The Rvv two-component regulatory system regulates biofilm formation and colonization in Vibrio cholerae.

The facultative human pathogen, Vibrio cholerae, employs two-component signal transduction systems (TCS) to sense and respond to environmental signals encountered during its infection cycle. TCSs consist of a sensor histidine kinase (HK) and a response regulator (RR); the V. cholerae genome encodes 43 HKs and 49 RRs, of which 25 are predicted to be cognate pairs. Using deletion mutants of each HK gene, we analyzed the transcription of vpsL, a biofilm gene required for Vibrio polysaccharide and biofilm formation. We found that a V. cholerae TCS that had not been studied before, now termed Rvv, controls biofilm gene transcription. The Rvv TCS is part of a three-gene operon that is present in 30% of Vibrionales species. The rvv operon encodes RvvA, the HK; RvvB, the cognate RR; and RvvC, a protein of unknown function. Deletion of rvvA increased transcription of biofilm genes and altered biofilm formation, while deletion of rvvB or rvvC lead to no changes in biofilm gene transcription. The phenotypes observed in ΔrvvA depend on RvvB. Mutating RvvB to mimic constitutively active and inactive versions of the RR only impacted phenotypes in the ΔrvvA genetic background. Mutating the conserved residue required for kinase activity in RvvA did not affect phenotypes, whereas mutation of the conserved residue required for phosphatase activity mimicked the phenotype of the rvvA mutant. Furthermore, ΔrvvA displayed a significant colonization defect which was dependent on RvvB and RvvB phosphorylation state, but not on VPS production. We found that RvvA's phosphatase activity regulates biofilm gene transcription, biofilm formation, and colonization phenotypes. This is the first systematic analysis of the role of V. cholerae HKs in biofilm gene transcription and resulted in the identification of a new regulator of biofilm formation and virulence, advancing our understanding of the role TCSs play in regulating these critical cellular processes in V. cholerae.

ECO: the Evidence and Conclusion Ontology, an update for 2022.

The Evidence and Conclusion Ontology (ECO) is a community resource that provides an ontology of terms used to capture the type of evidence that supports biomedical annotations and assertions. Consistent capture of evidence information with ECO allows tracking of annotation provenance, establishment of quality control measures, and evidence-based data mining. ECO is in use by dozens of data repositories and resources with both specific and general areas of focus. ECO is continually being expanded and enhanced in response to user requests as well as our aim to adhere to community best-practices for ontology development. The ECO support team engages in multiple collaborations with other ontologies and annotating groups. Here we report on recent updates to the ECO ontology itself as well as associated resources that are available through this project. ECO project products are freely available for download from the project website (https://evidenceontology.org/) and GitHub (https://github.com/evidenceontology/evidenceontology). ECO is released into the public domain under a CC0 1.0 Universal license.

Transcriptional rewiring of the GcrA/CcrM bacterial epigenetic regulatory system in closely related bacteria.

Transcriptional rewiring is the regulation of different target genes by orthologous regulators in different organisms. While this phenomenon has been observed, it has not been extensively studied, particularly in core regulatory systems. Several global cell cycle regulators are conserved in the Alphaproteobacteria, providing an excellent model to study this phenomenon. First characterized in Caulobacter crescentus, GcrA and CcrM compose a DNA methylation-based regulatory system that helps coordinate the complex life cycle of this organism. These regulators are well-conserved across Alphaproteobacteria, but the extent to which their regulatory targets are conserved is not known. In this study, the regulatory targets of GcrA and CcrM were analyzed by SMRT-seq, RNA-seq, and ChIP-seq technologies in the Alphaproteobacterium Brevundimonas subvibrioides, and then compared to those of its close relative C. crescentus that inhabits the same environment. Although the regulators themselves are highly conserved, the genes they regulate are vastly different. GcrA directly regulates 204 genes in C. crescentus, and though B. subvibrioides has orthologs to 147 of those genes, only 48 genes retained GcrA binding in their promoter regions. Additionally, only 12 of those 48 genes demonstrated significant transcriptional change in a gcrA mutant, suggesting extensive transcriptional rewiring between these organisms. Similarly, out of hundreds of genes CcrM regulates in each of these organisms, only 2 genes were found in common. When multiple Alphaproteobacterial genomes were analyzed bioinformatically for potential GcrA regulatory targets, the regulation of genes involved in DNA replication and cell division was well conserved across the Caulobacterales but not outside this order. This work suggests that significant transcriptional rewiring can occur in cell cycle regulatory systems even over short evolutionary distances.

Non-canonical LexA proteins regulate the SOS response in the Bacteroidetes.

Lesions to DNA compromise chromosome integrity, posing a direct threat to cell survival. The bacterial SOS response is a widespread transcriptional regulatory mechanism to address DNA damage. This response is coordinated by the LexA transcriptional repressor, which controls genes involved in DNA repair, mutagenesis and cell-cycle control. To date, the SOS response has been characterized in most major bacterial groups, with the notable exception of the Bacteroidetes. No LexA homologs had been identified in this large, diverse and ecologically important phylum, suggesting that it lacked an inducible mechanism to address DNA damage. Here, we report the identification of a novel family of transcriptional repressors in the Bacteroidetes that orchestrate a canonical response to DNA damage in this phylum. These proteins belong to the S24 peptidase family, but are structurally different from LexA. Their N-terminal domain is most closely related to CI-type bacteriophage repressors, suggesting that they may have originated from phage lytic phase repressors. Given their role as SOS regulators, however, we propose to designate them as non-canonical LexA proteins. The identification of a new class of repressors orchestrating the SOS response illuminates long-standing questions regarding the origin and plasticity of this transcriptional network.

Crowdsourcing biocuration: The Community Assessment of Community Annotation with Ontologies (CACAO).

Experimental data about gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5,000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a 10-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills.

ECO, the Evidence & Conclusion Ontology: community standard for evidence information.

The Evidence and Conclusion Ontology (ECO) contains terms (classes) that describe types of evidence and assertion methods. ECO terms are used in the process of biocuration to capture the evidence that supports biological assertions (e.g. gene product X has function Y as supported by evidence Z). Capture of this information allows tracking of annotation provenance, establishment of quality control measures and query of evidence. ECO contains over 1500 terms and is in use by many leading biological resources including the Gene Ontology, UniProt and several model organism databases. ECO is continually being expanded and revised based on the needs of the biocuration community. The ontology is freely available for download from GitHub (https://github.com/evidenceontology/) or the project's website (http://evidenceontology.org/). Users can request new terms or changes to existing terms through the project's GitHub site. ECO is released into the public domain under CC0 1.0 Universal.

Flexible comparative genomics of prokaryotic transcriptional regulatory networks.

BACKGROUND: Comparative genomics methods enable the reconstruction of bacterial regulatory networks using available experimental data. In spite of their potential for accelerating research into the composition and evolution of bacterial regulons, few comparative genomics suites have been developed for the automated analysis of these regulatory systems. Available solutions typically rely on precomputed databases for operon and ortholog predictions, limiting the scope of analyses to processed complete genomes, and several key issues such as the transfer of experimental information or the integration of regulatory information in a probabilistic setting remain largely unaddressed. RESULTS: Here we introduce CGB, a flexible platform for comparative genomics of prokaryotic regulons. CGB has few external dependencies and enables fully customized analyses of newly available genome data. The platform automates the merging of experimental information and uses a gene-centered, Bayesian framework to generate and integrate easily interpretable results. We demonstrate its flexibility and power by analyzing the evolution of type III secretion system regulation in pathogenic Proteobacteria and by characterizing the SOS regulon of a new bacterial phylum, the Balneolaeota. CONCLUSIONS: Our results demonstrate the applicability of the CGB pipeline in multiple settings. CGB's ability to automatically integrate experimental information from multiple sources and use complete and draft genomic data, coupled with its non-reliance on precomputed databases and its easily interpretable display of gene-centered posterior probabilities of regulation provide users with an unprecedented level of flexibility in launching comparative genomics analyses of prokaryotic transcriptional regulatory networks. The analyses of type III secretion and SOS response regulatory networks illustrate instances of convergent and divergent evolution of these regulatory systems, showcasing the power of formal ancestral state reconstruction at inferring the evolutionary history of regulatory networks.

Comparative genomics of the DNA damage-inducible network in the Patescibacteria.

Metagenomics provide unprecedented insights into the genetic diversity of uncultivated bacteria inhabiting natural environments. Recent surveys have uncovered a major radiation of candidate phyla encompassing the Patescibacteria superphylum. Patescibacteria have small genomes and a presumed symbiotic or parasitic lifestyle, but the difficulty in culturing representative members constrains the characterization of behavioural and adaptive traits. Here we combine in silico and in vitro approaches to characterize the SOS transcriptional response to DNA damage in the Patescibacteria superphylum. Leveraging comparative genomics methods, we identify and experimentally define a novel binding motif for the SOS transcriptional repressor LexA, and we use this motif to characterize the conserved elements of the SOS regulatory network in Patescibacteria. The Patescibacteria LexA-binding motif has unusual direct-repeat structure, and comparative analyses reveal sequence and structural similarities with the distant Acidobacteria LexA protein. Our results reveal a shared core SOS network, complemented by varying degrees of LexA regulation of other core SOS functions. This work illustrates how the combination of computational and experimental methods can leverage metagenomic data to characterize transcriptional responses in uncultivated bacteria. The report of an operational SOS response in presumed symbiotic and parasitic bacteria hints at an intermediate step in the process of genome reduction.

Assessment of transfer methods for comparative genomics of regulatory networks in bacteria.

BACKGROUND: Comparative genomics can leverage the vast amount of available genomic sequences to reconstruct and analyze transcriptional regulatory networks in Bacteria, but the efficacy of this approach hinges on the ability to transfer regulatory network information from reference species to the genomes under analysis. Several methods have been proposed to transfer regulatory information between bacterial species, but the paucity and distributed nature of experimental information on bacterial transcriptional networks have prevented their systematic evaluation. RESULTS: We report the compilation of a large catalog of transcription factor-binding sites across Bacteria and its use to systematically benchmark proposed transfer methods across pairs of bacterial species. We evaluate motif- and accuracy-based metrics to assess the results of regulatory network transfer and we identify the precision-recall area-under-the-curve as the best metric for this purpose due to the large class-imbalanced nature of the problem. Methods assuming conservation of the transcription factor-binding motif (motif-based) are shown to substantially outperform those assuming conservation of regulon composition (network-based), even though their efficiency can decrease sharply with increasing phylogenetic distance. Variations of the basic motif-based transfer method do not yield significant improvements in transfer accuracy. Our results indicate that detection of a large enough number of regulated orthologs is critical for network-based transfer methods, but that relaxing orthology requirements does not improve results. Using the transcriptional regulators LexA and Fur as case examples, we also show how DNA-binding domain sequence similarity can yield confounding results as an indicator of transfer efficiency for motif-based methods. CONCLUSIONS: Counter to standard practice, our evaluation of metrics to assess the efficiency of methods for regulatory network information transfer reveals that the area under precision-recall (PR) curves is a more precise and informative metric than that of receiver-operating-characteristic (ROC) curves, confirming similar findings in other class-imbalanced settings. Our systematic assessment of transfer methods reveals that simple approaches to both motif- and network-based transfer of regulatory information provide equal or better results than more elaborate methods. We also show that there are not effective predictors of transfer efficacy, substantiating the long-standing practice of manual curation in comparative genomics analyses.

Parametric bootstrapping for biological sequence motifs.

BACKGROUND: Biological sequence motifs drive the specific interactions of proteins and nucleic acids. Accordingly, the effective computational discovery and analysis of such motifs is a central theme in bioinformatics. Many practical questions about the properties of motifs can be recast as random sampling problems. In this light, the task is to determine for a given motif whether a certain feature of interest is statistically unusual among relevantly similar alternatives. Despite the generality of this framework, its use has been frustrated by the difficulties of defining an appropriate reference class of motifs for comparison and of sampling from it effectively. RESULTS: We define two distributions over the space of all motifs of given dimension. The first is the maximum entropy distribution subject to mean information content, and the second is the truncated uniform distribution over all motifs having information content within a given interval. We derive exact sampling algorithms for each. As a proof of concept, we employ these sampling methods to analyze a broad collection of prokaryotic and eukaryotic transcription factor binding site motifs. In addition to positional information content, we consider the informational Gini coefficient of the motif, a measure of the degree to which information is evenly distributed throughout a motif's positions. We find that both prokaryotic and eukaryotic motifs tend to exhibit higher informational Gini coefficients (IGC) than would be expected by chance under either reference distribution. As a second application, we apply maximum entropy sampling to the motif p-value problem and use it to give elementary derivations of two new estimators. CONCLUSIONS: Despite the historical centrality of biological sequence motif analysis, this study constitutes to our knowledge the first use of principled null hypotheses for sequence motifs given information content. Through their use, we are able to characterize for the first time differerences in global motif statistics between biological motifs and their null distributions. In particular, we observe that biological sequence motifs show an unusual distribution of IGC, presumably due to biochemical constraints on the mechanisms of direct read-out.

CollecTF: a database of experimentally validated transcription factor-binding sites in Bacteria.

The influx of high-throughput data and the need for complex models to describe the interaction of prokaryotic transcription factors (TF) with their target sites pose new challenges for TF-binding site databases. CollecTF (http://collectf.umbc.edu) compiles data on experimentally validated, naturally occurring TF-binding sites across the Bacteria domain, placing a strong emphasis on the transparency of the curation process, the quality and availability of the stored data and fully customizable access to its records. CollecTF integrates multiple sources of data automatically and openly, allowing users to dynamically redefine binding motifs and their experimental support base. Data quality and currency are fostered in CollecTF by adopting a sustainable model that encourages direct author submissions in combination with in-house validation and curation of published literature. CollecTF entries are periodically submitted to NCBI for integration into RefSeq complete genome records as link-out features, maximizing the visibility of the data and enriching the annotation of RefSeq files with regulatory information. Seeking to facilitate comparative genomics and machine-learning analyses of regulatory interactions, in its initial release CollecTF provides domain-wide coverage of two TF families (LexA and Fur), as well as extensive representation for a clinically important bacterial family, the Vibrionaceae.

Every Site Counts: Submitting Transcription Factor-Binding Site Information through the CollecTF Portal.

Experimentally verified transcription factor-binding sites represent an information-rich and highly applicable data type that aptly summarizes the results of time-consuming experiments and inference processes. Currently, there is no centralized repository for this type of data, which is routinely embedded in articles and extremely hard to mine. CollecTF provides the first standardized resource for submission and deposition of these data into the NCBI RefSeq database, maximizing its accessibility and prompting the community to adopt direct submission policies.

An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria.

UNLABELLED: The SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In the Betaproteobacteria, LexA is known to target a palindromic sequence with the consensus sequence CTGT-N8-ACAG. We report the characterization of a LexA regulon in the iron-oxidizing betaproteobacterium Sideroxydans lithotrophicus. In silico and in vitro analyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of the Bacillus subtilis LexA-binding motif. We confirm that the closely related Gallionella capsiferriformans shares the same LexA-binding motif, and in silico analyses indicate that this motif is also conserved in the Nitrosomonadales and the Methylophilales. Phylogenetic analysis of LexA and the alpha subunit of DNA polymerase III (DnaE) reveal that the organisms harboring this noncanonical LexA form a compact taxonomic cluster within the Betaproteobacteria. However, their lexA gene is unrelated to the standard Betaproteobacteria lexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon of S. lithotrophicus is comparable in size and function to that of many other Betaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA protein. Analysis of the DNA-binding domain of S. lithotrophicus LexA reveals little sequence similarity with that of other LexA proteins targeting similar binding motifs, suggesting that network structure may limit site evolution or that structural constrains make the B. subtilis-type motif an optimal interface for multiple LexA sequences. IMPORTANCE: Understanding the evolution of transcriptional systems enables us to address important questions in microbiology, such as the emergence and transfer potential of different regulatory systems to regulate virulence or mediate responses to stress. The results reported here constitute the first characterization of a noncanonical LexA protein regulating a standard SOS regulon. This is significant because it illustrates how a complex transcriptional program can be put under the control of a novel transcriptional regulator. Our results also reveal a substantial degree of plasticity in the LexA recognition domain, raising intriguing questions about the space of protein-DNA interfaces and the specific evolutionary constrains faced by these elements.

Identification and characterization of VpsR and VpsT binding sites in Vibrio cholerae.

UNLABELLED: The ability to form biofilms is critical for environmental survival and transmission of Vibrio cholerae, a facultative human pathogen responsible for the disease cholera. Biofilm formation is controlled by several transcriptional regulators and alternative sigma factors. In this study, we report that the two main positive regulators of biofilm formation, VpsR and VpsT, bind to nonoverlapping target sequences in the regulatory region of vpsL in vitro. VpsR binds to a proximal site (the R1 box) as well as a distal site (the R2 box) with respect to the transcriptional start site identified upstream of vpsL. The VpsT binding site (the T box) is located between the R1 and R2 boxes. While mutations in the T and R boxes resulted in a decrease in vpsL expression, deletion of the T and R2 boxes resulted in an increase in vpsL expression. Analysis of the role of H-NS in vpsL expression revealed that deletion of hns resulted in enhanced vpsL expression. The level of vpsL expression was higher in an hns vpsT double mutant than in the parental strain but lower than that in an hns mutant. In silico analysis of the regulatory regions of the VpsR and VpsT targets resulted in the identification of conserved recognition motifs for VpsR and VpsT and revealed that operons involved in biofilm formation and vpsT are coregulated by VpsR and VpsT. Furthermore, a comparative genomics analysis revealed substantial variability in the promoter region of the vpsT and vpsL genes among extant V. cholerae isolates, suggesting that regulation of biofilm formation is under active selection. IMPORTANCE: Vibrio cholerae causes cholera and is a natural inhabitant of aquatic environments. One critical factor that is important for environmental survival and transmission of V. cholerae is the microbe's ability to form biofilms, which are surface-associated communities encased in a matrix composed of the exopolysaccharide VPS (Vibrio polysaccharide), proteins, and nucleic acids. Two proteins, VpsR and VpsT, positively regulate VPS production and biofilm formation. We characterized the structural features of the promoter of the vpsL gene, determined the target sequences recognized by VpsT and VpsR, and analyzed their distribution and conservation patterns in multiple V. cholerae isolates. This work fills a fundamental gap in our understanding of the regulatory mechanisms employed by the master regulators VpsR and VpsT in controlling biofilm matrix production.

Characterization of the SOS meta-regulon in the human gut microbiome.

MOTIVATION: Data from metagenomics projects remain largely untapped for the analysis of transcriptional regulatory networks. Here, we provide proof-of-concept that metagenomic data can be effectively leveraged to analyze regulatory networks by characterizing the SOS meta-regulon in the human gut microbiome. RESULTS: We combine well-established in silico and in vitro techniques to mine the human gut microbiome data and determine the relative composition of the SOS network in a natural setting. Our analysis highlights the importance of translesion synthesis as a primary function of the SOS response. We predict the association of this network with three novel protein clusters involved in cell wall biogenesis, chromosome partitioning and restriction modification, and we confirm binding of the SOS response transcriptional repressor to sites in the promoter of a cell wall biogenesis enzyme, a phage integrase and a death-on-curing protein. We discuss the implications of these findings and the potential for this approach for metagenome analysis.

Genome-scale analyses of Escherichia coli and Salmonella enterica AraC reveal noncanonical targets and an expanded core regulon.

Escherichia coli AraC is a well-described transcription activator of genes involved in arabinose metabolism. Using complementary genomic approaches, chromatin immunoprecipitation (ChIP)-chip, and transcription profiling, we identify direct regulatory targets of AraC, including five novel target genes: ytfQ, ydeN, ydeM, ygeA, and polB. Strikingly, only ytfQ has an established connection to arabinose metabolism, suggesting that AraC has a broader function than previously described. We demonstrate arabinose-dependent repression of ydeNM by AraC, in contrast to the well-described arabinose-dependent activation of other target genes. We also demonstrate unexpected read-through of transcription at the Rho-independent terminators downstream of araD and araE, leading to significant increases in the expression of polB and ygeA, respectively. AraC is highly conserved in the related species Salmonella enterica. We use ChIP sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to map the AraC regulon in S. enterica. A comparison of the E. coli and S. enterica AraC regulons, coupled with a bioinformatic analysis of other related species, reveals a conserved regulatory network across the family Enterobacteriaceae comprised of 10 genes associated with arabinose transport and metabolism.

Complete genome and characteristics of cluster BC bacteriophage SoJo, isolated using Streptomyces mirabilis NRRL B-2400 in Columbia, MD.

Here, we present bacteriophage SoJo, a siphovirus infecting Streptomyces mirabilis, with a circularly permuted genome of 39 kbp and GC content of 71.5%. Its genome length and content are similar to that of other phages in the Actinobacteriophage Database BC cluster. SoJo was isolated from soil in Columbia, MD, USA.

Systematic In Silico Assessment of Antimicrobial Resistance Dissemination across the Global Plasmidome.

The emergence of pathogenic strains resistant to multiple antimicrobials is a pressing problem in modern healthcare. Antimicrobial resistance is mediated primarily by dissemination of resistance determinants via horizontal gene transfer. The dissemination of some resistance genes has been well documented, but few studies have analyzed the patterns underpinning the dissemination of antimicrobial resistance genes. Analyzing the %GC content of plasmid-borne antimicrobial resistance genes relative to their host genome %GC content provides a means to efficiently detect and quantify dissemination of antimicrobial resistance genes. In this work we automate %GC content analysis to perform a comprehensive analysis of known antimicrobial resistance genes in publicly available plasmid sequences. We find that the degree to which antimicrobial resistance genes are disseminated depends primarily on the resistance mechanism. Our analysis identifies conjugative plasmids as primary dissemination vectors and indicates that most broadly disseminated genes have spread from single genomic backgrounds. We show that resistance dissemination profiles vary greatly among antimicrobials, oftentimes reflecting stewardship measures. Our findings establish %GC content analysis as a powerful, intuitive and scalable method to monitor the dissemination of resistance determinants using publicly available sequence data.

Linking Copper-Associated Signal Transduction Systems with Their Environment in Marine Bacteria.

Copper is an essential trace element for living cells. However, copper can be potentially toxic for bacterial cells when it is present in excess amounts due to its redox potential. Due to its biocidal properties, copper is prevalent in marine systems due to its use in antifouling paints and as an algaecide. Thus, marine bacteria must possess means of sensing and responding to both high copper levels and those in which it is present at only typical trace metal levels. Bacteria harbor diverse regulatory mechanisms that respond to intracellular and extracellular copper and maintain copper homeostasis in cells. This review presents an overview of the copper-associated signal transduction systems in marine bacteria, including the copper efflux systems, detoxification, and chaperone mechanisms. We performed a comparative genomics study of the copper-regulatory signal transduction system on marine bacteria to examine the influence of the environment on the presence, abundance, and diversity of copper-associated signal transduction systems across representative phyla. Comparative analyses were performed among species isolated from sources, including seawater, sediment, biofilm, and marine pathogens. Overall, we observed many putative homologs of copper-associated signal transduction systems from various copper systems across marine bacteria. While the distribution of the regulatory components is mainly influenced by phylogeny, our analyses identified several intriguing trends: (1) Bacteria isolated from sediment and biofilm displayed an increased number of homolog hits to copper-associated signal transduction systems than those from seawater. (2) A large variability exists for hits to the putative alternate σ factor CorE hits across marine bacteria. (3) Species isolated from seawater and marine pathogens harbored fewer CorE homologs than those isolated from the sediment and biofilm.

Impact of mutagenesis and lateral gene transfer processes in bacterial susceptibility to phage in food biocontrol and phage therapy.

Introduction: The emergence of resistance and interference mechanisms to phage infection can hinder the success of bacteriophage-based applications, but the significance of these mechanisms in phage therapy has not been determined. This work studies the emergence of Salmonella isolates with reduced susceptibility to a cocktail of three phages under three scenarios: i) Salmonella cultures (LAB), ii) biocontrol of cooked ham slices as a model of food safety (FOOD), and iii) oral phage therapy in broilers (PT). Methods: S. Typhimurium ATCC 14028 RifR variants with reduced phage susceptibility were isolated from the three scenarios and conventional and molecular microbiology techniques were applied to study them. Results and discussion: In LAB, 92% of Salmonella isolates lost susceptibility to all three phages 24 h after phage infection. This percentage was lower in FOOD, with 4.3% of isolates not susceptible to at least two of the three phages after seven days at 4°C following phage treatment. In PT, 9.7% and 3.3 % of isolates from untreated and treated broilers, respectively, displayed some mechanism of interference with the life cycle of some of the phages. In LAB and FOOD scenarios, resistant variants carrying mutations in rfc and rfaJ genes involved in lipopolysaccharide synthesis (phage receptor) were identified. However, in PT, the significant decrease of EOP, ECOI, and burst size observed in isolates was prompted by lateral gene transfer of large IncI1 plasmids, which may encode phage defense mechanisms. These data indicate that the acquisition of specific conjugative plasmids has a stronger impact than mutagenesis on the emergence of reduced phage-susceptibility bacteria in certain environments. In spite of this, neither mechanism seems to significantly impair the success of Salmonella biocontrol and oral phage therapy.