Journal: iScience

Article Title: Multi-omics analyses provide insights into the sulfur metabolism of a novel deep-sea sulfate-reducing bacterium

doi: 10.1016/j.isci.2024.110095

Figure Lengend Snippet: Isolation, morphology, and phylogenetic analyses of Pseudodesulfovibrio serpens zrk46 (A) Diagram showing the strategy used to isolate deep-sea SRB. (B) TEM observation of the morphology of strain zrk46. Scale bar, 2.0 μm. (C) Phylogenetic analysis of strain zrk46. Phylogenetic placement of strain zrk46 within the genus Pseudodesulfovibrio , based on almost complete 16S rRNA gene sequences. The NCBI accession number for each 16S rRNA gene is indicated after each corresponding strain’s name. The tree was inferred and reconstructed using the maximum likelihood criterion. Bootstrap values are based on 1,000 replicates. The 16S rRNA gene sequence of Escherichia coli ATCC 11775 T was used as the outgroup. Bar, 0.02 substitutions per nucleotide position.

Article Snippet: The 16S rRNA gene sequence of Escherichia coli ATCC 11775 T was used as the outgroup.

Techniques: Isolation, Sequencing

Journal: Cell host & microbe

Article Title: Commensal Enterobacteriaceae Protect against Salmonella Colonization through Oxygen Competition.

doi: 10.1016/j.chom.2018.12.003

Figure Lengend Snippet: Figure 2. Virulence Factors Promote Pathogen Expansion through Aerobic Respiration (A) One-day-old chicks (n = 4–5) were infected with a 1:1 mixture of the indicated bacterial strains. Cecal content was collected 3 days after infection to determine the competitive index. See also Figure S2. (B–G) One-day-old chicks were mock-treated or infected with the indicated bacterial strains. (B) Composition of the gut microbiota at the phyla level (relative abundance) based on 16S rRNA gene sequencing of DNA isolated from cecal contents at the indicated time points after infection. See also Figure S1. (C–E) Total colony-forming units (CFUs) in cecal contents recovered on MacConkey agar (Enterobacteriaceae, black bars) or CFU of S. Enteritidis (red bars) were determined in groups of chicks that were mock-treated (C), infected with S. Enteritidis (wt) (D), or infected with a S. Enteritidis invA spiB mutant (invA spiB) (E). Bars represent geometric means ± SEM. (F and G) Normalized abundances of members of the family Lachnospiraceae (F) and Ruminococcaceae (G) in cecal contents were determined by microbiota profiling. Bars represent mean ± SD. (H and I) Representative images of pimonidazole (red, H) or MitoTracker (dark orange, I) staining detected in cecal sections counter stained with DAPI nuclear stain (blue). (B–G) The number of animals in each group (n) is indicated in Figure S1. Scale bars, 40 mm. *p < 0.05; ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-pimonidazole monoclonal antibody MAb1 Hydroxyprobe Hypoxyprobe-1 Kit, HP1-1000, lot 06242014 Alexa fluor 546 goat anti-mouse IgG antibody Life Technologies Cat# A11003, lot 1752017 Bacterial and Virus Strains NA See Table 1 NA Biological Samples Adult chicken microbiota This paper NA Neonate chicken microbiota This paper NA Chemicals, Peptides, and Recombinant Proteins DAPI Sigma P/N D9542 ImmuMount mounting reagent Thermo Scientific Cat# 9990402 TRI-Reagent Molecular Research Center Cat# TR118 SYBR safe Life Technologies P/N 533102 Critical Commercial Assays MitoTracker Red CMXRos Molecular Probes Cat# M7512 DNA-free kit Invitrogen Cat# AM1906 GoTaq Green Master Mix Promega Cat# M7123 QIAquick PCR Purification kit Qiagen Cat# 27106 Gibson Assembly Master Mix New England Biolabs Cat# E2611 Deposited Data Avian E. coli whole genome sequence NCBI PRJNA434124 16S ribosomal RNA gene sequencing data EBI ERP108716 Experimental Models: Organisms/Strains Leghorn layer chickens Hopkins Avian Facility, UC Davis Line UCD003 Swiss Webster germ free mice Bred inhouse, original breeders from Taconic Original breeders #GF-B6 Oligonucleotides NA See Table S2 NA Recombinant DNA NA See Table 2 NA Software and Algorithms QIIME version 1.9.1 Caporaso et al., 2010 http://qiime.org Spades Bankevich et al., 2012 http://cab.spbu.ru/software/spades/ prokka Seemann, 2014 http://www.vicbioinformatics.com/ software.prokka.shtml clustal omega Sievers and Higgins, 2014 http://www.clustal.org/omega/ R Team, 2011 https://www.r-project.org ggtree Yu et al., 2017 https://bioconductor.org/packages/ release/bioc/html/ggtree.html RaxML Stamatakis, 2014 https://sco.h-its.org/exelixis/software.html e1 Cell Host & Microbe 25, 128–139.e1–e5, January 9, 2019

Techniques: Infection, Sequencing, Isolation, Mutagenesis, Staining

Journal: Cell host & microbe

Article Title: Commensal Enterobacteriaceae Protect against Salmonella Colonization through Oxygen Competition.

doi: 10.1016/j.chom.2018.12.003

Figure Lengend Snippet: Figure 3. Bioluminescence Imaging of Luminal E. coli Requires a Live Host (A) Cecal contents of an adult bird were cultured on MacConkey and the identity of bacterial colonies determined using an EnteroPluri assay. (B) Rooted maximum likelihood tree, visualized as a cladogram, of select E. coli isolates indicating their relatedness to the avian commensal isolate YL178. Color coding denotes members of the same phylogroup (phylogroups A, B1, B2, D1, D2, or E1). Information on the biovar for each isolate is given in parentheses: ETEC, enterotoxigenic E. coli; EAEC, enteroaggregative E. coli; EHEC, Enterohemorrhagic E. coli; UPEC, uropathogenic E. coli; AIEC, adherent-invasive E. coli; ExPEC, etraintestinal pathogenic E. coli. (C–F) Chicks were infected 1 day after hatch (day 1) with E. coli strain YL178 transformed with a plasmid carrying a CP25::luxCDBAE transcriptional fusion (pBR2TTS:CP25::luxCDBAE) and bioluminescence imaging was determined 1 day later (day 2) in anesthetized birds (C), after euthanasia (D and E) or in the gastrointestinal tract open longitudinally (F). A representative image is displayed for each sample (n = 8). (E) Time course of bioluminescence imaging (n = 6) determined before (red dot) and after (black dots) euthanasia. (C), (D), and (F) The number of animals in each group (n) is indicated in Figure 4A. Data are shown as geometric means ± SEM (n = 6).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-pimonidazole monoclonal antibody MAb1 Hydroxyprobe Hypoxyprobe-1 Kit, HP1-1000, lot 06242014 Alexa fluor 546 goat anti-mouse IgG antibody Life Technologies Cat# A11003, lot 1752017 Bacterial and Virus Strains NA See Table 1 NA Biological Samples Adult chicken microbiota This paper NA Neonate chicken microbiota This paper NA Chemicals, Peptides, and Recombinant Proteins DAPI Sigma P/N D9542 ImmuMount mounting reagent Thermo Scientific Cat# 9990402 TRI-Reagent Molecular Research Center Cat# TR118 SYBR safe Life Technologies P/N 533102 Critical Commercial Assays MitoTracker Red CMXRos Molecular Probes Cat# M7512 DNA-free kit Invitrogen Cat# AM1906 GoTaq Green Master Mix Promega Cat# M7123 QIAquick PCR Purification kit Qiagen Cat# 27106 Gibson Assembly Master Mix New England Biolabs Cat# E2611 Deposited Data Avian E. coli whole genome sequence NCBI PRJNA434124 16S ribosomal RNA gene sequencing data EBI ERP108716 Experimental Models: Organisms/Strains Leghorn layer chickens Hopkins Avian Facility, UC Davis Line UCD003 Swiss Webster germ free mice Bred inhouse, original breeders from Taconic Original breeders #GF-B6 Oligonucleotides NA See Table S2 NA Recombinant DNA NA See Table 2 NA Software and Algorithms QIIME version 1.9.1 Caporaso et al., 2010 http://qiime.org Spades Bankevich et al., 2012 http://cab.spbu.ru/software/spades/ prokka Seemann, 2014 http://www.vicbioinformatics.com/ software.prokka.shtml clustal omega Sievers and Higgins, 2014 http://www.clustal.org/omega/ R Team, 2011 https://www.r-project.org ggtree Yu et al., 2017 https://bioconductor.org/packages/ release/bioc/html/ggtree.html RaxML Stamatakis, 2014 https://sco.h-its.org/exelixis/software.html e1 Cell Host & Microbe 25, 128–139.e1–e5, January 9, 2019

Techniques: Imaging, Cell Culture, Infection, Transformation Assay, Plasmid Preparation

Journal: Biochimica et biophysica acta

Article Title: Expansion of the aminoglycoside-resistance 16S rRNA (m 1 A1408) methyltransferase family: expression and functional characterization of four hypothetical enzymes of diverse bacterial origin

doi: 10.1016/j.bbapap.2014.06.012

Figure Lengend Snippet: Origins and identifiers for the four putative A1408 16S rRNA methyltransferases

Article Snippet: Reverse transcriptase (RT) primer extension was carried out using a 5’-end 32 P-labeled DNA primer complementary to E. coli 16S rRNA nucleotides 1457–1473 (5’-CAAAGTGGTAAGCGCCC) and AMV reverse transcriptase (NEB).

Techniques: Environmental Sampling

Journal: Biochimica et biophysica acta

Article Title: Expansion of the aminoglycoside-resistance 16S rRNA (m 1 A1408) methyltransferase family: expression and functional characterization of four hypothetical enzymes of diverse bacterial origin

doi: 10.1016/j.bbapap.2014.06.012

Figure Lengend Snippet: Reverse transcription primer extension analysis of in vitro methylation reactions using (A) 30S subunits and (B) naked 16S rRNA as substrate. With 30S subunit as the substrate, reverse transcription was strongly terminated at nucleotide C1409, corresponding to modification of A1408 for KamB (positive control), CacKam, TcuKam, CarKam and UncKam. Nucleotide positions were identified by sequencing of unmodified 16S rRNA with ddNTPs (shown on left for ddTTP). No termination is observed for the negative control (no enzyme) which lacks A1408 modification, or for any enzyme with naked 16S rRNA as the substrate.

Article Snippet: Reverse transcriptase (RT) primer extension was carried out using a 5’-end 32 P-labeled DNA primer complementary to E. coli 16S rRNA nucleotides 1457–1473 (5’-CAAAGTGGTAAGCGCCC) and AMV reverse transcriptase (NEB).

Techniques: In Vitro, Methylation, Modification, Positive Control, Sequencing, Negative Control

Journal: American journal of obstetrics and gynecology

Article Title: Does the human placenta delivered at term have a microbiota? Results of cultivation, quantitative real-time PCR, 16S rRNA gene sequencing, and metagenomics

doi: 10.1016/j.ajog.2018.10.018

Figure Lengend Snippet: a. Plot of similarity in profile composition among placental and control samples based on the Jaccard index; b. Plot of similarity in profile structure among placental and control samples based on the Bray-Curtis index. Operational taxonomic units (OTUs) were generated using a 97% sequence similarity cutoff and the primary 16S rRNA gene nested PCR data set.

Article Snippet: Standard curves for three 10-fold dilution series (2.82 × 10 6 – 2.82 × 10 1 copies, 2.12 × 10 6 – 2.12 × 10 1 copies, and 2.97 × 10 6 – 2.97 × 10 1 copies) of E. coli ATCC 25922 16S rDNA (mean Cq values across all qPCR runs); c. Standard curve for a 2-fold dilution series (mean Cq values) of E. coli ATCC 25922 DNA illustrating a limit of detection for the qPCR assay between 1.57 × 10 2 and 3.14 × 10 2 16S rDNA copies per reaction (20 μl), as indicated by a standard deviation of replicate dilution samples above 0.5 cycles; d. Comparison of mean 16S rDNA qPCR Cq values for placental and control samples; e. Amplification curves from placental samples, technical controls, and the serial dilution series of E. coli DNA described in Figure panel b. Quantitative real-time PCR revealed that 16S rDNA abundances within the majority of the placental and background technical control samples were beyond the detection and quantification limits of the qPCR assay ( , ).

Techniques: Control, Generated, Sequencing, Nested PCR

Journal: American journal of obstetrics and gynecology

Article Title: Does the human placenta delivered at term have a microbiota? Results of cultivation, quantitative real-time PCR, 16S rRNA gene sequencing, and metagenomics

doi: 10.1016/j.ajog.2018.10.018

Figure Lengend Snippet: Non-parametric MANOVA (NPMANOVA) analyses showing lack of variation in 16S rRNA gene profiles among amnion & chorionic plate, villous tree & basal plate, and room, hood, and blank extraction kit technical control samples. Operational taxonomic units (OTUs) were generated using a 97% sequence similarity cutoff. 16S profile composition and structure were characterized using Jaccard and Bray-Curtis indices, respectively. Results of overall global effect analyses are presented along with the results of pairwise comparisons involving placental samples. P-values for these permutation tests were not adjusted for multiple pairwise comparisons, as this can be overly conservative. However, for pairwise tests that were statistically significant, we do present the Bonferroni corrected p-value in parentheses.

Article Snippet: Standard curves for three 10-fold dilution series (2.82 × 10 6 – 2.82 × 10 1 copies, 2.12 × 10 6 – 2.12 × 10 1 copies, and 2.97 × 10 6 – 2.97 × 10 1 copies) of E. coli ATCC 25922 16S rDNA (mean Cq values across all qPCR runs); c. Standard curve for a 2-fold dilution series (mean Cq values) of E. coli ATCC 25922 DNA illustrating a limit of detection for the qPCR assay between 1.57 × 10 2 and 3.14 × 10 2 16S rDNA copies per reaction (20 μl), as indicated by a standard deviation of replicate dilution samples above 0.5 cycles; d. Comparison of mean 16S rDNA qPCR Cq values for placental and control samples; e. Amplification curves from placental samples, technical controls, and the serial dilution series of E. coli DNA described in Figure panel b. Quantitative real-time PCR revealed that 16S rDNA abundances within the majority of the placental and background technical control samples were beyond the detection and quantification limits of the qPCR assay ( , ).

Techniques: Extraction, Control, Generated, Sequencing

Journal: American journal of obstetrics and gynecology

Article Title: Does the human placenta delivered at term have a microbiota? Results of cultivation, quantitative real-time PCR, 16S rRNA gene sequencing, and metagenomics

doi: 10.1016/j.ajog.2018.10.018

Figure Lengend Snippet: Prominent OTUs were defined as those having an average relative abundance ≥ 1% among the placental samples. OTUs were generated using a 97% sequence similarity cutoff and the primary 16S rRNA gene nested PCR data set. Asterisks indicate OTUs that were prominent in placental samples but not in controls.

Article Snippet: Standard curves for three 10-fold dilution series (2.82 × 10 6 – 2.82 × 10 1 copies, 2.12 × 10 6 – 2.12 × 10 1 copies, and 2.97 × 10 6 – 2.97 × 10 1 copies) of E. coli ATCC 25922 16S rDNA (mean Cq values across all qPCR runs); c. Standard curve for a 2-fold dilution series (mean Cq values) of E. coli ATCC 25922 DNA illustrating a limit of detection for the qPCR assay between 1.57 × 10 2 and 3.14 × 10 2 16S rDNA copies per reaction (20 μl), as indicated by a standard deviation of replicate dilution samples above 0.5 cycles; d. Comparison of mean 16S rDNA qPCR Cq values for placental and control samples; e. Amplification curves from placental samples, technical controls, and the serial dilution series of E. coli DNA described in Figure panel b. Quantitative real-time PCR revealed that 16S rDNA abundances within the majority of the placental and background technical control samples were beyond the detection and quantification limits of the qPCR assay ( , ).

Techniques: Generated, Sequencing, Nested PCR

Journal: American journal of obstetrics and gynecology

Article Title: Does the human placenta delivered at term have a microbiota? Results of cultivation, quantitative real-time PCR, 16S rRNA gene sequencing, and metagenomics

doi: 10.1016/j.ajog.2018.10.018

Figure Lengend Snippet: a. Comparison of quantification cycle (Cq) values (mean ± SD) of serially diluted placental genomic DNA samples spiked with equal concentrations (5.7 × 103 copies per reaction) of genomic DNA from Echerichia coli ATCC 25922, illustrating that amplification inhibition is eliminated by diluting samples with nuclease-free water by a factor of 1:3 or more; b. Standard curves for three 10-fold dilution series (2.82 × 106 – 2.82 × 101 copies, 2.12 × 106 – 2.12 × 101 copies, and 2.97 × 106 – 2.97 × 101 copies) of E. coli ATCC 25922 16S rDNA (mean Cq values across all qPCR runs); c. Standard curve for a 2-fold dilution series (mean Cq values) of E. coli ATCC 25922 DNA illustrating a limit of detection for the qPCR assay between 1.57 × 102 and 3.14 × 102 16S rDNA copies per reaction (20 μl), as indicated by a standard deviation of replicate dilution samples above 0.5 cycles; d. Comparison of mean 16S rDNA qPCR Cq values for placental and control samples; e. Amplification curves from placental samples, technical controls, and the serial dilution series of E. coli DNA described in Figure panel b.

Article Snippet: Standard curves for three 10-fold dilution series (2.82 × 10 6 – 2.82 × 10 1 copies, 2.12 × 10 6 – 2.12 × 10 1 copies, and 2.97 × 10 6 – 2.97 × 10 1 copies) of E. coli ATCC 25922 16S rDNA (mean Cq values across all qPCR runs); c. Standard curve for a 2-fold dilution series (mean Cq values) of E. coli ATCC 25922 DNA illustrating a limit of detection for the qPCR assay between 1.57 × 10 2 and 3.14 × 10 2 16S rDNA copies per reaction (20 μl), as indicated by a standard deviation of replicate dilution samples above 0.5 cycles; d. Comparison of mean 16S rDNA qPCR Cq values for placental and control samples; e. Amplification curves from placental samples, technical controls, and the serial dilution series of E. coli DNA described in Figure panel b. Quantitative real-time PCR revealed that 16S rDNA abundances within the majority of the placental and background technical control samples were beyond the detection and quantification limits of the qPCR assay ( , ).

Techniques: Comparison, Amplification, Inhibition, Standard Deviation, Control, Serial Dilution

Journal: American journal of obstetrics and gynecology

Article Title: Does the human placenta delivered at term have a microbiota? Results of cultivation, quantitative real-time PCR, 16S rRNA gene sequencing, and metagenomics

doi: 10.1016/j.ajog.2018.10.018

Figure Lengend Snippet: Description of prior 16S rRNA gene or metagenomic studies of the human placental microbiota at term

Article Snippet: Standard curves for three 10-fold dilution series (2.82 × 10 6 – 2.82 × 10 1 copies, 2.12 × 10 6 – 2.12 × 10 1 copies, and 2.97 × 10 6 – 2.97 × 10 1 copies) of E. coli ATCC 25922 16S rDNA (mean Cq values across all qPCR runs); c. Standard curve for a 2-fold dilution series (mean Cq values) of E. coli ATCC 25922 DNA illustrating a limit of detection for the qPCR assay between 1.57 × 10 2 and 3.14 × 10 2 16S rDNA copies per reaction (20 μl), as indicated by a standard deviation of replicate dilution samples above 0.5 cycles; d. Comparison of mean 16S rDNA qPCR Cq values for placental and control samples; e. Amplification curves from placental samples, technical controls, and the serial dilution series of E. coli DNA described in Figure panel b. Quantitative real-time PCR revealed that 16S rDNA abundances within the majority of the placental and background technical control samples were beyond the detection and quantification limits of the qPCR assay ( , ).

Techniques: Infection, Sequencing, Extraction, Amplification, In Utero, Denaturing Gradient Gel Electrophoresis, Cell Culture, Real-time Polymerase Chain Reaction, Sterility, Control, Bacteria

Journal: American journal of obstetrics and gynecology

Article Title: Does the human placenta delivered at term have a microbiota? Results of cultivation, quantitative real-time PCR, 16S rRNA gene sequencing, and metagenomics

doi: 10.1016/j.ajog.2018.10.018

Figure Lengend Snippet: Genera indicated by Linear discriminant analysis Effect Size (LEfSe) as being more relatively abundant in placental tissues than technical controls

Article Snippet: Standard curves for three 10-fold dilution series (2.82 × 10 6 – 2.82 × 10 1 copies, 2.12 × 10 6 – 2.12 × 10 1 copies, and 2.97 × 10 6 – 2.97 × 10 1 copies) of E. coli ATCC 25922 16S rDNA (mean Cq values across all qPCR runs); c. Standard curve for a 2-fold dilution series (mean Cq values) of E. coli ATCC 25922 DNA illustrating a limit of detection for the qPCR assay between 1.57 × 10 2 and 3.14 × 10 2 16S rDNA copies per reaction (20 μl), as indicated by a standard deviation of replicate dilution samples above 0.5 cycles; d. Comparison of mean 16S rDNA qPCR Cq values for placental and control samples; e. Amplification curves from placental samples, technical controls, and the serial dilution series of E. coli DNA described in Figure panel b. Quantitative real-time PCR revealed that 16S rDNA abundances within the majority of the placental and background technical control samples were beyond the detection and quantification limits of the qPCR assay ( , ).

Techniques: Sequencing, Nested PCR, Bacteria, Touchdown PCR