Survival Strategies of Streptococcus mutans during Carbohydrate Starvation

Abstract

Streptococcus mutans is a facultative member of the oral plaque and is associated with dental caries. It is able to survive long periods of sugar starvation. The purpose of this project was to explore specific avenues that S. mutans may use in order to cope with carbohydrate deprivation. Intracellular polysaccharide (IPS) is accumulated by S. mutans when grown in excess sugar, and can contribute towards the cariogenicity of S. mutans. Inactivation of the glgA gene, encoding a putative glycogen synthase, prevented accumulation of IPS in batch cultures grown with excess glucose or sucrose. Inactivation of the pul gene, encoding a putative pullulanase which is thought to be involved in IPS catabolism, did not prevent IPS accumulation. IPS was found to be important for the persistence of S. mutans grown in batch culture with excess glucose, and then starved of glucose. In these conditions, the IPS was largely used up within one day of starvation, and yet survival of the parental strain was extended by at least 15 days beyond that of the glgA and pul mutants; potentially, some feature of IPS metabolism, distinct from providing nutrients, is important for persistence. IPS was not needed for persistence when sucrose was carbon source or when mucin was present in batch cultures. IPS accumulation was not clearly demonstrated in biofilm conditions. When grown in condition permissive for IPS accumulation, biofilms of the glgA and pul mutants did not show decreased survival, compared to the parental strain. It is plausible that, within a biofilm, S. mutans can use alternative sources of energy (like the extracellular matrix) to compensate for the lack of IPS. To look at specific genes upregulated by sugar starvation, microarrays analysis was performed on S. mutans batch cultures. Some of the genes upregulated by starved, stationary phase bacteria, appeared to be organized in an operon, thought to encode components of the pyruvate dehydrogenase (PDH) complex. Northern Blot analysis showed that pdhD and the downstream genes, pdhA, pdhB and pdhC, form an operon that is transcribed predominantly in stationary phase. Inactivation of pdhD impaired survival of both batch cultures and biofilms. Analysis with fluorescent reporters revealed a distinct expression pattern for the pdh promoter, with less than 1% of stationary phase bacteria displaying pdh expression. When first detected, after one day of sugar starvation, expression was in individual bacteria. At later times, expressing bacteria were often in chains. The lengths of chains increased with time suggesting growth and division. It is likely that the pdh-expressing sub-population is able to persist for extend times in stationary phase.