Journal: bioRxiv

Article Title: Identification and targeting of microbial putrescine acetylation in bloodstream infections

doi: 10.1101/2023.09.21.558834

Figure Lengend Snippet: (A) Volcano plot highlighting significant elevations in N -acetylputrescine and 4-acetamidobutanoate in humans with gram-negative septic shock (B) N -acetylpturescine and 4-acetomidobutanoate levels correlate with worse clinical outcomes as measured by APACHE II scores (C) Putrescine metabolic pathway outlining production of N -acetylputrescine and 4-acetamidobutanoate (D) Putrescine metabolites are elevated in the mouse cecal slurry model of septic shock/BSI; HK = heat killed, CS = cecal slurry; n = 8 PBS, n = 7 HK CS, n = 4 Live CS; p-values were determined by One-way ANOVA followed by Tukey’s multiple comparisons test (E)Septic shock/BSI with heat-killed cecal slurry rescued with E. coli BW25113 results in increased plasma putrescine metabolite levels; n = 5 HK CS, n = 9 HK CS + E. coli BW25113; Two-tailed p-values were determined by unpaired t test (F) Klebsiella pneumoniae pneumonia results in increased bronchial alveolar lavage (BAL) fluid levels of N - acetylputrescine; n = 4 PBS, n = 6 K. pneumoniae (KP9); Two-tailed p-values were determined by Mann Whitney test to include the outlier. (G) Klebsiella pneumoniae pneumonia results in increased plasma levels of N -acetylputrescine; n = 4 PBS, n = 6 K. pneumoniae (KP9); Two-tailed p-values were determined by Mann Whitney test to include the outlier. (H) Bacterial and mouse production of N -acetylputrescine + 4-acetamidobutanoate from putrescine; n = 3 for bacteria, representative data from 1-3 independent repeats; n = 6 mice; Blue = E. coli , Green = K. pneumoniae , Red = P. aeruginosa For all panels, data presented are means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p<0.0001

Article Snippet: SpeG was amplified from E. coli K12 MG1655 genomic DNA and SAT1 , PA4114, PA1472, and PA1377 were ordered as E. coli codon optimized sequences (ThermoFisher); all were cloned cloned into pET-28A-inducible expression vectors using Gibson assembly (including an in-frame either N-terminal or C-terminal polyhistidine sequence) (primers in Table S6 ).

Techniques: Clinical Proteomics, Two Tailed Test, MANN-WHITNEY, Bacteria

Journal: bioRxiv

Article Title: Identification and targeting of microbial putrescine acetylation in bloodstream infections

doi: 10.1101/2023.09.21.558834

Figure Lengend Snippet: (A) Complementation in E. coli BW25113 demonstrates SpeG can produce N -acetylputrescine; n = 3 per condition, representative data from 3 independent experiments (B) 1 hour production of N -acetylputrescine from putrescine by recombinant purified enzymes; n = 3 per condition, representative data from 3 independent experiments; NE = no enzyme, GFP = green fluorescent protein (C) Kinetics of putrescine acetylation by SpeG is consistent with previously demonstrated cooperative mechanism on spermidine, n = 2-3 per substrate concentration, representative data from 4 independent experiments; summary parameters includes all experiments (D) Maximum-likelihood phylogenetic tree of a representative member of each group of protein sequences sharing >80% amino acid ID (RepNode80); Blue = E. coli SpeG clade, Purple = mammalian SAT1 clade, Turquoise = B. subtilis BltD clade (E) Complementation in E. coli BW25113 demonstrates PA1472 can produce N -acetylputrescine; n = 3 per condition, representative data from 2-3 independent experiments (F) 1 hour production of N -acetylputrescine from putrescine by recombinant purified enzymes; n = 3 per condition, representative data from 2-3 independent experiments; NE = no enzyme, GFP = green fluorescent protein (G) Kinetics of PA1472 on putrescine; n = 2 per concentration, representative data from 3 independent experiments; summary parameters includes all experiments (H) SpeG (PDB: 3WR7) and PA1472 (AlphaFold2) dimers For all panels, data presented are means ± SEM

Article Snippet: SpeG was amplified from E. coli K12 MG1655 genomic DNA and SAT1 , PA4114, PA1472, and PA1377 were ordered as E. coli codon optimized sequences (ThermoFisher); all were cloned cloned into pET-28A-inducible expression vectors using Gibson assembly (including an in-frame either N-terminal or C-terminal polyhistidine sequence) (primers in Table S6 ).

Techniques: Recombinant, Purification, Concentration Assay

Journal: bioRxiv

Article Title: Identification and targeting of microbial putrescine acetylation in bloodstream infections

doi: 10.1101/2023.09.21.558834

Figure Lengend Snippet: (A) Suppression of speG expression with inducible CRISPRi in E. coli patient bloodstream isolate E23; n = 3 per condition, representative data from 2 independent experiments (B) Decreased levels of extracellular N -acetylputrescine with inducible CRISPRi of speG in E23, concentrations normalized to OD600 = 1.0; n = 3 per condition, representative data from 2 independent experiments (C) Increased relative intracellular putrescine levels (concentrations normalized to OD600 = 1.0 and then normalized to concentration of control condition) and decreased ratio of N -acetylputrescine/putrescine with inducible CRISPRi of speG in E23; n = 3 per condition, representative data from 2 independent experiments (D) Inducible CRISPRi of speG suppresses E23 growth in culture; n = 3 per condition, representative data from 3 independent experiments (E) Inducible CRISPRi of speG delays mortality in a cecal slurry model of sepsis with E23; n = 10 mice per group for all groups except n = 9 for RFP – aTC; p-value determined by Log-rank (Mantel-Cox) test For panels A-D, data presented are means ± SEM; Two-tailed p-values were determined by unpaired t test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p<0.0001 aTC = anhydrotetracycline

Article Snippet: SpeG was amplified from E. coli K12 MG1655 genomic DNA and SAT1 , PA4114, PA1472, and PA1377 were ordered as E. coli codon optimized sequences (ThermoFisher); all were cloned cloned into pET-28A-inducible expression vectors using Gibson assembly (including an in-frame either N-terminal or C-terminal polyhistidine sequence) (primers in Table S6 ).

Techniques: Expressing, Concentration Assay, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Identification and targeting of microbial putrescine acetylation in bloodstream infections

doi: 10.1101/2023.09.21.558834

Figure Lengend Snippet: (A) Checkerboard assays demonstrate synergy between diminazene and vancomycin in E. coli E23; representative data from 3 independent experiments (B) Inducible CRISPRi of speG reduces MIC of vancomycin in E. coli E23; mean growth of n = 3 per condition, representative data from 3 independent experiments (C) Inducible CRISPRi of speG enhances membrane permeability in E23; n = 3 per condition, representative data from 2 independent experiments; Two-tailed p-values were determined by unpaired t test (D) Diminazene treatment for 6 hours enhances membrane permeability in E. coli E23; n = 3 per condition, representative data from 2 independent experiments; p-values were determined by One-way ANOVA followed by Dunnett’s multiple comparisons test with all comparisons made against no drug control For panels B-D, data presented are means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p<0.0001 aTC = anhydrotetracycline

Article Snippet: SpeG was amplified from E. coli K12 MG1655 genomic DNA and SAT1 , PA4114, PA1472, and PA1377 were ordered as E. coli codon optimized sequences (ThermoFisher); all were cloned cloned into pET-28A-inducible expression vectors using Gibson assembly (including an in-frame either N-terminal or C-terminal polyhistidine sequence) (primers in Table S6 ).

Techniques: Membrane, Permeability, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Identification and targeting of microbial putrescine acetylation in bloodstream infections

doi: 10.1101/2023.09.21.558834

Figure Lengend Snippet: (A) Checkerboard assays demonstrate synergy between diminazene and antibiotics to which the assayed MDR strains are clinically resistant; representative data from 3-4 independent experiments per strain (B) Diminazene treatment enhances uptake of antibiotics to which MDR strains are resistant; n = 3 per condition, representative data from 1-3 independent experiments; Two-tailed p-values were determined by unpaired t test (C) Combination of diminazene and tetracycline treatment reduces mortality in a cecal slurry model of sepsis with the tetracycline resistant clinical isolate E. coli E1; n = 10 mice per group; p-value determined by Log-rank (Mantel-Cox) test For panels B, data presented are means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p<0.0001 aTC = anhydrotetracycline

Article Snippet: SpeG was amplified from E. coli K12 MG1655 genomic DNA and SAT1 , PA4114, PA1472, and PA1377 were ordered as E. coli codon optimized sequences (ThermoFisher); all were cloned cloned into pET-28A-inducible expression vectors using Gibson assembly (including an in-frame either N-terminal or C-terminal polyhistidine sequence) (primers in Table S6 ).

Techniques: Two Tailed Test

Journal: RSC Advances

Article Title: Microwave-assisted synthesis and in vitro and in silico studies of pyrano[3,2- c ]quinoline-3-carboxylates as dual acting anti-cancer and anti-microbial agents and potential topoisomerase II and DNA-gyrase inhibitors †

doi: 10.1039/d4ra06201a

Figure Lengend Snippet: Reported quinoline-, pyran- and pyranoquinoline-based anticancer and antibacterial derivatives as potential inhibitors of topoisomerase II and DNA gyrase.

Article Snippet: In vitro VEGFR-2 kinase inhibitory activity was evaluated using purified E. coli DNA Gyrase and Relaxed DNA Kit, Protocol TG2000GKIT, (TopoGEN, Inc.® Florida 32128, USA), following the manufacturer's instructions.

Techniques:

Journal: RSC Advances

Article Title: Microwave-assisted synthesis and in vitro and in silico studies of pyrano[3,2- c ]quinoline-3-carboxylates as dual acting anti-cancer and anti-microbial agents and potential topoisomerase II and DNA-gyrase inhibitors †

doi: 10.1039/d4ra06201a

Figure Lengend Snippet: Lead Topo II inhibitors, apoptosis inducers, DNA gyrase inhibitors and design rationale of the multitarget pyranoquinolones 3a–h.

Article Snippet: In vitro VEGFR-2 kinase inhibitory activity was evaluated using purified E. coli DNA Gyrase and Relaxed DNA Kit, Protocol TG2000GKIT, (TopoGEN, Inc.® Florida 32128, USA), following the manufacturer's instructions.

Techniques:

Journal: Genome Biology

Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

doi: 10.1186/gb-2012-13-3-r23

Figure Lengend Snippet: Performance evaluation of five rRNA depletion methods. (a) Shown is the distribution of RNA-seq reads aligning to protein-coding sequences (CDS; blue), rRNA (red), and other regions (tRNA, non-coding RNA, small RNA, and intergenic regions; gray) for undepleted total RNA (top) and five rRNA depletion protocols. (b) The lengths of the black bars represent the coefficient of determination (R2) for RPKM values before and after rRNA depletion using different rRNA-depletion methods. Ribo-Zero, normalization using duplex-specific nuclease (DSN) and Ovation were tested on a 1:1:1 pool (by mass) of total RNA prepared from P. marinus, E. coli, and R. sphaeroides. MICROBExpress and mRNA-ONLY were performed on individual RNA preparations without pooling.

Article Snippet: The second strand was synthesized by adding 1× of second strand buffer (5×; Invitrogen), 0.2 mM of dNTPs (10 mM; Invitrogen), 40 U of E. coli DNA polymerase I (10 U/μl; NEB, Ipswich, MA, USA), 10 U of E. coli DNA ligase (10 U/μl; NEB), 5 U of RNase H (5 U/μl; Invitrogen) to the first strand reaction (150 μl total volume).

Techniques: RNA Sequencing Assay

Journal: Genome Biology

Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

doi: 10.1186/gb-2012-13-3-r23

Figure Lengend Snippet: Depletion of rRNA in a mixture of total RNAs from E. coli, R. sphaeroides and P. marinus with Ribo-Zero is reproducible and works well with fragmented total RNA. (a) The pie charts represent the mapped read distributions of protein-coding genes (CDS; blue), rRNA (red), and other reads (tRNA, non-coding RNA, small RNA and intergenic regions; gray) for undepleted total RNA, two technical replicates of Ribo-Zero treatment of intact total RNA and for Ribo-Zero treatment of fragmented total RNA. (b,c) Double-log scatter plots of RPKM values and the coefficient of determination (R2) for the technical Ribo-Zero replicates (b) and for Ribo-Zero treatment of fragmented versus intact total RNA (c). Points on the axes represent CDSs with zero coverage in one of the two samples. The number of data points in the diagonal cloud and on the axes is indicated. The total number of annotated CDSs in the three bacterial genomes is 10,278.

Article Snippet: The second strand was synthesized by adding 1× of second strand buffer (5×; Invitrogen), 0.2 mM of dNTPs (10 mM; Invitrogen), 40 U of E. coli DNA polymerase I (10 U/μl; NEB, Ipswich, MA, USA), 10 U of E. coli DNA ligase (10 U/μl; NEB), 5 U of RNase H (5 U/μl; Invitrogen) to the first strand reaction (150 μl total volume).

Techniques:

Journal: Genome Biology

Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

doi: 10.1186/gb-2012-13-3-r23

Figure Lengend Snippet: Strand specificity of RNA-seq reads. Shown is a 17-kb window of the E. coli genome viewed with the Artemis browser [28]. The mapped reads aligning to the top strand (green) or bottom stand (purple) consistent with the direction of the annotated genes as represented by the blue boxes with arrows and corresponding gene ID numbers and operons below (for example, genes b3196 through b3206).

Article Snippet: The second strand was synthesized by adding 1× of second strand buffer (5×; Invitrogen), 0.2 mM of dNTPs (10 mM; Invitrogen), 40 U of E. coli DNA polymerase I (10 U/μl; NEB, Ipswich, MA, USA), 10 U of E. coli DNA ligase (10 U/μl; NEB), 5 U of RNase H (5 U/μl; Invitrogen) to the first strand reaction (150 μl total volume).

Techniques: RNA Sequencing Assay

Journal: Nucleic Acids Research

Article Title: A dynamic checkpoint in oxidative lesion discrimination by formamidopyrimidine–DNA glycosylase

doi: 10.1093/nar/gkv1092

Figure Lengend Snippet: Wild-type and mutant Escherichia coli Fpg kinetic parameters

Article Snippet: To obtain the AP substrate, 10 pmol of the duplex contained uracil in the required position was treated with 1 U E. coli uracil–DNA glycosylase (New England Biolabs, Ipswich, MA) immediately before reaction with Fpg.

Techniques: Mutagenesis, Activity Assay