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

During sequencing of an 11.5 kb genomic region of a serotype M49 group A st reptococcal (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 expres sion was found for the three open reading frames (ORFs) downstream and for the four ORFs upstream of nra, The deduced Nra protein sequence exhibited 6 2% homology to the GAS RofA positive regulator. In contrast to RofA, Nra wa s found to be a negative regulator of its own expression and that of the tw o adjacent operons by analysis of insertional inactivation mutants. By poly merase chain reaction and hybridization assays of 10 different GAS serotype s, the genomic presence of nra, rofA or both was demonstrated. Nra-regulate d genes include the fibronectin-binding protein F2 gene (prtF2) and a novel collagen-binding protein (cpa), The Cpa polypeptide was purified as a reco mbinant maltose-binding protein fusion and shown to bind type I collagen bu t not fibronectin, In accordance with nra acting as a negative regulator of prfF2 and cpa, levels of attachment of the nra mutant strain to immobilize d collagen and fibronectin was increased above wild-type levels. In additio n, nra was also found to regulate negatively (four- to 16-fold) the global positive regulator gene, mga, Using a strain carrying a chromosomally integ rated duplication of the nra 3' end and an nra-luciferase reporter gene tra nscriptional fusion, nra expression was observed to reach its maximum durin g late logarithmic growth phase, while no significant influence of atmosphe ric conditions could be distinguished clearly.