Influence of phosphate and ammonia on the growth, exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii TA1 with clover (Trifolium pratense).

The Rhizobium-legume interaction is sensitive to a number of environmental factors, among which phosphate (Pi) and ammonium availability are the most important. We investigated the effect of Pi and ammonia concentration on exopolysaccharide production and symbiosis Trifolium pratense with Rhizobium leguminosarum bv. trifolii TA1 (RtTA1). The optimal Pi concentration in the bacterial growth medium for RtTA1 growth and exopolysaccharide production was in the range from 0.9 mM to 8.1 mM. Independently of Pi concentration, ammonium (NH4Cl) concentration above 8.1 mM in the culture medium significantly decreased EPS production, indicating a regulatory role of this nutrient on the EPS production in the RtTA1 strain. Pi availability has a beneficial effect on both partners of symbiosis. Pi concentration in the plant medium in the range from 1.7 mM to 5 mM was optimal for nodule formation, nodule occupancy and nitrogen fixation ability. Despite of T. pratense cv. Bryza tolerance on high Pi concentration, 20 mM Pi occurs to be nearly phytotoxic, which negatively affects almost all symbiotic parameters. Large amounts of starch were accumulated in the nodules formed by clover grown on medium containing high Pi concentration.

Construction of improved vectors and cassettes containing gusA and antibiotic resistance genes for studies of transcriptional activity and bacterial localization.

Broad-host-range, conjugative vectors with a constitutively expressed gusA gene combined with different antibiotic resistance (tetracycline, gentamicin, kanamycin) genes have been constructed. These plasmids are designed for tracking Gram-negative bacterial strains without the risk of random mutagenesis. We also constructed a set of cassettes containing a promoterless gusA gene linked with different antibiotic resistance genes for making transcriptional fusions and for cassette mutagenesis. New plasmids and cassettes can be useful tools for studying gene expression, interaction of bacteria with plants and monitoring bacteria in the environment.

Molecular characterization of pssCDE genes of Rhizobium leguminosarum bv. trifolii strain TA1: pssD mutant is affected in exopolysaccharide synthesis and endocytosis of bacteria.

We have identified the three genes pssCDE in Rhizobium leguminosarum bv. trifolii TA1. Even though they were almost identical to earlier identified pssCDE genes of R. leguminosarum, they differed in gene lengths and gene overlaps. The predicted gene products of pssCDE genes shared significant homology to prokaryotic glycosyl transferases involved in exopolysaccharide synthesis. The Tn5 insertion in pssD created the nonmucoid mutant that induced non-nitrogen-fixing nodules. The microscopic analysis of the nodules, induced on Trifolium pratense by the pssD133 mutant, showed abnormally enlarged infection threads densely packed with bacteria, which were released from the infection threads in an unusual way. The symbiosomes were observed very rarely and the nodule remained almost empty. Symbiotic phenotype of the pssD133 suggested a correlation between this mutation and defective endocytosis of bacteria into nodule cells.

Molecular characterization and symbiotic importance of prsD gene of Rhizobium leguminosarum bv. trifolii TA1.

The prsD, prsE and orf3 genes of Rhizobium leguminosarum bv. trifolii strain TA1 encode the proteins which are significantly related to the family of bacterial ABC transporters type I secretion systems. The prsD:Km(r) mutant of strain TA1 induced non-nitrogen-fixing nodules on Trifolium pratense. Microscopic analysis of the nodules induced by prsD mutant did not reveal major abberations in the bacteroid appearance. The exopolysaccharide of prsD mutant was produced in increased amount and its level of polymerization was changed. SDS/PAGE of the proteins from the culture supernatants showed a lack of the 47-kDa protein in the culture of prsD mutant. Thus, PrsD may play a role in the export of this protein.