High genetic variability of the agr locus in Staphylococcus species

Abstract

The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.

FIG. 1.

Functional map of the staphylococcal agr (accessory gene regulator) system and consensus alignment for AgrB, AgrD, and AgrC. The schematic map of the agr locus shows the major transcripts RNA II and RNA III (arrows), and the genes are indicated by boxes. Hypervariable regions are shaded in gray.

FIG. 2.

Reconstruction of staphylococcal phylogenic tree based on agrB, agrD, agrC, and 16S rRNA genes. The nucleotide sequences of 16S rRNA from staphylococci were retrieved from GenBank (S. arlettae, AB009933; S. xylosus, D83374; S. simulans, D83373; S. lugdunensis, AB009941; S. intermedius, D83369; S. gallinarum, D83366; S. epidermidis, D83362; S. cohnii urealyticum, AB009936; S. cohnii cohnii, D83361; S. carnosus, AB009934; S. caprae, AB009935; S. capitis capitis, L37599; S. auricularis, D83358; and S. aureus, X68417). They were aligned by using the multisequence alignment program CLUSTAL X. Phylogenic relationships of agr sequences and 16S rRNA genes were inferred by using the PHYLIP software package. The evolutionary distances were determined by the method of Kimura, and these values were used to construct a dendrogram by use of the neighbor-joining method. The numbers at the nodes are the proportion of 1,000 bootstrap resamplings that support the topology shown. Only bootstrap values of >80% are indicated.