A genetic determinant in Streptococcus gordonii Challis encodes a peptide with activity similar to that of enterococcal sex pheromone cAM373, which facilitates intergeneric DNA transfer

Abstract

Enterococcus faecalis strains secrete multiple peptides representing different sex pheromones that induce mating responses by bacteria carrying specific conjugative plasmids. The pheromone cAM373, which induces a response by the enterococcal plasmid pAM373, has been of interest because a similar activity is also secreted by Streptococcus gordonii and Staphylococcus aureus. The potential to facilitate intergeneric DNA transfer from E. faecalis is of concern because of extensive multiple antibiotic resistance, including vancomycin resistance, that has emerged among enterococci in recent years. Here, we characterize the related pheromone determinant in S. gordonii and show that the peptide it encodes, gordonii-cAM373, does indeed induce transfer of plasmid DNA from E. faecalis into S. gordonii. The streptococcal determinant camG encodes a lipoprotein with a leader sequence, the last 7 residues of which represent the gordonii-cAM373 heptapeptide SVFILAA. Synthetic forms of the peptide had activity similar to that of the enterococcal cAM373 AIFILAS. The lipoprotein moiety bore no resemblance to the lipoprotein encoded by E. faecalis. We also identified determinants in S. gordonii encoding a signal peptidase and an Eep-like zinc metalloprotease (lspA and eep, respectively) similar to those involved in processing certain pheromone precursors in E. faecalis. Mutations generated in camG, lspA, and eep each resulted in the ablation of gordonii-cAM373 activity in culture supernatants. This is the first genetic analysis of a potential sex pheromone system in a commensal oral streptococcal species, which may have implications for intergeneric gene acquisition in oral biofilms.

FIG. 1.

Sequence of gordonii-cAM373 peptide and associated lipoprotein. (A) Deduced amino acid sequence of CamG from S. gordonii Challis CH1. The solid arrow marks the putative site of lipoprotein signal peptidase cleavage of the prelipoprotein, which removes the 19-amino-acid signal sequence. The proposed site of cleavage by the Eep homologue at the amino-terminal end of the gordonii-cAM373 peptide is marked by the open arrow. The gordonii-cAM373 peptide (SVFILAA) is underlined. Amino acid residues for the lipoprotein precursor molecule, consisting of 116 amino acids, are numbered. (B) Comparison of the amino acid sequences of the peptide pheromone molecules cAM373 of E. faecalis, staph-cAM373 of S. aureus, and gordonii-cAM373 of S. gordonii. Positions in staph-cAM373 and gordonii-cAM373 showing variation from cAM373 are boxed.

FIG. 2.

S. gordonii genome regions. The S. gordonii genome region is shown for each of the 3 genes of interest identified in these studies, SGO_1242 (camG) (A), SGO_1089 (lspA) (B), and SGO_1852 (eep) (C). The top line in each panel indicates the nucleotide numbers in the genome sequence in GenBank (accession number CP000725); the arrows at the end of each line show the ascending direction of the nucleotide numbers, indicating the orientation of the genome region pictured with respect to the chromosomal origin of replication. Beneath the genome nucleotide number line, each gene locus is indicated by the SGO number, which appears within an open arrow indicating the 5-prime-to-3-prime direction. The putative functional descriptions are based on the NCBI genome annotation and include conserved hypothetical (CHP) and hypothetical (HP) proteins. Putative bacterial promoters (forward arrows) and Rho-independent bacterial terminators (stem-loops) were identified in silico with the Bprom and FindTerm online software programs. Beneath camG, lspA, and eep are expansions that show the relevant features of the deduced CamG, LspA, and Eep proteins from the amino terminus (designated 1) to the carboxyl terminus. The filled blocks represent regions of conserved amino acids that share sequence homology to predicted functional domains identified in Prosite (PS) or pFam (PF) (22) or to signal sequences predicted by Signal P (41). The hatched bars represent the region of each encoded protein that was genetically manipulated in the construction of the isogenic mutant strains used in these studies (see Materials and Methods). (A) SGO_1242, which was designated camG, encodes a 116-amino-acid predicted lipoprotein with a processed signal sequence containing a peptide similar to cAM373. In silico analysis predicted that camG has its own promoter. Upstream of camG are two conserved hypothetical proteins, each with its own predicted promoter. Downstream of camG are two overlapping open reading frames with a single putative promoter; based upon sequence similarities, SGO_1241 encodes a 32.2-kDa protein similar to an aminoglycoside adenlytransferase (pFam04439) and SGO_1240 encodes a 27.2-kDa protein similar to CAAX amino-terminal proteases (pFam 02517) (42). No rho-independent terminators were detected in silico within the SGO_1244-through-SGO_1240 gene cluster. (B) SGO_1089, designated lspA, encodes a signal II peptidase signature motif (PF01252 and PS0085), as well as a conserved multicopper oxidase signature (PS00079). The lspA gene is located between two overlapping open reading frames with a single upstream promoter identified by both MacVector and Softberry promoter prediction software. In silico analysis of this operon indicated that the upstream gene (SGO_1088) encodes a putative transcriptional regulator of the LysR family. The downstream gene, SGO_1090, with its putative ribosomal binding site and start codon located within the lspA ORF, encodes a putative pseudouridine synthase. (C) SGO_1852, designated eep, encodes a 417-amino-acid protein with sequence similarity to E. faecalis Eep. The S. gordonii Eep carries a conserved zinc metalloprotease M-50 superfamily domain thought to be associated with the ability of a protein to cleave the transmembrane domains of substrate proteins, thereby transferring information and signals across bacterial membranes (38).

FIG. 3.

Donor, recipient, and transconjugant plasmid analyses. Plasmid preparations from donor, recipient, and nine representative transconjugant strains were digested with EcoRI and analyzed by agarose gel electrophoresis. Lane 1, “1 Kb Plus DNA Ladder” molecular size standard (Invitrogen); lane 2, donor E. faecalis JH2-2/pAM378/pAMS470; lane 3, recipient S. gordonii CH1S (plasmid free); lanes 4 to 6, transconjugants resistant to erythromycin; lanes 7 to 9, transconjugants resistant to tetracycline; lanes 10 to 12, transconjugants resistant to erythromycin and tetracycline. The expected EcoRI digestion fragments from pAM378 were 25.5, 13.3, 10.0, 4.8, and 0.9 kb; those from pAMS470 were 4.9, 0.46, and 0.05 kb.

FIG. 4.

Comparison of parC-parE chromosomal regions of streptococcal species. Comparison of genomic regions of representative streptococcal species indicated that the region flanking S. gordonii camG is potentially a hot spot for genetic integration and/or recombination. The top line is a nucleotide scale in kb for all the genomic regions. The parE and parC genes, encoding DNA topoisomerase IV beta and alpha subunits, respectively, and their immediate flanking genes and intergenic regions are shown for six representative streptococcal species: S. gordonii strain CH1 (GenBank CP000725; nucleotides 1293043 to 1282321), S. mutans strain UA159 (GenBank AE014133; nucleotides 1153799 to 1142879), S. sanguinis strain SK36 (GenBank CP000387; nucleotides 1257686 to 1247679); S. pneumoniae strain R6 (GenBank AE007317; nucleotides 749110 to 755873), S. pyogenes strain M1 GAS (GenBank AE004092; nucleotides 751576 to 757996), and S. agalactiae strain 2603 V/R (GenBank AE009948; nucleotides 1161100 to 1154670). The arrows indicate the size and direction of each ORF. The number beneath each ORF is the gene locus number. The name of each gene or annotation description is given above each arrow. Open arrows represent the genes with conserved gene order among all six species. The ORFs between parE and parC in the oral streptococcal species (S. gordonii, S. mutans, and S. sanguinis) were compared to each other by BLAST search at the amino acid and nucleotide levels. ORFs that showed no significant similarity to any of the ORFs in the intergenic regions of any of the three oral streptococcal species are shaded black. ORFs that showed significant similarity to each other are shaded gray (S. gordonii SGO_1243, S. mutans SMU.1208, and S. sanguinis SSA_1230) or hatched (SGO_1241 and SSA_1227). The region between parE and parC contains genes that suggest a phage or plasmid origin. The hypothetical protein (HP) encoded by SGO_1244 has amino acid regions with significant similarity to the Abi family proteins (PF07751) involved in bacteriophage resistance mediated by abortive infection in lactococcal species (29). The fic gene of S. mutans, which despite its location has no homology to camG, encodes a putative mobilization protein similar to that in lactococcal plasmid pNZ4000 (58).