The isolation and characterization of Rhizobium loti exopolysaccharide mutants : a thesis presented in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand
PN1 84, a streptomycin resistant derivative of the broad host range Rhizobium
loti strain NZP2037, was shown to be Calcofluor-bright due to the production of a
Calcofluor-binding exopolysaccharide (EPS) with both high and low molecular
weight forms, the low molecular weight form being predominant
Eight Calcofluor-dark EPS mutants (three smooth and five rough) were generated
by Tn5 mutagenesis of PN1 84. Each mutant was shown to carry a single ,
independent Tn5 insertion. Cosmids that complemented the mutation carried by
each of the rough, PN1 84-derived EPS mutants were isolated from a pLAFRl gene
library to NZP2037 by complementation of the Calcofluor-dark phenotype. The
genetic regions identified were shown to be located on the chromosome, and were
not closely linked. The mutants were divided into three (complementation) groups.
While the rough, PN1 84-derived EPS mutants failed to synthesize EPS, the
smooth, PN1 84-derived EPS mutants were found to synthesize an EPS which
failed to bind Calcofluor, and which was shown, by 1 H-NMR spectroscopy, to be
significantly less acetylated than the EPS produced by PN1 84. Furthermore,
PN1 177, one of the smooth, PN1 84-derived EPS mutants, was shown to produce
only a small amount of high molecular weight EPS compared to PN1 84.
All the PN1 84-derived EPS mutants induced the formation of fully effective
(Nod+Fix+) nodules on Lotus pedunculatus, a determinate nodulating host legume,
but, in contrast, induced the formation of ineffective (Nod+Fix-) nodules on
Leucaena leucocephala, an indeterminate nodulating host legume. Each rough,
PN1 84-derived EPS mutant, c arrying its complementing cosmid, was fully
effective on L. leucocephala.
PN4 1 15, a streptomycin resistant derivative of the restricted, effective host range
R . loti strain NZP2213, was shown to be Calcofluor-dark. PN4 1 1 5 was shown to
produce an EPS, which fails to bind Calcofluor, that is acetylated to approximately
the same extent as the EPS produced by PN1 84. Like PN1 1 77, PN4 1 15 was
shown to produce only a small amount of high molecular weight EPS. Examination
of 1 H-NMR spectra of EPS from PN4 1 1 5 and the smooth, PN1 84-derived EPS
mutants suggests that these strains produce an EPS of similar structure, with the
exception of the degree of 0- acetylation.
Three non-mucoid, Calcofluor-bright, EPS mutants were generated by Tn5
mutagenesis of PN4 1 15. Each mutant was shown to carry a single, independent
Tn5 insertion. Cosmids could not be isolated which stably complemented the
mutation carried by each mutant. None of the mutants produced EPS, but all three
mutants produce a Calcofluor-binding EPS, possibly cellulose.
All three PN4 1 15-derived EPS mutants induced the formation of fully effective
nodules on Lotus corniculatus, a determinate nodulating host legume. On L.
leucocephala, PN4 1 15 induced the formation of both small, ineffective, nodular
swellings and large, ineffective, tumour-like structures. Occasionally, a low level
of nitrogen fixation was observed. In contrast, the PN41 15-derived EPS mutants
all induced the formation of only small, ineffective, nodular swellings.
These results, obtained in isogenic Rhizobium backgrounds, support suggestions
that EPS is required for effective nodulation of indeterminate nodulating legumes,
but is not required for effective nodulation of determinate nodulating legumes.