2D proteome analysis initiates new insights on the Salmonella Typhimurium LuxS protein.
BACKGROUND
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 Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2.


RESULTS
Differential proteome analysis of wildtype S. Typhimurium versus a luxS 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 S. Typhimurium genome contains only one luxS gene. This observation was further explored and we show that the S. Typhimurium LuxS protein can undergo posttranslational modification at a catalytic cysteine residue. Additionally, by constructing LuxS-betala and LuxS-PhoA fusion proteins, we demonstrate that S. Typhimurium LuxS can substitute the cognate signal peptide sequences of beta-lactamase and alkaline phosphatase for translocation across the cytoplasmic membrane in S. Typhimurium. This was further confirmed by fractionation of S. Typhimurium protein extracts, followed by Western blot analysis.


CONCLUSIONS
2D-DIGE analysis of a luxS mutant vs. wildtype Salmonella Typhimurium did not reveal new insights into the role of AI-2/LuxS in Salmonella 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 S. 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 S. Typhimurium.
Kint
Gwendoline
G
Centre of Microbial and Plant Genetics, K, U, Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium. gwendoline.kint@biw.kuleuven.be
Sonck
Kathleen Aj
KA
Schoofs
Geert
G
De Coster
David
D
Vanderleyden
Jos
J
De Keersmaecker
Sigrid Cj
SC
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006714:Homoserine / Q000031:analogs & derivatives / Q000096:biosynthesis; D007783:Lactones; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D053038:Quorum Sensing; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D007783:Lactones; C091088:N-octanoylhomoserine lactone; D020543:Proteome; D006714:Homoserine; D013437:Carbon-Sulfur Lyases; C117753:LuxS protein, Bacteria