Copeland, S. Lucas, A. Lapidus, unpublished data; Sanford et al., 2002; Goldman et al., 2006; Huntley et al., 2011; Li et al., 2011; Huntley et al., 2012). A potential ortholog of nla6S was present in all genomes except those of the Anaeromyxobacter species, which are the only members of this group that do not form fruiting bodies (Sanford et al., 2002). The genomes of two myxobacteria from other suborders have been sequenced: Sorangium cellulosum (Schneiker et al., 2007)

and Haliangium ochracium (Ivanova et al., 2010). Tacrolimus solubility dmso We did not find a potential ortholog of nla6S in the genome sequences of these myxobacteria nor did we find a potential nla6S ortholog in any other sequenced bacterial genome. Furthermore, a phylogenetic comparison of the putative protein products of the five nla6S orthologs with representatives of previously described HK families revealed that the Nla6S-like proteins form a cluster that is separate from the previously characterized

HK families (Fig. 6b). These findings, together with our previous results, suggest that Nla6S is the prototype for a new family of HKs found in fruiting Cystobacterineae. Myxococcus xanthus has a large repertoire of signal transduction proteins to regulate its complex multicellular lifecycle. Many of these signal transduction proteins are HKs (Goldman et al., 2006; Shi et al., 2008), suggesting that M. xanthus cells have the capacity to detect and respond to a Alisertib solubility dmso variety of intracellular and extracellular signals. Here, we report the characterization of an M. xanthus HK that has a CA domain that appears to be found in only a subset of fruiting myxobacteria. The transmitter domain of Nla6S has a highly conserved DHp domain, but lacks a recognizable CA domain (Fig. 2). However, we have shown that the Nla6S transmitter domain is capable of hydrolyzing ATP (Fig. 3a)and autophosphorylating in vitro with kinetic parameters similar to those of known HKs (Figs 4a and Carbachol 5), indicating that Nla6S is a functional HK.

Although the putative CA domain of Nla6S has little similarity to the CA domains of known HKs, it does appear to have the conserved D-Box (Fig. 2). The conserved D-box Asp in the CA domain of HKs plays an important role in ATP binding by directly interacting with ATP via a hydrogen bond with the N6-amine of the adenine moiety (Dutta & Inouye, 2000). In Nla6S, the Asp204 residue is the putative D-Box Asp. Thus, our results showing that a D204A substitution in Nla6S causes strong defects in its ATPase and autophosphorylation activities (Figs 3b and 4b) suggest that the Asp204 residue and the putative CA domain of Nla6S are important for ATP binding and hydrolysis. Furthermore, the putative CA domain of Nla6S is predicted to have the secondary structure elements that are crucial for the formation of the α/β sandwich Bergerat fold, the signature motif of CA domains (Bergerat et al., 1997; Dutta & Inouye, 2000).