Molecular analysis of the capsule gene region of group A Streptococcus: the hasAB genes are sufficient for capsule expression

Abstract

Enzymes directing the biosynthesis of the group A streptococcal hyaluronic acid capsule are encoded in the hasABC gene cluster. Inactivation of hasC, encoding UDP-glucose pyrophosphorylase in the heavily encapsulated group A streptococcal strain 87-282, had no effect on capsule production, indicating that hasC is not required for hyaluronic acid synthesis and that an alternative source of UDP-glucose is available for capsule production. Nucleotide sequence and deletion mutation analysis of the 5.5 kb of DNA upstream of hasA revealed that this region is not required for capsule expression. Many (10 of 23) group A streptococcal strains were found to contain insertion element IS1239' approximately 50 nucleotides upstream of the -35 site of the hasA promoter. The presence of IS1239' upstream of hasA did not prevent capsule expression. These results elucidate the molecular architecture of the group A streptococcal chromosomal region upstream of the has operon, indicate that hasABC are the sole components of the capsule gene cluster, and demonstrate that hasAB are sufficient to direct capsule synthesis in group A streptococci.

FIG. 1

Schematic map of the GAS capsule gene region and subclones. The hasABC genes, the open reading frames upstream of hasA, and the subclones from the capsule gene region are shown in black. Arrows indicate the direction of transcription for the hasABC genes and the upstream open reading frames. Selected restriction endonuclease sites are indicated: B, BamHI; Bg, BglII; H, HindIII; E, EcoRI; A, Asp718; X, XbaI.

FIG. 2

Schematic representation comparing the 87-282 and Vaughn capsule gene regions and sequence analysis defining the boundaries and insertion site of the insertion element IS1239′. (A) Comparison of the site of IS1239′ insertion in GAS strain Vaughn to the homologous chromosomal region in GAS strain 87-282. The insertion element is shown in black. The genes hasABC, the putative insertion element transposase, and orf1 are identified; arrows indicate the direction of gene transcription. (B) Comparison of the nucleotide sequences beginning 71 nucleotides upstream of the hasA initiation codon in GAS strains 87-282 and Vaughn. Alignment of the homologous sequences is shown; identical nucleotides are indicated by vertical bars. The hasA −35 promoter site is shown in boldface type and indicated. Inverted (IR) and direct (DR) repeat sequences are shown in boldface type. The putative initiation codon for the IS1239′ transposase is indicated (Start). Dashes in the 87-282 sequence indicate continuity with the 87-282 sequence in panel C. Dots in the Vaughn sequence indicate the continuation of the IS1239′ nucleotide sequence, which is not shown. (C) Comparison of the nucleotide sequences approximately 250 bp upstream of the putative initiation codon for orf1 in GAS strains 87-282 and Vaughn. Alignment of the homologous sequences is shown; identical nucleotides are indicated by vertical bars. Dashes in the 87-282 sequence indicate continuity with the 87-282 sequence shown in panel B. Dots in the Vaughn sequence indicate preceding nucleotides present in IS1239′. Inverted (IR) and direct (DR) repeat sequences are shown in boldface type. The putative termination codon for the IS1239′ transposase is indicated (stop).