Molecular analysis of lipopolysaccharide and membrane associated proteins in Rhizobium leguminosarum.
This study describes the use of monoclonal antibodies to investigate
molecular components of the Rhizobium cell surface that might be important for
symbiotic interactions with the host legume. Components that have been identified
include lipopolysaccharide and both membrane-associated and secreted proteins.
Differences were observed in the structure and antigenicity of
lipopolysaccharide (LPS) from free-living rhizobia compared with that of
endosymbiotic bacteroids. Culture pH, oxygen concentration and carbon source
were all found to be important factors that could affect the expression of LPS
antigens within the nodule. Mutants with altered LPS demonstrated that complete
LPS structures are necessary for effective symbiosis.
Monoclonal antibody nM 25 identified a protease-sensitive epitope that
appeared to be attached to a particular species of LPS macromolecule (identified
by MAC 114 antibody). Immunocytochemical localization studies of pea nodule
sections revealed that JIM 25 antigen, present on the cell surface, was expressed in
infection threads but its expression was low in the symbiotic zone containing
mature bacteroids. A 38kDa secreted protein was identified by JIM 24 antibody.
Fractionation of nodule extracts by differential centrifugation suggested that the
protein was present in the peribacteroid space.
A 55kDa membrane protein recognized by MAC 115 antibody proved to be
a species-specific marker for R. leguminosarum. The structural gene(s) for this
protein are encoded on a 2.8kb EcoRI fragment. Localised mutagenesis of this
DNA region with the transposon TnPhoA (which carries a promoter-less gene for
alkaline phosphatase) provided evidence that the 55kDa protein was' membraneassociated.
However, attempts to "marker-exchange" the transposon-induced
mutations from cosmid DNA into the Rhizobium genome were unsuccessful,
suggesting that the 55kDa protein is essential for growth of free-living rhizobia.
In a DNA region adjoining this 2.8kb EcoRI fragment that encodes the
55kDa protein, a new gene has been described, termed muc. When present on a
cosmid the muc gene from R. leguminosarum conferred non-mucoid colony
morphology on R. meliloti strain B287. "Marker-exchange" of muc::Tn5 mutations
from the plasmid to R. ieguminosarum 8002 (bv. phaseo/i) resulted in derivatives
that had lost the ability to nodulate Phaseolus beans. However, marker-exchange
into R. leguminosarum B556 (bv. viciae) resulted in mutants that showed no
abnormal symbiotic phenotypes on peas.
The cosmid carrying the muc and 55kDa protein determinants (pIJ1639)
was subjected to saturation transposon mutagenesis with TnPhoA This study
revealed several new genes that probably encode membrane-associated or secreted
proteins. In some cases gene transcription was dependent on the presence of
hesperitin [which is known to be an activator of Rhizobium nodulation (nod)
genes]. A 4.6kb EcoRI fragment adjacent to the 2.8kb fragment described above
was also found to encode essential functions that prevented the construction of
chromosomal mutants by marker-exchange.