The small regulatory RNA molecule MicA is involved in Salmonella enterica serovar Typhimurium biofilm formation

<p>Abstract</p> <p>Background</p> <p>LuxS is the synthase enzyme of the quorum sensing signal AI-2. In <it>Salmonella </it>Typhimurium, it was previously shown that a <it>luxS </it>deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.</p> <p>Results</p> <p>Analysis of additional <it>S. </it>Typhimurium <it>luxS </it>mutants indicated that the LuxS enzyme itself is not a prerequisite for a wild type mature biofilm. However, in close proximity of the <it>luxS </it>coding sequence, a small RNA molecule, MicA, is encoded on the opposite DNA strand. Interference with the MicA expression level showed that a balanced MicA level is essential for mature <it>Salmonella </it>biofilm formation. Several MicA targets known to date have previously been reported to be implicated in biofilm formation in <it>Salmonella </it>or in other bacterial species. Additionally, we showed by RT-qPCR analysis that MicA levels are indeed altered in some <it>luxS </it>mutants, corresponding to their biofilm formation phenotype.</p> <p>Conclusions</p> <p>We show that the <it>S. </it>Typhimurium biofilm formation phenotype of a <it>luxS </it>mutant in which the complete coding region is deleted, is dependent on the sRNA molecule MicA, encoded in the <it>luxS </it>adjacent genomic region, rather than on LuxS itself. Future studies are required to fully elucidate the role of MicA in <it>Salmonella </it>biofilm formation.</p

FabR regulates Salmonella biofilm formation via its direct target FabB

Background: Biofilm formation is an important survival strategy of Salmonella in all environments. By mutant screening, we showed a knock-out mutant of fabR, encoding a repressor of unsaturated fatty acid biosynthesis (UFA), to have impaired biofilm formation. In order to unravel how this regulator impinges on Salmonella biofilm formation, we aimed at elucidating the S. Typhimurium FabR regulon. Hereto, we applied a combinatorial high-throughput approach, combining ChIP-chip with transcriptomics. Results: All the previously identified E. coli FabR transcriptional target genes (fabA, fabB and yqfA) were shown to be direct S. Typhimurium FabR targets as well. As we found a fabB overexpressing strain to partly mimic the biofilm defect of the fabR mutant, the effect of FabR on biofilms can be attributed at least partly to FabB, which plays a key role in UFA biosynthesis. Additionally, ChIP-chip identified a number of novel direct FabR targets (the intergenic regions between hpaR/hpaG and ddg/ydfZ) and yet putative direct targets (i.a. genes involved in tRNA metabolism, ribosome synthesis and translation). Next to UFA biosynthesis, a number of these direct targets and other indirect targets identified by transcriptomics (e.g. ribosomal genes, ompA, ompC, ompX, osmB, osmC, sseI), could possibly contribute to the effect of FabR on biofilm formation. Conclusion: Overall, our results point at the importance of FabR and UFA biosynthesis in Salmonella biofilm formation and their role as potential targets for biofilm inhibitory strategies

The complex cellular role of the Salmonella Typhimurium LuxS protein studied by omics and targeted approaches

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) is één van de meest voorkomende Salmonella serovars geassocieerd met gastroenteritis bij mensen. Bovendien is Salmonella één van de meest voorkomende oorzaken van een bacteriële voedselvergiftiging. Het stijgende aantal rapporten over zogenaamde multi-drug resistente Salmonella isolaten vereist het zoeken naar alternatieve manieren ter preventie en bestrijding van Salmonella. Eén van de processen die in dit opzicht worden onderzocht, is een bacterieel communicatiesysteem, algemeen omschreven onder de naam quorum sensing .Quorum sensing wordt gekenmerkt door de vorming en herkenning van kleine signaalmoleculen ( autoinducers ). Het stelt bacteriën in staat diverse eigenschappen, zoals bijvoorbeeld virulentie, te coördineren afhankelijk van de bacteriële populatiedichtheid. Type II quorum sensing is gebaseerd op het autoinducer 2 (AI-2) synthase eiwit LuxS, dat evolutionair geconserveerd is in verschillende Gram-positieve en Gram-negatieve bacteriën. Hierdoor wordt type II quorum sensing gezien als een interspecies quorum sensing systeem. De exacte rol van type II quorum sensing in S. Typhimurium is voorlopig nog niet volledig in kaart gebracht.Dit thesisonderzoek bouwt voort op onderzoek aan het Centrum voor Microbiële en Plantengenetica (CMPG) op het type II quorum sensing systeem van S. Typhimurium. Hoge doorvoer ( high throughput ) proteoomanalyse van verschillende quorum sensing mutanten wees op een complexere rol voor het type II quorum sensing synthase LuxS dan algemeen aanvaard. Dit was de start van een meer diepgaande analyse van dit intrigerende synthase in S. Typhimurium.In dit doctoraatsonderzoek worden verschillende nieuwe eigenschappen van luxS in S. Typhimurium aangetoond, die de complexiteit van het geheel aanzienlijk verhogen. Deze geheel nieuwe inzichten situeren zich op verschillende biologische niveaus: DNA, RNA, eiwit en fenotypisch niveau. Er wordt aangetoond dat mutaties in de luxS coderende sequentie kunnen interfereren met de expressie van een nabij gelegen klein niet-coderend regulatorisch RNA molecule micA. Hierdoor worden ook MicA gereguleerde fenotypes zoals biofilmvorming indirect beïnvloed. Daarnaast wordt ook een posttranslationele modificatie van LuxS beschreven. Deze kon reeds gedetecteerd worden in voorgaand onderzoek uitgevoerd aan het CMPG, maar in dit doctoraatsonderzoek wordt ook het katalytisch cysteïneresidu dat het doelwit vormt van deze modificatie geïdentificeerd. Ten slotte wordt aangetoond dat LuxS meerdere subcellulaire locaties kan hebben in S. Typhimurium. Verder onderzoek leert dat er een regulatorische regio voorkomt, stroomopwaarts van de luxS coderende sequentie, die betrokken is in de translocatie van LuxS over het cytoplasmamembraan.Samengevat kan gesteld worden dat dit doctoraatsonderzoek wijst op een veel complexere regulatie van het quorum sensing synthase LuxS in S. Typhimurium dan algemeen aanvaard. Deze nieuwe inzichten vormen de basis voor verder uitdagend onderzoek naar LuxS-gebaseerde quorum sensing in zowel S. Typhimurium als andere bacteriële species. Op (middel)lange termijn zal deze kennis mogelijk kunnen worden aangewend om accurate strategieën te ontwikkelen voor de bestrijding en de preventie van pathogene bacteriën.nrpages: 149status: publishe

Integration of 'omics' data: does it lead to new insights into host-microbe interactions?

The interaction between both beneficial and pathogenic microbes and their host has been the subject of many studies. Although the field of systems biology is rapidly evolving, the use of a systems biology approach by means of high-throughput techniques to study host-microbe interactions is just beginning to be explored. In this review, we discuss some of the most recently developed high-throughput 'omics' techniques and their use in the context of host-microbe interaction. Moreover, we highlight studies combining several techniques that are pioneering the integration of 'omics' data related to host-microbe interactions. Finally, we list the major challenges ahead for successful systems biology research on host-microbe interactions

2D proteome analysis initiates new Insights on the <it>Salmonella </it>Typhimurium LuxS protein

<p>Abstract</p> <p>Background</p> <p>Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen <it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype <it>S</it>. Typhimurium with that of a <it>luxS </it>mutant, unable to produce AI-2.</p> <p>Results</p> <p>Differential proteome analysis of wildtype <it>S</it>. Typhimurium versus a <it>luxS </it>mutant revealed relatively few changes beyond the known effect on phase 2 flagellin. However, two highly differentially expressed protein spots with similar molecular weight but differing isoelectric point, were identified as LuxS whereas the <it>S</it>. Typhimurium genome contains only one <it>luxS </it>gene. This observation was further explored and we show that the <it>S</it>. Typhimurium LuxS protein can undergo posttranslational modification at a catalytic cysteine residue. Additionally, by constructing LuxS-βla and LuxS-PhoA fusion proteins, we demonstrate that <it>S</it>. Typhimurium LuxS can substitute the cognate signal peptide sequences of β-lactamase and alkaline phosphatase for translocation across the cytoplasmic membrane in <it>S</it>. Typhimurium. This was further confirmed by fractionation of <it>S</it>. Typhimurium protein extracts, followed by Western blot analysis.</p> <p>Conclusion</p> <p>2D-DIGE analysis of a <it>luxS </it>mutant <it>vs</it>. wildtype <it>Salmonella </it>Typhimurium did not reveal new insights into the role of AI-2/LuxS in <it>Salmonella </it>as only a small amount of proteins were differentially expressed. However, subsequent in depth analysis of the LuxS protein itself revealed two interesting features: posttranslational modification and potential translocation across the cytoplasmic membrane. As the <it>S</it>. Typhimurium LuxS protein does not contain obvious signal motifs, it is speculated that LuxS is a new member of so called moonlighting proteins. These observations might have consequences in future studies on AI-2 quorum signaling in <it>S</it>. Typhimurium.</p

FabR regulates Salmonella biofilm formation via its direct target FabB

Biofilm formation is an important survival strategy of Salmonella in all environments. By mutant screening, we showed a knock-out mutant of fabR, encoding a repressor of unsaturated fatty acid biosynthesis (UFA), to have impaired biofilm formation. In order to unravel how this regulator impinges on Salmonella biofilm formation, we aimed at elucidating the S. Typhimurium FabR regulon. Hereto, we applied a combinatorial high-throughput approach, combining ChIP-chip with transcriptomics.status: publishe