Journal: mSystems

Article Title: A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis

doi: 10.1128/mSystems.00224-21

Figure Lengend Snippet: C T from quantitative PCR (qPCR) of genomic DNA extracted by DNeasy Blood and Tissue Kits and cells treated by direct PCR methods (method 1, IGEPAL only; method 2, IGEPAL+freeze-thaw; method 3, IGEPAL+freeze-thaw+proteinase K). The strains are Escherichia coli K-12 MG1655, Pseudomonas putida KT2440, Lactococcus lactis cremoris MG1363, and Lactobacillus brevis ATCC 14869. A plus sign indicates that the C T of the direct PCR method is greater than the C T of the extracted gDNA; a minus sign indicates that the C T of the direct PCR method is less than the C T of the extracted gDNA; C T with no symbols are similar between extracted gDNA and direct PCR methods.

Article Snippet: The cell lysis efficiency of direct PCR methods was compared to that of the Qiagen DNeasy Blood and Tissue Kit using the following strains in both stationary phase and exponential phase: Escherichia coli strain K-12 substrain MG1655 (referred to here as E. coli ), Pseudomonas putida KT2440 (referred to here as P. putida ), Lactococcus lactis subsp. cremoris strain MG1363 (referred to here as L. lactis ), and Lactobacillus brevis ATCC 14869 (referred to here as L. brevis ).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Microbiology

Article Title: Use of Transposon Directed Insertion-Site Sequencing to Probe the Antibacterial Mechanism of a Model Honey on E. coli K-12

doi: 10.3389/fmicb.2021.803307

Figure Lengend Snippet: Antibacterial effect of model honeys on E. coli . Colony counts, showing the cell survival (A) , and mean fluorescence intensity (MFI) of PI, showing the extent of cell wall damage (B) , are plotted for 30, 60, 90, and 120 min post-exposure to honey model honey A (plain bars) and B (black bars). Colony counts (C) show the survival of the four treated populations after 2 h outgrowth in LB and the MFI of PI (D) of 30 min- and 90 min-treated population post- 2 h outgrowth in fresh LB. Error bars show standard deviations, ( n = 3; biological replicates).

Article Snippet: A separate study using qPCR of selected genes in exponential-growing E. coli Crooks ATCC 8739 (which like E. coli K-12 is in phylogroup A) exposed to clover, citrus, and marjoram honey gave similar results, showing particularly strong down-regulation of tnaA , the gene for indole biosynthesis; indole plays a signaling role in biofilm formation ( ).

Techniques: Fluorescence

Journal: Frontiers in Microbiology

Article Title: Use of Transposon Directed Insertion-Site Sequencing to Probe the Antibacterial Mechanism of a Model Honey on E. coli K-12

doi: 10.3389/fmicb.2021.803307

Figure Lengend Snippet: Genome-wide transposon insertion sites mapped to E. coli strain MG1655 in the TraDIS library. Frequency and location of transposon junction sequences from a mini-Tn 5 transposon library in strain MG1655, mapped to the MG1655 genome (CP009273.1). The outermost track marks the MG1655 genome in base pairs starting at the annotation origin. The next two inner tracks correspond to sense and antisense CDS, respectively. The innermost circles correspond to the frequency and location of transposons mapped to the MG1655 genome. The circles represent the initial MG1655 transposon library (black), and the library after 30 min (blue) and 90 min (red) exposure to model honey. The figure was created using DNAPlotter.

Article Snippet: A separate study using qPCR of selected genes in exponential-growing E. coli Crooks ATCC 8739 (which like E. coli K-12 is in phylogroup A) exposed to clover, citrus, and marjoram honey gave similar results, showing particularly strong down-regulation of tnaA , the gene for indole biosynthesis; indole plays a signaling role in biofilm formation ( ).

Techniques: Genome Wide

Journal: Frontiers in Microbiology

Article Title: Use of Transposon Directed Insertion-Site Sequencing to Probe the Antibacterial Mechanism of a Model Honey on E. coli K-12

doi: 10.3389/fmicb.2021.803307

Figure Lengend Snippet: Significantly enriched gene ontology (GO) terms for the seventeen mutants that show significant loss of fitness following 30- and 90-min exposure to model honey. Genes included in the analysis were these whose corresponding mutants had a logFC >1 or <–1 with adjusted p -values < 0.005. Comparisons were made using the number of genes associated with each term in the gene set versus the total number of genes associated with each term in the ( E. coli MG1655) genome. The respective percentage is given in x axis.

Article Snippet: A separate study using qPCR of selected genes in exponential-growing E. coli Crooks ATCC 8739 (which like E. coli K-12 is in phylogroup A) exposed to clover, citrus, and marjoram honey gave similar results, showing particularly strong down-regulation of tnaA , the gene for indole biosynthesis; indole plays a signaling role in biofilm formation ( ).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Use of Transposon Directed Insertion-Site Sequencing to Probe the Antibacterial Mechanism of a Model Honey on E. coli K-12

doi: 10.3389/fmicb.2021.803307

Figure Lengend Snippet: Selection rates for strains deleted for individual genes. Genes classified in the TraDIS dataset as having (A) negative fitness or (B) positive fitness when containing transposon insertions. Strains were mixed with represented their wild type parent ( E. coli MG1655, marked with a lacZ mutation) in model honey for 30 min (plain bar) or 90 min (black bar), before plating on MacConkey lactose to determine relative survival rates. The WT control shows the data for MG1655 lacZ mixed with MG1655 LacZ + . Data were analyzed using two-way ANOVA: asterisks show significance levels of Sidak’s multiple comparisons test (**** p ≤ 0.0001, *** p ≤ 0.0005, ** p ≤ 0.005, * p ≤ 0.05); all bars without asterisks are not significant ( p > 0.05). Error bars represent the average ± SD ( n = 3; independent biological replicates).

Article Snippet: A separate study using qPCR of selected genes in exponential-growing E. coli Crooks ATCC 8739 (which like E. coli K-12 is in phylogroup A) exposed to clover, citrus, and marjoram honey gave similar results, showing particularly strong down-regulation of tnaA , the gene for indole biosynthesis; indole plays a signaling role in biofilm formation ( ).

Techniques: Selection, Mutagenesis, Control

Journal: bioRxiv

Article Title: Mycobacterium tuberculosis MutT4 is an RNA pyrophosphohydrolase that forms biomolecular condensates and sensitizes mRNAs to degradation

doi: 10.1101/2025.05.13.653832

Figure Lengend Snippet: (A) Schematic of splinted ligation. The adapter is ligated only if the target mRNA has a 5’ monophosphate. Ligation is performed with and without treatment by E. coli RppH to convert 5’ triphosphates to monophosphates. Quantitative PCR reveals the abundance of ligated mRNA (primers 1 and 3) relative to total mRNA (primers 2 and 3). (B-C) Screening of Mtb Nudix gene deletion mutants and complemented strains by splinted ligation of a transcript in the endogenous mRNA pool. ΔΔCt indicates the impact of in vitro treatment with E. coli RppH on the ligatability of the Rv3248c transcript, after normalizing to total transcript abundance. Higher ΔΔCt values indicate a higher proportion of the endogenous Rv3248c mRNA was triphosphorylated. Mean and standard deviation of three biological replicates are shown for panel C. (D) MutT4 has RNA pyrophosphohydrolase activity in vitro . Splinted ligation of in vitro transcribed Rv3248c mRNA (5’ monophosphorylated) treated with purified MutT4, MutT4 E118Q, or no enzyme (mock). Each value represents results from the assay performed with an independent protein preparation. A one-way ANOVA was performed with Dunnett’s test for panel B and Tukey’s test for panel C and D. *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: 5 µg of DNase-treated total RNA extracted from Mtb in exponential phase was incubated with 10 U of E. coli RNA 5’ Pyrophosphohydrolase (RppH) (New England Biolabs, M0356), or with no enzyme as a control, in NEBuffer 2 for 1 h at 37 °C.

Techniques: Ligation, Real-time Polymerase Chain Reaction, In Vitro, Standard Deviation, Activity Assay, Purification

Journal: bioRxiv

Article Title: Mycobacterium tuberculosis MutT4 is an RNA pyrophosphohydrolase that forms biomolecular condensates and sensitizes mRNAs to degradation

doi: 10.1101/2025.05.13.653832

Figure Lengend Snippet: (A) Schematic of 5’-end directed RNASeq. Total RNA was used to construct 5’-end-directed libraries with or without pre-treatment with E. coli RppH to convert 5’ triphosphates to monophosphates. Only transcripts with 5’ monophosphates (whether endogenous or produced by E. coli RppH) can be ligated to adapters and therefore be captured in the libraries. Comparison of the number of reads obtained in libraries made from RppH-treated vs untreated RNA indicates the extent to which the RNA was endogenously monophosphorylated. The “log 2 coverage ratio” (green) and “ mutT4 impact” (blue) are metrics used in subsequent panels. (B) Log 2 coverage ratio of +/-RppH libraries for reads derived from RNA 5’ ends previously classified as transcription start sites (TSS, teal curve, n=1687) or RNase cleavage sites (pink, n=590) (Zhou and Sun et al 2023). The RNase cleavage sites produce RNAs with monophosphorylated 5’ ends and are therefore expected to have log 2 coverage ratios of ∼0, while TSSs produce RNAs with triphosphorylated 5’ ends and are therefore expected to have log 2 coverage ratios >0. The median TSS log 2 coverage ratio for each strain is shown in teal. (C-F) For each TSS quantified in panel B, the impact of mutT4 on 5’ end phosphorylation status was quantified by calculating the difference in log 2 coverage ratio +/- RppH between the deletion strain and the WT strain (see panel A). A higher number indicates a larger impact by mutT4 . (C) Transcripts with 5’ ends mapping to previously published TSSs were grouped according to their first two nt. As most transcripts initiate with A or G, those initiating with C or U are not shown. (D-E) Transcripts with 5’ ends mapping to published TSSs that initiated with G were binned into quartiles according to the impact of mutT4 on their 5’ end phosphorylation status. Secondary structure characteristics of the quartile least affected by mutT4 (Q1) and the quartile most affected by mutT4 (Q4) were compared. The first 20 nt of each transcript were computationally folded and the probability that the first 5 nt were unpaired in these structures (D) as well as the minimum free energy (MFE) of the structures (E) were determined. (F) The impact of mutT4 on leadered vs leaderless transcripts was compared. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. A one-way ANOVA was performed with Tukey’s test for panel C. Mann-Whitney test was performed for panels D, E and F.

Article Snippet: 5 µg of DNase-treated total RNA extracted from Mtb in exponential phase was incubated with 10 U of E. coli RNA 5’ Pyrophosphohydrolase (RppH) (New England Biolabs, M0356), or with no enzyme as a control, in NEBuffer 2 for 1 h at 37 °C.

Techniques: Construct, Produced, Comparison, Derivative Assay, Phospho-proteomics, MANN-WHITNEY