Streptococcus mutans is the primary agent of Dental caries. We think that stationary-phase bacteria are critical to the behavior and survival of S. mutans in the Dental plaque biofilm, and hence to its role in Dental caries. They are surely a critical part of the feast-or-famine lifestyle of S. mutans, as famine causes entry into the stationary phase. Yet, most studies of S. mutans have been of exponentially growing bacteria. We propose to test the general hypothesis that stationary-phase bacteria in biofilms behave differently from vegetative bacteria and have an important role in determining the properties of mature biofilms, and in particular in their persistence. We propose using single-species biofilms formed in flow cells to test this hypothesis. We propose to test how well stationary-phase bacteria survive prolonged carbon-source starvation in biofilms and how they respond to nutrient restoration and acid shock. We will test the effect of different treatments on the shedding of bacteria from stationary-phase biofilms, and test how effectively the shed bacteria can initiate secondary biofilm formation in a second flow cell. We propose to use the fluorescing protein GFPmut3b* as a probe to locate bacteria expressing known stationary-phase-specific genes and those expressing vegetative-phase specific genes. This will help clarify the difference in properties between the two bacterial types. We will screen for new stationary-phase expressed genes. These genes will enable testing if there are different types of stationary-phase bacteria with different resistances and different responses to nutrient. They will give an indication of the sorts of function that are expressed during stationary phase. We propose to test the roles of these genes. We propose to identify and characterize the regulators of stationary phase gene expression. These studies should lead to a better understanding of how S. mutans in mature Dental plaque biofilm persists and responds to fluctuations in its environment.
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