e coli genomic dna  (ATCC)

Journal: Journal of Bacteriology

Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression

doi: 10.1128/jb.00280-25

Figure Lengend Snippet: The multi-functional DHAP shunt of E. coli . ( A ) Genomic organization of the DHAP shunt operon and neighboring genes in E. coli ATCC 25922. The operon specifically consists of mtnK , mtnA , ald2 , and an annotated permease of unknown function . The pfs gene, conserved across all E. coli , is encoded in a different region of the E. coli genome. ( B ) Multiple sequence alignment of the DHAP shunt 5′-UTR region from 1,569 E. coli genomes containing the DHAP shunt operon. Positions matching the ATCC 25922 reference are shown in gray, while mismatches are shown in red. ( C and D ) Biochemical reactions catalyzed by the DHAP shunt pathway enzymes, with 5′-deoxyadenosine ( C ) or 5′-methylthioadenosine ( D ) as initial substrate.

Article Snippet: Regions upstream of the DHAP shunt operon were amplified from E. coli ATCC 25922 genomic DNA as templates for sequencing ladders.

Techniques: Functional Assay, Sequencing

Journal: Journal of Bacteriology

Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression

doi: 10.1128/jb.00280-25

Figure Lengend Snippet: Regulation of E. coli ATCC 25922 transcriptome during growth on glucose, 5dR, or carbon starvation. Volcano plots of changes in ATCC 25922 transcript abundance for ( A ) wild-type ATCC 25922 grown with 5dR versus glucose, ( B ) MtnR deletion strain (∆ mtnR ) versus wild-type ATCC 25922 grown with glucose, and ( C ) ATCC 25922 incubated under carbon starvation conditions versus carbon replete (glucose) conditions. Gray lines indicate 2.5 log 2 -fold change thresholds; DHAP shunt genes are highlighted in pink. ( A ) DHAP shunt genes are strongly upregulated (~7.5 log 2 ) in the presence of 5dR. ( B ) Deletion of MtnR (∆ mtnR ) results in ~5 log 2 increased expression of DHAP shunt genes compared to the wild-type strain in the presence of glucose. ( C ) Carbon starvation only moderately upregulated DHAP shunt gene expression. ( D ) Inventory of genes differentially expressed under 5dR growth and carbon starvation relative to growth on glucose. Carbon starvation triggers a large change in the proteome compared to growth on 5dR, indicating the proteome change during growth on 5dR is specific to the substrate and not a general stress response.

Article Snippet: Regions upstream of the DHAP shunt operon were amplified from E. coli ATCC 25922 genomic DNA as templates for sequencing ladders.

Techniques: Incubation, Expressing, Gene Expression

Journal: Journal of Bacteriology

Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression

doi: 10.1128/jb.00280-25

Figure Lengend Snippet: DHAP shunt 5′-UTR regions required for operon expression and repression by MtnR. ( A ) E. coli ATCC 25922 DHAP shunt genomic context and sections of 5′-UTR used for construction of plasmids (pLacZ###) with promoter– lacZ fusions. ( B, D ) LacZ activity assays from cell extracts of wild-type E. coli ATCC 25922 and ( C, E ) LacZ activity assays from cell extracts of MtnR deletion strain (Δ mtnR ) containing the indicated DHAP shunt 5′-UTR– lacZ fusion plasmids when grown with either 5 mM glucose (Glc), 5 mM glucose plus 5 mM 5dR (Glc + 5dR), or 5dR as the carbon source. The pLacZ control is a Lac promoter– lacZ fusion without added IPTG. Average and standard deviation error bars are for n = 3 independent replicates. ** P > 0.05, * P > 0.1; two-tailed t -test. ( F ) Log 2 -fold change in mtnK mRNA expression from transcriptomic analysis ( ; ) and measured by qRT-PCR for wild-type ATCC 25922 (WT), mtnR deletion strain (Δ mtnR ), and mtnR deletion strain complemented with mtnR from a plasmid (Δ mtnR + pmtnR). Tetracycline (10 ng/mL) was added to induce expression of mtnR from pmtnR plasmid. qRT-PCR average and standard deviation error bars are for n = 4 independent biological replicates except for WT Glc + 5dR versus Glc ( n = 8) and Δ mtnR + pmtnR Glc + 5dR versus Glc ( n = 3). ** P > 0.05; two-tailed t -test.

Article Snippet: Regions upstream of the DHAP shunt operon were amplified from E. coli ATCC 25922 genomic DNA as templates for sequencing ladders.

Techniques: Expressing, Activity Assay, Control, Standard Deviation, Two Tailed Test, Quantitative RT-PCR, Plasmid Preparation

Journal: Journal of Bacteriology

Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression

doi: 10.1128/jb.00280-25

Figure Lengend Snippet: DHAP shunt expression is not activated by other alternative growth substrates. LacZ activity assays from cell extracts of E. coli ATCC 25922 containing the DHAP shunt 5′-UTR– lacZ fusion plasmid placZ255 and grown with either 5 mM glucose, NANA, l -fucose, l -arabinose, l -rhamnose, or 5-deoxy- d -ribose (5dR) as the sole carbon source. Average and standard deviation error bars are for n = 3 independent replicates. ** P > 0.05.

Article Snippet: Regions upstream of the DHAP shunt operon were amplified from E. coli ATCC 25922 genomic DNA as templates for sequencing ladders.

Techniques: Expressing, Activity Assay, Plasmid Preparation, Standard Deviation

Journal: Journal of Bacteriology

Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression

doi: 10.1128/jb.00280-25

Figure Lengend Snippet: CRP is required for growth of E. coli ATCC 25922 using 5dR as a substrate. ( A and B ) Growth of wild-type ATCC 25922 (diamonds), CRP deletion strain (Δ crp ; triangles), and CRP deletion strain complemented with crp expressed from a plasmid (Δ crp + pCRP; circles) with ( A ) 5dR or ( B ) glucose as the sole carbon source. Average and standard deviation error bars are for n = 4 for growth on 5dR and n = 5 for growth on glucose. ( C ) Diauxic growth observed when wild-type ATCC 25922 is grown on a combination of 1 mM glucose plus 8 mM 5dR (diamonds), as compared to cultures grown with 1 mM glucose (triangles) or 8 mM 5dR (circles) as the sole carbon source. Dotted lines and fit parameters are for exponential regression fit to the growth data. Average and standard deviation error bars are for n = 5 independent biological replicates.

Article Snippet: Regions upstream of the DHAP shunt operon were amplified from E. coli ATCC 25922 genomic DNA as templates for sequencing ladders.

Techniques: Plasmid Preparation, Standard Deviation

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: a . Diagram of key molecules in the chemotaxis network. Extracellular ligands (black semicircles) bind to receptors and modulate the activity of CheA, the kinase that phosphorylates the diffusible messenger CheY. CheA activity adaptation is mediated by the methylase CheR and de-methylase CheB. CheY-P binds to the C-ring and triggers a conformational change that results in BFM changing direction (CCW → CW). The swimming bacterium switches from a smooth run to a tumble. b . AlphaFold structure predictions of chimeric molecules. Grey is E. coli CheY, blue is cpAsLOV2, black is a short linker, and orange is the FliM peptide. Cartoons depicting the “caged” dark-state of Opto-CheY and the constitutively active control are shown with the same color code. c . Blue-light absorption triggers unfolding of the J α helix, and CheY activation by removal of the FliM plug from the CheY active site. Active CheY binds to the C-ring and promotes the rotational switch of the BFM from CCW → CW. Light pulses (blue dots, ~100 µ J/cm 2 ) are applied every 6 seconds while monitoring BFM rotational state. Each response is a CW rotation (vertical black line) following a blue light pulse (blue dot) in an otherwise mostly CCW motor (bottom trace)

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Chemotaxis Assay, Activity Assay, Control, Activation Assay

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet: Fluorescent signal autocorrelation (black dots) as a function of lag time for a bacterial cell. Data is fitted with a single component diffusion model (red line) with 2 parameters, diffusion time (used to compute the diffusion coefficient) and average number of molecules in the observed volume. Right Scatter plot for the computed upper bound and lower bound concentration for 8 cells (individual cell measurements are shown as gray circles). Mean upper bound is 2.14 ± 0.25 µM and mean lower bound is 1.43 ± 0.17 µM. Assuming the average volume of an E. coli bacterium to be 1 femto-L, each cell contains ~1000-2000 molecules of fluorescent protein.

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Diffusion-based Assay, Concentration Assay

Journal: bioRxiv

Article Title: The dynamic response of the bacterial flagellar motor to its direct intracellular input signal

doi: 10.1101/2025.10.28.684865

Figure Lengend Snippet:

Article Snippet: cheY DNA coding sequence was amplified from E. coli genomic DNA; cpAsLOV2 synthesized as a G block by Invitrogen ( ).

Techniques: Transformation Assay, Expressing, Variant Assay