Analysis of AI-2/LuxS–Dependent Transcription inCampylobacter jejuniStrain 81-176

Abstract

Autoinducer-2 (AI-2) is a quorum-sensing signal molecule that controls a variety of cellular activities in response to cell density in both gram-negative and gram-positive bacteria. The production of AI-2 is dependent upon LuxS, the last enzyme in the AI-2 biosynthesis pathway. For this study, we constructed a luxS null mutation (Delta luxS) in Campylobacter jejuni strain 81-176, and showed that it abolished AI-2 production. The Delta luxS mutant had a longer doubling time in Mueller-Hinton (MH) broth and reduced swarming on MH soft agar at 37 degrees C compared to the wild type (wt), whereas growth rate or swarming at 42 degrees C was not affected. The Delta luxS mutant was also more sensitive to hydrogen peroxide (H(2)O(2)) and cumene hydroperoxide than the wt by disc inhibition assays at 42 degrees C, though minimum inhibitory concentration comparisons were inconclusive. Differences in genome-wide gene expression between wt and Delta luxS mutant with and without H(2)O(2) treatments were compared using DNA microarrays. The genes that showed differential expressions (wt/Delta luxS) include operons/pathways involved in AI-2 synthesis and S-adenosylmethionine (SAM) metabolism (metE, metF, and pfs), flagellar assembly/regulation, stress response (ahpC, tpx, and groES), ABC transporters/efflux systems, and two genes of unknown function located downstream of luxS (Cj1199 and Cj1200). The wt/Delta luxS expression ratios of ahpC (encoding alkyl hydroperoxide reductase) and tpx (encoding thiol peroxidase) were increased only with H(2)O(2) treatment, consistent with our finding that the Delta luxS mutant exhibits higher sensitivity to oxidative stress than wt. Our microarray results agreed with the Delta luxS mutant phenotypes, and suggested that LuxS plays a role in central metabolism involving SAM metabolism, but it is uncertain whether AI-2 functions as a true quorum-sensing signal in C. jejuni.

Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch

“…Increased expression of perR, which is known to negatively regulate ahpC, is somewhat paradoxical, and may indicate the existence of alternative regulators of ahpC (van Vliet et al, 1999). Recent microarray data suggest that the luxS gene, putatively encoding an autoinducer-2 biosynthetic enzyme, increases the expression of ahpC and tpx, suggesting that these genes could be under the control of quorum-sensing signals (He et al, 2008). Phenotypic studies of C. jejuni stress responses in different growth phases have, however, proved inconclusive and further work is needed to establish the details of C. jejuni stress responses during stationary phase and their regulation Klanĉnik et al, 2006;Murphy et al, 2003Murphy et al, , 2005.…”

Quorum sensing dependent phenotypes and their molecular mechanisms inCampylobacterales

“…sembly/regulation genes including flgD, flgE, fliD, fliS, flgR, flgI, flgK, flaA, flgG2 but not flaB or flhA [14]. In contrast, microarray analysis of a C. jejuni NCTC 11168 luxS mutant grown for 8 h at 42 °C performed by Holmes et al [43] indicated a reduced expression profile of 15 flagellar genes including 12 genes which were shown to be up-regulated in the study by He et al [14].…”

“…While C. jejuni luxS mutants showed nearly the same growth rate and motility as wild type strains at 42 °C, a minor reduction of growth rate and decreased motility on soft agar was described at 37 °C [14,32,35,41,42]. It has been shown that flaA but not flaB transcription was reduced in a C. jejuni 81116 luxS mutant, grown for 24 h at 42 °C, although the FlaA and FlaB proteins as well as the total flagellar protein amounts and the flagellar structure seemed to be unaffected [41].…”

“…The SAM-dependent methyltransferase cleaves a methyl residue of SAM thereby producing the toxic product S-adenosylhomocysteine (SAH). SAH is metabolised to homocysteine by a two step reaction, where the enzyme Pfs (5´-methylthioadenosine/S-adenosylhomocysteine nucleoidase) catalyses the cleavage of SAH to adenine and S-ribosylhomocysteine (SRH) followed by the LuxS catalysed cleavage of SRH to homocysteine and DPD, which is the precursor of AI-2 (as described above) [14]. Eukaryotes, archaebacteria and some eubacteria are able to metabolise SAH to homocysteine in a one step reaction by the SAH hydrolase [12] …”

“…The LuxR-type/AI-1 complex functions as transcriptional activator of the luxI-type as well as the target genes. The AI-2 signalling are best described for V. harveyi and E. coli: In these systems, the enzyme LuxS cleaves S-ribosylhomocysteine to produce homocysteine and 4,5-dihydroxyl-2,3-pentanedione (DPD), which is the precursor of AI-2 [14]. DPD undergoes spontaneous cyclisation, hydration and boronate ester formation, thereby forming different derivates such as S-THMF-borate (V. harveyi) and R-THMF (E. coli), which are reported to be AI-2 signalling molecules [6].…”