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

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

Relationship between the SOS system and the chemoreceptors clustering in Salmonella enterica sv. Typhimurium

La proteïna RecA és coneguda per ser la principal recombinasa bacteriana I també l’activador del sistema SOS. RecA no només s’associa amb processos de reparació del DNA sinó que també està relacionada amb altres funcions com ara el control d’integrons, la transferència de resistències antibiòtiques i d’elements de virulència o la inducció de pròfags. A més, en els últims anys s’ha associat a la proteïna RecA un nou paper com a modulador del moviment en eixam o swarming a E. Coli i a S. Typhimurium. A dia d’avui, es coneix que tant la deficiència com l’excés de RecA causen una disminució dràstica de la capacitat de desplaçar-se mitjançant swarming. També es coneix que, en una soca de S. Typhimurium que sobreexpressa recA i que per tant és incapaç de desplaçar-se per swarming, aquest moviment es pot recuperar a través de l’expressió concomitant del gen cheW, indicant que in vivo aquestes dues proteïnes poden estar relacionades. A més, hi ha evidències experimentals de la interacció entre les proteïnes RecA i CheW in vitro. El gen cheW és un dels gens que componen el nucli de l’aparell de quimiotaxi. El seu producte, la proteïna CheW, és la encarregada de l’acoblament entre la proteïna CheA (una histidina quinasa) i els trímers de dímers de quimioreceptors formant la unitat de senyalització bàsica per la quimiotaxi. Es coneix que diverses unitats de senyalització són capaces d’agregar als pols de la cèl·lula donant lloc a una estructura macromolecular coneguda com a clústers de quimioreceptors que, a part de tenir un paper en la transducció de la senyal quimiotàctica, són necessaris per el moviment en swarming. De fet, mutants de E. Coli que sobreexpressen o presenten deficiències en el gen cheW presenten també una estructuració aberrant dels clústers de quimioreceptors, fet que s’ha relacionat amb els defectes en la quimiotaxis i el swarming també observats en aquestes cèl·lules. El mecanisme molecular a través del qual els sistema SOS, i més concretament RecA, modula el moviment per swarming encara es desconeix. De tota manera, hi ha suficients evidències que apunten a una connexió entre el sistema SOS i el sistema de quimiotaxi a través de la interacció entre les proteïnes RecA i CheW. Per això, el principal objectiu d’aquest treball és aclarir el paper del sistema SOS, i de la proteïna RecA, en el moviment per swarming de S. Typhimurium. Els resultats presentats en aquest treball demostren que les proteïnes RecA i CheW de S. Typhimurium són capaces d’interaccionar tant in vivo com in vitro. A més, també es demostra la tenir una estequiometria concreta entre aquestes dues proteïnes constitueix un factor clau a la hora de desplaçar-se per swarming. El mecanisme molecular que permet al sistema SOS controlar la motilitat per swarming encara no ha pogut ser descobert però en aquest treball es demostra que soques de S. Typhimurium que sobreexpressen recA i mutants deficients en el mateix gen presenten severes deficiències en l’estructuració dels clústers polars de quimioreceptors. En conclusió, el present treball clarifica la relació existent entre el sistema SOS i els sistema de quimiotaxi a S. Typhimurium a través de la interacció entre les proteïnes RecA i CheW. Malgrat la demostració de que la variació de la concentració cel·lular de RecA a les cèl·lules ocasiona defectes en l’estructuració dels clústers de quimioreceptors, el mecanisme molecular que permet la regulació del swarming a través de RecA encara no s’ha establert. En aquest treball, s’hipotetitza que RecA afecta el procés de formació de clústers de quimoreceptors a S. Typhimurium i que l’impediment de formar aquests clústers correctament és el motiu principal per el qual els mutants recA presenten defectes en el moviment per swarming.The RecA protein is known to be the main bacterial recombinase and the activator of the SOS system. RecA is associated not only with DNA repair but also with several other functions such as the control of integron dynamics, prophage induction and the transfer of antibiotic resistances and virulence factors. Furthermore, in the last years a novel role of the RecA protein as a modulator of the swarming motility in E. coli and S. Typhimurium has been revealed. Up to date, it is known that the lack or the excess of RecA causes a dramatic depletion of the swarming motility in the aforementioned bacterial species. Also, the ability to swarm of an S. Typhimurium strain that overexpresses the recA gene can be recovered by concomitantly overexpressing the cheW gene. Moreover, there are experimental evidences of the interaction between RecA and CheW proteins. The cheW gene is one of the chemotaxis system core genes. Its product, the CheW protein, is known to serve in the cell as the coupling protein between the CheA histidine kinase and the chemoreceptors trimers of dimers to form the basic chemotaxis signaling unit. Several chemotaxis signaling units are known to aggregate at the cell poles forming a macromolecular structure known as chemoreceptor signaling arrays that, apart from their role in signal transduction during chemotaxis, are known to be required for swarming motility. E. coli mutants that either overexpress or lacks the cheW gene are known to have severe impairments in the formation of this chemoreceptor clusters. This, have been linked with the depletion of the swarming and chemotactic abilities displayed by those mutants The molecular mechanism by which the SOS system modulates the swarming motility through RecA still remains unknown. There are sufficient evidences pointing towards a link between the chemotaxis and the SOS systems through a RecA-CheW interaction. Thus, the main aim of this work is to elucidate the role of the SOS system through the RecA protein in the swarming motility of S. Typhimurium. Results presented here demonstrate that RecA and CheW proteins of S. Typhimurium are able to interact both in vivo and in vitro thus establishing a link between the SOS system and the bacterial motility. Also, the importance of a concrete stoichiometric relationship between both proteins have been established as a key factor for swarming motility. The molecular mechanism that exactly allows the SOS system to control the swarming motility still remains poorly understood but in this work it has been demonstrated that strains that either overexpress or lack the recA gene present a severe impairment to successfully structuring the chemoreceptor clusters arrays at its cell poles. In conclusion, the present work clarifies the relationship between the SOS and chemotaxis systems of S. Typhimurium through the interaction between the RecA and CheW proteins. The molecular mechanism behind the RecA modulation of the swarming motility still needs to be further investigated but in this work the affectation of the ability to form chemoreceptor signaling arrays in cells with an excess or lack of RecA is reported. Thus, it is hypothesized that RecA affects the clustering process in S. Typhimurium and that the inability to successfully form this clusters is at the core of the swarming impairment shown by the recA mutants of this specie

Proposed model for the control of swarming motility by the SOS response during bacterial surface colonization in the presence of a DNA damaging compound.

<p>Proposed model for the control of swarming motility by the SOS response during bacterial surface colonization in the presence of a DNA damaging compound.</p

Swarming ability of the <i>S</i>. <i>enterica</i> wild-type and the <i>ΔsulA</i>, <i>recAo</i>, <i>lexA3</i>(Ind⁻), and <i>lexA3</i>(Ind⁻) <i>recAo</i> mutant derivatives in the absence or presence of mitomycin C.

<p>Representative images of a bacterial colony swarming on a semisolid agar surface following incubation of the culture for 14 h at 37°C. When indicated, 0.08 μg mitomycin C/mL was added to the semisolid agar plates.</p

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

Evolution of the percentage of <i>S</i>. <i>enterica</i> cells that developed polar chemoreceptor clusters after cessation of SOS response induction in a culture growing in liquid medium.

<p>Cultures treated for 300 min with either 0.08 (<math><mrow><mi>○</mi></mrow></math>) or 10 (△) μg mitomycin C/mL were centrifuged to remove the inducer. Samples were periodically taken thereafter and the presence of polar clusters was determined. As controls, a non-treated culture (□) and two cultures treated again after centrifugation with either 0.08 (<math><mrow><mi>●</mi></mrow></math>, discontinuous line) or 10 (▲, discontinuous line) μg mitomycin C/mL are also shown. The results are the mean of three independent imaging experiments. Error bars represent the standard deviation.</p

A) Swarming ability of either wild-type or <i>lexA3</i>(Ind⁻) strains in the presence of a mitomycin C concentration gradient. Swarming plates with a disk soaked with mitomycin C solution (2 mg/mL) were inoculated with the corresponding strain. Colony growth was followed by imaging the same representative swarming plate 6, 10, 13, and 14 h after plate inoculation. B) The susceptibility to mitomycin C of each strain was also evaluated.

<p>It must be noted that, as indicated in Material and Methods section, the swarming plates were point inoculated with the corresponding bacterial strain using a sterile toothpick while the susceptibility assays were carried out applying the bacterial inoculum using a sterile swab all over the plate surface.</p

Swarming ability of the <i>S</i>. <i>enterica</i> wild-type and the <i>ΔsulA</i>, <i>recAo</i>, <i>lexA3</i>(Ind⁻), and <i>lexA3</i>(Ind⁻) <i>recAo</i> mutant derivatives in the absence or presence of mitomycin C.

<p>Representative images of a bacterial colony swarming on a semisolid agar surface following incubation of the culture for 14 h at 37°C. When indicated, 0.08 μg mitomycin C/mL was added to the semisolid agar plates.</p