Functional Analysis in Type Ia Group B Streptococcusof a Cluster of Genes Involved in Extracellular Polysaccharide Production by Diverse Species of Streptococci

Abstract

Several species of streptococci produce extracellular polysaccharides in the form of secreted exopolysaccharides or cell-associated capsules. Although the biological properties and repeating unit structures of these polysaccharides are diverse, sequence analysis of the genes required for their production has revealed a surprising degree of conservation among five genes found in the capsule gene cluster of each of several polysaccharide-producing streptococci. To determine the function of these conserved genes, we characterized a series of isogenic mutants derived from a wild-type strain of type Ia group B Streptococcus by selectively inactivating each gene. Inactivation of cpsIaE resulted in an acapsular phenotype, consistent with previous work that identified the cpsIaE product as the glycosyltransferase that initiates synthesis of the polysaccharide repeating unit. Mutants in cpsIaA, cpsIaB, cpsIaC, or cpsIaD produced type Ia capsular polysaccharide, but in reduced amounts compared with the wild type. Analysis of the mutant polysaccharides and of capsule gene transcription in the mutant strains provided evidence that cpsIaA encodes a transcriptional activator that regulates expression of the capsule gene operon. Mutants in cpsIaC or cpsIaD produced polysaccharide of reduced molecular size but with an identical repeating unit structure as the wild-type strain. We conclude that CpsA to -D are not required for polysaccharide repeating unit biosynthesis but rather that they direct the coordinated polymerization and export of high molecular weight polysaccharide.

Streptococcus agalactiae Capsule Polymer Length and Attachment Is Determined by the Proteins CpsABCD

“…Bacterial Strains and Growth Conditions-GBS strains 515 and 515⌬cpsE were provided by Dr. Dennis Kasper (Harvard Medical School, Boston, MA) and Dr. Michael Cieslewicz (14), respectively. GBS strains were grown in Todd-Hewitt broth (THB) at 37°C, 5% CO 2 .…”

“…Constructs for chromosomal complementation were prepared by cloning the respective WT loci into pJRS233. The various pJRS233 constructs were used for insertion/duplication and excision mutagenesis (14,20). Briefly, pJRS233-derived plasmids were used to transform electrocompetent GBS 515 cells by electroporation (23).…”

“…Strains-CpsA has previously been reported to be involved in cps operon transcription (14,15). We used real-time quantitative RT-PCR to analyze the transcription of the operon in all of the mutant strains.…”

“…Their function in GBS has been previously investigated through the construction and characterization of knock-out mutants, suggesting that CpsA is required for transcription of the cps operon, whereas CpsC and CpsD have a more undefined role in polymerization/export of CPS (14). Moreover, recombinant CpsA was shown to bind the cps operon promoter in vitro (15).…”

“…The last four genes of the operon (neuA-neuD) encode enzymes that synthesize sialic acid. Finally, the first genes of the operon (cpsA-D) are not directly involved in the biosynthesis of the CPS repeating units (14).…”