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Zusammenfassung:
Myxococcus xanthus arranges into two morphologically distinct biofilms
depending on its nutritional status, i.e., coordinately spreading
colonies in the presence of nutrients and spore-filled fruiting bodies
in the absence of nutrients. A secreted polysaccharide, referred to as
exopolysaccharide (EPS), is a structural component of both biofilms and
is also important for type IV pilus-dependent motility and fruiting body
formation. Here, we characterize the biosynthetic machinery responsible
for EPS biosynthesis using bioinformatics, genetics, heterologous
expression, and biochemical experiments. We show that this machinery
constitutes a Wzx/Wzy-dependent pathway dedicated to EPS biosynthesis.
Our data support that EpsZ (MXAN_7415) is the polyisoprenyl-phosphate
hexose-1-phosphate transferase responsible for the initiation of the
repeat unit synthesis. Heterologous expression experiments support that
EpsZ has galactose-1-P transferase activity. Moreover, MXAN_7416,
renamed Wzx(EPS), and MXAN_7442, renamed Wzy(EPS), are the Wzx flippase
and Wzy polymerase responsible for translocation and polymerization of
the EPS repeat unit, respectively. In this pathway, EpsV (MXAN_7421)
also is the polysaccharide copolymerase and EpsY (MXAN_7417) the outer
membrane polysaccharide export (OPX) protein. Mutants with single
in-frame deletions in the five corresponding genes had defects in type
IV pilus-dependent motility and a conditional defect in fruiting body
formation. Furthermore, all five mutants were deficient in type IV pilus
formation, and genetic analyses suggest that EPS and/or the EPS
biosynthetic machinery stimulates type IV pilus extension. Additionally,
we identify a polysaccharide biosynthesis gene cluster, which together
with an orphan gene encoding an OPX protein make up a complete
Wzx/Wzy-dependent pathway for synthesis of an unknown polysaccharide.
IMPORTANCE The secreted polysaccharide referred to as exopolysaccharide
(EPS) has important functions in the social life cycle of M. xanthus;
however, little is known about how EPS is synthesized. Here, we
characterized the EPS biosynthetic machinery and showed that it makes up
a Wzx/Wzy-dependent pathway for polysaccharide biosynthesis. Mutants
lacking a component of this pathway had reduced type IV pilus-dependent
motility and a conditional defect in development. These analyses also
suggest that EPS and/or the EPS biosynthetic machinery is important for
type IV pilus formation.
