POSTERS - BLAST X - University of Utah

BLAST X Poster #74
EXAMINATION OF PHOSPHORYLATION IN THE Dif CHEMOTAXIS-LIKE SYSTEM IN
MYXOCOCCUS XANTHUS
Wesley P. Black and Zhaomin Yang
Department of Biological Sciences, Virginia Polytechnic and State University, Life Sciences I,
Washington Street, Blacksburg, VA 24061
Myxococcus xanthus is able to move on surfaces using social (S) gliding motility, which
is powered by the retraction of type IV pili (Tfp). Exopolysaccharides (EPS), also essential for S
motility, have been proposed to function as the anchor and trigger for Tfp retraction in M.
xanthus. EPS production in M. xanthus is regulated by the Dif chemotaxis-like signal
transduction pathway. DifA, DifC and DifE, homologous to methyl-accepting chemotaxis
proteins (MCPs), CheW and CheA respectively, are positive regulators of EPS production. DifD
and DifG, which are respective homologs of CheY and CheC, are negative regulators of EPS
production. It was demonstrated previously that DifD (CheY-like) does not function downstream
of DifE (CheA-like) in the regulation of EPS production.
The purpose of this study was to examine the phosphorylation of heterologously
expressed and purified Dif proteins in vitro. Protein phosphorylation, phosphate transfer and
dephosporylation were monitored using γ- 32 P-ATP. We demonstrate the autophosphorylation of
DifE, which is an atypical CheA-like kinase with an extra domain. Unlike observations in other
systems, the presence of both DifA (MCP-like) and DifC (CheW-like) had inhibitory rather than
stimulatory effects on DifE autophosphorylation. In addition, we show that the phosphate from
DifE-phosphate can be transferred to DifD (CheY-like). Lastly, DifG, which is similar to the CheC
phosphatase, accelerates the dephosphorylation of DifD. These results are consistent with a
model where the DifE kinase positively regulates EPS production, while DifD and DifG function
collectively to divert phosphate away from an unidentified downstream phosphorylation
substrate of DifE.
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