GUEST COMMENTARIES A Unique Sensor Histidine Kinase

Sporulation initiation in Bacillus species is controlled by an extended version of two-component signal transduction systems, termed phosphorelay (4). The cascade of His3Asp3 His3Asp phosphotransfer is typically initiated by the ATPdependent autophosphorylation of the histidine kinase domain. In this issue, Scaramozzino et al. (11) reported that a sporulation histidine kinase, BA2291, in Bacillus anthracis exclusively utilizes GTP for this purpose. BA2291, while complementing the sporulation deficiency of Bacillus subtilis kinase mutants, was shown to inhibit sporulation in B. subtilis when cloned on a multicopy plasmid (1), and the prediction that BA2291 can function as a phosphorylated Spo0F (Spo0F P) phosphatase is confirmed in this study (11). The ability of BA2291 to specifically generate GTP through the reverse flow of a phosphoryl group from Spo0F P is also demonstrated (11). Interestingly, a point mutation in the linker region that connects the sensor domain to the kinase domain converts BA2291 to a sporulation inhibitor. The identification of the reversible GTPase activities together with other unique regulatory features that modulate the enzymatic activity of BA2291 provides new insight into the sophisticated nature of bacterial signal transduction mechanisms and raises an intriguing question, whether the BA2291 kinase plays a role in both sporulation initiation and inhibition in B. anthracis. In B. subtilis, upon sensing as-yet-unidentified signals, five histidine kinases phosphorylate the single-domain response regulator Spo0F, with the phosphoryl group being transferred to the ultimate master regulator, Spo0A. Spo0A then controls the expression of over 500 genes either directly or indirectly (2, 6). The dominant cytoplasmic kinase KinA is responsible for more than 90% of signal input under laboratory conditions. Negative regulatory inputs function via two aspartyl-phosphate phosphatase family proteins that target either Spo0F P or Spo0A P and are mediated by growth conditions antithetical to sporulation (8, 9). Similarly, five histidine kinases regulate sporulation, with BA2291 being the most actively transcribed one in B. anthracis (1). Although inactivating any of the kinases does not result in a significant sporulation deficiency, the deletion of BA2291 kinase caused the most noticeable sporulation delay in B. anthracis (1). Curiously, BA2291 is found in all B. anthracis strains but has no ortholog in B. subtilis. Due to its cytoplasmic localization, the direct activating ligand of BA2291 must reside intracellularly, and the activation results in the GTP-dependent autophosphorylation of BA2291. No other nucleotides, including the commonly used ATP, serve as substrates for the autophosphorylation. Attempts to understand the selectivity toward GTP have revealed a prominent role of a D-to-N substitution in the G1 box of the nucleotide binding domain in excluding ATP binding (11). As pointed out by those authors, 5% of histidine kinases have an asparagine at this position, and whether this substitution can serve as an indicator for GTP selectivity remains to be explored. Interestingly, BA2291 on a multicopy plasmid completely blocked sporulation in the wild-type B. subtilis strain, suggesting that it elicits a dominant negative role when artificially elevated in the cells. Thus, the ability of BA2291, when overexpressed, to efficiently deplete Spo0F P and possibly sequester Spo0F from receiving phosphoryl groups from other sporulation kinases provides a new strategy for dampening sporulation initiation. Many histidine kinases exhibit phosphatase activity toward their cognate response regulators under certain physiological conditions, but this is the first example showing that merely overproducing the kinase protein converts it from a positive regulator to a negative regulator. Scaramozzino et al. also showed that an S147L mutation of BA2291, located in the linker region between the sensor domain and the kinase domain, yielded a slower autophosphorylation rate than that of the wild-type kinase and, as a result, changed the BA2291 kinase to a sporulation inhibitor even when expressed from a single-copy gene. A possible explanation is that the mutation altered the equilibrium state between the kinase and the phosphatase activities of BA2291 with respect to signal activation. It remains to be determined whether choosing GTP over ATP as the phosphoryl group donor provides mechanism feasibility for such functional switching and if the signal relay communications from the sensor domain to the catalytic domain differ between ATPand GTP-dependent autokinases. The propensity of the BA2291 kinase to switch to a negative sporulation regulator appears to be specifically targeted by virulence plasmid-encoded sporulation inhibitors. Two singledomain proteins with high degrees of sequence similarity to the sensor domain of BA2291 were demonstrated to inhibit sporulation initiation in B. subtilis in a BA2291-dependent manner (13). Both proteins are encoded on the virulence plasmids: one gene, the pXO1-118 gene, is encoded divergently from the atxA gene on pXO1 and is controlled by the toxin regulator AtxA, while the other, the pXO2-61 gene, is located on plasmid pXO2 and adjacent to the cap operon, coding for capsule expression. The acquisition of pXO1 and pXO2, which are * Mailing address: Department of Medicine, Emory University School of Medicine, Woodruff Memorial Research Building, Room 2101, 1639 Pierce Drive, Atlanta, GA 30322. Phone: (404) 727-8393. Fax: (404) 712-2278. E-mail: [email protected]. Published ahead of print on 21 November 2008.