Distinct Subpopulations in Biofilms of Streptococcus mutans and their Response to Sugar Starvation and Restoration

Abstract

Streptococcus mutans is a secondary colonizer of the dental plaque biofilm and is the primary causative agent of dental caries. Sugar metabolism is central to S. mutans growth and survival. S. mutans produces lactic acid as an end product of sugar metabolism, which results in dissolution of the tooth enamel, leading to dental cavities. Sucrose metabolism also results in the formation of extracellular dextrans that are a key component of the extracellular matrix that encases the bacteria in the biofilm. The availability of sugars is dependent on diet, on competition with other bacteria and on the location of the bacteria within the dental plaque. I hypothesize there are distinct subpopulations of S. mutans within biofilms that respond differently to environmental conditions. I have identified several genetic markers that are helping us identify and characterize some of these subpopulations, and how they react to starvation and to the restoration of nutrients in single species biofilms of S. mutans. Two of the loci that were identified as markers via microarray analysis are rpsT and pdh. rpsT encodes a small ribosomal protein which is strongly expressed during exponential growth, when the cells are producing high levels of ribosomes. The other marker, pdh, is a four-gene operon encoding the pyruvate dehydrogenase complex; pdh is upregulated in late stationary phase. Our laboratory has recently shown that expression of the pdh operon is important for long-term survival in stationary phase, where a subpopulation (~0.5%) is dividing, forms long chains and expresses pdh. In the current studies, rpsT and pdh promoters driving expression of gfp were used to identify the exponential phase subpopulation (rpsT) and a subpopulation capable of surviving in late stationary phase (pdh). In addition, I developed an unstable variant of GFP by fusing a proteolytic tag sequence to the C-terminus of GFP (encoded by ugfp). When the rpsT promoter was inserted upstream, uGFP was produced and subsequently degraded within about 1.5 hours of translation. This behavior allowed us to distinguish exponentially growing cells, as the signal diminishes once the cells entered stationary phase. In biofilms that had been starved for 10 days, there was no expression of PrpsTugfp. I observed that when sucrose was added to these biofilms, some bacteria within the biofilm microcolonies underwent fast exponential-like growth indicated by expression of PrpsT-ugfp. Within 24 hours of the sucrose addition, most growth had ceased and fluorescence had decreased. Using a Ppdh-gfp construct in bacteria in 10-day starved biofilms, fluorescence was observed in long chains of cells within the biofilms indicating slow growth. I hypothesized that the pdh-expressing cells were capable of responding to sucrose restoration and would be one of the principal subpopulations to do so. However, when sucrose was added, these fluorescing chains did not exhibit any growth, while other non-fluorescing bacteria within the biofilm clearly responded to the sucrose by growing. This was unexpected since inactivating the pdh operon leads to drastically reduced survival. It is concluded that pdh plays a role in long term survival, but pdhexpressers do not appear to respond to sugar restoration. This led me to hypothesize that the pdh-expressing population is interacting with other populations of cells in some capacity, enabling them to survive. To determine if this was the case, we performed a mixed culture experiment with wild-type S. mutans and the pdh null mutant. I observed that when these two strains were grown in co-culture, the pdh null mutant survived at low levels, for over 30 days, while this mutant by itself typically did not survive past ten days. This result indicates that the wild-type strain was able to interact with the mutant, leading to increased survival. In biofilms, it seems possible that the pdh-expressing cells secrete a substance or directly interact with other cells, somehow promoting their survival in the starved biofilm. The fluorescent constructs appear to mark distinct populations of cells that respond in different ways to sugar availability, suggesting that S. mutans forms a mixed population of cells able to grow in the presence of sugar or survive prolonged sugar starvation. These studies demonstrate that indeed subpopulations of cells do exist within biofilms, and their interactions may be more complex than previously thought.