Biological and Biochemical Roles of Two Distinct Cyclic Dimeric Adenosine 3',5'-Monophosphate- Associated Phosphodiesterases in Streptococcus mutans

Cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP), a recently identified secondary messenger in bacteria, plays a role in several bacterial processes, including biofilm formation. It is enzymatically produced by diadenylate cyclase and cleaved by c-di-AMP phosphodiesterase. c-di-AMP is believed to be essential for the viability of bacterial cells that produce it. In the current study, the biochemical and biological roles of GdpP (SMU_2140c) and DhhP (SMU_1297), two distinct phosphodiesterases involved in the pathway producing AMP from c-di-AMP, were investigated. Liquid chromatography-tandem mass spectrometry revealed that c-di-AMP was degraded to phosphoadenylyl adenosine (pApA) by truncated recombinant GdpP, and pApA was cleaved by recombinant DhhP to yield AMP. In-frame deletion mutants lacking the gene (Δ ) and both the and genes (Δ Δ ) displayed significantly more biofilm formation than the wild-type and a mutant strain lacking the gene (Δ ; < 0.01). Furthermore, biofilm formation was restored to the level of the wild type strain upon complementation with . Optical and electron microscopy observations revealed that Δ and Δ Δ mutants self-aggregated into large cell clumps, correlated with increased biofilm formation, but cell clumps were not observed in cultures of wild-type, Δ , or strains complemented with and . Thus, deletion of presumably leads to the formation of bacterial cell aggregates and a subsequent increase in biofilm production.