Nitrate-Dependent Activation of the Dif Signaling Pathway ofMyxococcus xanthusMediated by a NarX-DifA Interspecies Chimera

Abstract

Myxococcus xanthus fibril exopolysaccharide (EPS), essential for the social gliding motility and development of this bacterium, is regulated by the Dif chemotaxis-like pathway. DifA, an MCP homolog, is proposed to mediate signal input to the Dif pathway. However, DifA lacks a prominent periplasmic domain, which in classical chemoreceptors is responsible for signal perception and for initiating transmembrane signaling. To investigate the signaling properties of DifA, we constructed a NarX-DifA (NafA) chimera from the sensory module of Escherichia coli NarX and the signaling module of M. xanthus DifA. We report here the first functional chimeric signal transducer constructed using genes from organisms in two different phylogenetic subdivisions. When expressed in M. xanthus, NafA restored fruiting body formation, EPS production, and S-motility to difA mutants in the presence of nitrate. Studies with various double mutants indicate that NafA requires the downstream Dif proteins to function. We propose that signal inputs to the Dif pathway and transmembrane signaling by DifA are essential for the regulation of EPS production in M. xanthus. Despite the apparent structural differences, DifA appears to share similar transmembrane signaling mechanisms with enteric sensor kinases and chemoreceptors.

DifA, a Methyl-Accepting Chemoreceptor Protein-Like Sensory Protein, Uses a Novel Signaling Mechanism to Regulate Exopolysaccharide Production inMyxococcus xanthus

“…Recent structural studies of the NarX sensory module clearly demonstrated a piston-like displacement in its transmembrane signaling helix or TM2 with and without nitrate (14). Since the NarX-DifA chimera included the entire NarX sensory module, including TM1 and TM2 (47), our previous observation is in agreement with a transmembrane-dependent signaling mechanism by DifA (14,39,47). More definitive and prudent conclusions regarding the various mechanisms of intramolecular signal transmission by DifA require additional experimentation.…”

“…It has been proposed that PE molecules may function as signals to distinguish between self and nonself during vegetative hunting and developmental aggregation (15,26). The dual roles of Dif proteins in S motility and PE taxis may explain why EPS Ϫ dif mutants show no developmental aggregation (5,8,47,48,50); this also underscores the importance of the Dif pathway in the biology of M. xanthus.…”

“…The resulting transformants were plated onto CTT agar containing 60 g/ml Congo red to identify EPS Ϫ colonies, as indicated by the lack of Congo red binding (8). The EPS Ϫ transformants that produced full-length DifA protein were identified by immunoblotting using antiDifA antibodies (47). The region targeted for mutagenesis from these transformants was PCR amplified and sequenced to identify mutations.…”

“…This speculative TM1-to-TM2 signal transmission could involve the short periplasmic region or be mediated by direct interactions between the TMs. The Dif pathway was previously engineered to respond to nitrate by use of a NarX-DifA chimera (47). Recent structural studies of the NarX sensory module clearly demonstrated a piston-like displacement in its transmembrane signaling helix or TM2 with and without nitrate (14).…”

“…More definitive and prudent conclusions regarding the various mechanisms of intramolecular signal transmission by DifA require additional experimentation. DifA and other 44H class MCP transducers (2) have the longest C-terminal signaling domain (10,47,48). It is known that this domain forms a coiled-coil hairpin that folds into a four-helix bundle in a receptor dimer (Fig.…”