Gene expression in Streptococcus mutans biofilms

Abstract

Streptococcus mutans is considered the major aetiological agent of human dental caries. It is an obligate biofilm-forming bacterium, which resides on teeth and forms, together with other species, an oral biofilm that is often designated as supragingival plaque. This thesis consists of three distinct parts. The first part describes, using microarray analysis, how S. mutans modulates gene expression when grown under different conditions in biofilms. The goal of this analysis was to identify genes that are required for establishment and survival in biofilms. When grown in exponential biofilms S. mutans increased the expression of uptake systems for Mn2+, suggesting a requirement for this element for proper establishment in biofilm. Furthermore, S. mutans down-regulated genes associated with sucrose-dependent adhesion (spaP, gtfB). When exponential biofilms were cultivated in the presence of sucrose, a specific sucrose-transporter was activated. Compared with planktonic growth, stationary phase biofilms showed a greater change in their gene expression profile than biofilms harvested from the exponential growth phase, indicating that different regulation modes of growth processes are operating under altered environmental conditions. Again compared with planktonic growth, stationary phase biofilms grown with glucose showed up-regulation of genes involved in carbohydrate metabolism and signal transduction, and downregulation of genes associated with translation, energy production and amino-acid transport, and metabolism. A gene cluster comprising the lrgA and lrgB genes, which are predicted to be involved in cell death, was also highly up-regulated in these biofilms. Comparison of the transcriptome of a sucrose-grown stationary biofilm with that of a glucosegrown stationary biofilm culture suggested that there may be more DNA damage in the former, since several genes coding for repair enzymes were up-regulated. Energy consumption and the metabolism of amino-acids and sugars were decreased. When comparing starved biofilms with fed biofilms of S. mutans, the latter showed an increase in expression of genes required for transcription and translation, as well as of genes incoding a specific PTS system responsible for the uptake of sucrose. The opposite pattern was observed when biofilms were transfered from replete medium to starving conditions. In the second part of this work, the involvement of the two gene clusters lrgAB and cidAB in cell death was investigated. Using real-time RT-PCR, it was found that expression of lrgAB increased with rising cell density. A similar pattern was observed for lytSR, which encodes a two-component 5 signal transduction system. Further analysis suggested that the lrgAB genes were negatively regulated by LytSR. The lrgAB genes appear to be important for long-term survival, since a lrgAB knock-out mutant exhibited a 3log10-decrease in viable cell count after 13 days of incubation. The expression of lrgAB increased in response to lactic acid, the predominant organic acid found in culture media from bacteria grown with sucrose as the sole carbon source. The exposure of S. mutans to CCCP, an ionophore that affects both components of the proton motive force, resulted in high expression of lrgAB. Biofilm formation was not affected in both lrgAB and cidAB mutants. Overall, it was shown that the lrgAB gene cluster encodes an important factor for S. mutans survival in stationary phase. This factor is probably required to withstand low pH, starvation, or membrane-damaging agents. Bacteria can detect, transmit and react to signals from the outside world by two-component systems and by serine-threonine kinases and phosphatases first detected eukaryotic cells, but later recognized to occur widespread in bacteria as well. S. mutans contains one such serine-threonine kinase, encoded by pknB. A gene encoding serine-threonine phosphatase, pppL, is located upstream of pknB. In the third part of the work, the phenotypes of single mutants in pknB and pppL and of a pknB - pppL double mutant were characterized. All mutants showed abnormal cell shapes and grew slower than the wild type. Whole genome transcriptome analysis revealed that a pknB mutant showed reduced expression of genes in bacteriocin production and genetic competence. Among the genes that were differentially regulated in the pknB mutant several were likely involved in cell-wall metabolism. One such gene, SMU.2146c, and two genes encoding bacteriocins were shown to be also down-regulated in a mutant in vicK, a sensor kinase involved in response to oxidative stress. Collectively, the results indicate that PknB can modulate the activity of the two-component signal transduction systems VicKR and ComDE.