimurium, S . Typhi and S . Paratyphi A in minimal medium containing glucose-6-phosphate, 3-phosphoglyceric acid, glucarate, tartrate, aspartate, or L-histidine as the sole carbon source. Bacteria were grown in M9 medium supplemented with casamino acids (0.05%) and containing 20 mM of the different metabolites and the OD600 was measured at 30 min intervals. Values indicate the mean ± SEM of n= 3-4 replicates per condition.

    Techniques Used: Bacteria

    Alignment of portions of the amino acid sequence of S . Typhi STY3536 ( A ), STY3537 ( B ), and STY3538 ( C ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.
    Figure Legend Snippet: Alignment of portions of the amino acid sequence of S . Typhi STY3536 ( A ), STY3537 ( B ), and STY3538 ( C ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Techniques Used: Sequencing

    Alignment of portions of the amino acid sequence of S . Typhi DcuS ( A ), Tar ( B ), and AsnB ( C ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.
    Figure Legend Snippet: Alignment of portions of the amino acid sequence of S . Typhi DcuS ( A ), Tar ( B ), and AsnB ( C ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Techniques Used: Sequencing

    Alignment of portions of the amino acid sequence of S . Typhi HutU ( A ) and HutC ( B ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.
    Figure Legend Snippet: Alignment of portions of the amino acid sequence of S . Typhi HutU ( A ) and HutC ( B ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Techniques Used: Sequencing

    reference strain e coli atcc 25922  (ATCC)


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    reference strain escherichia  (ATCC)


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    ATCC reference strain atcc 49226
    a TEM of gonococcal OMV secretion during invasion and exocytosis of HeLa cells. Images are representative of 2 independent experiments. b Enhanced intracellular survival and exocytosis of gonococcal Δ vacJ mutant in gentamicin protection assays with HeLa cells. Data are mean ± s.d. of log-normalized colony forming units (CFU) per well and relative differences in survival between WT and Δ vacJ are provided within the bars; n = 4 independent experiments, two-way ANOVA with posthoc Bonferroni test. c Δ vacJ mutant shows reduced LAMP1-colocalization after a one-hour challenge of HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 53 cells, unpaired two-tailed t -test. d TEM images of purified gonococcal OMVs. Images are representative of 4 (ATCC 49226) and 3 (ZJXSH86) independent experiments. e LC3 Western blots show induction of autophagy flux in HeLa cells stimulated with gonococcal OMVs from strains <t>ATCC</t> <t>49226</t> and ZJXSH86. f Gonococcal OMVs induce accumulation of LC3 puncta in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Vehicle Mock-ZJXSH86 OMVs: P < 10 −15 ). g OMVs from the gonococcal Δ ldcA mutant induce accumulation of LC3 puncta in HeLa cells, but largely lost colocalization with LC3 puncta. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, one-way ANOVA with post-hoc Tukey test for quantification of LC3 puncta (Mock-WT OMVs: P = 3 × 10 −14 ; Mock-Δ ldcA OMVs: P = 6 × 10 −14 ), unpaired two-tailed t -test for quantification of LC3-positive OMVs ( P = 5 × 10 −13 ). h LC3 Western blots of OMV-stimulated HeLa cells show reduced accumulation of LC3 for the Δ ldcA mutant. Cells in c , f , g are from 3 independent experiments. Western blots in e , h are representative of 3 independent experiments. Source data are provided as a Source Data file.
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    ATCC reference strains b longum subsp longum atcc 15707
    a TEM of gonococcal OMV secretion during invasion and exocytosis of HeLa cells. Images are representative of 2 independent experiments. b Enhanced intracellular survival and exocytosis of gonococcal Δ vacJ mutant in gentamicin protection assays with HeLa cells. Data are mean ± s.d. of log-normalized colony forming units (CFU) per well and relative differences in survival between WT and Δ vacJ are provided within the bars; n = 4 independent experiments, two-way ANOVA with posthoc Bonferroni test. c Δ vacJ mutant shows reduced LAMP1-colocalization after a one-hour challenge of HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 53 cells, unpaired two-tailed t -test. d TEM images of purified gonococcal OMVs. Images are representative of 4 (ATCC 49226) and 3 (ZJXSH86) independent experiments. e LC3 Western blots show induction of autophagy flux in HeLa cells stimulated with gonococcal OMVs from strains <t>ATCC</t> <t>49226</t> and ZJXSH86. f Gonococcal OMVs induce accumulation of LC3 puncta in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Vehicle Mock-ZJXSH86 OMVs: P < 10 −15 ). g OMVs from the gonococcal Δ ldcA mutant induce accumulation of LC3 puncta in HeLa cells, but largely lost colocalization with LC3 puncta. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, one-way ANOVA with post-hoc Tukey test for quantification of LC3 puncta (Mock-WT OMVs: P = 3 × 10 −14 ; Mock-Δ ldcA OMVs: P = 6 × 10 −14 ), unpaired two-tailed t -test for quantification of LC3-positive OMVs ( P = 5 × 10 −13 ). h LC3 Western blots of OMV-stimulated HeLa cells show reduced accumulation of LC3 for the Δ ldcA mutant. Cells in c , f , g are from 3 independent experiments. Western blots in e , h are representative of 3 independent experiments. Source data are provided as a Source Data file.
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    ATCC reference s typhimurium strain lt2
    Glucose-6-phosphate sensing and transcriptional regulation are impaired in Salmonella Typhi. ( A ) Diagram of the glucose-6-phosphate (G6P) sensing, transcription regulation, and transport in Salmonella enterica . G6P (purple hexagon) is imported into the bacterial cell by the UhpT antiporter, whose expression is strictly controlled by an unconventional two-component regulatory system composed of the membrane-associated UhpC sensor and UhpB kinase/phosphatase, and the cytoplasmic response regulator UhpA. In this system, UhpC directly senses G6P triggering a conformational change in the interacting kinase UhpB, which in turn phosphorylates and activates the response regulator UhpA. Phosphorylation increases the affinity of UhpA for the uhpT promoter (P uhpT ) thus initiating transcription. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . <t>Typhimurium</t> and S . Typhi G6P biosensors after growth in LB medium containing G6P (500 µM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of G6P is shown. ( C ) Investigation of the functionality of UhpA, UhpB, and UhpC regulatory proteins. Each S . Typhi gene was swapped for its S . Typhimurium homolog in the plasmid backbone containing the G6P biosensor as indicated in the diagram. The resulting plasmids were then introduced into the S . Typhi or S. Typhimurium ΔuhpABC mutant backgrounds, and the resulting strains were grown in M9 medium in the presence or absence of G6P (1mM). ( D ) Investigation of the functionality of each of the different uhpA mutations present in S . Typhi in comparison of its functional homolog in S . Typhimurium. Plasmids encoding the G6P biosensor and expressing S . Typhi UhpA mutants in which residues had been individually changed to the amino acid present in its S . Typhimurium homolog as indicated (i. e. UhpA Met77Val , UhpA Glu146Ala , or UhpA Met168Thr ) were introduced into the S. Typhi Δ uhpABC mutant background. The resulting strains were grown into M9 medium in the presence of G6P (1mM). In ( B - D ) the sfGFP fluorescence signal normalized to cell density (sfGFP RFU / OD600) was measured. Values indicate the mean ± SEM of n= 3 or 4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test ***p < 0.001, ( B and D ), or unpaired two-tailed t-test ****p < 0.0001 ( C ). ( E ) Functionality of different UhpA variants expressed in S . Typhi in the context of cultured epithelial cell infection. HeLa cells were infected with wild-type S . Typhi or derivative strains expressing the uhpA allele from S. Typhimurium ( uhpA STm ) or an S . Typhi allele, which had been rendered functional by introducing changes based on the S . Typhimurium sequence (i. e. uhpA Glu146Ala,Met168Thr ). Hela cells were infected with the different S. Typhi strains for 6 hours, fixed, stained with DAPI, and examined under a fluorescence microscope. In all the images, brightness and contrast were optimized for each of the individual color channels to maximize visual clarity. Scale bars, 10 μm. ( F ) Alignment of the modeled atomic structures of S . Typhi UhpA (light blue) and S. Typhimurium UhpA (light pink) showing structural variations in the C-terminal portion at the helix-turn-helix motif (boxed). The models were predicted using AlphaFold2 and aligned with Pymol. The amino acid differences between the two structures (Val77Met, Ala146Glu, and Thr168Met) are shown in green.
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    ATCC reference strain escherichia
    Glucose-6-phosphate sensing and transcriptional regulation are impaired in Salmonella Typhi. ( A ) Diagram of the glucose-6-phosphate (G6P) sensing, transcription regulation, and transport in Salmonella enterica . G6P (purple hexagon) is imported into the bacterial cell by the UhpT antiporter, whose expression is strictly controlled by an unconventional two-component regulatory system composed of the membrane-associated UhpC sensor and UhpB kinase/phosphatase, and the cytoplasmic response regulator UhpA. In this system, UhpC directly senses G6P triggering a conformational change in the interacting kinase UhpB, which in turn phosphorylates and activates the response regulator UhpA. Phosphorylation increases the affinity of UhpA for the uhpT promoter (P uhpT ) thus initiating transcription. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . <t>Typhimurium</t> and S . Typhi G6P biosensors after growth in LB medium containing G6P (500 µM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of G6P is shown. ( C ) Investigation of the functionality of UhpA, UhpB, and UhpC regulatory proteins. Each S . Typhi gene was swapped for its S . Typhimurium homolog in the plasmid backbone containing the G6P biosensor as indicated in the diagram. The resulting plasmids were then introduced into the S . Typhi or S. Typhimurium ΔuhpABC mutant backgrounds, and the resulting strains were grown in M9 medium in the presence or absence of G6P (1mM). ( D ) Investigation of the functionality of each of the different uhpA mutations present in S . Typhi in comparison of its functional homolog in S . Typhimurium. Plasmids encoding the G6P biosensor and expressing S . Typhi UhpA mutants in which residues had been individually changed to the amino acid present in its S . Typhimurium homolog as indicated (i. e. UhpA Met77Val , UhpA Glu146Ala , or UhpA Met168Thr ) were introduced into the S. Typhi Δ uhpABC mutant background. The resulting strains were grown into M9 medium in the presence of G6P (1mM). In ( B - D ) the sfGFP fluorescence signal normalized to cell density (sfGFP RFU / OD600) was measured. Values indicate the mean ± SEM of n= 3 or 4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test ***p < 0.001, ( B and D ), or unpaired two-tailed t-test ****p < 0.0001 ( C ). ( E ) Functionality of different UhpA variants expressed in S . Typhi in the context of cultured epithelial cell infection. HeLa cells were infected with wild-type S . Typhi or derivative strains expressing the uhpA allele from S. Typhimurium ( uhpA STm ) or an S . Typhi allele, which had been rendered functional by introducing changes based on the S . Typhimurium sequence (i. e. uhpA Glu146Ala,Met168Thr ). Hela cells were infected with the different S. Typhi strains for 6 hours, fixed, stained with DAPI, and examined under a fluorescence microscope. In all the images, brightness and contrast were optimized for each of the individual color channels to maximize visual clarity. Scale bars, 10 μm. ( F ) Alignment of the modeled atomic structures of S . Typhi UhpA (light blue) and S. Typhimurium UhpA (light pink) showing structural variations in the C-terminal portion at the helix-turn-helix motif (boxed). The models were predicted using AlphaFold2 and aligned with Pymol. The amino acid differences between the two structures (Val77Met, Ala146Glu, and Thr168Met) are shown in green.
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    a TEM of gonococcal OMV secretion during invasion and exocytosis of HeLa cells. Images are representative of 2 independent experiments. b Enhanced intracellular survival and exocytosis of gonococcal Δ vacJ mutant in gentamicin protection assays with HeLa cells. Data are mean ± s.d. of log-normalized colony forming units (CFU) per well and relative differences in survival between WT and Δ vacJ are provided within the bars; n = 4 independent experiments, two-way ANOVA with posthoc Bonferroni test. c Δ vacJ mutant shows reduced LAMP1-colocalization after a one-hour challenge of HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 53 cells, unpaired two-tailed t -test. d TEM images of purified gonococcal OMVs. Images are representative of 4 (ATCC 49226) and 3 (ZJXSH86) independent experiments. e LC3 Western blots show induction of autophagy flux in HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86. f Gonococcal OMVs induce accumulation of LC3 puncta in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Vehicle Mock-ZJXSH86 OMVs: P < 10 −15 ). g OMVs from the gonococcal Δ ldcA mutant induce accumulation of LC3 puncta in HeLa cells, but largely lost colocalization with LC3 puncta. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, one-way ANOVA with post-hoc Tukey test for quantification of LC3 puncta (Mock-WT OMVs: P = 3 × 10 −14 ; Mock-Δ ldcA OMVs: P = 6 × 10 −14 ), unpaired two-tailed t -test for quantification of LC3-positive OMVs ( P = 5 × 10 −13 ). h LC3 Western blots of OMV-stimulated HeLa cells show reduced accumulation of LC3 for the Δ ldcA mutant. Cells in c , f , g are from 3 independent experiments. Western blots in e , h are representative of 3 independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

    doi: 10.1038/s41467-024-45961-1

    Figure Lengend Snippet: a TEM of gonococcal OMV secretion during invasion and exocytosis of HeLa cells. Images are representative of 2 independent experiments. b Enhanced intracellular survival and exocytosis of gonococcal Δ vacJ mutant in gentamicin protection assays with HeLa cells. Data are mean ± s.d. of log-normalized colony forming units (CFU) per well and relative differences in survival between WT and Δ vacJ are provided within the bars; n = 4 independent experiments, two-way ANOVA with posthoc Bonferroni test. c Δ vacJ mutant shows reduced LAMP1-colocalization after a one-hour challenge of HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 53 cells, unpaired two-tailed t -test. d TEM images of purified gonococcal OMVs. Images are representative of 4 (ATCC 49226) and 3 (ZJXSH86) independent experiments. e LC3 Western blots show induction of autophagy flux in HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86. f Gonococcal OMVs induce accumulation of LC3 puncta in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Vehicle Mock-ZJXSH86 OMVs: P < 10 −15 ). g OMVs from the gonococcal Δ ldcA mutant induce accumulation of LC3 puncta in HeLa cells, but largely lost colocalization with LC3 puncta. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, one-way ANOVA with post-hoc Tukey test for quantification of LC3 puncta (Mock-WT OMVs: P = 3 × 10 −14 ; Mock-Δ ldcA OMVs: P = 6 × 10 −14 ), unpaired two-tailed t -test for quantification of LC3-positive OMVs ( P = 5 × 10 −13 ). h LC3 Western blots of OMV-stimulated HeLa cells show reduced accumulation of LC3 for the Δ ldcA mutant. Cells in c , f , g are from 3 independent experiments. Western blots in e , h are representative of 3 independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Therefore, we generated a gonococcal Δ vacJ deletion mutant of the international reference strain ATCC 49226, which indeed showed increased secretion of OMVs (Supplementary Fig. ).

    Techniques: Mutagenesis, Two Tailed Test, Purification, Western Blot

    a Western blots of OMV-stimulated HeLa cells show specific autophagosome/lysosome-dependent degradation of mitochondrial marker proteins (TOM20 and TIM23), but not of Golgi (GM130) or endoplasmic reticulum (PDI). Western blots are representative of 3 independent experiments. b DiO-labeled gonococcal OMVs from strain ATCC 49226 colocalize with MitoBright-labeled mitochondria in HeLa cells. Scale bar, 5 μm. c Live HeLa cell microscopy and 3D image reconstruction shows OMVs remain associated with Cascade Blue-labeled endosomes when delivered to MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. d Gonococcal OMVs from strain ATCC 49226 dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P < 10 −15 . e TEM of HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86 show mitochondrial disruption and capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, one-way ANOVA with post-hoc Tukey test for mitochondrial disruption (Mock-ATCC 49226 OMVs: P = 2 × 10 −11 ; Mock-ZJXSH86 OMVs: P = 2 × 10 −11 ), Kruskal–Wallis with posthoc Dunn test for mitochondria in mitophagy-like structures (Mock-ATCC 49226 OMVs: P = 1 × 10 −6 ; Mock-ZJXSH86 OMVs: P = 7 × 10 −6 ). f Quantitative real-time PCR showing a reduced mitochondrial to genomic DNA ratio in ATCC 49226 OMV-stimulated HeLa cells. Data are mean ± s.d.; n = 3 independent biological replicates, one-way ANOVA with post-hoc Tukey test. g Increased HSP60 and LAMP1 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P = 6 × 10 −9 . h Increased HSP60 and LC3 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test (LC3 puncta Mock-OMVs: P = 7 × 10 −12 ; LC3 colocalized HSP60 Mock-ZJXSH86 OMVs: P = 4 × 10 −15 ). Cells in d , e , g , h are from 3 independent experiments. Images in b , c are representative of 3 independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

    doi: 10.1038/s41467-024-45961-1

    Figure Lengend Snippet: a Western blots of OMV-stimulated HeLa cells show specific autophagosome/lysosome-dependent degradation of mitochondrial marker proteins (TOM20 and TIM23), but not of Golgi (GM130) or endoplasmic reticulum (PDI). Western blots are representative of 3 independent experiments. b DiO-labeled gonococcal OMVs from strain ATCC 49226 colocalize with MitoBright-labeled mitochondria in HeLa cells. Scale bar, 5 μm. c Live HeLa cell microscopy and 3D image reconstruction shows OMVs remain associated with Cascade Blue-labeled endosomes when delivered to MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. d Gonococcal OMVs from strain ATCC 49226 dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P < 10 −15 . e TEM of HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86 show mitochondrial disruption and capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, one-way ANOVA with post-hoc Tukey test for mitochondrial disruption (Mock-ATCC 49226 OMVs: P = 2 × 10 −11 ; Mock-ZJXSH86 OMVs: P = 2 × 10 −11 ), Kruskal–Wallis with posthoc Dunn test for mitochondria in mitophagy-like structures (Mock-ATCC 49226 OMVs: P = 1 × 10 −6 ; Mock-ZJXSH86 OMVs: P = 7 × 10 −6 ). f Quantitative real-time PCR showing a reduced mitochondrial to genomic DNA ratio in ATCC 49226 OMV-stimulated HeLa cells. Data are mean ± s.d.; n = 3 independent biological replicates, one-way ANOVA with post-hoc Tukey test. g Increased HSP60 and LAMP1 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P = 6 × 10 −9 . h Increased HSP60 and LC3 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test (LC3 puncta Mock-OMVs: P = 7 × 10 −12 ; LC3 colocalized HSP60 Mock-ZJXSH86 OMVs: P = 4 × 10 −15 ). Cells in d , e , g , h are from 3 independent experiments. Images in b , c are representative of 3 independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Therefore, we generated a gonococcal Δ vacJ deletion mutant of the international reference strain ATCC 49226, which indeed showed increased secretion of OMVs (Supplementary Fig. ).

    Techniques: Western Blot, Marker, Labeling, Microscopy, Fluorescence, Membrane, Two Tailed Test, Disruption, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    a TEM of HeLa cells expressing gonococcal PorB from strain ATCC 49226 show mitochondrial capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, two-tailed Mann–Whitney test, P = 2 × 10 −10 . b Western blots showing degradation of mitochondrial proteins TOM20 and TIM23 in HeLa cells expressing gonococcal PorB from strains ATCC 49226 and ZJXSH86, but not for PorB from Neisseria mucosa . c Western blots showing that gonococcal OMVs expressing PorB from N. mucosa lost the ability to induce degradation of TOM20 and TIM23 in HeLa cells. d Gonococcal OMVs expressing PorB from N. mucosa lost the ability to dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 55 cells from 4 independent experiments, Kruskal–Wallis with posthoc Dunn test (Mock-N.g. PorB: P = 8 × 10 −13 ; N.g. PorB-N.m. PorB: P < 10 −15 ). e Flow cytom e try analysis of TMRM fluorescence intensity in HeLa cells stimulated with gonococcal OMVs expressing gonococcal PorB or PorB from N. mucosa . f Gonococcal PorB structure (pdb entry 4AUI) and sequence of PorB from strain ATCC 49226. Lysines are indicated in blue, or in magenta when located within the PorB channel and associated with ATP (orange) binding. g Western blots showing reduced degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q. h Reduced HSP60 and LC3 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 2 × 10 −9 ; WT PorB-PorB K117Q: P = 1 × 10 −5 ). i Reduced HSP60 and LAMP1 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 9 × 10 −8 ). j Western blots of HeLa 4KO cells showing p62 expression restores PorB K117Q-dependent degradation of TOM20 and TIM23, while expression of OPTN or NDP52 restores PorB K5-dependent degradation of TOM20 and TIM23. k Western blots showing impaired degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q, K5 or K null . l Western blots showing that knock-down of PINK1 in HeLa cells inhibits PorB K5-dependent degradation of TOM20 and TIM23, but not for PorB K117Q. Cells in a , h , i are from 3 independent experiments. Western blots in b , c , g , j , k , l are representative of 3 independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

    doi: 10.1038/s41467-024-45961-1

    Figure Lengend Snippet: a TEM of HeLa cells expressing gonococcal PorB from strain ATCC 49226 show mitochondrial capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, two-tailed Mann–Whitney test, P = 2 × 10 −10 . b Western blots showing degradation of mitochondrial proteins TOM20 and TIM23 in HeLa cells expressing gonococcal PorB from strains ATCC 49226 and ZJXSH86, but not for PorB from Neisseria mucosa . c Western blots showing that gonococcal OMVs expressing PorB from N. mucosa lost the ability to induce degradation of TOM20 and TIM23 in HeLa cells. d Gonococcal OMVs expressing PorB from N. mucosa lost the ability to dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 55 cells from 4 independent experiments, Kruskal–Wallis with posthoc Dunn test (Mock-N.g. PorB: P = 8 × 10 −13 ; N.g. PorB-N.m. PorB: P < 10 −15 ). e Flow cytom e try analysis of TMRM fluorescence intensity in HeLa cells stimulated with gonococcal OMVs expressing gonococcal PorB or PorB from N. mucosa . f Gonococcal PorB structure (pdb entry 4AUI) and sequence of PorB from strain ATCC 49226. Lysines are indicated in blue, or in magenta when located within the PorB channel and associated with ATP (orange) binding. g Western blots showing reduced degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q. h Reduced HSP60 and LC3 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 2 × 10 −9 ; WT PorB-PorB K117Q: P = 1 × 10 −5 ). i Reduced HSP60 and LAMP1 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 9 × 10 −8 ). j Western blots of HeLa 4KO cells showing p62 expression restores PorB K117Q-dependent degradation of TOM20 and TIM23, while expression of OPTN or NDP52 restores PorB K5-dependent degradation of TOM20 and TIM23. k Western blots showing impaired degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q, K5 or K null . l Western blots showing that knock-down of PINK1 in HeLa cells inhibits PorB K5-dependent degradation of TOM20 and TIM23, but not for PorB K117Q. Cells in a , h , i are from 3 independent experiments. Western blots in b , c , g , j , k , l are representative of 3 independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Therefore, we generated a gonococcal Δ vacJ deletion mutant of the international reference strain ATCC 49226, which indeed showed increased secretion of OMVs (Supplementary Fig. ).

    Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Western Blot, Membrane, Fluorescence, Sequencing, Binding Assay

    Glucose-6-phosphate sensing and transcriptional regulation are impaired in Salmonella Typhi. ( A ) Diagram of the glucose-6-phosphate (G6P) sensing, transcription regulation, and transport in Salmonella enterica . G6P (purple hexagon) is imported into the bacterial cell by the UhpT antiporter, whose expression is strictly controlled by an unconventional two-component regulatory system composed of the membrane-associated UhpC sensor and UhpB kinase/phosphatase, and the cytoplasmic response regulator UhpA. In this system, UhpC directly senses G6P triggering a conformational change in the interacting kinase UhpB, which in turn phosphorylates and activates the response regulator UhpA. Phosphorylation increases the affinity of UhpA for the uhpT promoter (P uhpT ) thus initiating transcription. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . Typhimurium and S . Typhi G6P biosensors after growth in LB medium containing G6P (500 µM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of G6P is shown. ( C ) Investigation of the functionality of UhpA, UhpB, and UhpC regulatory proteins. Each S . Typhi gene was swapped for its S . Typhimurium homolog in the plasmid backbone containing the G6P biosensor as indicated in the diagram. The resulting plasmids were then introduced into the S . Typhi or S. Typhimurium ΔuhpABC mutant backgrounds, and the resulting strains were grown in M9 medium in the presence or absence of G6P (1mM). ( D ) Investigation of the functionality of each of the different uhpA mutations present in S . Typhi in comparison of its functional homolog in S . Typhimurium. Plasmids encoding the G6P biosensor and expressing S . Typhi UhpA mutants in which residues had been individually changed to the amino acid present in its S . Typhimurium homolog as indicated (i. e. UhpA Met77Val , UhpA Glu146Ala , or UhpA Met168Thr ) were introduced into the S. Typhi Δ uhpABC mutant background. The resulting strains were grown into M9 medium in the presence of G6P (1mM). In ( B - D ) the sfGFP fluorescence signal normalized to cell density (sfGFP RFU / OD600) was measured. Values indicate the mean ± SEM of n= 3 or 4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test ***p < 0.001, ( B and D ), or unpaired two-tailed t-test ****p < 0.0001 ( C ). ( E ) Functionality of different UhpA variants expressed in S . Typhi in the context of cultured epithelial cell infection. HeLa cells were infected with wild-type S . Typhi or derivative strains expressing the uhpA allele from S. Typhimurium ( uhpA STm ) or an S . Typhi allele, which had been rendered functional by introducing changes based on the S . Typhimurium sequence (i. e. uhpA Glu146Ala,Met168Thr ). Hela cells were infected with the different S. Typhi strains for 6 hours, fixed, stained with DAPI, and examined under a fluorescence microscope. In all the images, brightness and contrast were optimized for each of the individual color channels to maximize visual clarity. Scale bars, 10 μm. ( F ) Alignment of the modeled atomic structures of S . Typhi UhpA (light blue) and S. Typhimurium UhpA (light pink) showing structural variations in the C-terminal portion at the helix-turn-helix motif (boxed). The models were predicted using AlphaFold2 and aligned with Pymol. The amino acid differences between the two structures (Val77Met, Ala146Glu, and Thr168Met) are shown in green.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Glucose-6-phosphate sensing and transcriptional regulation are impaired in Salmonella Typhi. ( A ) Diagram of the glucose-6-phosphate (G6P) sensing, transcription regulation, and transport in Salmonella enterica . G6P (purple hexagon) is imported into the bacterial cell by the UhpT antiporter, whose expression is strictly controlled by an unconventional two-component regulatory system composed of the membrane-associated UhpC sensor and UhpB kinase/phosphatase, and the cytoplasmic response regulator UhpA. In this system, UhpC directly senses G6P triggering a conformational change in the interacting kinase UhpB, which in turn phosphorylates and activates the response regulator UhpA. Phosphorylation increases the affinity of UhpA for the uhpT promoter (P uhpT ) thus initiating transcription. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . Typhimurium and S . Typhi G6P biosensors after growth in LB medium containing G6P (500 µM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of G6P is shown. ( C ) Investigation of the functionality of UhpA, UhpB, and UhpC regulatory proteins. Each S . Typhi gene was swapped for its S . Typhimurium homolog in the plasmid backbone containing the G6P biosensor as indicated in the diagram. The resulting plasmids were then introduced into the S . Typhi or S. Typhimurium ΔuhpABC mutant backgrounds, and the resulting strains were grown in M9 medium in the presence or absence of G6P (1mM). ( D ) Investigation of the functionality of each of the different uhpA mutations present in S . Typhi in comparison of its functional homolog in S . Typhimurium. Plasmids encoding the G6P biosensor and expressing S . Typhi UhpA mutants in which residues had been individually changed to the amino acid present in its S . Typhimurium homolog as indicated (i. e. UhpA Met77Val , UhpA Glu146Ala , or UhpA Met168Thr ) were introduced into the S. Typhi Δ uhpABC mutant background. The resulting strains were grown into M9 medium in the presence of G6P (1mM). In ( B - D ) the sfGFP fluorescence signal normalized to cell density (sfGFP RFU / OD600) was measured. Values indicate the mean ± SEM of n= 3 or 4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test ***p < 0.001, ( B and D ), or unpaired two-tailed t-test ****p < 0.0001 ( C ). ( E ) Functionality of different UhpA variants expressed in S . Typhi in the context of cultured epithelial cell infection. HeLa cells were infected with wild-type S . Typhi or derivative strains expressing the uhpA allele from S. Typhimurium ( uhpA STm ) or an S . Typhi allele, which had been rendered functional by introducing changes based on the S . Typhimurium sequence (i. e. uhpA Glu146Ala,Met168Thr ). Hela cells were infected with the different S. Typhi strains for 6 hours, fixed, stained with DAPI, and examined under a fluorescence microscope. In all the images, brightness and contrast were optimized for each of the individual color channels to maximize visual clarity. Scale bars, 10 μm. ( F ) Alignment of the modeled atomic structures of S . Typhi UhpA (light blue) and S. Typhimurium UhpA (light pink) showing structural variations in the C-terminal portion at the helix-turn-helix motif (boxed). The models were predicted using AlphaFold2 and aligned with Pymol. The amino acid differences between the two structures (Val77Met, Ala146Glu, and Thr168Met) are shown in green.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Expressing, Membrane, Fluorescence, Plasmid Preparation, Mutagenesis, Comparison, Functional Assay, Two Tailed Test, Cell Culture, Infection, Sequencing, Staining, Microscopy

    ( A ) Diagram of the G6P Biosensor for detection of glucose-6-phosphate in S . Typhimurium and S . Typhi. The membrane UhpC sensor detects G6P on the extracellular environment and interacts with the membrane UhpB histidine kinase that phosphorylates the cytosolic UhpA response regulator, which activates the P uhpT promoter. The S. Typhi P uhpT promoter was coupled to a sfGFP reporter gene on a plasmid system and transformed into S . Typhimurium and S . Typhi. In the presence of G6P, the cognate promoter P uhpT STy is activated leading to sfGFP expression. The possible steps where the sensing and regulation could be impaired in S . Typhi are noted. ( B ) Test of the response of the S . Typhimurium and S . Typhi G6P biosensors after growth in media containing increasing concentrations of G6P. The sfGFP fluorescense signal over time for the S . Typhimurium (upper graph) and S . Typhi (lower graph) G6P biosensors are shown. Values indicate the mean ± SEM of n= 3 replicates per condition. ( C ) Functionality of the G6P Biosensor in S. Typhimurium and S. Typhi in the context of cultured epithelial cell infection. Fluorescence microscopy of HeLa cells infected with bacterial strains encoding the G6P biosensors. HeLa cells were infected with wild type S . Typhimurium and S . Typhi strains encoding the G6P biosensors for 20 hs, fixed, stained with DAPI, and examined under a fluorescence microscope. For all images, brightness and contrast were adjusted for each of the individual channels to maximize visual clarity using the same parameters for both strains. Scale bars, 10 μm.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: ( A ) Diagram of the G6P Biosensor for detection of glucose-6-phosphate in S . Typhimurium and S . Typhi. The membrane UhpC sensor detects G6P on the extracellular environment and interacts with the membrane UhpB histidine kinase that phosphorylates the cytosolic UhpA response regulator, which activates the P uhpT promoter. The S. Typhi P uhpT promoter was coupled to a sfGFP reporter gene on a plasmid system and transformed into S . Typhimurium and S . Typhi. In the presence of G6P, the cognate promoter P uhpT STy is activated leading to sfGFP expression. The possible steps where the sensing and regulation could be impaired in S . Typhi are noted. ( B ) Test of the response of the S . Typhimurium and S . Typhi G6P biosensors after growth in media containing increasing concentrations of G6P. The sfGFP fluorescense signal over time for the S . Typhimurium (upper graph) and S . Typhi (lower graph) G6P biosensors are shown. Values indicate the mean ± SEM of n= 3 replicates per condition. ( C ) Functionality of the G6P Biosensor in S. Typhimurium and S. Typhi in the context of cultured epithelial cell infection. Fluorescence microscopy of HeLa cells infected with bacterial strains encoding the G6P biosensors. HeLa cells were infected with wild type S . Typhimurium and S . Typhi strains encoding the G6P biosensors for 20 hs, fixed, stained with DAPI, and examined under a fluorescence microscope. For all images, brightness and contrast were adjusted for each of the individual channels to maximize visual clarity using the same parameters for both strains. Scale bars, 10 μm.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Membrane, Plasmid Preparation, Transformation Assay, Expressing, Cell Culture, Infection, Fluorescence, Microscopy, Staining

    Investigation of the S . Typhi P uhpABC promoter. ( A ) Wild-type and mutant strains of S . Typhimurium containing the P uhpABC promoter from S . Typhi (P uhpABC STy ), or S . Typhi containing the P uhpABC promoter from S . Typhimurium (P uhpABC STm ), both harboring the G6P biosensor (P uhpT -sfGFP) were grown in presence of G6P for 20 hs and analyzed by flow-cytometry. Histograms show the sfGFP fluorescence intensities of individual bacteria for the indicated concentration of G6P. ( B ) Diagram of the transcriptional reporters in which the P uhpABC STm or the P uhpABC STy (containing an 80 bp insert indicated with a green rectangle) promoters drive the expression of a NanoLuc luciferase (NLuc). ( C ) S . Typhimurium and S . Typhi harboring the P uhpABC STm - NLuc or P uhpABC STy - NLuc reporters were grown for 20 hs, lysed and the luminescence was measured on a microplate reader. The signal in Relative Luminescence (RLU) for the indicated strains is shown. No statistically significative difference was observed between the P uhpABC STm - NLuc or P uhpABC STy - NLuc reporters as determined by Anova with Dunnett’s multiple comparisons test.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Investigation of the S . Typhi P uhpABC promoter. ( A ) Wild-type and mutant strains of S . Typhimurium containing the P uhpABC promoter from S . Typhi (P uhpABC STy ), or S . Typhi containing the P uhpABC promoter from S . Typhimurium (P uhpABC STm ), both harboring the G6P biosensor (P uhpT -sfGFP) were grown in presence of G6P for 20 hs and analyzed by flow-cytometry. Histograms show the sfGFP fluorescence intensities of individual bacteria for the indicated concentration of G6P. ( B ) Diagram of the transcriptional reporters in which the P uhpABC STm or the P uhpABC STy (containing an 80 bp insert indicated with a green rectangle) promoters drive the expression of a NanoLuc luciferase (NLuc). ( C ) S . Typhimurium and S . Typhi harboring the P uhpABC STm - NLuc or P uhpABC STy - NLuc reporters were grown for 20 hs, lysed and the luminescence was measured on a microplate reader. The signal in Relative Luminescence (RLU) for the indicated strains is shown. No statistically significative difference was observed between the P uhpABC STm - NLuc or P uhpABC STy - NLuc reporters as determined by Anova with Dunnett’s multiple comparisons test.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Mutagenesis, Flow Cytometry, Fluorescence, Bacteria, Concentration Assay, Expressing, Luciferase

    Alignment of portions of the amino acid sequence of the transcriptional regulator UhpA showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Alignment of portions of the amino acid sequence of the transcriptional regulator UhpA showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Sequencing

    Glucose-6-Phosphate transport is impaired in S . Typhi due to a loss-of-function mutation in UhpT. ( A - C ) Growth kinetics of wild-type (WT) S . Typhimurium and S . Typhi or the indicated mutant strains. Bacteria were grown in M9 medium containing 10 mM G6P as the only carbon source. The OD600 values measured at 30 min intervals are shown. Values indicate the mean ± SEM of n=3 replicates per condition. ( D ) Alignment of the modeled atomic structures of S . Typhi (dark blue) and S . Typhimurium (pink) UhpT predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Asp18Asn, Pro27Ser, Gly61Arg, and Ala395Asp) are shown in green.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Glucose-6-Phosphate transport is impaired in S . Typhi due to a loss-of-function mutation in UhpT. ( A - C ) Growth kinetics of wild-type (WT) S . Typhimurium and S . Typhi or the indicated mutant strains. Bacteria were grown in M9 medium containing 10 mM G6P as the only carbon source. The OD600 values measured at 30 min intervals are shown. Values indicate the mean ± SEM of n=3 replicates per condition. ( D ) Alignment of the modeled atomic structures of S . Typhi (dark blue) and S . Typhimurium (pink) UhpT predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Asp18Asn, Pro27Ser, Gly61Arg, and Ala395Asp) are shown in green.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Mutagenesis, Bacteria

    Alignment of portions of the amino acid sequence of the antiporter UhpT showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Alignment of portions of the amino acid sequence of the antiporter UhpT showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Sequencing

    Intracellular growth of different S . Typhimurium or S. Typhi uhp mutants. Cultured HeLa cells were infected with indicated bacterial strains and the CFU 2 and 8 hours after infection were determined by plate dilutions. Values represent the ratio between the CFUs measured at 8 and 2 hours post infection and are the mean ± SEM of n= 7 - 10 replicates per condition. No statistically significative difference was observed as determined using Anova with Dunnett’s multiple comparisons test.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Intracellular growth of different S . Typhimurium or S. Typhi uhp mutants. Cultured HeLa cells were infected with indicated bacterial strains and the CFU 2 and 8 hours after infection were determined by plate dilutions. Values represent the ratio between the CFUs measured at 8 and 2 hours post infection and are the mean ± SEM of n= 7 - 10 replicates per condition. No statistically significative difference was observed as determined using Anova with Dunnett’s multiple comparisons test.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Cell Culture, Infection

    ( A ) Diagram of 3PG sensing, transcription regulation, and transport in Salmonella enterica . PgtC senses 3PG (red trapezoid) triggering its interaction with the PgtB histidine kinase/phosphatase that phosphorylates the PgtA response regulator. Phosphorylated PgtA, in turn, activates the P gtpP promoter leading to the expression of the PgtP transporter. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . Typhimurium and S . Typhi strains encoding the 3PG biosensors after growth in LB medium containing 3PG (1 mM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of 3PG is shown. Values indicate the mean ± SEM of n=3 replicates per condition. ( C ) Investigation of the functionality of the S. Typhi PgtA and PgtB regulatory proteins. The indicated S . Typhi genes were swapped for their S . Typhimurium homologs and the resulting mutant strains were tested for their ability to sense 3PG with the P pgtP-sfGFP biosensor. Bacteria were grown in LB medium for 20 h in the presence or absence of 3PG (1mM), and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values in ( B ) and ( C ) are the mean ± SEM of n=3-4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test. ***p < 0.001. ( D ) Alignment of the modeled atomic structures of S . Typhi (blue) and S . Typhimurium (pink) PgtA predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Glu141Asp, Gly173Asp, and His223Tyr) are shown in green. ( E and F ) Growth kinetics of wild type (WT) S . Typhimurium and S . Typhi or the indicated mutant strains. Bacteria were grown in M9 + 0.05% casamino acids medium containing 10 mM 3PG as the only carbon source. The OD600 values measured at 30 min intervals are shown. Values indicate the mean ± SEM of n=3 replicates per condition.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: ( A ) Diagram of 3PG sensing, transcription regulation, and transport in Salmonella enterica . PgtC senses 3PG (red trapezoid) triggering its interaction with the PgtB histidine kinase/phosphatase that phosphorylates the PgtA response regulator. Phosphorylated PgtA, in turn, activates the P gtpP promoter leading to the expression of the PgtP transporter. The potential steps where the sensing, regulation and transport could be impaired in S . Typhi are noted. ( B ) Transcriptional response of the S . Typhimurium and S . Typhi strains encoding the 3PG biosensors after growth in LB medium containing 3PG (1 mM). The sfGFP fluorescence signal for S . Typhimurium (circle on red bar) or S . Typhi (square on blue bar) in the presence or absence of 3PG is shown. Values indicate the mean ± SEM of n=3 replicates per condition. ( C ) Investigation of the functionality of the S. Typhi PgtA and PgtB regulatory proteins. The indicated S . Typhi genes were swapped for their S . Typhimurium homologs and the resulting mutant strains were tested for their ability to sense 3PG with the P pgtP-sfGFP biosensor. Bacteria were grown in LB medium for 20 h in the presence or absence of 3PG (1mM), and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values in ( B ) and ( C ) are the mean ± SEM of n=3-4 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Tukey’s multiple comparisons test. ***p < 0.001. ( D ) Alignment of the modeled atomic structures of S . Typhi (blue) and S . Typhimurium (pink) PgtA predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Glu141Asp, Gly173Asp, and His223Tyr) are shown in green. ( E and F ) Growth kinetics of wild type (WT) S . Typhimurium and S . Typhi or the indicated mutant strains. Bacteria were grown in M9 + 0.05% casamino acids medium containing 10 mM 3PG as the only carbon source. The OD600 values measured at 30 min intervals are shown. Values indicate the mean ± SEM of n=3 replicates per condition.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Expressing, Fluorescence, Mutagenesis, Bacteria

    Investigation of the functionality of the PgtA and PgtB regulatory proteins. The S. Typhi pgtA and pgtB genes were swapped for their S . Typhimurium homologs in the 3PG biosensor plasmid. The resulting constructs were introduced into the S . Typhi ΔpgtAB mutant background and tested on the presence or absence of 3PG at 2 mM. The fluorescent gene expression normalized to cell density (sfGFP RFU / OD600) is shown. Values indicate the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Investigation of the functionality of the PgtA and PgtB regulatory proteins. The S. Typhi pgtA and pgtB genes were swapped for their S . Typhimurium homologs in the 3PG biosensor plasmid. The resulting constructs were introduced into the S . Typhi ΔpgtAB mutant background and tested on the presence or absence of 3PG at 2 mM. The fluorescent gene expression normalized to cell density (sfGFP RFU / OD600) is shown. Values indicate the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Plasmid Preparation, Construct, Mutagenesis, Expressing

    Alignment of portions of the amino acid sequence of the transcriptional regulator PgtA showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Alignment of portions of the amino acid sequence of the transcriptional regulator PgtA showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Sequencing

    Investigation of the functionality of S . Typhi PgtA amino acid substitutions. ( A ) S . Typhi PgtA amino acids at the indicated positions were substituted for the equivalent S . Typhimurium residues in the plasmid backbone containing the 3PG biosensor. The resulting constructs were introduced into the S . Typhi ΔpgtAB mutant background and the transcriptional response was tested in the presence or absence of 3PG at 2mM. Values represent the fluorescence signal normalized to cell density (sfGFP RFU / OD600) and are the mean ± SEM of n= 4 replicates per condition. ( B ) The S. Typhi pgtA or pgtB were individually swapped for their S. Typhimurium homologues (S. Typhi pgtA Stm and S. Typhi pgtB Stm ). The resulting strains and an S . Typhi strain encoding the pgtA Asp173Gly , all encoding the 3PG biosensor, were tested for their transcriptional response to 3PG at 10 mM. The fluorescence signal normalized to cell density (sfGFP RFU / OD600) is shown. Values indicate the mean ± SEM of n= 3 replicates per condition. For all panels, asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Investigation of the functionality of S . Typhi PgtA amino acid substitutions. ( A ) S . Typhi PgtA amino acids at the indicated positions were substituted for the equivalent S . Typhimurium residues in the plasmid backbone containing the 3PG biosensor. The resulting constructs were introduced into the S . Typhi ΔpgtAB mutant background and the transcriptional response was tested in the presence or absence of 3PG at 2mM. Values represent the fluorescence signal normalized to cell density (sfGFP RFU / OD600) and are the mean ± SEM of n= 4 replicates per condition. ( B ) The S. Typhi pgtA or pgtB were individually swapped for their S. Typhimurium homologues (S. Typhi pgtA Stm and S. Typhi pgtB Stm ). The resulting strains and an S . Typhi strain encoding the pgtA Asp173Gly , all encoding the 3PG biosensor, were tested for their transcriptional response to 3PG at 10 mM. The fluorescence signal normalized to cell density (sfGFP RFU / OD600) is shown. Values indicate the mean ± SEM of n= 3 replicates per condition. For all panels, asterisks denote statistically significant differences relative to the corresponding uninduced sample determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Plasmid Preparation, Construct, Mutagenesis, Fluorescence

    ( A ) Diagram of glucarate and galactarate sensing, transcription regulation, transport, and metabolism in Salmonella enterica . Glucarate (green circles) and galactarate (yellow squares) are transported into the bacterial cell by the permease GudT and converted to 5-dehydro-4-deoxy-d-glucarate (orange star) by the dehydratases GudD and GarD, respectively. 5-dehydro-4-deoxy-d-glucarate is further metabolized into D-glycerate (red triangles) by the aldolase GarL and the reductase GarR. The expression of these genes is controlled by the transcription factor CdaR, which senses glucarate, galactarate and D-glycerate in the cytosol and activates the P gudTYD , P garD and P garLRK promoters. The potential steps where the sensing, regulation, transport, or metabolism could be impaired in S . Typhi are noted. ( B and C ) Wild-type S . Typhimurium and S. Typhi or the indicated mutant strains all harboring the PgarL_ sfGFP transcriptional reporter were grown in LB medium containing glucarate, galactarate or glycerate (as indicated) at 10 mM (or water as a negative control) and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values indicate the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences determined using Anova with Tukey’s multiple comparisons test. ***p < 0.001. ( D ) Growth kinetics of wild-type S. Typhimurium and S. Typhi or their isogenic mutants ΔgudT, ΔgudT and gudT as indicated. Growth was monitored in M9 + 0.05% casamino acids medium containing 10 mM glucarate as the only carbon source. The OD600 measured at 30 min intervals are shown and values represent the mean ± SEM of n=3-4 replicates per condition. ( E ) Alignment of the modeled atomic structures of S . Typhi (Teal) and S . Typhimurium (Beige) GudT predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Ser321Phe and Ala357Val) are shown in green. ( F ) Growth kinetics of S. Typhi gudT STm expressing plasmid-borne gudD STm and gudD STy , as indicated. Growth was monitored in M9 medium + 0.05% casamino acids containing 10 mM glucarate as the only carbon source. The OD600 measured at 30 min intervals are shown and values represent the mean ± SEM of n=3-4 replicates per condition.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: ( A ) Diagram of glucarate and galactarate sensing, transcription regulation, transport, and metabolism in Salmonella enterica . Glucarate (green circles) and galactarate (yellow squares) are transported into the bacterial cell by the permease GudT and converted to 5-dehydro-4-deoxy-d-glucarate (orange star) by the dehydratases GudD and GarD, respectively. 5-dehydro-4-deoxy-d-glucarate is further metabolized into D-glycerate (red triangles) by the aldolase GarL and the reductase GarR. The expression of these genes is controlled by the transcription factor CdaR, which senses glucarate, galactarate and D-glycerate in the cytosol and activates the P gudTYD , P garD and P garLRK promoters. The potential steps where the sensing, regulation, transport, or metabolism could be impaired in S . Typhi are noted. ( B and C ) Wild-type S . Typhimurium and S. Typhi or the indicated mutant strains all harboring the PgarL_ sfGFP transcriptional reporter were grown in LB medium containing glucarate, galactarate or glycerate (as indicated) at 10 mM (or water as a negative control) and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values indicate the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences determined using Anova with Tukey’s multiple comparisons test. ***p < 0.001. ( D ) Growth kinetics of wild-type S. Typhimurium and S. Typhi or their isogenic mutants ΔgudT, ΔgudT and gudT as indicated. Growth was monitored in M9 + 0.05% casamino acids medium containing 10 mM glucarate as the only carbon source. The OD600 measured at 30 min intervals are shown and values represent the mean ± SEM of n=3-4 replicates per condition. ( E ) Alignment of the modeled atomic structures of S . Typhi (Teal) and S . Typhimurium (Beige) GudT predicted with AlphaFold2 and aligned with Pymol. The amino acids that differ between the two structures (Ser321Phe and Ala357Val) are shown in green. ( F ) Growth kinetics of S. Typhi gudT STm expressing plasmid-borne gudD STm and gudD STy , as indicated. Growth was monitored in M9 medium + 0.05% casamino acids containing 10 mM glucarate as the only carbon source. The OD600 measured at 30 min intervals are shown and values represent the mean ± SEM of n=3-4 replicates per condition.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Expressing, Mutagenesis, Negative Control, Fluorescence, Plasmid Preparation

    ( A ) ( A ) Diagram of the biosensor for the detection of glucarate, galactarate and glycerate based on the CdaR transcription factor and the cognate promoters P gudTYD , P garD and P garLRK driving expression of sfGFP . ( B ) The indicated wild-type strains of S . Typhimurium (S. Tm) and S . Typhi (S. Ty) harboring the indicated biosensors were tested in LB medium containing glucarate or galactarate (1 mM), as indicated, or water as a negative control, and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. ( C ) The indicated S . Typhimurium and S . Typhi mutant strains all harboring the P garL _ sfGFP transcriptional reporter were grown in LB medium containing galactarate at 10mM, and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values are the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001. ( D ) Growth kinetics of wild-type S. Typhimurium and S. Typhi or their isogenic mutants ΔgudT, ΔgudT and gudT STm as indicated. Growth was monitored in M9 +0.05% casamino acids medium containing 10 mM galactarate as the only carbon source. The OD600 was measured at 30 min intervals and values represent the mean ± SEM of n=3-4 replicates per condition.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: ( A ) ( A ) Diagram of the biosensor for the detection of glucarate, galactarate and glycerate based on the CdaR transcription factor and the cognate promoters P gudTYD , P garD and P garLRK driving expression of sfGFP . ( B ) The indicated wild-type strains of S . Typhimurium (S. Tm) and S . Typhi (S. Ty) harboring the indicated biosensors were tested in LB medium containing glucarate or galactarate (1 mM), as indicated, or water as a negative control, and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. ( C ) The indicated S . Typhimurium and S . Typhi mutant strains all harboring the P garL _ sfGFP transcriptional reporter were grown in LB medium containing galactarate at 10mM, and the fluorescence signal (normalized to cell density) was measured (sfGFP RFU/OD600) for each strain. Values are the mean ± SEM of n= 3 replicates per condition. Asterisks denote statistically significant differences determined using Anova with Dunnett’s multiple comparisons test. ***p < 0.001. ( D ) Growth kinetics of wild-type S. Typhimurium and S. Typhi or their isogenic mutants ΔgudT, ΔgudT and gudT STm as indicated. Growth was monitored in M9 +0.05% casamino acids medium containing 10 mM galactarate as the only carbon source. The OD600 was measured at 30 min intervals and values represent the mean ± SEM of n=3-4 replicates per condition.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Expressing, Negative Control, Fluorescence, Mutagenesis

    Alignment of portions of the amino acid sequence of the glucarate/galactarate GudT transporter showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Alignment of portions of the amino acid sequence of the glucarate/galactarate GudT transporter showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Sequencing

    Alignment of portions of the amino acid sequences of the GudD (A) and GarD (B) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Alignment of portions of the amino acid sequences of the GudD (A) and GarD (B) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques:

    Growth kinetics of S . Typhimurium, S . Typhi and S . Paratyphi A in minimal medium containing glucose-6-phosphate, 3-phosphoglyceric acid, glucarate, tartrate, aspartate, or L-histidine as the sole carbon source. Bacteria were grown in M9 medium supplemented with casamino acids (0.05%) and containing 20 mM of the different metabolites and the OD600 was measured at 30 min intervals. Values indicate the mean ± SEM of n= 3-4 replicates per condition.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Growth kinetics of S . Typhimurium, S . Typhi and S . Paratyphi A in minimal medium containing glucose-6-phosphate, 3-phosphoglyceric acid, glucarate, tartrate, aspartate, or L-histidine as the sole carbon source. Bacteria were grown in M9 medium supplemented with casamino acids (0.05%) and containing 20 mM of the different metabolites and the OD600 was measured at 30 min intervals. Values indicate the mean ± SEM of n= 3-4 replicates per condition.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Bacteria

    Alignment of portions of the amino acid sequence of S . Typhi STY3536 ( A ), STY3537 ( B ), and STY3538 ( C ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Alignment of portions of the amino acid sequence of S . Typhi STY3536 ( A ), STY3537 ( B ), and STY3538 ( C ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Sequencing

    Alignment of portions of the amino acid sequence of S . Typhi DcuS ( A ), Tar ( B ), and AsnB ( C ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Alignment of portions of the amino acid sequence of S . Typhi DcuS ( A ), Tar ( B ), and AsnB ( C ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Sequencing

    Alignment of portions of the amino acid sequence of S . Typhi HutU ( A ) and HutC ( B ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Journal: bioRxiv

    Article Title: Loss of function of metabolic traits in typhoidal Salmonella without apparent genome degradation

    doi: 10.1101/2024.02.14.580360

    Figure Lengend Snippet: Alignment of portions of the amino acid sequence of S . Typhi HutU ( A ) and HutC ( B ) showing differences between S . Typhi CT18 and nontyphoidal S. enterica serovars S. Typhimurium LT2, S. Enteritidis P125109 and S. Agona SL483. The rest of the sequences not shown are identical between the different serovars.

    Article Snippet: Degraded coding sequences (pseudogenes) for metabolic genes in S. Paratyphi A (strains ATCC 9150 and AKU_12601) that are putatively intact in S. Typhi (strains CT18 and Ty2) compared to the sequences in the reference S. Typhimurium strain LT2 were listed at Table S1 based on previously reported genomic comparisons( , , , ).

    Techniques: Sequencing