The intracellular bacterium Listeria monocytogenes can invade several types of normally non-phagocytic cells. Entry into cultured epithelial cells requires the expression of inIA, the first gene of an operon, comprising two genes: inIA, which encodes internalin, an 800-amino-acid protein, and inIB, which encodes a 630-amino-acid protein. Several genes homologous to inIA are detected in the genome of L. monocytogenes; InIB is one of them. We have assessed the role of inIB in invasiveness of L. monocytogenes by constructing isogenic chromosomal deletion mutants in the inIAB locus. Our findings indicate that: i) inIB is required for entry of L. monocytogenes into hepatocytes, but not into intestinal epithelial cells; ii) inIB encodes a surface protein; iii) internalin plays a role for entry into some hepatocyte cell lines. These results provide the first insight into the cell tropism displayed by L. monocytogenes.

A system for high-efficiency single-and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG'host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37°C. A nested set of L. lactis chromosomal fragments cloned onto pG'host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37°C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28°C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40%o of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.Numerous gram-positive bacteria are targets of study as biological models (e.g., Bacillus subtilis), industrially important fermenter strains (the lactic acid bacteria), or pathogens (e.g., clostridia, listeria, staphylococci, and streptococci). Many strains of industrial or medical importance have been characterized physiologically, but relatively few have been studied or modified genetically. The study or modification of strains could be facilitated by the use of delivery vectors for the introduction of directed or nonspecific insertions in the bacterial chromosome. Delivery systems that rely on nonreplicative vectors are limited to bacteria that can be transformed at a high frequency, and those with conditionally active replicons are often restricted in their host range. Thus, the construction of recombinant strains requires substantial effort and can be applied efficiently only to particular organisms.We previously described a broad-host-range thermosensitive (Ts) plasmid, pVE6002 (22), which was isolated from pGK12 (15). Such plasmids replicate by a rolling-circle (rc) mechanism (rc plasmids) (21). pVE6002 was shown to have potential use as a delivery vector in numerous gram-positive bacteria (22). Ts plasmids are nonreplicative at 37°C, making them particularly useful in bacteria with a low-temperature growth range or when a drastic thermal shock is un...

Streptococcus mutans is an important etiological agent of dental caries in humans. The extracellular polysaccharides synthesized by cell-associated glucosyltransferases (encoded by gtfBC) from sucrose have been recognized as one of the important virulence factors that promote cell aggregation and adherence to teeth, leading to dental plaque formation. In this study, we have characterized the effect of CovR, a global response regulator, on glucosyltransferase expression. Inactivation of covR in strain UA159 resulted in a marked increase in the GtfB and GtfC proteins, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With the use of a transcriptional reporter system of a single chromosomal copy of the PgtfB-gusA and PgtfC-gusA fusions, we confirmed the transcriptional regulation of these promoters by CovR. By in vitro electrophoretic mobility shift assays with purified CovR protein, we showed that CovR regulates these promoters directly. DNase I footprinting analyses suggest that CovR binds to large regions on these promoters near the transcription start sites. Taken together, our results indicate that CovR negatively regulates the expression of the gtfB and gtfC genes by directly binding to the promoter region.

A set of shuttle plasmids containing four different constitutive promoters was generated to facilitate overexpression of foreign and native genes in streptococci, such as Streptococcus mutans. The four promoters that were chosen were: Pami, Pspac, P23 and Pveg. These promoters are active in many Gram-positive bacteria, and allow various levels of gene expression depending on the host bacterium. Shuttle plasmids were constructed based on two types of broad-host-range replication origins: a rolling-circle replicon (pSH71) and a theta replicon (pAMβ1). Shuttle plasmids derived from the pAMβ1 replicon were generated to avoid the structural and segregational stability problems associated with rolling-circle replication, since these problems may be encountered during large gene cloning. In a complementation assay, we used one such plasmid to express a gene in trans to show the utility of these plasmids. In addition, a series of plasmids was generated for the expression of recombinant proteins with an N-terminal 6×His tag or a C-terminal Strep-tag fusion, and, using a gene derived from S. mutans, we showed a high level of recombinant protein expression in S. mutans and Streptococcus pyogenes. Since these plasmids contain broad-host-range replication origins, and because the selected promoters are functional in many bacteria, they can be used for gene expression studies, such as complementation and recombinant protein expression.

The gram-positive bacterium Streptococcus mutans is the primary causative agent in the formation of dental caries in humans. The ability of S. mutans to adapt and to thrive in the hostile environment of the oral cavity suggests that this cariogenic pathogen is capable of sensing and responding to different environmental stimuli. This prompted us to investigate the role of two-component signal transduction systems (TCS), particularly the sensor kinases, in response to environmental stresses. Analysis of the annotated genome sequence of S. mutans indicates the presence of 13 putative TCS. Further bioinformatics analysis in our laboratory has identified an additional TCS in the genome of S. mutans. We verified the presence of the 14 sensor kinases by using PCR and Southern hybridization in 13 different S. mutans strains and found that not all of the sensor kinases are encoded by each strain. To determine the potential role of each TCS in the stress tolerance of S. mutans UA159, insertion mutations were introduced into the genes encoding the individual sensor kinases. We were successful in inactivating all of the sensor kinases, indicating that none of the TCS are essential for the viability of S. mutans. The mutant S. mutans strains were assessed for their ability to withstand various stresses, including osmotic, thermal, oxidative, and antibiotic stress, as well as the capacity to produce mutacin. We identified three sensor kinases, Smu486, Smu1128, and Smu1516, which play significant roles in stress tolerance of S. mutans strain UA159.