The ability of numerous microorganisms to cause disease relies upon the highly regulated expression of secreted proteinases. In this study, mutagenesis with a novel derivative of Tn4001 was used to identify genes required for the expression of the secreted cysteine proteinase (SCP) of the pathogenic Gram-positive bacterium Streptococcus pyogenes. Designated as Rop loci (regulation of proteinase), ropB is a rgg-like transcriptional activator required for transcription of the gene which encodes the proteinase. In contrast, ropA contributes post-transcriptionally to the secretion and processing of SCP and encodes a homologue of Trigger Factor, a peptidyl-prolyl isomerase and putative chaparone which is highly conserved in most bacterial species, but of unknown function. Analysis of additional ropA mutants demonstrated that RopA acts both to assist in targeting SCP to the secretory pathway and to promote the ability of the proprotein to establish an active conformation upon secretion. This latter function was dependent upon the peptidyl-prolyl isomerase domain of RopA and mutants that lacked this domain exhibited a bipartite deficiency manifested as a kinetic defect in autologous processing of the proprotein to the mature proteinase, and as a catalytic defect in the mature proteinase. These results provide insight into the function of Trigger Factor, the regulation of proteinase activity and the mechanism of secretion in Gram-positive bacteria.

The gram-positive microorganism Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous infections of the skin and pharynx ranging from superficial diseases including erysipelas, impetigo, and pharyngitis to those characterized by extensive tissue destruction, such as necrotizing fasciitis. The initial stage of all streptococcal infections involves the attachment of the organism to epithelial cells of the nasopharynx or epidermis (49), and considerable evidence suggests that the ability to sense an aerobic environment and survive plays an important role in this process (17,47,48). A good example of this is streptococcal fibronectin-binding protein F, which is regulated in response to oxidative stress (16,48).The mechanisms and gene products that allow S. pyogenes to survive in aerobic environments remain largely unknown. While S. pyogenes produces a single Mn-containing superoxide dismutase (SOD) that is essential for aerobic streptococcal growth (16), it lacks many of the proteins known to be important for aerobic growth. Since the lactic acid bacteria (including those in the genera Streptococcus, Enterococcus, and Lactococcus) cannot synthesize heme (11), S. pyogenes lacks the catalases and cytochrome oxidases required for oxidative energy-linked metabolism and instead depends on substrate level phosphorylation for growth. In addition, streptococci lack the moderate-to-high levels of intracellular glutathione found in gram-negative bacteria (12). Without such mechanisms for handling oxidative stress, it seems that aerobic conditions should severely restrict streptococcal growth, yet O 2 seems to have a positive effect on the growth yields of some other lactic acid bacteria (25,30). This suggests the existence of other enzymes that are important for aerobic streptococcal growth.Recently, other lactic acid bacteria have been found to contain unique flavoproteins involved in oxidative metabolism that are very different from the respiratory redox enzymes of cytochrome-containing bacteria like Escherichia coli (8,20,41,42). One such flavoprotein, NADH peroxidase (NPXase), has been characterized extensively in Enterococcus faecalis, where it uses H 2 O 2 as an electron acceptor, thereby providing an enzymatic defense against peroxide stress (41). Another E. faecalis flavoprotein, NADH oxidase (NOXase), catalyzes the direct four-electron reduction of O 2 to water and serves as an electron acceptor during active aerobic metabolism in this organism (42). These two flavoproteins have 44% amino acid identity to one another, with the most highly conserved segments containing the nonflavin redox center and the flavin adenine dinucleotide (FAD)-and NADH-binding regions. The nonflavin redox center in each of these enzymes is an unusual stabilized cysteine-sulfenic acid that cycles between oxidized and reduced states (33).A role for these two flavoproteins in facilitating the aerobic metabolism of lactic acid bacteria may require the regeneration of one NAD ϩ molecule by NPXase, and the regeneration ...

Binding of the Gram-positive pathogenic bacterium Streptococcus pyogenes (group A streptococcus) to respiratory epithelium is mediated by the fibronectin-binding adhesin, protein F. Most strains of streptococci regulate the expression of protein F in response to oxygen levels and redox potential; however, JRS4 constitutively binds high levels of fibronectin under all environmental conditions. In this study, we have examined the regulation of protein F expression in JRS4 using a shuttle mutagenesis strategy novel to S. pyogenes. Cloned DNA representing the chromosomal loci adjacent to the gene which encodes protein F (prtF) was subjected to transposon mutagenesis in Escherichia coli using a derivative of transposon m gamma delta that was modified to contain a streptococcal antibiotic-resistance gene. mutagenized DNA was then returned to the streptococcal chromosome by allelic replacement. Analysis of the resulting fibronectin-binding phenotypes revealed that insertions in a region upstream of prtF abolished the constitutive phenotype. However, these mutants now demonstrated regulation in response to both oxygen levels and redox potential. Because these insertions define a locus responsible for the constitutive phenotype, it has been designated rofA (regulator of F). Chromosomal interruption studies using integrational plasmids together with complementation data from a previous study (VanHeyningen et al., 1993) suggested that rofA acts as a positive trans-acting regulator of prtF. Construction of prtF-lacZ fusions indicated that transcription of prtF is constitutive in JRS4 but is regulated in rofA mutants. Analysis of the DNA sequence defined by the rofA insertions revealed a 1495 bp open reading frame, whose predicted product (RofA) possessed both a putative helix-turn-helix motif and limited homology to two other transcriptional activators (Mry, PrgR) of Gram-positive surface proteins. Sequences homologous to rofA were found in regulated strains of S. pyogenes, which suggests that rofA may act as an activator of prtF in response to an unidentified environmental signal. We speculate that the allele reported here contains a mutation that renders it constitutively active.

In establishing an infection, Streptococcus pyogenes has the capacity to bind to the host extracellular matrix protein fibronectin via its protein F adhesin. Previous studies have suggested that the expression of protein F is stimulated during aerobic growth or upon addition of superoxide-generating agents to the culture under O 2 -limited conditions. To further explore the role of superoxide, we have examined the transcription of the gene which encodes protein F (prtF), as well as the expression of superoxide dismutase (SOD) under conditions which promote or repress protein F expression. These studies show that prtF transcription is regulated in response to superoxide concentration and that SOD is regulated in different environments in a manner which directly parallels the expression of protein F. A mutant deficient in SOD activity was constructed by insertional mutation into the gene which encodes SOD (sod). The resulting mutant was sensitive to superoxide and aerobic conditions, showed hypersensitive induction of prtF in response to superoxide, and expressed prtF under normally unfavorable O 2 -limited conditions. These findings suggest that a streptococcal signal transduction system which senses superoxide may coordinately control expression of prtF and sod.

We describe the construction of TnFuZ, a genetic tool for the discovery and mutagenesis of proteins exported from gram-positive bacteria. This tool combines a transposable element (Tn4001) of broad host range in gram-positive bacteria and an alkaline phosphatase gene (phoZ) derived from a gram-positive bacterium that has been modified by removal of the region encoding its export signal. Mutagenesis of Streptococcus pyogenes with TnFuZ ("FuZ" stands for fusions to phoZ) identified genes encoding secreted proteins whose expression was enhanced during growth in an aerobic environment. Thus, TnFuZ should be valuable for analysis of protein secretion, gene regulation, and virulence in gram-positive bacteria.