In Bacillus subtilis, the Sirtuin Protein Deacetylase, Encoded by the srtN Gene (Formerly yhdZ), and Functions Encoded by the acuABC Genes Control the Activity of Acetyl Coenzyme A Synthetase

Acetate is abundant in soil, the natural habitat of the gram-positive bacterium Bacillus subtilis. This short-chain fatty acid accumulates in soil and other environments because it is the product of many fermentative processes (7). Many prokaryotes use acetate as a source of carbon and energy. The first step in the utilization of acetate is its activation into acetyl coenzyme A (Ac-CoA). Once Ac-CoA is made, it is used to synthesize lipids, can enter the tricarboxylic acid cycle to generate precursors of amino acids, or can be oxidized to generate reducing power via diverse pathways (9, 15, 26, 57). In B. subtilis, like in many other prokaryotes, acetate is excreted into the environment until the compound from which it is derived (e.g., glucose) is depleted. At that point, the acetate switch (55) is flipped, and acetate recapture is initiated (1, 10, 35). Two pathways are central to acetate catabolism in prokaryotes. Both of these pathways activate acetate into Ac-CoA, but they do it via different intermediates and in response to different concentrations of acetate in the environment. The pathway relevant to this work is the one illustrated in reactions 1 and 2 below, both of which are catalyzed by the Ac-CoA synthetase (AcsA) EC 6.2.1.1 (49). (1) (2) The second pathway is comprised of the acetate kinase (AckA; EC 2.7.2.1) and the phosphotransacetylase (Pta; EC 2.3.1.8) enzymes (6). AckA catalyzes reaction 3, while Pta catalyzes reaction 4. (3) (4) Although both pathways are reversible, hydrolysis of pyrophosphate generated by reaction 1 prevents the AcsA from converting Ac-CoA to acetate. The role of these pathways is to carefully maintain a physiological balance of Ac-CoA and free CoA (13, 14, 55). In B. subtilis, the Pta/AckA pathway appears to be a carbon overflow pathway used for the excretion of acetate (21). In contrast, the AcsA enzyme is preferentially used to capture acetate from the environment (53). The interplay of these acetate utilization pathways is carefully controlled at the transcription level by the global regulators CcpA and CodY. The latter represses acsA while activating ackA expression (34, 43, 44, 46). Previous work from our laboratory identified some of the functions required for the posttranslational control of the B. subtilis AcsA enzyme in vitro. Enzymes encoded by the acuA and acuC genes were shown to inactivate and reactivate the Ac-CoA-forming activity of AcsA (17). In B. subtilis, the acuA and acuC loci are adjacent to the acsA gene. A gene of unknown function, acuB, is located between acuA and acuC (16, 17). In this report, we provide evidence that the AcsA enzyme of B. subtilis is posttranslationally regulated in vivo, and we identify an additional protein deacetylase enzyme involved in the control of the activity of AcsA. At present, expression of acuABC genes appears to be responsive to conditions relevant to spore germination and outgrowth and during anaerobic respiration to nitrate (8). The data showing AcuA involvement in spore germination are relevant because they suggest that posttranslational modification is a mechanism used by the cell to control central metabolic functions other than AcsA. The regulation of expression of the acuABC genes is complicated by the fact that the operon is under the control of the global regulatory protein CcpA, which, as mentioned above, also regulates the expression of the acsA gene. The tight regulation at the transcriptional level of the acsA gene and the effective posttranslational control of AcsA activity reflect on the importance of maintaining CoA homeostasis in this bacterium. The posttranslational control system that regulates the AcsA enzyme of B. subtilis appears to be a variation of the one first described for Salmonella enterica, whose Acs enzyme is under acetylation control (48). In S. enterica, a protein acetyltransferase (Pat) enzyme homologous to the Gcn-5 protein of Saccharomyces cerevisiae (30, 50) uses Ac-CoA to acetylate a lysyl residue in the active site of Acs, which stops the first step of Ac-CoA formation (48) (reaction 1). The cognate deacetylase in S. enterica is a class III protein deacetylase that uses NAD+ as a substrate (not as a coenzyme), yielding O-acetyl-ADP-ribose (O-AADPR) and nicotinamide as by-products (25, 29, 39, 40, 51). Class III histone deacetylases are called sirtuins because they are homologous to the founding member of this family of proteins, the SIR2 protein of Saccharomyces cerevisiae (5, 36). In the gram-negative enterobacterium S. enterica, sirtuin-type deacetylase activity is associated with the CobB protein (45, 48, 52). In this paper, we provide in vitro and in vivo evidence that the YhdZ protein of B. subtilis has sirtuin-like activity; that is, YhdZ has NAD+-dependent protein deacetylase activity. To avoid confusion generated by the use of a gene name that reflects unknown function (i.e., yhdZ), hereinafter we refer to yhdZ as srtN (for sirtuin). We present evidence that indicates that the posttranslational modification system controlling the B. subtilis AcsA enzyme is more complex than the one in S. enterica, because two deacetylases appear to be involved in the deacetylation of AcsAAc.