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1. Three genes controlling streptomycin susceptibility in Agrobacterium fabrum

   https://doi.org/10.1128/jb.00165-23  

   Howarth, Robyn E; Pattillo, Curtis M; Griffitts, Joel S; Calvopina-Chavez, Diana G (September 2023, Journal of Bacteriology)

   Becker, Anke (Ed.)

   ABSTRACT Streptomycin (Sm) is a commonly used antibiotic for its efficacy against diverse bacteria. The plant pathogenAgrobacterium fabrumis a model for studying pathogenesis and interkingdom gene transfer. Streptomycin-resistant variants ofA. fabrumare commonly employed in genetic analyses, yet mechanisms of resistance and susceptibility to streptomycin in this organism have not previously been investigated. We observe that resistance to a high concentration of streptomycin arises at high frequency inA. fabrum, and we attribute this trait to the presence of a chromosomal gene (strB) encoding a putative aminoglycoside phosphotransferase. We show howstrB, along withrpsL(encoding ribosomal protein S12) andrsmG(encoding a 16S rRNA methyltransferase), modulates streptomycin sensitivity inA. fabrum. IMPORTANCEThe plant pathogenAgrobacterium fabrumis a widely used model bacterium for studying biofilms, bacterial motility, pathogenesis, and gene transfer from bacteria to plants. Streptomycin (Sm) is an aminoglycoside antibiotic known for its broad efficacy against gram-negative bacteria.A. fabrumexhibits endogenous resistance to somewhat high levels of streptomycin, but the mechanism underlying this resistance has not been elucidated. Here, we demonstrate that this resistance is caused by a chromosomally encoded streptomycin-inactivating enzyme, StrB, that has not been previously characterized inA. fabrum. Furthermore, we show how the genesrsmG,rpsL, andstrBjointly modulate streptomycin susceptibility inA. fabrum. 

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2. Conserved lipid-facing basic residues promote the insertion of the porin OmpC into the E. coli outer membrane

   https://doi.org/10.1128/mbio.03319-24  

   Peterson, Janine H; Yang, Lixinhao; Gumbart, James C; Bernstein, Harris D (March 2025, mBio)

   Trent, M Stephen; Konovalova, Anna (Ed.)

   ABSTRACT Almost all integral membrane proteins that reside in the outer membrane (OM) of gram-negative bacteria contain a closed amphipathic β sheet (“β barrel”) that serves as a membrane anchor. The membrane integration of β barrel structures is catalyzed by a highly conserved heterooligomer called thebarrelassemblymachine (BAM). Although charged residues that are exposed to the lipid bilayer are infrequently found in outer membrane protein β barrels, the β barrels of OmpC/OmpF-type trimeric porins produced by Enterobacterales contain multiple conserved lipid-facing basic residues located near the extracellular side of the OM. Here, we show that these residues are required for the efficient insertion of theEscherichia coliOmpC protein into the OMin vivo. We found that the mutation of multiple basic residues to glutamine or alanine slowed insertion and reduced insertion efficiency. Furthermore, molecular dynamics simulations provided evidence that the basic residues promote the formation of hydrogen bonds and salt bridges with lipopolysaccharide, a unique glycolipid located exclusively in the outer leaflet of the OM. Taken together, our results support a model in which hydrophilic interactions between OmpC and LPS help to anchor the protein in the OM when the local environment is perturbed by BAM during membrane insertion and suggest a surprising role for membrane lipids in the insertion reaction.IMPORTANCEThe assembly (folding and membrane insertion) of bacterial outer membrane proteins (OMPs) is an essential cellular process that is a potential target for novel antibiotics. A heterooligomer called thebarrelassemblymachine (BAM) plays a major role in catalyzing OMP assembly. Here, we show that a group of highly conserved lipid-facing basic residues inEscherichia coliOmpC, a member of a major family of abundant OMPs known as trimeric porins, is required for the efficient integration of the protein into the outer membrane (OM). Based on our work and previous studies, we propose that the basic residues form interactions with a unique OM lipid (lipopolysaccharide) that promotes the insertion reaction. Our results provide strong evidence that interactions between specific membrane lipids and at least a subset of OMPs are required to supplement the activity of BAM and facilitate the integration of the proteins into the membrane. 

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3. High prevalence of lipopolysaccharide mutants and R2-pyocin susceptible variants in Pseudomonas aeruginosa populations sourced from cystic fibrosis lung infections

   https://doi.org/10.1128/spectrum.01773-23  

   Mei, Madeline; Pheng, Preston; Kurzeja-Edwards, Detriana; Diggle, Stephen P (December 2023, Microbiology Spectrum)

   Van_Tyne, Daria (Ed.)

   ABSTRACT Chronic, highly antibiotic-resistant infections in cystic fibrosis (CF) lungs contribute to increasing morbidity and mortality.Pseudomonas aeruginosa, a common CF pathogen, exhibits resistance to multiple antibiotics, contributing to antimicrobial resistance (AMR). These bacterial populations display genetic and phenotypic diversity, but it is unclear how this diversity affects susceptibility to bacteriocins. R-pyocins, i.e., bacteriocins produced byP. aeruginosa, are phage-tail-like antimicrobials. R-pyocins have potential as antimicrobials, however, recent research suggests the diversity ofP. aeruginosavariants within CF lung infections leads to varying susceptibility to R-pyocins. This variation may be linked to changes in lipopolysaccharide (LPS), acting as the R-pyocin receptor. Currently, it is unknown how frequently R-pyocin-susceptible strains are in chronic CF lung infection, particularly when considering the heterogeneity within these strains. In this study, we tested the R2-pyocin susceptibility of 139P.aeruginosavariants from 17 sputum samples of 7 CF patients and analyzed LPS phenotypes. We found that 83% of sputum samples did not have R2-pyocin-resistant variants, while nearly all samples contained susceptible variants. There was no correlation between LPS phenotype and R2-pyocin susceptibility, though we estimate that about 76% of sputum-derived variants lack an O-specific antigen, 40% lack a common antigen, and 24% have altered LPS cores. The absence of a correlation between LPS phenotype and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Our research supports the potential of R-pyocins as therapeutic agents, as many infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations. IMPORTANCECystic fibrosis (CF) patients often experience chronic, debilitating lung infections caused by antibiotic-resistantPseudomonas aeruginosa, contributing to antimicrobial resistance (AMR). The genetic and phenotypic diversity ofP. aeruginosapopulations in CF lungs raises questions about their susceptibility to non-traditional antimicrobials, like bacteriocins. In this study, we focused on R-pyocins, a type of bacteriocin with high potency and a narrow killing spectrum. Our findings indicate that a large number of infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations, supporting their potential use as therapeutic agents. The absence of a clear correlation between lipopolysaccharide (LPS) phenotypes and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Understanding the relationship between LPS phenotypes and R-pyocin susceptibility is crucial for developing effective treatments for these chronic infections. 

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4. A single phosphorylatable amino acid residue is essential for the recognition of multiple potyviral HCPro effectors by potato Ny_tbr

   https://doi.org/10.1111/mpp.70027  

   Alex, Bryce G; Zhang, Zong‐Ying; Lasky, Danny; Garcia‐Ruiz, Hernan; Dewberry, Ronnie; Allen, Caitilyn; Halterman, Dennis; Rakotondrafara, Aurélie M (November 2024, Molecular Plant Pathology)

   Abstract Potato virus Y(PVY,Potyviridae) is among the most important viral pathogens of potato. The potato resistance geneNy_tbrconfers hypersensitive resistance to the ordinary strain of PVY (PVY^O), but not the necrotic strain (PVY^N). Here, we unveil that residue 247 of PVY helper component proteinase (HCPro) acts as a central player controllingNy_tbrstrain‐specific activation. We found that substituting the serine at 247 in the HCPro of PVY^O(HCPro^O) with an alanine as in PVY^NHCPro (HCPro^N) disruptsNy_tbrrecognition. Conversely, an HCPro^Nmutant carrying a serine at position 247 triggers defence. Moreover, we demonstrate that plant defences are induced against HCPro^Omutants with a phosphomimetic or another phosphorylatable residue at 247, but not with a phosphoablative residue, suggesting that phosphorylation could modulateNy_tbrresistance. Extending beyond PVY, we establish that the same response elicited by the PVY^OHCPro is also induced by HCPro proteins from other members of thePotyviridaefamily that have a serine at position 247, but not by those with an alanine. Together, our results provide further insights in the strain‐specific PVY resistance in potato and infer a broad‐spectrum detection mechanism of plant potyvirus effectors contingent on a single amino acid residue. 

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5. Comparison of methods proposed for monitoring cefotaxime-resistant Escherichia coli in the water environment

   https://doi.org/10.1128/aem.02128-23  

   Calarco, Jeanette; Pruden, Amy; Harwood, Valerie J (May 2024, Applied and Environmental Microbiology)

   Semrau, Jeremy D (Ed.)

   ABSTRACT Escherichia coliis a promising subject for globally coordinated surveillance of antimicrobial resistance (AMR) in water environments due to its clinical relevance and widespread use as an indicator of fecal contamination. Cefotaxime-resistantE. coliwas recently evaluated favorably for this purpose by the World Health Organization TriCycle Protocol, which specifies tryptone bile x-glucuronide (TBX) medium and incubation at 35°C. We assessed comparability with the U.S. Environmental Protection Agency-approved method forE. coliquantification, which uses membrane-thermotolerantE. coli(mTEC) agar and incubation at 44.5°C, in terms of recovery ofE. coliand cefotaxime-resistantE. colifrom wastewater influent and surface waters. TotalE. coliconcentrations in wastewater influent were 10⁶–10⁸CFU/100 mL, while cefotaxime-resistantE. coliwere ~100-fold lower. TotalE. coliin surface waters were ~10²CFU/100 mL, and cefotaxime-resistant isolates were near the limit of detection (0.4 CFU/100 mL). Total and putative cefotaxime-resistantE. coliconcentrations did not differ significantly between media or by incubation method; however, colonies isolated on mTEC were more frequently confirmed to species (97.1%) compared to those from TBX (92.5%). Incubation in a water bath at 44.5°C significantly decreased non-specific background growth and improved confirmation frequency on both media (97.4%) compared to incubation at 35°C (92.3%). This study helps to advance globally coordinated AMR in water environments and suggests that the TriCycle Protocol is adaptable to other standard methods that may be required in different locales, while also offering a means to improve specificity by decreasing the frequency of false-positive identification of cefotaxime-resistantE. coliby modifying incubation conditions.IMPORTANCEAs antibiotic-resistant bacteria in water environments are increasingly recognized as contributors to the global antibiotic resistance crisis, the need for a monitoring subject that captures antibiotic resistance trends on a global scale increases. The World Health Organization TriCycle Protocol proposes the use of cefotaxime-resistantEscherichia coliisolated on tryptone bile x-glucuronide agar. The U.S. Environmental Protection Agency (USEPA) criteria for safe recreational waters also useE. colias an indicator but specify the use of mTEC agar at a higher incubation temperature (44.5°C vs 35°C). We assessed the comparability of these methods for isolating total and cefotaxime-resistantE. coli, finding overall good agreement and performance, but significantly higher specificity towardE. coliselection with the use of the USEPA incubation protocol and mTEC agar. This study is the first to directly compare these methods and provides evidence that the methods may be used interchangeably for global surveillance of antibiotic resistance in the environment. 

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