SOS system induction inhibits the assembly of chemoreceptor signaling clusters in Salmonella enterica

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 interaction of RecA with both CheA and CheW is required for chemotaxis

Altres ajuts: CERCA Programme/Generalitat de CatalunyaSalmonella 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

RecA Protein Plays a Role in the Chemotactic Response and Chemoreceptor Clustering of Salmonella enterica

The RecA protein is the main bacterial recombinase and the activator of the SOS system. In Escherichia coli and Salmonella enterica sv. Typhimurium, RecA is also essential for swarming, a flagellar-driven surface translocation mechanism widespread among bacteria. In this work, the direct interaction between RecA and the CheW coupling protein was confirmed, and the motility and chemotactic phenotype of a S. Typhimurium ΔrecA mutant was characterized through microfluidics, optical trapping, and quantitative capillary assays. The results demonstrate the tight association of RecA with the chemotaxis pathway and also its involvement in polar chemoreceptor cluster formation. RecA is therefore necessary for standard flagellar rotation switching, implying its essential role not only in swarming motility but also in the normal chemotactic response of S. Typhimurium.National Institutes of Health (U.S.) (Grant 1R01GM100473

Expansion of the SOS regulon of Vibrio cholerae through extensive transcriptome analysis and experimental validation

The SOS response is an almost ubiquitous response of cells to genotoxic stresses. The full complement of genes in the SOS regulon for Vibrio species has only been addressed through bioinformatic analyses predicting LexA binding box consensus and in vitro validation. Here, we perform whole transcriptome sequencing from Vibrio cholerae treated with mitomycin C as an SOS inducer to characterize the SOS regulon and other pathways affected by this treatment. Comprehensive transcriptional profiling allowed us to define the full landscape of promoters and transcripts active in V. cholerae. We performed extensive transcription start site (TSS) mapping as well as detection/quantification of the coding and non-coding RNA (ncRNA) repertoire in strain N16961. To improve TSS detection, we developed a new technique to treat RNA extracted from cells grown in various conditions. This allowed for identification of 3078 TSSs with an average 5'UTR of 116 nucleotides, and peak distribution between 16 and 64 nucleotides; as well as 629 ncRNAs. Mitomycin C treatment induced transcription of 737 genes and 28 ncRNAs at least 2 fold, while it repressed 231 genes and 17 ncRNAs. Data analysis revealed that in addition to the core genes known to integrate the SOS regulon, several metabolic pathways were induced. This study allowed for expansion of the Vibrio SOS regulon, as twelve genes (ubiEJB, tatABC, smpA, cep, VC0091, VC1190, VC1369-1370) were found to be co-induced with their adjacent canonical SOS regulon gene(s), through transcriptional read-through. Characterization of UV and mitomycin C susceptibility for mutants of these newly identified SOS regulon genes and other highly induced genes and ncRNAs confirmed their role in DNA damage rescue and protection. We show that genotoxic stress induces a pervasive transcriptional response, affecting almost 20% of the V. cholerae genes. We also demonstrate that the SOS regulon is larger than previously known, and its syntenic organization is conserved among Vibrio species. Furthermore, this specific co-localization is found in other γ-proteobacteria for genes recN-smpA and rmuC-tatABC, suggesting SOS regulon conservation in this phylum. Finally, we comment on the limitations of widespread NGS approaches for identification of all RNA species in bacteria

Elucidation of the RecA-mediated mechanisms governing swarming motility in Salmonella enterica

RecA es una proteína multifuncional que, aparte de ser la recombinasa principal implicada en los pasos cenrrales de la recombinación homóloga y en los mecanismos de reparación de DNA,también es el activador de la respuesta SOS.RecA actúa como sensor de lesiones en el DNA.Al unirse a DNA monocarenario, la proteína se activa (RecA*)y promueve la auto-hidrólisis del represor lexA,induciendo asíla expresión de los genes de la respuesta SOS.Además,se ha descrito que la proteína RccA está también vinculada con la motilidad en enjambre. El movimiento en enjambre o swarming, que está. ampliamente distribuido en el dominio Bacteria, se defin.e como una translocación multicelular rápida y organiz.ada de lasbacterias sobresuperficies sólidas o semi.sólidasmediada por la rotación Aagclar. Varios estudios asocian la proteína RecA con CheW, un componente davc en el ensamblaje de los quirniorrecepcores y de las matrices de señalización formadas por éstos, queson.esenciales para el swarming. los resultadospresentados en la presente Tesis Doctoral demuestran inequívocamente la interacción entre RecA y CheW. En el desarrollo de éste trabajo se ha caracterizado el complejo RecA-CheW, permitiendo la identificación de las interfaces crúicas implicadas en la interacción entre ambas proteín.as y su papel en la forrn.ación de las matrices de señalización . Además, se han podido identificar como esenciales para la interacción los residuos Gln20,Arg222,Argl 76 y Lys250 de RecA,que se encuentran en.losdomin.ios esttucmrales N-terminal ycentral de dicha proteína,y los residuos Phe21,Lys55, Asp83 y Phe121 de CbeW,que por dónde se ubican no parecen interferir con ninguna otra región de unión descrita para ChcW. Además, los experimentos realizados demuestran que la pérdida de swarming es consecuencia de la disrupción de las matricesde seúalización generada por el aumemo en la concentración innacelular de la proteína RecA.Los ensayos llevados a cabo mediante microscopía de alta resolución, han permitido rasnear la distribución i nnacelular de las proteínas CheW y RecA durante la inducción de la respuesta SOS,y elucidar el papel de la proteína RecA en.la distribución de CheW y en el ensamblajedelasmatrices de sef1alización. Final meme, los resultados obre.nidos permiten proponer un modelo que explica cómo las células bacterianas adaptan su motilidad sobre superficies en respuesta a la presencia de agentes nocivos para el DNA mediante la detección de ésrns a través. de la induccióndel sis.tema SOS. Durantela coloniz.ación desuperficies,lascélulas bacterianas pueden estar expuestas a una amplia gama de compuestos dañinosy poten.dalm.ente letales. Sin embargo, las células pueden eludirlos gracias a la inducción de la respuesta SOS y la consiguiente inhibición del swarmíng. Así, cuando concentraciones sub-letales de compuestos tóxicosgeneran lesionesen el DNA,la proteína RecA se activa induciendo la respuesta SOS, y,dado que recA es uno de Los primeros genesen lajerarquía de la activación SOS,su concen.tración aumenta rápidamence.Éste incremento de la concentración intracelular RecA perturba el equilibrio entre esta proteína y CheW,concretamente,RecA secuestra a la proteína CheW, evitando el correcto ensamblaje de las matrices de seña!i:?ación polar, causando el cese del movimiento swarmingcuando seinduce la respuesta SOS.Mediante este mecanismo ,las bacterias evitan la exposición a concentraciones mayores del agente nocivo,y por lo tanto, la muerte celular. Una vez se reparan las lesiones en el DNA dañado, la concentración de RecA decrece hasta su nivel basal, evitando el secue5tro de la proteína CheW, re5taurándose así el ensamblaje de las matrices quimiosensoriales y también el movimiento en enjanbre.Así pues, los daros presentados han permitido la caracterización del mecanismo molecular que gobierna la modulación de swttrming mediada por RecA, mediante d cualSafmoneüapuede adaptar su motilidad en superficie en respuesta a condiciones ambientales adversas.We characterized the RecA-CheW protein complex, that allowed the identification of the critical interfaces implied in the interaction and its role in the signaling array assembly. RecA residues Gln20, Arg222, Arg176 and Lys250 that are located in the multi-functional N-terminal and central structural domains of the protein, were described as essential for the interaction. In the case of CheW protein, residues Phe21, Lys55, Asp83 and Phe121 were involved in the RecA-binding, that do not seem to interfere with any other CheW-biding targets. Further, the obtained results demonstrate that the loss of swarming ability when there is an increase of RecA concentration was the consequence of chemosensing array assembly disruption, that previous works have established as essential for swarming in temperate swarmers. Using high resolution microscopy assays we were able to track CheW and RecA protein distribution within the cell during SOS response induction, elucidating the role of the RecA protein in the distribution of CheW and the assembly of chemoreceptor signaling arrays. The obtained results head to the proposal of a model that explains how bacterial cells adapt their surface motility in response to the presence of DNA-damaging agents by sensing them via SOS system induction. During surface colonization, bacterial cells will likely be exposed to a wide range of injurious, and potentially lethal, compounds that are avoided through SOS response induction and consequent swarming ability impairment. When DNA injuries are generated, RecA activates the SOS machinery, and its concentration rises swi��ly since recA is one of the first genes to be induced in the hierarchy of SOS activation. The increase of intracellular RecA concentration during SOS-response disturbs the equilibrium between this protein and CheW, causing the cessation of swarming. RecA prompts the titration of CheW protein, preventing polar signaling array assembly during SOS response, and thereby inhibiting motility. By this mechanism, bacteria avoid exposure to higher concentrations of the DNA damaging agent, and so, cell death. Following DNA damage repair, RecA concentration returns to its basal level, releasing CheW, that restores chemosensory array assembly, returning the cell to a non-DNA damage motile condition. Therefore, the present work characterizes the molecular mechanisms that govern RecAmediated swarming modulation, by which using RecA as a sensor, Salmonella cells can adapt their surface motility in response to adverse environmental conditions

The Transient Multidrug Resistance Phenotype of Salmonella enterica Swarming Cells Is Abolished by Sub-inhibitory Concentrations of Antimicrobial Compounds

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

Peptidoglycan Muropeptides : Release, Perception, and Functions as Signaling Molecules

Peptidoglycan (PG) is an essential molecule for the survival of bacteria, and thus, its biosynthesis and remodeling have always been in the spotlight when it comes to the development of antibiotics. The peptidoglycan polymer provides a protective function in bacteria, but at the same time is continuously subjected to editing activities that in some cases lead to the release of peptidoglycan fragments (i.e., muropeptides) to the environment. Several soluble muropeptides have been reported to work as signaling molecules. In this review, we summarize the mechanisms involved in muropeptide release (PG breakdown and PG recycling) and describe the known PG-receptor proteins responsible for PG sensing. Furthermore, we overview the role of muropeptides as signaling molecules, focusing on the microbial responses and their functions in the host beyond their immunostimulatory activity

Elucidation of the RecA-mediated mechanisms governing swarming motility in Salmonella enterica /

RecA es una proteína multifuncional que, aparte de ser la recombinasa principal implicada en los pasos cenrrales de la recombinación homóloga y en los mecanismos de reparación de DNA,también es el activador de la respuesta SOS.RecA actúa como sensor de lesiones en el DNA.Al unirse a DNA monocarenario, la proteína se activa (RecA*)y promueve la auto-hidrólisis del represor lexA,induciendo asíla expresión de los genes de la respuesta SOS.Además,se ha descrito que la proteína RccA está también vinculada con la motilidad en enjambre. El movimiento en enjambre o swarming, que está. ampliamente distribuido en el dominio Bacteria, se defin.e como una translocación multicelular rápida y organiz.ada de lasbacterias sobresuperficies sólidas o semi.sólidasmediada por la rotación Aagclar. Varios estudios asocian la proteína RecA con CheW, un componente davc en el ensamblaje de los quirniorrecepcores y de las matrices de señalización formadas por éstos, queson.esenciales para el swarming. los resultadospresentados en la presente Tesis Doctoral demuestran inequívocamente la interacción entre RecA y CheW. En el desarrollo de éste trabajo se ha caracterizado el complejo RecA-CheW, permitiendo la identificación de las interfaces crúicas implicadas en la interacción entre ambas proteín.as y su papel en la forrn.ación de las matrices de señalización . Además, se han podido identificar como esenciales para la interacción los residuos Gln20,Arg222,Argl 76 y Lys250 de RecA,que se encuentran en.losdomin.ios esttucmrales N-terminal ycentral de dicha proteína,y los residuos Phe21,Lys55, Asp83 y Phe121 de CbeW,que por dónde se ubican no parecen interferir con ninguna otra región de unión descrita para ChcW. Además, los experimentos realizados demuestran que la pérdida de swarming es consecuencia de la disrupción de las matricesde seúalización generada por el aumemo en la concentración innacelular de la proteína RecA.Los ensayos llevados a cabo mediante microscopía de alta resolución, han permitido rasnear la distribución i nnacelular de las proteínas CheW y RecA durante la inducción de la respuesta SOS,y elucidar el papel de la proteína RecA en.la distribución de CheW y en el ensamblajedelasmatrices de sef1alización. Final meme, los resultados obre.nidos permiten proponer un modelo que explica cómo las células bacterianas adaptan su motilidad sobre superficies en respuesta a la presencia de agentes nocivos para el DNA mediante la detección de ésrns a través. de la induccióndel sis.tema SOS. Durantela coloniz.ación desuperficies,lascélulas bacterianas pueden estar expuestas a una amplia gama de compuestos dañinosy poten.dalm.ente letales. Sin embargo, las células pueden eludirlos gracias a la inducción de la respuesta SOS y la consiguiente inhibición del swarmíng. Así, cuando concentraciones sub-letales de compuestos tóxicosgeneran lesionesen el DNA,la proteína RecA se activa induciendo la respuesta SOS, y,dado que recA es uno de Los primeros genesen lajerarquía de la activación SOS,su concen.tración aumenta rápidamence.Éste incremento de la concentración intracelular RecA perturba el equilibrio entre esta proteína y CheW,concretamente,RecA secuestra a la proteína CheW, evitando el correcto ensamblaje de las matrices de seña!i:?ación polar, causando el cese del movimiento swarmingcuando seinduce la respuesta SOS.Mediante este mecanismo ,las bacterias evitan la exposición a concentraciones mayores del agente nocivo,y por lo tanto, la muerte celular. Una vez se reparan las lesiones en el DNA dañado, la concentración de RecA decrece hasta su nivel basal, evitando el secue5tro de la proteína CheW, re5taurándose así el ensamblaje de las matrices quimiosensoriales y también el movimiento en enjanbre.Así pues, los daros presentados han permitido la caracterización del mecanismo molecular que gobierna la modulación de swttrming mediada por RecA, mediante d cualSafmoneüapuede adaptar su motilidad en superficie en respuesta a condiciones ambientales adversas.We characterized the RecA-CheW protein complex, that allowed the identification of the critical interfaces implied in the interaction and its role in the signaling array assembly. RecA residues Gln20, Arg222, Arg176 and Lys250 that are located in the multi-functional N-terminal and central structural domains of the protein, were described as essential for the interaction. In the case of CheW protein, residues Phe21, Lys55, Asp83 and Phe121 were involved in the RecA-binding, that do not seem to interfere with any other CheW-biding targets. Further, the obtained results demonstrate that the loss of swarming ability when there is an increase of RecA concentration was the consequence of chemosensing array assembly disruption, that previous works have established as essential for swarming in temperate swarmers. Using high resolution microscopy assays we were able to track CheW and RecA protein distribution within the cell during SOS response induction, elucidating the role of the RecA protein in the distribution of CheW and the assembly of chemoreceptor signaling arrays. The obtained results head to the proposal of a model that explains how bacterial cells adapt their surface motility in response to the presence of DNA-damaging agents by sensing them via SOS system induction. During surface colonization, bacterial cells will likely be exposed to a wide range of injurious, and potentially lethal, compounds that are avoided through SOS response induction and consequent swarming ability impairment. When DNA injuries are generated, RecA activates the SOS machinery, and its concentration rises swily since recA is one of the first genes to be induced in the hierarchy of SOS activation. The increase of intracellular RecA concentration during SOS-response disturbs the equilibrium between this protein and CheW, causing the cessation of swarming. RecA prompts the titration of CheW protein, preventing polar signaling array assembly during SOS response, and thereby inhibiting motility. By this mechanism, bacteria avoid exposure to higher concentrations of the DNA damaging agent, and so, cell death. Following DNA damage repair, RecA concentration returns to its basal level, releasing CheW, that restores chemosensory array assembly, returning the cell to a non-DNA damage motile condition. Therefore, the present work characterizes the molecular mechanisms that govern RecAmediated swarming modulation, by which using RecA as a sensor, Salmonella cells can adapt their surface motility in response to adverse environmental conditions

Concentration of RecA and CheW proteins in <i>S</i>. <i>enterica</i> mitomycin-C-treated cells growing in liquid medium.

<p>ELISA quantification of RecA (♦, continuous line) and CheW (<math><mrow><mi>■</mi></mrow></math>, continuous line) proteins of <i>S</i>. <i>enterica ΔcheR</i> cells harboring plasmid pUA1127 (eYFP::<i>cheR</i>) and treated with mitomycin C (0.08 μg/mL). The amounts of RecA (◇, discontinuous line) and CheW (□, discontinuous line) in a non-treated culture are also shown. The concentration is expressed as the number of RecA or CheW molecules per μg of total protein. The results are the mean of three independent experiments. Error bars represent the standard deviation. The relative RecA concentration (boxed) was calculated as the mean RecA concentration at each time point with respect to the mean initial RecA concentration [1.16 (±0.17) x 10¹¹ molecules per μg of total protein].</p

SOS System Induction Inhibits the Assembly of Chemoreceptor Signaling Clusters in <i>Salmonella enterica - Fig 2 </i>

<p><b>A)</b> Percentage of cells of <i>S</i>. <i>enterica ΔcheR</i> harboring plasmid pUA1127 (wild type) and of <i>ΔsulA</i>, <i>recAo</i>, <i>lexA3</i>(Ind⁻) or <i>lexA3</i>(Ind⁻) <i>recAo</i> mutant derivatives that developed polar clusters while growing on swarming plates in the absence (-) or presence (+) of mitomycin C. The cells were harvested from the edge of the swarming colony growing on soft agar plates. When indicated, 0.08 μg mitomycin C/mL was added to the plates. The results are the mean of at least four independent imaging studies. Error bars represent the standard deviation. ***<i>p</i><0.001 as determined by a one-way ANOVA with a Bonferroni correction. <b>B)</b> Representative fluorescence microscopy images of cells from wild-type, <i>lexA3</i>(Ind⁻), and <i>recAo</i> strains grown in the presence or absence mitomycin C.</p