The symbiotic phenotypes of exopolysaccharide-defective mutants of Rhizobium sp. strain TAL1145 do not differ on determinate- and indeterminate-nodulating tree legumes

Abstract

Three classes of exopolysaccharide (EPS) defective mutants were isolated by Tn3Hogus-insertion mutagenesis of Rhizobium sp. strain TALI 145, which nodulates tree legumes. The class I and class 111 mutants produced 10-22% of the EPS produced by TALI145 and appeared partially mucoid while the class II mutants formed small, opaque and non-mucoid colonies. Size-fractionation of the soluble EPSs made by these mutants in the culture supernatant indicated that the class I and the class 111 mutants produced reduced levels of both highand low-molecular-mass EPSs while the class II mutants lacked both these EPSs but produced a small amount of a medium-molecular-mass anthrone-reactive EPS. The succinyl and acetyl substituents observed in the TALI145 EPS were absent in the EPS of the class II mutants. When examined under UV, the class I and class 111 mutants grown on Calcofluor-containing YEM agar showed dim blue fluorescence, compared to the bright blue fluorescence of the wild-type strain, whereas the class II mutants did not fluoresce. While the dim blue fluorescence of the class 111 mutants changed to yellow-green after 10 d, the fluorescence of the class I mutants did not change after prolonged incubation. Unlike the EPS-defective mutants of other rhizobia, these mutants did not show different symbiotic phenotypes on determinate-and indeterminatenodulating tree legumes. The class I and the class 111 mutants formed small ineffective nodules on both types of legumes whereas the class II mutants formed normal nitrogen-fixing nodules on both types. The genes disrupted in the class I and class 111 mutants form a single complementation group while those disrupted in the class II mutants constitute another. All the three classes of EPS-defective mutants were located within a 108 kb region and complemented by two overlapping cosmids.

Regulation of exopolysaccharide synthesis in Rhizobium sp. strain TAL1145 involves an alternative sigma factor gene, rpoH2

“…On YEM agar containing Calcofluor, the morphologies of the RUH102 colonies were similar to those of class III EPS-defective mutants of TAL1145 described previously (Parveen et al, 1997). These morphological features included partially mucoid colonies with reduced amount of EPS and dim blue fluorescence under UV.…”

“…strain TAL1145 forms nitrogen-fixing nodules on the tree-legume Leucaena leucocephala (leucaena). TAL1145 produces large amounts of EPS in medium containing mannitol and the EPS-deficient Tn3Hogus-insertion mutants of TAL1145 are defective in nodulation of leucaena (Parveen et al, 1997). We have also isolated one spontaneous EPS-deficient mutant, which was complemented by a DNA region of TAL1145, that is outside the exo gene cluster.…”

“…Under similar growth conditions TAL1145 yielded a larger peak area for the high-molecular-mass EPS and a smaller peak for the lower-molecular-mass EPS (data not shown). Thus, RUH102 showed a similarity to class III mutants in terms of EPS profile (Parveen et al, 1997). The identities of the polysaccharide components in the Bio-Gel A-5M fractions were not investigated further in the present study.…”

“…Cosmid pUHR223 containing a 24 kb region from strain TAL1145 complemented several EPS-defective mutants of TAL1145 (Parveen et al, 1997). A 7?6 kb HindIII fragment of pUHR223, subcloned in plasmid pUHR290, complemented some of these EPS-defective mutants.…”

“…AY536267). Another 7?9 kb EcoRI fragment of plasmid pUHR223 (Parveen et al, 1997) was gel purified The rpoH2 gene of S. meliloti Rm1021 was amplified by PCR as a 1323 bp fragment, using primers 59-CGCTTAAGCACTCCTTGCG-TAA-39 and 59-AAATGACCTCGCGAAGCCTTAC-39. The amplified fragment was first cloned into the TOPO cloning vector to verify the sequence.…”