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The genome sequence of multidrug-resistant Raoultella terrigena RT01-5M1 strain isolated from Canadian farmed salmon was determined using Oxford nanopore and Illumina MiSeq sequencers. The assembled chromosome was estimated at 5,699,993 bp in size, with two plasmids, 164,879 bp and 82,046 bp. The chromosome and smaller plasmid contained antimicrobial resistance genes.

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In marine sediments surrounding salmon aquaculture sites, organic matter (OM) enrichment has been shown to influence resident bacterial community composition; however, additional effects on these communities due to combined use of the sea-lice therapeutant emamectin benzoate (EMB) and the widely used antibiotic oxytetracycline (OTC) are unknown. Here, we use sediment microcosms to assess the influence of OM, EMB, and OTC on benthic bacterial communities. Microcosms consisted of mud or sand sediments enriched with OM (fish and feed wastes) and spiked with EMB and OTC at environmentally-relevant concentrations. Samples were collected from initial matrices at the initiation of the trial and after 110 days for 16 S rRNA gene sequencing of the V3-V4 region and microbiome profiling. The addition of OM in both mud and sand sediments reduced alpha diversities; for example, an average of 1106 amplicon sequence variants (ASVs) were detected in mud with no OM addition, while only 729 and 596 ASVs were detected in mud with low OM and high OM, respectively. Sediments enriched with OM had higher relative abundances of Spirochaetota, Firmicutes, and Bacteroidota. For instance, Spirochaetota were detected in sediments with no OM with a relative abundance range of 0.01-1.2%, while in sediments enriched with OM relative abundance varied from 0.16% to 26.1%. In contrast, the addition of EMB (60 ng/g) or OTC (150 ng/g) did not result in distinct taxonomic shifts in the bacterial communities compared to un-spiked sediments during the timeline of this experiment. EMB and OTC concentrations may have been below effective inhibitor concentrations for taxa in these communities; further work should explore gene content and the presence of antibiotic resistance genes (ARGs) in sediment-dwelling bacteria.

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Three types of DNA methyl modifications have been detected in bacterial genomes, and mechanistic studies have demonstrated roles for DNA methylation in physiological functions ranging from phage defense to transcriptional control of virulence and host-pathogen interactions. Despite the ubiquity of methyltransferases and the immense variety of possible methylation patterns, epigenomic diversity remains unexplored for most bacterial species. Members of the Bacteroides fragilis group (BFG) reside in the human gastrointestinal tract as key players in symbiotic communities but also can establish anaerobic infections that are increasingly multi-drug resistant. In this work, we utilize long-read sequencing technologies to perform pangenomic (n = 383) and panepigenomic (n = 268) analysis of clinical BFG isolates cultured from infections seen at the NIH Clinical Center over four decades. Our analysis reveals that single BFG species harbor hundreds of DNA methylation motifs, with most individual motif combinations occurring uniquely in single isolates, implying immense unsampled methylation diversity within BFG epigenomes. Mining of BFG genomes identified more than 6000 methyltransferase genes, approximately 1000 of which were associated with intact prophages. Network analysis revealed substantial gene flow among disparate phage genomes, implying a role for genetic exchange between BFG phages as one of the ultimate sources driving BFG epigenome diversity.

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Erwinia amylovora is the causal agent of fire blight of apple and pear trees. Several bacteria have been shown to produce antibiotics that antagonize E. amylovora, including pantocins, herbicolins, dapdiamides, and the vinylglycines, 4-formylaminooxyvinylglycine (FVG) and 4-aminoethoxyvinylglycine (AVG). Pantoea ananatis BRT175 was previously shown to exhibit antibiotic activity against E. amylovora via the production of Pantoea natural product 1 (PNP-1), later shown to be FVG; however, exposure of E. amylovora to FVG results in spontaneously resistant mutants. To identify the mechanism of resistance, we used genome variant analysis on spontaneous FVG-resistant mutants of E. amylovora and identified null mutations in the l-asparagine permease gene ansP Heterologous expression of ansP in normally resistant Escherichia coli was sufficient to impart FVG susceptibility, suggesting that FVG is imported through this permease. Because FVG and AVG are structurally similar, we hypothesized that resistance to AVG would also be conferred through inactivation of ansP; however, ansP mutants were not resistant to AVG. We found that spontaneously resistant Ea321 mutants also arise in the presence of AVG, with whole-genome variant analysis revealing that resistance was due to inactivation of the arginine ABC transporter permease subunit gene artQ Heterologous expression of the predicted lysE-like transporter encoded within the Pantoea ananatis BRT175 FVG biosynthetic cluster, which is likely responsible for antibiotic export, was sufficient to confer resistance to both FVG and AVG. This work highlights the important roles of amino acid transporters in antibiotic import into bacteria and the potential utility of antimicrobial amino acid analogs as antibiotics.IMPORTANCE The related antibiotics formylaminooxyvinylglycine (FVG) and aminoethoxyvinylglycine (AVG) have been shown to have activity against the fire blight pathogen Erwinia amylovora; however, E. amylovora can develop spontaneous resistance to these antibiotics. By comparing the genomes of mutants to those of the wild type, we found that inactivation of the l-asparagine transporter conferred resistance to FVG, while inactivation of the l-arginine transporter conferred resistance to AVG. We also show that the transporter encoded by the FVG biosynthetic cluster can confer resistance to both FVG and AVG. Our work indicates the important role that amino acid transporters play in the import of antibiotics and highlights the possible utility in designer antibiotics that enter the bacterial cell through amino acid transporters.

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Pantoea is a versatile genus of bacteria with both plant- and animal-pathogenic strains, some of which have been suggested to cause human infections. There is, however, limited knowledge on the potential determinants used for host association and pathogenesis in animal systems. In this study, we used the model host Dictyostelium discoideum to show that isolates of Pantoea ananatis exhibit differential grazing susceptibility, with some being resistant to grazing by the amoebae. We carried out a high-throughput genetic screen of one grazing-resistant isolate, P. ananatis BRT175, using the D. discoideum pathosystem to identify genes responsible for the resistance phenotype. Among the 26 candidate genes involved in grazing resistance, we identified rhlA and rhlB, which we show are involved in the biosynthesis of a biosurfactant that enables swarming motility in P. ananatis BRT175. Using liquid chromatography-mass spectrometry (LC-MS), the biosurfactant was shown to be a glycolipid with monohexose-C10-C10 as the primary congener. We show that this novel glycolipid biosurfactant is cytotoxic to the amoebae and is capable of compromising cellular integrity, leading to cell lysis. The production of this biosurfactant may be important for bacterial survival in the environment and could contribute to the establishment of opportunistic infections. IMPORTANCE The genetic factors used for host interaction by the opportunistic human pathogen Pantoea ananatis are largely unknown. We identified two genes that are important for the production of a biosurfactant that confers grazing resistance against the social amoeba Dictyostelium discoideum. We show that the biosurfactant, which exhibits cytotoxicity toward the amoebae, is a glycolipid that incorporates a hexose rather than rhamnose. The production of this biosurfactant may confer a competitive advantage in the environment and could potentially contribute to the establishment of opportunistic infections.

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Fire Blight is a destructive disease of apple and pear caused by the enteric bacterial pathogen, Erwinia amylovora. E. amylovora initiates infection by colonizing the stigmata of apple and pear trees, and entering the plants through natural openings. Epiphytic populations of the related enteric bacterium, Pantoea, reduce the incidence of disease through competition and antibiotic production. In this study, we identify an antibiotic from Pantoea ananatis BRT175, which is effective against E. amylovora and select species of Pantoea. We used transposon mutagenesis to create a mutant library, screened approximately 5,000 mutants for loss of antibiotic production, and recovered 29 mutants. Sequencing of the transposon insertion sites of these mutants revealed multiple independent disruptions of an 8.2 kb cluster consisting of seven genes, which appear to be coregulated. An analysis of the distribution of this cluster revealed that it was not present in any other of our 115 Pantoea isolates, or in any of the fully sequenced Pantoea genomes, and is most closely related to antibiotic biosynthetic clusters found in three different species of Pseudomonas. This identification of this biosynthetic cluster highlights the diversity of natural products produced by Pantoea.

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Pantoea agglomerans is an enteric bacterium that is capable of causing both plant and human disease. Here, we report the genome sequence of a cystic fibrosis isolate, P. agglomerans Tx10, which produces an antibiotic that is effective against Staphylococcus aureus.

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Pantoea is a member of the Enterobacteriaceae, whose members have been shown to produce novel antibiotics. Here, we report the 4.8-Mb genome sequence of Pantoea ananatis strain BRT175, an epiphytic isolate from strawberries that produces an antibiotic that is effective against the fire blight pathogen, Erwinia amylovora.