The luxS gene of Lactobacillus reuteri 100-23C was amplified by PCR, cloned, and then sequenced. To define a physiological and ecological role for the luxS gene in L. reuteri 100-23C, a luxS mutant was constructed by insertional mutagenesis. The luxS mutant did not produce autoinducers AI-2 or AI-3. Complementation of the luxS mutation by a plasmid construct containing luxS restored AI-2 and AI-3 synthesis. In vitro experiments revealed that neither the growth rate, nor the cell yield, nor cell survival in the stationary phase were compromised in the luxS mutant relative to the wild type and complemented mutant. The ATP content of exponentially growing cells of the luxS mutant was, however, 65% of that of wild-type cells. Biofilms formed by the luxS mutant on plastic surfaces in a bioreactor were thicker than those formed by the wild type. Biofilm thickness was not restored to wild-type values by the addition of purified AI-2 to the culture medium. In vivo experiments, conducted with ex-Lactobacillus-free mice, showed that biofilms formed by the mutant strain on the epithelial surface of the forestomach were approximately twice as thick as those formed by the wild type. The ecological performance of the luxS mutant, when in competition with L. reuteri strain 100-93 in the mouse cecum, was reduced compared to that of a xylA mutant of 100-23C. These results demonstrate that LuxS influences important ecological attributes of L. reuteri 100-23C, the consequences of which are niche specific.The proximal region of the gastrointestinal tracts of rodents, pigs, chickens, and horses is inhabited by a Lactobacillus population. These bacteria adhere to, and proliferate on, the surface of nonsecretory epithelia present in the forestomachs, pars oesophagea, crops, and nonglandular stomachs of mice and rats, pigs, chickens, and horses, respectively (7,32,39). These epithelial associations can be termed biofilms because the bacteria appear to be encased in carbohydrate material and layers of bacterial cells several cells thick can be demonstrated in stained cryosections on the surface of proximal gut epithelia (7,24). Shed from these biofilms, lactobacilli can be detected throughout the gut of the animal, including in the large bowel (23).Lactobacillus reuteri strain 100-23 is an autochthonous inhabitant of the rodent gut. Recent molecular biological studies have begun to unravel the molecular mechanisms that enable this bacterial strain to reside in the guts of mice (34,35). These studies have shown that L. reuteri 100-23 and the murine gut together provide an excellent paradigm to study the ecologically important characteristics, at the molecular level, of a gram-positive bacterial species that can form an epitheliumassociated biofilm and of coping with ecological competition in the large bowel environment (34,35).Several examples of the ability of bacterial cells to alert their kindred to increasing cell density (quorum sensing) have been described (17). Typically, bacterial cells produce a small extracellular signal molecu...

Mycobacterium smegmatis is able to grow and survive at acidic pH, and exhibits intracellular pH homeostasis under these conditions. In this study, the authors have identified low proton permeability of the cytoplasmic membrane, and high cytoplasmic buffering capacity, as determinants of intrinsic acid resistance of M. smegmatis. To identify genes encoding proteins involved in protecting cells from acid stress, a screening method was developed using the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP was used to suppress intrinsic acid resistance of M. smegmatis. The screen involved exposing cells to pH 5·0 in the presence of CCCP, and survivors were rescued at various time intervals on solid medium at pH 7·5. Cells capable of responding to intracellular acidification (due to CCCP-induced proton equilibration) will survive longer under these conditions than acid-sensitive cells. From a total pool of 5000 transposon (Tn611) insertion mutants screened, eight acid-sensitive M. smegmatis mutants were isolated. These acid-sensitive mutants were unable to grow at pH 5·0 in the presence of 1–5 μM CCCP, a concentration not lethal to the wild-type strain mc2155. The DNA flanking the site of Tn611 was identified using marker rescue in Escherichia coli, and DNA sequencing to identify the disrupted locus. Acid-sensitive mutants of M. smegmatis were disrupted in genes involved in phosphonate/phosphite assimilation, methionine biosynthesis, the PPE multigene family, xenobiotic-response regulation and lipid biosynthesis. Several of the acid-sensitive mutants were also defective in stationary-phase survival, suggesting that overlapping stress protection systems exist in M. smegmatis.