Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae

Abstract

The production of bacteriocins can be favorable for colonization of the host by eliminating other bacterial species that share the same environment. In Streptococcus pneumoniae, the pnc (blp) locus encoding putative bacteriocins, immunity, and export proteins is controlled by a two-component system similar to the comCDE system required for the induction of genetic competence. A detailed comparison of the pnc clusters of four genetically distinct isolates confirmed the great plasticity of this locus and documented several repeat sequences. Members of the multiple-antibiotic-resistant Spain23F-1 clone, one member of the Spain9V-3 clone, sensitive 23F strain 2306, and the TIGR4 strain produced bactericidal substances active against other gram-positive bacteria and in some cases against S. pneumoniae as well. However, other strains did not show activity against the indicator strains despite the presence of a bacteriocin cluster, indicating that other factors are required for bacteriocin activity. Analysis of strain 2306 and mutant derivatives of this strain confirmed that bacteriocin production was dependent on the two-component regulatory system and genes involved in bacteriocin transport and processing. At least one other bacteriocin gene, pncE, is located elsewhere on the chromosome and might contribute to the bacteriocin activity of this strain.

FIG. 1.

Bacteriocin cluster and regulatory components in S. pneumoniae strains. The gene encoding the pheromone BlpC/SpiP is indicated by arrowheads with horizontal stripes. Genes in the bacteriocin cluster are indicated as follows: solid arrows, genes encoding putative bacteriocins with the typical double-glycine-type leader sequence; dotted arrows, CAAX peptidases; arrows with vertical stripes, immunity proteins; bold type, IS1381 element and truncated derivatives; open arrows, hypothetical genes of unknown function. Asterisks indicate SpiR2 recognition motifs, and small arrows indicate transcription units. Genes are designated as described by Reichmann et al. (38). Except for the first pnc gene the pnc genes in the bacteriocin clusters are indicated by only uppercase letters; annotated genes in the TIGR genome are indicated by brackets. A Hu15 spiA gene is present, but it has not been fully sequenced.

FIG. 2.

Amino acid sequences of bacteriocins, immunity proteins, and proteins of unknown function in the bacteriocin cluster of S. pneumoniae strains. Only divergent amino acids are shown in homologous peptides of different strains. The double-glycine leader peptide of the bacteriocin genes is indicated by bold type. The nomenclature is the same as that in Fig. 1. T4, TIGR4.

FIG. 3.

Bacteriocin-like activity in S. pneumoniae. Different S. pneumoniae strains were tested for bacteriocin production with L. lactis (A, C, E, and G) or M. luteus (B, D, F, and H) as the indicator strain, using the double-layer technique as described in Materials and Methods. The test strains included R6 (A and B), TIGR4 (C and D), 2306 (E and F), and 632 (G and H). Strain 632 also showed activity against S. mitis NCTC10712 (I) and S. pneumoniae R6 (K) as indicator strains. The bacteriocin activities of different S. pneumoniae mutants against L. lactis were also determined; the mutants used were S. pneumoniae 2306spiR::pJDR1 (L), S. pneumoniae 2306spiB::pJDB2 (M), S. pneumoniae 2306pncO::pJDO3 (N), S. pneumoniae 2306ΔpncR-K (O), S. pneumoniae 2306pncP::pJDP4 (P), and S. pneumoniae TIGR4ΔpncA-N (Q). The mutants with mutations in spiR2, spiB, and pncO are bacteriocin negative, and the pncP mutant is bacteriocin positive. The 2306ΔpncR-K mutant is bacteriocin positive, but the zones of inhibition are slightly smaller than those of the wild type. In contrast, the TIGR4ΔpncA-N mutant is bacteriocin negative. Bar = 1 cm.

FIG. 4.

(A) Comparison of the upstream region of the comA gene containing the pncE gene in S. pneumoniae R6 and TIGR4. The pncE gene is indicated by a solid arrowhead. The genes according to the annotation of the R6 and TIGR4 genomes are indicated. (B) Alignment of the peptide sequences. The arrow indicates the C39 processing site.

FIG. 5.

Expression of the bacteriocin genes pncE and pncJ in strain 2306 and mutants. Gene expression was determined by RT-PCR. The bars indicate CP values relative to the CP values of the control gene ldh (lactate dehydrogenase), which was defined as 100%. Minus and plus signs indicate the absence and presence of SpiP during cellular growth, respectively. Dark gray bars, pncE; light gray bars, pncJ product.