The globally disseminated Streptococcus pyogenes M1T1 clone causes a number of highly invasive human diseases. The transition from local to systemic infection occurs by an unknown mechanism; however invasive M1T1 clinical isolates are known to express significantly less cysteine protease SpeB than M1T1 isolates from local infections. Here, we show that in comparison to the M1T1 strain 5448, the isogenic mutant ∆speB accumulated 75-fold more human plasmin activity on the bacterial surface following incubation in human plasma. Human plasminogen was an absolute requirement for M1T1 strain 5448 virulence following subcutaneous infection of humanized plasminogen transgenic mice. S. pyogenes M1T1 isolates from the blood of infected humanized plasminogen transgenic mice expressed reduced levels of SpeB in comparison with the parental 5448 used as inoculum. We propose that the human plasminogen system plays a critical role in group A streptococcal M1T1 systemic disease initiation. SpeB is required for S. pyogenes M1T1 survival at the site of local infection, however, SpeB also disrupts the interaction of S. pyogenes M1T1 with the human plasminogen activation system. Loss of SpeB activity in a sub-population of S. pyogenes M1T1 at the site of infection results in accumulation of surface plasmin activity thus triggering systemic spread. ABSTRACT The globally disseminated Streptococcus pyogenes M1T1 clone causes a number of highly invasive human diseases. The transition from local to systemic infection occurs by an unknown mechanism; however invasive M1T1 clinical isolates are known to express significantly less cysteine protease SpeB than M1T1 isolates from local infections. Here, we show that in comparison to the M1T1 strain 5448, the isogenic mutant ∆speB accumulated 75-fold more human plasmin activity on the bacterial surface following incubation in human plasma. Human plasminogen was an absolute requirement for M1T1 strain 5448 virulence following subcutaneous infection of humanized plasminogen transgenic mice. S. pyogenes M1T1 isolates from the blood of infected humanized plasminogen transgenic mice expressed reduced levels of SpeB in comparison with the parental 5448 used as inoculum. We propose that the human plasminogen system plays a critical role in group A streptococcal M1T1 systemic disease initiation. SpeB is required for S. pyogenes M1T1 survival at the site of local infection, however, SpeB also disrupts the interaction of S. pyogenes M1T1 with the human plasminogen activation system. Loss of SpeB activity in a sub-population of S. pyogenes M1T1 at the site of infection results in accumulation of surface plasmin activity thus triggering systemic spread.

Streptococcus sanguinis is a commensal oral bacterium producing hydrogen peroxide (H 2 O 2 ) that is dependent on pyruvate oxidase (Spx) activity. In addition to its well-known role in bacterial antagonism during interspecies competition, H 2 O 2 causes cell death in about 10% of the S. sanguinis population. As a consequence of H 2 O 2 -induced cell death, largely intact chromosomal DNA is released into the environment. This extracellular DNA (eDNA) contributes to the self-aggregation phenotype under aerobic conditions. To further investigate the regulation of spx gene expression, we assessed the role of catabolite control protein A (CcpA) in spx expression control. We report here that CcpA represses spx expression. An isogenic ⌬ccpA mutant showed elevated spx expression, increased Spx abundance, and H 2 O 2 production, whereas the wild type did not respond with altered spx expression in the presence of glucose and other carbohydrates. Since H 2 O 2 is directly involved in the release of eDNA and bacterial cell death, the presented data suggest that CcpA is a central control element in this important developmental process in S. sanguinis.

Certain oral streptococci produce H 2 O 2 under aerobic growth conditions to inhibit competing species like Streptococcus mutans. Additionally, H 2 O 2 production causes the release of extracellular DNA (eDNA). eDNA can participate in several important functions: biofilm formation and cell-cell aggregation are supported by eDNA, while eDNA can serve as a nutrient and as an antimicrobial agent by chelating essential cations. eDNA contains DNA fragments of a size that has the potential to transfer genomic information. By using Streptococcus gordonii as a model organism for streptococcal H 2 O 2 production, H 2 O 2 -dependent eDNA release was further investigated. Under defined growth conditions, the eDNA release process was shown to be entirely dependent on H 2 O 2 . Chromosomal DNA damage seems to be the intrinsic signal for the release, although only actively growing cells were proficient eDNA donors. Interestingly, the process of eDNA production was found to be coupled with the induction of the S. gordonii natural competence system. Consequently, the production of H 2 O 2 triggered the transfer of antibiotic resistance genes. These results suggest that H 2 O 2 is potentially much more than a simple toxic metabolic by-product; rather, its production could serve as an important environmental signal that facilitates species evolution by transfer of genetic information and an increase in the mutation rate.

Control over mRNA stability is an essential part of gene regulation that involves both endo-and exoribonucleases. RNase Y is a recently identified endoribonuclease in Gram-positive bacteria, and an RNase Y ortholog has been identified in Streptococcus pyogenes (group A streptococcus [GAS]). In this study, we used microarray and Northern blot analyses to determine the S. pyogenes mRNA half-life of the transcriptome and to understand the role of RNase Y in global mRNA degradation and processing. We demonstrated that S. pyogenes has an unusually high mRNA turnover rate, with median and mean half-lives of 0.88 min and 1.26 min, respectively. A mutation of the RNase Y-encoding gene (rny) led to a 2-fold increase in overall mRNA stability. RNase Y was also found to play a significant role in the mRNA processing of virulence-associated genes as well as in the rapid degradation of rnpB read-through transcripts. From these results, we conclude that RNase Y is a pleiotropic regulator required for mRNA stability, mRNA processing, and removal of read-through transcripts in S. pyogenes. RNA degradation is a strictly regulated process that involves both endo-and exoribonucleases (1). In prokaryotes, mRNA degradation is initiated by endonucleolytic cleavage and followed by exonuclease digestion (2-4). The first step is relatively slow and rate limiting, while the second step proceeds rapidly (1). In Escherichia coli, RNase E functions as the major endoribonuclease that initiates the bulk of mRNA degradation (5). Although RNase E is absent in Bacillus subtilis, the recently identified RNase Y is considered its functional analog (6). RNase E and RNase Y do not share sequence homology but are strikingly similar in function (7,8). Both RNases are membrane-bound proteins that interact with other components to form a complex called the RNA degradosome (7, 9). These components include other RNases, an RNA helicase, and two glycolytic enzymes (10, 11). Both RNase Y and RNase E prefer 5= monophosphorylated RNA substrates with downstream secondary structures (6, 12). The depletion of B. subtilis RNase Y results in the accumulation of about 550 mRNAs, including important transcriptional regulators for stress response and biofilm formation and metabolic operons for tryptophan biosynthesis and glycolytic enzymes (8). B. subtilis RNase Y also interacts with RNases J1 and III to control the abundance of total mRNAs (13). RNase Y of Staphylococcus aureus plays a major role in virulence gene regulation and is involved in the processing and stabilization of a global regulator system, SaePQRS (14). These observations suggest that RNase Y is the major endoribonuclease in mRNA degradation in B. subtilis and perhaps also in other Gram-positive pathogens, such as Streptococcus pyogenes.S. pyogenes (group A streptococcus [GAS]) causes a variety of human diseases ranging from mild local infections such as pharyngitis and impetigo to life-threatening systemic diseases such as toxic shock syndrome and necrotizing fasciitis (15). GAS infections often cau...

Streptococcus gordonii is an important member of the oral biofilm. One of its phenotypic traits is the production of hydrogen peroxide (H 2 O 2 ). H 2 O 2 is an antimicrobial component produced by S. gordonii that is able to antagonize the growth of cariogenic Streptococcus mutans. Strategies that modulate H 2 O 2 production in the oral cavity may be useful as a simple therapeutic mechanism to improve oral health, but little is known about the regulation of H 2 O 2 production. The enzyme responsible for H 2 O 2 production is pyruvate oxidase, encoded by spxB. The functional studies of spxB expression and SpxB abundance presented in this report demonstrate a strong dependence on environmental oxygen tension and carbohydrate availability. Carbon catabolite repression (CCR) modulates spxB expression carbohydrate dependently. Catabolite control protein A (CcpA) represses spxB expression by direct binding to the spxB promoter, as shown by electrophoretic mobility shift assays (EMSA). Promoter mutation studies revealed the requirement of two catabolite-responsive elements (CRE) for CcpA-dependent spxB regulation, as evaluated by spxB expression and phenotypic H 2 O 2 production assays. Thus, molecular mechanisms for the control of S. gordonii spxB expression are presented for the first time, demonstrating the possibility of manipulating H 2 O 2 production for increased competitive fitness.

Summary Streptococcus gordonii is an important member of the oral biofilm community. As oral commensal streptococci, S. gordonii is considered beneficial in promoting biofilm homeostasis. CcpA is known as central regulator of carbon catabolite repression in Gram-positive bacteria and is also involved in the control of virulence gene expression. To further establish the role of CcpA as central regulator in S. gordonii, the effect of CcpA on biofilm formation and natural competence of S. gordonii was investigated. These phenotypic traits have been suggested to be important to oral streptococci in coping with environmental stress. Here we demonstrate that a CcpA mutant was severely impaired in its biofilm forming ability, showed a defect in extracellular polysaccharide production and reduced competence. The data suggest that CcpA is involved in the regulation of biofilm formation and competence development in S. gordonii.

This neglected pathogen causes a large portion of these infections.

SUMMARY In both prokaryotes and eukaryotes insight into gene function is typically obtained by in silico homology searches and/or phenotypic analyses of strains bearing mutations within open reading frames. However, the studies herein illustrate how mRNA function is not limited to the expression of a cognate protein. We demonstrate that a stress-induced protein-encoding mRNA (irvA) from the dental caries pathogen Streptococcus mutans directly modulates target mRNA (gbpC) stability through seed pairing interactions. The 5’ untranslated region of irvA mRNA is a trans-riboregulator of gbpC and a critical activator of the DDAG stress response, whereas IrvA functions independently in the regulation of natural competence. The irvA riboregulatory domain controls GbpC production by forming irvA-gbpC hybrid mRNA duplexes that prevent gbpC degradation by an RNase J2-mediated pathway. These studies implicate a potentially ubiquitous role for typical protein-encoding mRNAs as riboregulators, which could alter current concepts in gene regulation.