Total RNA was isolated using RNAprotect Bortezomib mouse Bacteria Reagent and RNeasy Plus Mini kit (Qiagen). cDNA was generated using iScript

cDNA synthesis kit (Bio-Rad). Expression of nla6S was normalized to that of rpoD, which is expressed at similar levels during growth and development (Fig. S1). Primers for QPCR were designed to produce 178- and 169-bp amplicons of the nla6S and rpoD genes, respectively. QPCR experiments were performed in triplicate. The annotated genome sequence of M. xanthus indicates that the nla6S gene (MXAN4043) encodes a putative HK (Goldman et al., 2006). To examine whether nla6S may function during the formation of fruiting bodies, developmental expression of nla6S in wild-type M. xanthus cells was monitored using QPCR. As shown in Fig. 1, nla6S mRNA is induced in two phases, with the first induction phase starting between 0.5- and 1-h poststarvation and the second induction buy Palbociclib phase starting between 2.5- and 3-h poststarvation. The peak nla6S mRNA level in both phases is about sixfold higher

than that observed in growing cells (0 h), indicating that nla6S is developmentally regulated and that Nla6S is likely to be involved in fruiting body development. We also attempted to examine the development function of nla6S via mutational analysis. However, we were unable to generate an nla6S deletion mutant, and the nla6 insertion mutant that we generated had a severe growth defect and was unstable (data not shown). These findings suggest that nla6S plays a role in vegetative growth out and fruiting body development in M. xanthus. Nla6S is predicted to be a cytoplasmic protein. An alignment of the putative Nla6S transmitter domain with those of known HKs suggests that Nla6S has a DHp domain (Fig. 2).

However, Nla6S lacks most of the conserved motifs found in the CA domain of HKs; the D-Box is the only conserved sequence motif that was identified (Fig. 2). The putative secondary structure of Nla6S was examined using the Jpred3 secondary structure prediction server (Cole et al., 2008), and the C-terminal domain of the protein that contains the D-box motif was predicted to have four α helices and five β strands arranged in the following order: α1, β1, β2, α2, β3, β4, α3, β5, α5. This secondary structure composition and arrangement is similar to that of previously characterized CA domains (Tanaka et al., 1998; Marina et al., 2001; Song et al., 2004), suggesting that the region containing the D-box motif might be a CA domain. When an HK senses a particular signal, the CA region of the transmitter domain binds and hydrolyzes ATP. To determine whether the putative Nla6S transmitter domain has ATPase activity, we used a colorimetric assay that couples the hydrolysis of ATP to the oxidation of NADH (Lascu et al., 1983). A polyhistidine-tagged version of the well-characterized E.