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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.

Acinetobacter baumannii is a pathogen of increasing clinical importance worldwide, especially given its ability to readily acquire resistance determinants. Motile strains of this bacterium can move by either or both of two types of motility: (i) twitching, driven by type IV pili, and (ii) surface-associated motility, an appendage-independent form of movement. A. baumannii strain MAR002 possesses both twitching and surface-associated motility. In this study, we isolated spontaneous rifampin-resistant mutants of strain MAR002 in which point mutations in the rpoB gene were identified that resulted in an altered motility pattern. Transcriptomic analysis of mutants lacking twitching, surface-associated motility, or both led to the identification of deregulated genes within each motility phenotype, based on their level of expression and their biological function. Investigations of the corresponding knockout mutants revealed several genes involved in the motility of A. baumannii strain MAR002, including two involved in twitching (encoding a minor pilin subunit and an RND [resistance nodulation division] component), one in surface-associated motility (encoding an amino acid permease), and eight in both (encoding RND and ABC components, the energy transducer TonB, the porin OprD, the T6SS component TagF, an IclR transcriptional regulator, a PQQ-dependent sugar dehydrogenase, and a putative pectate lyase). Virulence assays showed the reduced pathogenicity of mutants with impairments in both types of motility or in surface-associated motility alone. By contrast, the virulence of twitching-affected mutants was not affected. These results shed light on the key role of surface-associated motility and the limited role of twitching in the pathogenicity of A. baumannii.

Ribonucleotide reductases (RNRs) are key enzymes in living cells that provide the precursors of DNA synthesis. The three characterized classes of RNRs differ by their metal cofactor and their stable organic radical. We have purified to near homogeneity the enzymatically active Mn-containing RNR of Corynebacterium ammoniagenes, previously claimed to represent a fourth RNR class. N-terminal and internal peptide sequence analyses clearly indicate that the C. ammoniagenes RNR is a class Ib enzyme. In parallel, we have cloned a 10-kilobase pair fragment from C. ammoniagenes genomic DNA, using primers specific for the known class Ib RNR. The cloned class Ib locus contains the nrdHIEF genes typical for class Ib RNR operon. The deduced amino acid sequences of the nrdE and nrdF genes matched the peptides from the active enzyme, demonstrating that C. ammoniagenes RNR is composed of R1E and R2F components typical of class Ib. We also show that the Mn-containing RNR has a specificity for the NrdH-redoxin and a response to allosteric effectors that are typical of class Ib RNRs. Electron paramagnetic resonance and atomic absorption analyses confirm the presence of Mn as a cofactor and show, for the first time, insignificant amounts of iron and cobalt found in the other classes of RNR. Our discovery that C. ammoniagenes RNR is a class Ib enzyme and possesses all the highly conserved amino acid side chains that are known to ligate two ferric ions in other class I RNRs evokes new, challenging questions about the control of the metal site specificity in RNR. The cloning of the entire NrdHIEF locus of C. ammoniagenes will facilitate further studies along these lines.

Trimethoprim is a synthetic antibacterial agent that targets folate biosynthesis by competitively binding to the di-hydrofolate reductase enzyme (DHFR). Trimethoprim is often administered synergistically with sulfonamide, another chemotherapeutic agent targeting the di-hydropteroate synthase (DHPS) enzyme in the same pathway. Clinical resistance to both drugs is widespread and mediated by enzyme variants capable of performing their biological function without binding to these drugs. These mutant enzymes were assumed to have arisen after the discovery of these synthetic drugs, but recent work has shown that genes conferring resistance to sulfonamide were present in the bacterial pangenome millions of years ago. Here, we apply phylogenetics and comparative genomics methods to study the largest family of mobile trimethoprim-resistance genes (dfrA). We show that most of the dfrA genes identified to date map to two large clades that likely arose from independent mobilization events. In contrast to sulfonamide resistance (sul) genes, we find evidence of recurrent mobilization in dfrA genes. Phylogenetic evidence allows us to identify novel dfrA genes in the emerging pathogen Acinetobacter baumannii , and we confirm their resistance phenotype in vitro. We also identify a cluster of dfrA homologues in cryptic plasmid and phage genomes, but we show that these enzymes do not confer resistance to trimethoprim. Our methods also allow us to pinpoint the chromosomal origin of previously reported dfrA genes, and we show that many of these ancient chromosomal genes also confer resistance to trimethoprim. Our work reveals that trimethoprim resistance predated the clinical use of this chemotherapeutic agent, but that novel mutations have likely also arisen and become mobilized following its widespread use within and outside the clinic. Hence, this work confirms that resistance to novel drugs may already be present in the bacterial pangenome, and stresses the importance of rapid mobilization as a fundamental element in the emergence and global spread of resistance determinants.

2.AbstractTrimethoprim is a synthetic antibacterial agent that targets folate biosynthesis by competitively binding to the di-hydrofolate reductase enzyme (DHFR). Trimethoprim is often administered synergistically with sulfonamide, another chemotherapeutic agent targeting the di-hydro-pteroate synthase (DHPS) enzyme in the same pathway. Clinical resistance to both drugs is widespread and mediated by enzyme variants capable of performing their biological function without binding to these drugs. These mutant enzymes were assumed to have arisen after the discovery of these synthetic drugs, but recent work has shown that genes conferring resistance to sulfonamide were present in the bacterial pangenome millions of years ago. Here we apply phylogenetics and comparative genomics methods to study the largest family of mobile trimethoprim resistance genes (dfrA). We show that most of the dfrA genes identified to date map to two large clades that likely arose from independent mobilization events. In contrast to sulfonamide resistance (sul) genes, we find evidence of recurrent mobilization in dfrA genes. Phylogenetic evidence allows us to identify novel dfrA genes in the emerging pathogen Acinetobacter baumannii, and we confirm their resistance phenotype in vitro. We also identify a cluster of dfrA homologs in cryptic plasmid and phage genomes, but we show that these enzymes do not confer resistance to trimethoprim. Our methods also allow us to pinpoint the chromosomal origin of previously reported dfrA genes, and we show that many of these ancient chromosomal genes also confer resistance to trimethoprim. Our work reveals that trimethoprim resistance predated the clinical use of this chemotherapeutic agent, but that novel mutations have likely also arisen and become mobilized following its widespread use within and outside the clinic. This work hence confirms that resistance to novel drugs may already be present in the bacterial pangenome, and stresses the importance of rapid mobilization as a fundamental element in the emergence and global spread of resistance determinants.3.Impact statementAntibiotic resistance is a pressing and global phenomenon. It is well-established that resistance to conventional antibiotics emerged millions of years ago in either antibiotic-producing bacteria or their competitors. Resistance to synthetic chemotherapeutic agents cannot be explained by this paradigm, since these drugs are not naturally produced. Resistance is hence assumed to have evolved rapidly following the clinical introduction of these drugs. Recently we showed that resistance to one such drug, sulfonamide, evolved not recently, but millions of years ago, suggesting that the diversity of bacterial genomes may well contain genes conferring resistance to drugs yet to be developed. Here we analyze the origin of resistance to trimethoprim, another chemotherapeutic agent developed in the 1960’s. Using phylogenetic methods, we identify new variants of the trimethoprim resistance genes that had not previously been reported, and we trace the chromosomal origins for a number of already known resistance variants. Our results show that resistance to trimethoprim is very diverse and has originated both from recent mutations and from preexisting ancient variants. These results stress the importance of gene mobilization mechanisms as the main drivers of the current antibiotic resistance phenomenon.4.Data summary- The scripts used for data collection and analysis can be obtained at the GitHub ErillLab repository (https://github.com/ErillLab/).- The Bayesian phylogenetic tree can be visualitzed online on iTOL (https://itol.embl.de/tree/855674159451585133078) [1].- The authors confirm all other supporting data has been provided within the article or through supplementary data files.

Altres ajuts: CERCA Programme/Generalitat de Catalunya Salmonella enterica is the most frequently reported cause of foodborne illness. As in other microorganisms, chemotaxis affords key physiological benefits, including enhanced access to growth substrates, but also plays an important role in infection and disease. Chemoreceptor signaling core complexes, consisting of CheA, CheW and methyl-accepting chemotaxis proteins (MCPs), modulate the switching of bacterial flagella rotation that drives cell motility. These complexes, through the formation of heterohexameric rings composed of CheA and CheW, form large clusters at the cell poles. RecA plays a key role in polar cluster formation, impairing the assembly when the SOS response is activated. In this study, we determined that RecA protein interacts with both CheW and CheA. The binding of these proteins to RecA is needed for wild-type polar cluster formation. In silico models showed that one RecA molecule, attached to one signaling unit, fits within a CheA-CheW ring without interfering with the complex formation or array assembly. Activation of the SOS response is followed by an increase in RecA, which rises up the number of signaling complexes associated with this protein. This suggests the presence of allosteric inhibition in the CheA-CheW interaction and thus of heterohexameric ring formation, impairing the array assembly. STED imaging demonstrated that all core unit components (CheA, CheW, and MPCs) have the same subcellular location as RecA. Activation of the SOS response promotes the RecA distribution along the cell instead of being at the cell poles. CheA- and CheW- RecA interactions are also crucial for chemotaxis, which is maintained when the SOS response is induced and the signaling units are dispersed. Our results provide new molecular-level insights into the function of RecA in chemoreceptor clustering and chemotaxis determining that the impaired chemoreceptor clustering not only inhibits swarming but also modulates chemotaxis in SOS-induced cells, thereby modifying bacterial motility in the presence of DNA-damaging compounds, such as antibiotics.

Acinetobacter baumannii is a nosocomial pathogen that causes multi-drug resistant infections mainly in immunocompromised patients. Although this gram-negative species lacks flagella, it is able to move over wet surfaces through a not well characterized type of movement known as surface-associated motility. In this study we demonstrate through the inactivation of the A1S_2813 gene (coding a CheW-like protein) and recA (coding a DNA damage repair and recombination protein) that both genes are involved in the surface-associated motility and chemotaxis of A. baumannii ATCC 17978 strain. In addition, we also point out that the lack of either RecA or CheW-like proteins reduces its virulence in the Caenorhabditis elegans and the Galleria mellonella animal models. Furthermore, we show through co-immunoprecipitation assays that the CheW-like protein and RecA interact and that this interaction is abolished by the introduction of the mutation S97A in one of the domains of CheW-like protein that is structurally conserved in Salmonella enterica and necessary for the RecA-CheW interaction in this bacterial species. Finally, we show that the replacement of the wild-type CheW-like protein by that presenting the S97A mutation impairs surface-associated motility, chemotaxis and virulence of A. baumannii strain ATCC 17978.

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.

Ralstonia solanacearum is a bacterial plant pathogen causing important economic losses worldwide. In addition to the polar flagella responsible for swimming motility, this pathogen produces type IV pili (TFP) that govern twitching motility, a flagellum-independent movement on solid surfaces. The implication of chemotaxis in plant colonization, through the control flagellar rotation by the proteins CheW and CheA, has been previously reported in R. solanacearum. In this work, we have identified in this bacterium homologues of the Pseudomonas aeruginosa pilI and chpA genes, suggested to play roles in TFP-associated motility analogous to those played by the cheW and cheA genes, respectively. We demonstrate that R. solanacearum strains with a deletion of the pilI or the chpA coding region show normal swimming and chemotaxis but altered biofilm formation and reduced twitching motility, transformation efficiency, and root attachment. Furthermore, these mutants displayed wild-type growth in planta and impaired virulence on tomato plants after soil-drench inoculations but not when directly applied to the xylem. Comparison with deletion mutants for pilA and fliC—encoding the major pilin and flagellin subunits, respectively—showed that both twitching and swimming are required for plant colonization and full virulence. This work proves for the first time the functionality of a pilus-mediated pathway encoded by pil-chp genes in R. solanacearum, demonstrating that pilI and chpA genes are bona fide motility regulators controlling twitching motility and its three related phenotypes: virulence, natural transformation, and biofilm formation., This study was funded by grants BIO2016-77011-R and AGL2016-78002-R (Spanish Ministry of Economy and Competitiveness). We acknowledge financial support from the Severo Ochoa Program for Centers of Excellence in R&D (SEV‐2015‐0533) and the CERCA Program from the Catalan Government (Generalitat de Catalunya). J.A. is a Serra Húnter Fellow (Generalitat de Catalunya). The project that gave rise to these results received the support of a fellowship (code is LCF/BQ/IN17/11620004) from la Caixa Foundation (identifier [ID] 100010434). This project has received funding from the European Union's Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement no. 713673.

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 behavioral 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. This article is protected by copyright. All rights reserved.

Although the relationship between Acinetobacter baumannii efflux pumps and antimicrobial resistance is well documented, less is known about the involvement of these proteins in the pathogenicity of this nosocomial pathogen. In previous work, we identified the AbaQ major facilitator superfamily (MFS) efflux pump and demonstrated its participation in the motility and virulence of A. baumannii In the present study, we examined the role in these processes of A. baumannii transporters belonging to different superfamilies of efflux pumps. Genes encoding known or putative permeases belonging to efflux pump superfamilies other than the MFS were selected, and the corresponding knockouts were constructed. The antimicrobial susceptibilities of these mutants were consistent with previously reported data. In mutants of A. baumannii strain ATCC 17978 carrying inactivated genes encoding the efflux pumps A1S_2736 (resistance nodulation division [RND]), A1S_3371 (multidrug and toxic compound extrusion [MATE]), and A1S_0710 (small multidrug resistance [SMR]), as well as the newly described ATP-binding cassette (ABC) permeases A1S_1242 and A1S_2622, both surface-associated motility and virulence were reduced compared to the parental strain. However, inactivation of the genes encoding the known ABC permeases A1S_0536 and A1S_1535, the newly identified putative ABC permeases A1S_0027 and A1S_1057, or the p roteobacterial a ntimicrobial c ompound e fflux (PACE) transporters A1S_1503 and A1S_2063 had no effects on bacterial motility or virulence. Our results demonstrate the involvement of antimicrobial transporters belonging at least to five of the six known efflux pump superfamilies in both surface-associated motility and virulence in A. baumannii ATCC 17978.

Sulfonamides are synthetic chemotherapeutic agents that work as competitive inhibitors of the di-hydro-pteroate synthase (DHPS) enzyme, encoded by the folP gene. Resistance to sulfonamides is widespread in the clinical setting and predominantly mediated by plasmid- and integron-borne sul1-3 genes encoding mutant DHPS enzymes that do not bind sulfonamides. In spite of their clinical importance, the genetic origin of sul1-3 genes remains unknown. Here we analyze sul genes and their genetic neighborhoods to uncover sul signature elements that enable the elucidation of their genetic origin. We identify a protein sequence Sul motif associated with sul-encoded proteins, as well as consistent association of a phosphoglucosamine mutase gene (glmM) with the sul2 gene. We identify chromosomal folP genes bearing these genetic markers in two bacterial families: the Rhodobiaceae and the Leptospiraceae. Bayesian phylogenetic inference of FolP/Sul and GlmM protein sequences clearly establishes that sul1-2 and sul3 genes originated as a mobilization of folP genes present in, respectively, the Rhodobiaceae and the Leptospiraceae, and indicate that the Rhodobiaceae folP gene was transferred from the Leptospiraceae. Analysis of %GC content in folP/sul gene sequences supports the phylogenetic inference results and indicates that the emergence of the Sul motif in chromosomally-encoded FolP proteins is ancient and considerably predates the clinical introduction of sulfonamides. In vitro assays reveal that both the Rhodobiaceae and the Leptospiraceae, but not other related chromosomally-encoded FolP proteins confer resistance in a sulfonamide-sensitive Escherichia coli background, indicating that the Sul motif is associated with sulfonamide resistance. Given the absence of any known natural sulfonamides targeting DHPS, these results provide a novel perspective on the emergence of resistance to synthetic chemotherapeutic agents, whereby preexisting resistant variants in the vast bacterial pangenome may be rapidly selected for and mobilized upon the clinical introduction of novel chemotherapeuticals.

Acinetobacter baumannii has emerged as an important multidrug-resistant nosocomial pathogen. In previous work, we identified a putative MFS transporter, AU097_RS17040, involved in the pathogenicity of A. baumannii (M. Pérez-Varela, J. Corral, J. A. Vallejo, S. Rumbo-Feal, G. Bou, J. Aranda, and J. Barbé, Infect Immun 85:e00327-17, 2017, https://doi.org/10.1128/IAI.00327-17). In this study, we analyzed the susceptibility to diverse antimicrobial agents of A. baumannii cells defective in this transporter, referred to as AbaQ. Our results showed that AbaQ is mainly involved in the extrusion of quinolone-type drugs in A. baumannii.

Acinetobacter baumannii , a worldwide emerging nosocomial pathogen, acquires antimicrobial resistances in response to DNA-damaging agents, which increase the expression of multiple error-prone DNA polymerase components. Here we show that the aminocoumarin novobiocin, which inhibits the DNA damage response in Gram-positive bacteria, also inhibits the expression of error-prone DNA polymerases in this Gram-negative multidrug-resistant pathogen and, consequently, its potential acquisition of antimicrobial resistance through DNA damage-induced mutagenesis.

Acinetobacter baumannii is a major cause of antibiotic-resistant nosocomial infections worldwide. In this study, several rifampin-resistant spontaneous mutants obtained from the A. baumannii ATCC 17978 strain that differed in their point mutations in the rpoB gene, encoding the β-subunit of the RNA polymerase, were isolated. All the mutants harboring amino acid substitutions in position 522 or 540 of the RpoB protein were impaired in surface-associated motility and had attenuated virulence in the fertility model of Caenorhabditis elegans . The transcriptional profile of these mutants included six downregulated genes encoding proteins homologous to transporters and metabolic enzymes widespread among A. baumannii clinical isolates. The construction of knockout mutants in each of the six downregulated genes revealed a significant reduction in the surface-associated motility and virulence of four of them in the A. baumannii ATCC 17978 strain, as well as in the virulent clinical isolate MAR002. Taken together, our results provide strong evidence of the connection between motility and virulence in this multiresistant nosocomial pathogen.

Swarming motility is the rapid and coordinated multicellular migration of bacteria across a moist surface. During swarming, bacterial cells exhibit increased resistance to multiple antibiotics, a phenomenon described as adaptive or transient resistance. In this study, we demonstrate that sub-inhibitory concentrations of cefotaxime, ciprofloxacin, trimethoprim, or chloramphenicol, but not that of amikacin, colistin, kanamycin or tetracycline, impair Salmonella enterica swarming. Chloramphenicol-treated S. enterica cells exhibited a clear decrease in their flagellar content, while treatment with other antibiotics that reduced swarming (cefotaxime, ciprofloxacin, and trimethoprim) inhibited polar chemoreceptor array assembly. Moreover, the increased resistance phenotype acquired by swarming cells was abolished by the presence of these antimicrobials. The same occurred in cells treated with these antimicrobial agents in combination with others that had no effect on swarming motility. Our results reveal the potential of inhibiting swarming ability to enhance the therapeutic effectiveness of antimicrobial agents.

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.

The effect of antimicrobials on SOS-mediated mutagenesis induction depends on the bacterial species and the antimicrobial group. In this work, we studied the effect of different families of antimicrobial agents used in clinical therapy against Acinetobacter baumannii in the induction of mutagenesis in this multiresistant Gram-negative pathogen. The data showed that ciprofloxacin and tetracycline induce SOS-mediated mutagenesis, whereas colistin and meropenem, which are extensively used in clinical therapy, do not.

This paper addresses the use of bacteriophages immobilized on magnetic particles for the biorecognition of the pathogenic bacteria, followed by electrochemical magneto-genosensing of the bacteria. The P22 bacteriophage specific to Salmonella (serotypes A, B, and D1) is used as a model. The bacteria are captured and preconcentrated by the bacteriophage-modified magnetic particles through the host interaction with high specificity and efficiency. DNA amplification of the captured bacteria is then performed by double-tagging polymerase chain reaction (PCR). Further detection of the double-tagged amplicon is achieved by electrochemical magneto-genosensing. The strategy is able to detect in 4 h as low as 3 CFU mL(-1) of Salmonella in Luria-Bertani (LB) media. This approach is compared with conventional culture methods and PCR-based assay, as well as with immunological screening assays for bacteria detection, highlighting the outstanding stability and cost-efficient and animal-free production of bacteriophages as biorecognition element in biosensing devices.

The SOS response is the primary bacterial mechanism to address DNA damage, coordinating multiple cellular processes that include DNA repair, cell division, and translesion synthesis. In contrast to other regulatory systems, the composition of the SOS genetic network and the binding motif of its transcriptional repressor, LexA, have been shown to vary greatly across bacterial clades, making it an ideal system to study the co-evolution of transcription factors and their regulons. Leveraging comparative genomics approaches and prior knowledge on the core SOS regulon, here we define the binding motif of the Verrucomicrobia, a recently described phylum of emerging interest due to its association with eukaryotic hosts. Site directed mutagenesis of the Verrucomicrobium spinosum recA promoter confirms that LexA binds a 14 bp palindromic motif with consensus sequence TGTTC-N4-GAACA. Computational analyses suggest that recognition of this novel motif is determined primarily by changes in base-contacting residues of the third alpha helix of the LexA helix-turn-helix DNA binding motif. In conjunction with comparative genomics analysis of the LexA regulon in the Verrucomicrobia phylum, electrophoretic shift assays reveal that LexA binds to operators in the promoter region of DNA repair genes and a mutagenesis cassette in this organism, and identify previously unreported components of the SOS response. The identification of tandem LexA-binding sites generating instances of other LexA-binding motifs in the lexA gene promoter of Verrucomicrobia species leads us to postulate a novel mechanism for LexA-binding motif evolution. This model, based on gene duplication, successfully addresses outstanding questions in the intricate co-evolution of the LexA protein, its binding motif and the regulatory network it controls.

Swarming, a flagellar-driven multicellular form of motility, is associated with bacterial virulence and increased antibiotic resistance. In this work we demonstrate that activation of the SOS response reversibly inhibits swarming motility by preventing the assembly of chemoreceptor-signaling polar arrays. We also show that an increase in the concentration of the RecA protein, generated by SOS system activation, rather than another function of this genetic network impairs chemoreceptor polar cluster formation. Our data provide evidence that the molecular balance between RecA and CheW proteins is crucial to allow polar cluster formation in Salmonella enterica cells. Thus, activation of the SOS response by the presence of a DNA-injuring compound increases the RecA concentration, thereby disturbing the equilibrium between RecA and CheW and resulting in the cessation of swarming. Nevertheless, when the DNA-damage decreases and the SOS response is no longer activated, basal RecA levels and thus polar cluster assembly are reestablished. These results clearly show that bacterial populations moving over surfaces make use of specific mechanisms to avoid contact with DNA-damaging compounds.

The role of Acinetobacter baumannii ATCC 17978 UmuDC homologs A1S_0636-A1S_0637, A1S_1174-A1S_1173, and A1S_1389 (UmuD Ab ) in antibiotic resistance acquired through UV-induced mutagenesis was evaluated. Neither the growth rate nor the UV-related survival of any of the three mutants was significantly different from that of the wild-type parental strain. However, all mutants, and especially the umuD Ab mutant, were less able to acquire resistance to rifampin and streptomycin through the activities of their error-prone DNA polymerases. Furthermore, in the A. baumannii mutant defective in the umuD Ab gene, the spectrum of mutations included a dramatic reduction in the frequency of transition mutations, the mutagenic signature of the DNA polymerase V encoded by umuDC .

Many cell surface-associated, divalent cation-regulated proteins are immunogenic, and some of them confer protection against the bacterial species from which they are derived. In this work, two Streptococcus suis divalent cation uptake regulator genes controlling zinc/manganese and iron uptake (adcR and fur, respectively) were inactivated in order to study the protective capacities of their cell surface-associated proteins. The results obtained showed overexpression of a set of immunogenic proteins (including members of the pneumococcal histidine triad family previously reported to confer protection against streptococcal pathogens) in S. suis adcR mutant cell surface extracts. Likewise, genes encoding zinc transporters, putative virulence factors and a ribosomal protein paralogue related to zinc starvation appeared to be derepressed in this mutant strain. Moreover, protection assays in mice showed that although neither adcR- nor fur-regulated cell surface-associated proteins were sufficient to confer protection in mice, the combination of both adcR- and fur-regulated cell surface-associated proteins is able to confer significant protection (50 %, P=0.038) against a challenge to mice vaccinated with them.

A novel material for electrochemical biosensing based on rigid conducting gold nanocomposite (nano-AuGEC) is presented. Islands of chemisorbing material (gold nanoparticles) surrounded by nonreactive, rigid, and conducting graphite epoxy composite are thus achieved to avoid the stringent control of surface coverage parameters required during immobilization of thiolated oligos in continuous gold surfaces. The spatial resolution of the immobilized thiolated DNA was easily controlled by merely varying the percentage of gold nanoparticles in the composition of the composite. As low as 9 fmol (60 pM) of synthetic DNA were detected in hybridization experiments when using a thiolated probe. Moreover, for the first time a double tagging PCR strategy was performed with a thiolated primer for the detection of Salmonella sp., one of the most important foodborne pathogens affecting food safety. This assay was performed by double-labeling the amplicon during the PCR with a -DIG and -SH set of labeled primers. The thiolated end allows the immobilization of the amplicon on the nano-AuGEC electrode, while digoxigenin allows the electrochemical detection with the antiDIG-HRP reporter in the femtomole range. Rigid conducting gold nanocomposite represents a good material for the improved and oriented immobilization of biomolecules with excellent transducing properties for the construction of a wide range of electrochemical biosensors such as immunosensors, genosensors, and enzymosensors.

Double-tagged DNA coming from PCR amplification of a Salmonella spp. sample was detected by an electrochemical impedimetric genosensor based on avidin bulk-modified graphite-epoxy biocomposite (Av-GEB). The double-tagging PCR strategy provided the amplicon with both biotin and digoxigenin (DIG) moieties. The immobilization of the double-tagged DNA was based on its biotin moiety, while the DIG label was used for signal amplification. Impedance spectra were recorded to detect the change in interfacial charge transfer resistance (R(ct)), experimented by the redox marker ferri-/ferro-cyanide after the avidin-biotin fixation of the sample DNA onto the electrode surface. A further step in the genosensing strategy was the amplification of impedimetric signal by the use of an enhancing procedure. The latter was based on the reaction of the DIG moiety belonging to the amplicon with an anti-DIG antibody from mouse. Two different secondary enhancing steps based both on gold nanoparticle-labelled anti-mouse IgG or on Protein G were performed and compared for improving assay sensitivity.

In contrast to the vast majority of the members of the domain Bacteria , several Pseudomonas and Xanthomonas species have two lexA genes, whose products have been shown to recognize different LexA binding motifs, making them an interesting target for studying the interplay between cohabiting LexA regulons in a single species. Here we report an analysis of the genetic composition of the two LexA regulons of Pseudomonas putida KT2440 performed with a genomic microarray. The data obtained indicate that one of the two LexA proteins (LexA1) seems to be in control of the conventional Escherichia coli -like SOS response, while the other LexA protein (LexA2) regulates only its own transcriptional unit, which includes the imuA, imuB , and dnaE2 genes, and a gene (PP_3901) from a resident P. putida prophage. Furthermore, PP_3901 is also regulated by LexA1 and is required for DNA damage-mediated induction of several P. putida resident prophage genes. In silico searches suggested that this marked asymmetry in regulon contents also occurs in other Pseudomonas species with two lexA genes, and the implications of this asymmetry in the evolution of the SOS network are discussed.

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.

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-N 8 -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.

By complementation of an Escherichia coli fur mutant, the Haemophilus parasuis fur gene has been isolated from a genomic library of this organism. The H. parasuis fur gene is the distal one of a three-gene operon. Two genes placed upstream of the H. parasuis fur open-reading frame encode for a hypothetical protein and a flavodoxin, respectively. Attempts performed to isolate an H. parasuis fur-defective mutant either through manganese-resistance selection or exchange markers were unsuccessful. Likewise, anaerobic growth conditions do not enable the attainment of H. parasuis fur-defective mutants either. Nevertheless, H. parasuis clones carrying a knockout mutation in the chromosomal fur gene by insertion of a KmR cassette were obtained when a stable plasmid, containing an additional copy of the transcriptional unit to which the fur gene belongs, was present. Likewise, the presence of a plasmid in which the H. parasuis fur gene is under the control of the Escherichia coli tac promoter allows for the isolation of fur::Km mutants of this organism. Nonetheless, no fur-defective mutants may be isolated from H. parasuis cells harbouring a stable plasmid in which only the single fur gene is contained. These data clearly indicate that H. parasuis cell viability requires the presence of a wild-type fur gene.

The lexA genes of Thermotoga maritima and Petrotoga miotherma, both members of the Order Thermotogales, have been cloned and their transcriptional organization, as well as the functional characteristics of their encoded products, analyzed. In both bacterial species, the lexA gene was found to be co-transcribed together with another four (T. maritima) or three (P. miotherma) upstream open-reading frames. The P. miotherma LexA was able to bind promoters of both the cognate lexA encoding operon and the uvrA gene but not to that of the recA. Conversely, LexA protein and crude cell extracts from T. maritima were unable to bind promoters governing the expression of either its lexA or recA genes. In agreement with these observations, no functional copy of the P. miotherma LexA box, corresponding to the GANTN(6)GANNAC motif, seems to be present in the T. maritima genome. Giving support to the proposal that the evolutionary branching order of the Order Thermotogales is very close to that of Gram-positive bacteria, the P. miotherma LexA protein was still able to recognize the previously described LexA-binding sequence for Gram-positive bacteria.

A temperate bacteriophage (F108) has been isolated through mitomycin C induction of a Pasteurella multocida serogroup A strain. F108 has a typical morphology of the family Myoviridae , presenting a hexagonal head and a long contractile tail. F108 is able to infect all P. multocida serogroup A strains tested but not those belonging to other serotypes. Bacteriophage F108, the first P. multocida phage sequenced so far, presents a 30,505-bp double-stranded DNA genome with cohesive ends (CTTCCTCCCC cos site). The F108 genome shows the highest homology with those of Haemophilus influenzae HP1 and HP2 phages. Furthermore, an F108 prophage attachment site in the P. multocida chromosome has been established to be inside a gene encoding tRNA Leu . By using several chromosomal markers that are spread along the P. multocida chromosome, it has been demonstrated that F108 is able to perform generalized transduction. This fact, together with the absence of pathogenic genes in the F108 genome, makes this bacteriophage a valuable tool for P. multocida genetic manipulation.

The recA gene of Paracoccus denitrificans has been isolated from a genomic library by hybridization with the Rhodobacter sphaeroides recA gene. Its complete nucleotide sequence consists of 1071 bp encoding a polypeptide of 356 amino acids. Nucleotide sequence analysis of the P. denitrificans recA gene revealed the closest identities with the R. sphaeroides and the Rhodobacter capsulatus recA genes. Nevertheless, and surprisingly, recA genes of these two phototrophic bacteria are not DNA damage-inducible when introduced into P. denitrificans cells, whereas recA genes of both P. denitrificans and Rhizobium etli are. These results suggest that the promoters of P. denitrificans and R. etli recA genes have a similar regulatory sequence. A recA-defective mutant of P. denitrificans has also been constructed by replacement of the active recA gene by an in vitro inactivated gene copy.

Recently, a multiple gene cassette with mutagenic translation synthesis activity was identified and shown to be under LexA regulation in several proteobacteria species. In this work, we have traced down instances of this multiple gene cassette across the bacteria domain. Phylogenetic analyses show that this cassette has undergone several reorganizations since its inception in the actinobacteria, and that it has dispersed across the bacterial domain through a combination of vertical inheritance, lateral gene transfer and duplication. In addition, our analyses show that LexA regulation of this multiple gene cassette is persistent in all the phyla in which it has been detected, and suggest that this regulation is prompted by the combined activity of two of its constituent genes: a polymerase V homolog and an alpha subunit of the DNA polymerase III.

The LexA regulon encompasses an ensemble of genes involved in preserving cell viability under massive DNA damage and is present in most bacterial phyla. Up to date, however, the scope of this network had only been assessed in the Gamma Proteobacteria. Here, we report the structure of the LexA regulon in the Alpha Proteobacteria, using a combined approach that makes use of in vitro and in vivo techniques to assist and validate the comparative genomics in silico methodology. This leads to the first experimentally validated description of the LexA regulon in the Alpha Proteobacteria, and comparison of regulon core structures in both classes suggests that a least common multiple set of genes (recA, ssb, uvrA and ruvCAB) might be a defining property of the Proteobacteria LexA network.

In recent years, the recognition sequence of the SOS repressor LexA protein has been identified for several bacterial clades, such as the Gram-positive, green non-sulfur bacteria and Cyanobacteria phyla, or the ‘Alphaproteobacteria’, ‘Deltaproteobacteria’ and ‘Gammaproteobacteria’ classes. Nevertheless, the evolutionary relationship among these sequences and the proteins that recognize them has not been analysed.Fibrobacter succinogenesis an anaerobic Gram-negative bacterium that branched from a common bacterial ancestor immediately before the Proteobacteria phylum. Taking advantage of its intermediate position in the phylogenetic tree, and in an effort to reconstruct the evolutionary history of LexA-binding sequences, theF. succinogenes lexAgene has been isolated and its product purified to identify its DNA recognition motif through electrophoretic mobility assays and footprinting experiments. After comparing the available LexA DNA-binding sequences with theF. succinogenesone, reported here, directed mutagenesis of theF. succinogenesLexA-binding sequence and phylogenetic analyses of LexA proteins have revealed the existence of two independent evolutionary lanes for the LexA recognition motif that emerged from the Gram-positive box: one generating the Cyanobacteria and ‘Alphaproteobacteria’ LexA-binding sequences, and the other giving rise to theF. succinogenesandMyxococcus xanthusones, in a transitional step towards the current ‘Gammaproteobacteria’ LexA box. The contrast between the results reported here and the phylogenetic data available in the literature suggests that, some time after its emergence as a distinct bacterial class, the ‘Alphaproteobacteria’ lost its vertically receivedlexAgene, but received later through lateral gene transfer a newlexAgene belonging to either a cyanobacterium or a bacterial species closely related to this phylum. This constitutes the first report based on experimental evidence of lateral gene transfer in the evolution of a gene governing such a complex regulatory network as the bacterial SOS system.