A functional analysis of the formyl-coenzyme A (frc) gene from Lactobacillus reuteri 100-23C.

AIM: To examine the role of the Lactobacillus reuteri 100-23C frc gene product in oxalate metabolism, host colonization and the acid stress response. METHODS AND RESULTS: Genes encoding putative formyl-CoA transferase (frc) and oxalyl-CoA decarboxylase (oxc) enzymes are present in the genome sequences of Lact. reuteri strains. Two strains isolated from humans harboured an IS200 insertion sequence in the frc ORF and a group 2 intron-associated transposase downstream of the frc gene, both of which were lacking in two strains of animal origin, which contained intact frc and oxc genes. An frc(-) insertional mutant of Lact. reuteri 100-23C was compared with the parent strain with respect to oxalate degradation, colonization of an RLF-mouse host model and growth in the presence of acids. Neither parent nor mutant degraded oxalate in vitro or in vivo. However, the parent outcompeted the frc(-) mutant in the mouse intestine during co-colonization and the frc(-) mutant showed a reduced growth rate in the presence of hydrochloric acid. CONCLUSIONS: Intact oxc and frc genes do not ensure oxalate degradation under the conditions tested. The frc gene product is important during host colonization and survival of acid stress by Lact. reuteri 100-23C. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxalate metabolism by oxalate-degrading intestinal bacterial strains may be important in preventing urolithiasis and might lead to the derivation of probiotic products. To produce safe and efficacious probiotics, however, an understanding of the genetic characteristics of potential oxalate degraders must be obtained, together with knowledge of their functional ramifications.

Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.

Influence of different functional elements of plasmid pGT232 on maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro and in vivo.

Plasmid pGT232 (5.1 kb), an indigenous plasmid of Lactobacillus reuteri 100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein, repA. An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an L. reuteri population under laboratory growth conditions but was poorly maintained (<33% resistant cells) in the L. reuteri population inhabiting the murine gastrointestinal tract. Stable maintenance (>90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner.

Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties.

It has been shown previously that inactivation of the cshA gene, encoding a major cell surface polypeptide (259 kDa) in the oral bacterium Streptococcus gordonii, generates mutants that are markedly reduced in hydrophobicity, deficient in binding to oral Actinomyces species and to human fibronectin, and unable to colonize the oral cavities of mice. We now show further that surface fibrils 60.7 +/- 14.5 nm long, which are present on wild-type S. gordonii DL1 (Challis) cells, bind CshA-specific antibodies and are absent from the cell surfaces of cshA mutants. To more precisely determine the structural and functional properties of CshA, already inferred from insertional-mutagenesis experiments, we have cloned the entire cshA gene into the replicative plasmid pAM401 and expressed full-length CshA polypeptide on the cell surface of heterologous Enterococcus faecalis JH2-2. Enterococci expressing CshA exhibited a 30-fold increase in cell surface hydrophobicity over E. faecalis JH2-2 carrying the pAM401 vector alone and 2.4-fold-increased adhesion to human fibronectin. CshA expression in E. faecalis also promoted cell-cell aggregation and increased the ability of enterococci to bind Actinomyces naeslundii cells. Electron micrographs of negatively stained E. faecalis cells expressing CshA showed peritrichous surface fibrils 70.3 +/- 9.1 nm long that were absent from control E. faecalis JH2-2(pAM401) cells. The fibrils bound CshA-specific antibodies, as detected by immunoelectron microscopy, and the antibodies inhibited the adhesion of E. faecalis cells to fibronectin. The results demonstrate that the CshA polypeptide is the structural and functional component of S. gordonii adhesive fibrils, and they provide a molecular basis for past correlations of surface fibril production, cell surface hydrophobicity, and adhesion in species of oral "sanguis-like" streptococci.

Lipoprotein receptors in oral streptococci.

Streptococcus gordonii produces cell-surface lipopolypeptides that have been implicated in the determination of cell adherence and aggregation properties. SarA lipopolypeptide produced by S. gordonii is highly similar to the oligopeptide-binding protein AmiA in Streptococcus pneumoniae and to the OppA and SpoOKA oligopeptide-binding proteins in Bacillus subtilis. Insertional mutagenesis was used to inactivate the genes encoding SarA (76kDa) lipoprotein and a related 78-kDa lipoprotein denoted SarG. SarA- mutants were defective in serum-induced aggregation, competence, growth on complex nitrogen sources, and ability to colonize the oral cavity. Conversely, SarG- mutants were unaltered in the above properties, but were deficient in growth on simple nitrogen sources. It is proposed that SarA plays a central role in environmental sensing of extracellular factors by streptococci leading to modulation of cell-surface composition and growth responses of cells.

Cell-surface-associated polypeptides CshA and CshB of high molecular mass are colonization determinants in the oral bacterium Streptococcus gordonii.

The human oral bacterium Streptococcus gordonii expresses, on the cell surface, two antigenically related high-molecular-mass polypeptides denoted CshA and CshB, encoded by genes at separate chromosomal loci. The precursor form of CshA is composed of four distinct segments: (i) a 41-amino-acid residue leader peptide, (ii) N-terminal 42-878 residues, (iii) residues 879-2417 comprising 13 repeat blocks of 101 amino acid residues and three shorter blocks, and (iv) a C-terminal anchor domain similar to those present in some other Gram-positive bacterial cell-wall polypeptides. Insertional mutations within cshA reduced both cell-surface hydrophobicity and ability to adhere to oral Actinomyces naeslundii. Insertional mutations in cshB had less effect on hydrophobicity and coadherence. However, expression of both polypeptides was found to be necessary for streptococci to colonize the murine oral cavity.

Colonization of the murine oral cavity by Streptococcus gordonii.

Streptococcus gordonii DL1 (Challis) colonized the oral cavities of BALB/c mice that lacked streptococci, enterococci, and lactobacilli (LF mice) as members of an otherwise complex digestive tract microflora. Conventional mice, in comparison, were refractory to colonization by S. gordonii. Mice that harbored lactobacilli but were free of streptococci and enterococci (EF mice) had a lower incidence of colonization by S. gordonii than LF animals. The LF mouse system should be useful in the study of the molecular mechanisms that enable S. gordonii to inhabit the oral cavity.