Multiple Two-Component Systems Modulate Alkali Generation in Streptococcus gordonii in Response to Environmental Stresses

Streptococcus gordonii colonizes the oral cavity very early in life (10, 27, 52), and its presence in oral biofilms is generally associated with oral health (1, 7, 48, 49). Colonization by S. gordonii is believed to be beneficial to the host in large part because of the contribution of this microorganism to pH homeostasis in oral biofilms through the hydrolysis of arginine in saliva and the diet (11). The pH of the oral cavity fluctuates in response to the diet and diurnal rhythms of the host, and adaptation to acidic conditions is important for the survival of S. gordonii and other oral biofilm organisms (58). Residents of oral biofilms also experience other environmental stresses, including modest temperature fluctuations, substantial changes in nutrient source and abundance, and wide variation in oxygen tension and redox potential as oral biofilms mature (26). Despite these challenging conditions, S. gordonii is able to persist as a significant proportion of the biofilm populations of the oral cavity (20, 56) and, like some other viridans streptococci, can cause endocarditis (6, 24, 42). Thus, adaptations by S. gordonii to conditions in multiple intraoral sites or in blood and infected heart valves are essential for persistence. The arginine deiminase system (ADS), which is present in S. gordonii and a number of other abundant commensal oral streptococci, is a three-enzyme pathway that converts arginine to ornithine, CO2, and ammonia, with the concomitant generation of ATP (13). The ADS augments acid tolerance in S. gordonii by neutralizing the cytoplasm and environment, and the ATP generated can be used for growth, anabolism, and to extrude protons (11, 14, 15, 38). A strong association with the arginolytic potential of human dental biofilms and resistance to caries has been documented (45, 60), and organisms that are ADS positive are believed to have a beneficial impact on oral microbial ecology. Mechanistically, the presence of the ADS in oral biofilms is believed to provide a selective advantage to those organisms that possess it and to moderate biofilm acidification, both of which favor the persistence of a microflora that is compatible with dental health and discourage the emergence of aciduric organisms associated with dental caries. In S. gordonii, the genes for the three enzymes of the pathway, arginine deiminase (AD; arcA), ornithine carbamyltransferase (arcB) and carbamate kinase (arcC) are cotranscribed in an operon with arcD (arginine:ornithine antiporter) and arcT (arginine aminopeptidase). Induction of the ADS genes (arcABCDT) by arginine is mediated by a transcriptional activator encoded by the divergently transcribed arcR gene, located immediately 3′ to the ADS operon. Immediately 5′ to the ADS operon is a gene for an Fnr-like protein (flp) (14), which activates expression from the arcA promoter (ParcA) under anaerobic conditions. Carbohydrate catabolite repression of the operon by preferred carbohydrate sources, such as glucose, is exerted primarily through CcpA (14, 15). In addition, ADS expression is optimal under acidic conditions, but the basis for pH-dependent control of transcription has not been investigated (38). Other complexities in ADS regulation exist in S. gordonii, including that queosine modification of tRNA may impact translation of the genes (38), ADS expression is higher in stationary phase (38), and other mechanisms for posttranscriptional regulation of expression appear to exist (38). Because of the spectrum of control mechanisms governing ADS production and the wide distribution of this system in abundant commensal organisms and pathogens, analysis of ADS expression has provided many insights into genetic regulation in streptococci. There is also a high degree of relevance of ADS regulation to oral biofilm ecology, oral health, and disease (11). In spite of substantial progress in understanding control of the ADS, critical gaps remain in our knowledge of how this system is regulated. Transcriptional control of gene expression by two-component signal transduction systems (TCSs) is a common mechanism used by bacteria to modulate cell behaviors in response to environmental conditions (57). A TCS is composed of a histidine kinase that usually detects an environmental signal and a response regulator that can be phosphorylated by the sensor kinase (25). TCSs are involved in the stress responses of many bacteria, including Escherichia coli (16, 50), Pseudomonas aeruginosa (51, 59), Staphylococcus aureus (19, 59), and Streptococcus mutans (4, 9, 29, 54). In S. gordonii, the ComDE TCS was shown to regulate development of genetic competence (23, 41) and was required for efficient in vitro biofilm formation (40). The BfrAB TCS of S. gordonii affects biofilm development and the expression of multiple ABC transporters (28, 64, 65). In addition to ComDE, the CiaRH and VicRK TCSs of oral streptococci and some pathogenic streptococci seem particularly important for regulation of traits associated with colonization, growth in the host, and pathogenesis (4, 35, 37, 54). We investigated here whether the CiaRH, ComDE, and VicRK TCSs are able to influence the expression of the ADS in response to pH and oxidative stress. In addition, we begin to explore whether these TCSs, which play key regulatory roles in a variety of critical cellular functions in low G+C gram-positive bacteria (18, 44, 46), affect traits of S. gordonii that are known to be important for establishment and persistence in the oral cavity.