Characterization of nra, a global negative regulator gene in group A streptococci

Abstract

SummaryDuring sequencing of an 11.5 kb genomic region of a serotype M49 group A streptococcal (GAS) strain, a series of genes were identified including nra (negative regulator of GAS). Transcriptional analysis of the region revealed that nra was primarily monocistronically transcribed. Polycistronic expression was found for the three open reading frames (ORFs) downstream and for the four ORFs upstream of nra. The deduced Nra protein sequence exhibited 62% homology to the GAS RofA positive regulator. In contrast to RofA, Nra was found to be a negative regulator of its own expression and that of the two adjacent operons by analysis of insertional inactivation mutants. By polymerase chain reaction and hybridization assays of 10 different GAS serotypes, the genomic presence of nra, rofA or both was demonstrated. Nra-regulated genes include the fibronectin-binding protein F2 gene (prtF2) and a novel collagen-binding protein (cpa). The Cpa polypeptide was purified as a recombinant maltose-binding protein fusion and shown to bind type I collagen but not fibronectin. In accordance with nra acting as a negative regulator of prtF2 and cpa, levels of attachment of the nra mutant strain to immobilized collagen and fibronectin was increased above wild-type levels. In addition, nra was also found to regulate negatively (four-to 16-fold) the global positive regulator gene, mga. Using a strain carrying a chromosomally integrated duplication of the nra 3Ј end and an nra -luciferase reporter gene transcriptional fusion, nra expression was observed to reach its maximum during late logarithmic growth phase, while no significant influence of atmospheric conditions could be distinguished clearly.

Streptococcus pyogenes prtFII, but not sfbI, sfbII or fbp54, is represented more frequently among invasive-disease isolates of tropical Australia

“…This result suggests that SfbI-mediated internalization of host cells is not likely to be a necessary characteristic of group A streptococcus for invasive diseases. This interpretation is consistent with the lack of sfbI among M types 1 and 18 that are known to be associated with severe invasive diseases elsewhere [24].…”

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: Population genetics, human serologic response, and gene transcription

“…These data provide information about gene regulatory circuits in GAS. Additional regulatory genes have been described in GAS (27,(44)(45)(46), and other probable regulators are present in the four genomes analyzed (data not shown). Hence, high-throughput TaqMan assays will permit rapid assessment of the influence of each regulatory gene on target genes such as those encoding virulence factors, extracellular molecules, and other transcriptional regulators.…”

Streptococcus pyogenes Collagen Type I-binding Cpa Surface Protein

“…Nucleic Acid Techniques and Sequence Analysis-Chromosomal and plasmid DNA preparations, genetic manipulations, and other conventional DNA techniques, including electroporation of GAS and E. coli strains, were done as described in Podbielski et al (51). Nucleic acid sequences of the S. pyogenes serotypes M1, M3, M5, M6, M12, M18, and M49 FCT regions were obtained from Refs.…”

“…The resulting PCR product was cloned into the multiple cloning site of pFW5-luc (51). The resulting recombinant pFW-luc plasmid was integrated by a site-specific single crossover event into the strain 591 genome as described by Podbielski et al (51), thereby duplicating the 3Јend of the cpa operon. The correct insertion site was confirmed by using Southern blot hybridizations and appropriate PCR assays on genomic DNA preparations from wt and mutant strains (data not shown).…”