Evidence of localized prophage-host recombination in the lytA gene, encoding the major pneumococcal autolysin

Abstract

According to a highly polymorphic region in the lytA gene, encoding the major autolysin of Streptococcus pneumoniae, two different families of alleles can be differentiated by PCR and restriction digestion. Here, we provide evidence that this polymorphic region arose from recombination events with homologous genes of pneumococcal temperate phages.

FIG. 1.

Polymorphic sites of lytA_Spn and the LytA_Spn NAM-amidase alleles. (A and B) Only full-length (957-bp) lytA_Spn alleles (17 alleles) and the corresponding 318-amino-acid-long LytA_Spn NAM-amidases were aligned using Clustal W 2. Polymorphism within a window of 31 nucleotides (or 11 amino acid residues), while advancing through the sequence, was calculated from a multiple sequence alignment. Overlapping windows were displaced from each other by one nucleotide (or residue). The sum of polymorphic positions found was plotted at the middle position of the window. The blackened bar or the stippled boxes, respectively, represent the N-terminal domain or the C-terminal repeats of NAM-amidase. (C) Nucleotide sequence around position 453 (taking as position 1 the first nucleotide of the ATG initiation codon) that identifies two different lytA_Spn families. The deduced amino acid sequence and its corresponding positions are boldfaced and italicized. Asterisks indicate nucleotides conserved in all lytA_Spn alleles described in Table 1. Nucleotides distinctive of each family of lytA_Spn alleles are highlighted on a gray (Fam_A) or black (Fam_B) background. The BamHI and HincII restriction sites characteristic of Fam_A and Fam_B lytA alleles, respectively, are boxed. (D) Phylogenetic tree of the S. pneumoniae lytA alleles. The neighbor-joining phylogenetic tree (phylogram) shows evolutionary relationships among lytA alleles. Sequences were trimmed so that the length of the alignment was adjusted to 906 nucleotides. The statistical significance of the tree branches was assessed by bootstrap analysis involving the construction of 1,000 trees from resampled data. Bootstrap values of >50% are indicated. The gray box indicates the positions of Fam_B lytA alleles. The scale bar represents the number of nucleotide substitutions per site.

FIG. 2.

Distributions of polymorphic sites in bacterial and phage lytA alleles and their encoded NAM-amidases. Only full-length lytA alleles were analyzed. Polymorphic sites were calculated as indicated in Fig. 1. Nucleotide (nt) and amino acid (aa) positions appear as black or red symbols, respectively. (A) Pneumococcal temperate phages. (B) S. pneumoniae alleles. (C) Streptococci of the mitis group (SMG). A hatched bar indicates the position of the 6-nucleotide deletion distinctive of lytA_SMG alleles.

FIG. 3.

Multiple alignments of pneumococcal, phage, and SMG lytA alleles. Nucleotides distinctive of the Fam_A and Fam_B lytA alleles are shown on a red or on a blue background, respectively. Other polymorphic nucleotides are shown on a yellow background. Asterisks indicate nucleotides identical in all the sequences. The hyphen corresponds to a 1-bp deletion. Nucleotide (and amino acid) positions (taking 1 as the first nucleotide of the ATG initiation codon or the first residue) are shown. Amino acid polymorphisms are indicated.

FIG. 4.

Dendrogram of genetic relationships among 109 strains belonging to 30 different STs based on multilocus sequence typing data (http://www.mlst.net/). Linkage distance is indicated by a scale at the bottom. The lytA_Spn allele present in pneumococcal strains of each ST is shown. Fam_A and Fam_B alleles are shown on a red or on a blue background, respectively.