Molecular characterization of a locus required for hyaluronic acid capsule production in group A streptococci.

Abstract

SummaryTo characterize the production of hyaluronate capsule by the membrane-associated enzyme hyaluronate synthase (HAS), group A streptococci from a recent outbreak of acute rheumatic fever were mutagenized via Tn916 insertion. Acapsular transconjugants harboring multiple, nontandem copies of the transposon were identified and found to lack HAS activity (<1% of wild-type levels). Generalized transduction was then performed to determine which Tn916 insertion was responsible for the HAS-phenotype. These marker exchange experiments resulted in the isolation of two distinct classes of acapsular transductants, designated WF61 and WF62. Both transductants also lacked significant HAS activity, and excision of the transposon from WF62 restored capsular hyaluronate production. Southern analysis of WF61 DNA demonstrated a large deletion of genomic DNA adjacent to the Tn916 insertion. This deletion event is presumably responsible for the observed stability of the acapsular phenotype of WF61. Further analyses of transductant whole-cell DNA indicated that the transposon insertions of WF61 and WF62 were separated by 2.5 kb. These studies define a locus required for hyaluronate capsule production in group A streptococci. Further genetic analysis of this locus has identified a gene required for HAS activity which was inactivated by Tn916 in WF62 and deleted in WF61.

Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization

“…The ExoO protein from R meliloti (17) is involved in the synthesis of the exopolysaccharide succinoglycan and catalyzes the formation of a ,B-1,6 glycosidic linkage. HasA has been identified as a hyaluronate synthase in the group A streptococci and is predicted to function in the 13-1,4 transfer of N-acetylglu- cosamine to glucuronic acid during hyaluronate biosynthesis (15). Multiple alignments of these polypeptides with the AcsAB and BcsA polypeptides identify regions of conserved residues present among these various glycosyltransferases that may be involved in formation of 3-glycosidic linkages (Fig.…”

“…The AcsC polypeptide was found to be homologous to bacterial proteins that are components of membrane channels or pores. Even though pore-like structures have been proposed for the extrusion of the cellulose microfibrils (56) (15), and ExoO (17) polypeptides were aligned using the GCG program PILEUP, and a region of conserved residues is shown. The amino acids displayed correspond to residues 151 to 241 of AcsAB, 151 to 241 of BcsA, 51 to 145 of NodC, 66 to 163 of HasA, and proteins involved in the secretion of macromolecules from bacteria suggests that the cellulose-synthesizing and extrusion complex has functions other than the polymerization of glucans.…”

Polymorphism, duplication, and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA and capsular K antigens

“…The K30 polysaccharide contains mannose, galactose, and glucuronic acid (14), and based on other bacterial systems, UDP-glucuronic acid is an expected biosynthetic precursor (40). The predicted Ugd protein from E. coli E69 is 388 amino acids in length and displays 86.6% amino acid similarity to KfiD from E. coli K5 (41) and 72.2% similarity to HasB from Streptococcus pyogenes (11). KfiD and HasB are involved in capsular polymer synthesis, and UDPglucose dehydrogenase enzymatic activity has been confirmed for both enzymes.…”

Bacterial polysaccharides in sickness and in health

“…The use of microbial fermentation offers considerable financial and ethical advantages over tissue extraction. The recent cloning and expression of the hyaluronic acid synthase gene from Streptococcxr pyugenes (Dougherty & van de Rijn, 1994) may permit the expression of this polymer in other hosts (see below).…”

“…The recent cloning and expression in E. coli of the gene from S. PJyogeneS encoding the hyaluronic acid synthase (Dougherty & van de Rijn, 1994) may permit the expression of large amounts of hyaluronic acid in E. coli. Whether this will offer any commercial advantages over the current production methods using group A and C streptococci is not yet known.…”