Environmental Triggers of lrgA Expression in Streptococcus mutans

Abstract

The lrgAB and cidAB operons of Streptococcus mutans encode proteins that are structurally similar to the bacteriophage lambda family of holin-antiholin proteins, which are believed to facilitate cell death in other bacterial species. Although their precise function is not known, cidAB and lrgAB are linked to multiple virulence traits of S. mutans, including oxidative stress tolerance, biofilm formation, and autolysis. The regulation of cidAB and lrgAB is still not understood, as these operons show opposite patterns of expression as well as a complex dependence on growth conditions. We have used a microfluidic approach, together with single-cell imaging of a fluorescent gene reporter, to identify with greater precision the cues that trigger lrgA expression and characterize cell-to-cell heterogeneity in lrgA activity. lrgA activates very abruptly at stationary phase, with a high degree of synchrony across the population. We find this activation is controlled by a small number of inputs that are sensitive to growth phase: Extracellular pyruvate, glucose, and molecular oxygen. Further, activation of lrgA appears to be self-limiting, so that lrgA is strongly expressed only for a very short interval of time. Consequently, lrgA is programmed to switch on briefly at the end of exponential growth, as glucose and molecular oxygen are exhausted and extracellular pyruvate is available. Our findings are consistent with studies showing that homologs of lrgAB are linked, together with lytST, to the reimport of pyruvate for anaerobic fermentative growth.\n\nImportanceThe function and regulation of cidAB and lrgAB in Streptococcus mutans is not understood, although these operons have been clearly linked to stress responses and they show a complex dependence on environmental inputs and growth phase. Identifying specific environmental cues that trigger activation of lrgAB has been difficult owing to the cells own modification of key inputs such as glucose and oxygen: In S. mutans the lrgAB operon is strongly upregulated at the end of exponential phase, where growth conditions in a bulk culture become poorly defined. Here we have used microfluidics to apply precise control of environmental inputs to S. mutans and identify specific chemical cues that activate lrgAB. We find that rigorously anaerobic conditions and the presence of extracellular pyruvate are sufficient to induce lrgAB expression, suggesting that lrgAB is timed to activate just as pyruvate fermentation becomes favorable.