Paul Deangelis

Rapid detection of hyaluronic acid capsules on group a streptococci by buoyant density centrifugation

One of the virulence factors of group A streptococci is the hyaluronic acid polysaccharide capsule. A rapid method for ascertaining the status of the capsule phenotype in Streptococcus pyogenes is described. Bacteria with a capsule have a lower buoyant density than acapsular or hyaluronidase-treated cells. Early log phase cultures were underlaid with 65% Percoll and centrifuged at 500-1000 g for 5 min. Upon visual examination, encapsulated cells were observed at the interface, whereas acapsular cells appeared in the pellet. Cultures that produced at least 7 micrograms/ml of hyaluronic acid per A600 unit of cells were detected at the interface; this level of polysaccharide is only about 0.5%-4% of that found for most mucoid strains. Therefore, this procedure can detect capsules around strains that do not appear to be encapsulated by light microscopy or do not possess mucoid colony morphology. Furthermore, this method reduces dependence on other expensive assays that use labile radioactive reagents to detect hyaluronic acid.

Molecular cloning of the gene encoding RecF, a DNA repair enzyme, from Streptococcus pyogenes

RecF is a component of one of the bacterial DNA repair systems. The start codon of the recF gene of Streptococcus pyogenes resides about 4.4 kb downstream from the end of hasA, the hyaluronan synthase-encoding gene, which is transcribed in the opposite orientation. The deduced 368-amino-acid RecF protein is 43.5% identical to its homolog from Bacillus subtilis. When cloned on a plasmid, recF could complement a S. pyogenes deletion mutant that was sensitive to ultraviolet irradiation and chemical mutagens. This is the first report of a recF sequence from a Gram+ pathogen.