Siderophore activity in Rhizobium species isolated from different legumes.

Rhizobium strains isolated from nodules of the different legumes including wild-growing plants were examined for their siderophore activity. Fifteen of the 84 screened rhizobial strains were able to grow under conditions of limited iron supply. Nine of them gave orange halos in the assay with Chrom azurol S. Among these strains were Rhizobium sp. (Ononis) and Rhizobium (Genista), producing hydroxamates and phenolates. These compounds could promote the growth of siderophore-negative bacteria on iron-deficient media. The results imply that the hydroxamates from G1 and O1 strains may belong to the monohydroxamate class of siderophores.

Homology of genes for exopolysaccharide synthesis in Rhizobium leguminosarum and effect of cloned exo genes on nodule formation.

A 5.4 kb BamHI fragment of R. leguminosarum bv. trifolii TA1 was found to carry genes involved in exopolysaccharide synthesis (exo genes). This fragment was strongly hybridized to the total DNA from R. l. bv. viciae and bv. phaseoli digested with EcoRI. No homology was found with total DNA of R. meliloti and Rhizobium sp. NGR 234. The exo genes from R. l. bv. trifolii TA1 conjugally introduced into R. l. bv. viciae 1302 considerably affected the symbiosis: the nodules induced on vetch were abortive and did not fix nitrogen. On the other hand, Phaseolus beans infected with R. l. bv. phaseoli harbouring R. l. bv. trifolii exo genes formed the nitrogen-fixing nodules. It can be concluded that additional copies of exo genes introduced into wild type Rhizobium leguminosarum strains can disturb the synthesis of acidic exopolysaccharides and affect symbiosis of the plants forming indeterminate nodules, but do not affect symbiosis of the plants forming the determinate nodules.

Exopolysaccharides of Rhizobium leguminosarum biovar trifolii harbouring cloned exo region.

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) which plays an important role in the development of nitrogen-fixing nodules. Tn5 mutant of R. trifolii 93 defective in EPS production (Exo-) forms ineffective (Fix-) nodules on red clover. This Exo- mutation is complemented by the pARF1368 and pARF25 cosmids isolated from gene bank of Rhizobium trifolii TA1, but the complementation is not correlated with restoration of Fix+ phenotype. Furthermore, these cosmids introduced to wild-type of R. trifolii 24 repress its ability to form nitrogen-fixing nodules. These results might suggest that bacteria with cosmids carrying the exo region form EPS of altered structure. It has been shown by 1H-n.m.r. that exopolysaccharides produced by R. trifolii 93pARF-1368 and 93pARF25 contain less non-carbohydrate residues (acetyl, pyruvyl and 3-hydroxybutanoyl) than the wild type EPS. These data suggest that the biological activity of the exopolysaccharide of R. trifolii depends on the contents of the non-carbohydrate substitutions.

Biological activity of rhizobial siderophore.

Non-nodulating mutant of Rhizobium leguminosarum biovar trifolli produces the phenolate type of siderophore consisting of 2,3-dihydroxybenzoic acid and threonine. The activity of this compound against the various bacteria was tested. Only, the growth of R. leguminosarum strains was stimulated by siderophore. The other species of Rhizobium, especially R. meliloti, were sensitive to this agent. The growth of R. meliloti was also inhibited by agrobactin and pseudobactin. This effect was reversed by ferric iron.

The region for exopolysaccharide synthesis in Rhizobium leguminosarum biovar trifolii is located on the non-symbiotic plasmid.

An Exo- mutant of Rhizobium leguminosarum biovar trifolii was isolated which did not produce acidic exopolysaccharide and induced defective, non-fixing nodules on clover plants. The nodules were defective at a late stage of development, they contained infection threads and bacteria were released into the host cells. Cosmid pARF136 capable of complementing the Exo- mutation was isolated from a cosmid bank made from total R. trifolii DNA. Hybridization between DNA of pARF136 and plasmids of R. trifolii strains separated by Eckhardt's technique suggested that the exo locus is located on a 300 kb megaplasmid, and nodDABC and nifKDH genes are located on another 180 kb pSym plasmid. A 5.4 kb BamH1 fragment of the recombinant cosmid pARF136 was able to restore exopolysaccharide synthesis in Exo- mutant of R. trifolii 93 but it did not complement the symbiotic defect.

Siderophore containing 2,3-dihydroxybenzoic acid and threonine formed by Rhizobium trifolli.

An iron-binding compound was isolated from ethyl acetate extract of culture supernatant fluid of Rhizobium trifolii AR6 and was purified by iron-exchange chromatography. The compound was characterized by UV and IR. It contained 2,3-dihydroxy-benzoic acid and threonine and was accumulated during stationary phase of growth in iron-deficient media. Synthesis of the siderophore was repressed by FeCl3. In iron limited medium the compound promoted growth of R. trifolii strains.

Characterization of the in vitro constructed plasmid composed of the nif gene cluster of Lignobacter and RP4.

The nitrogen fixation of Lignobacter K17 is plasmid mediated. Nif plasmid was transferred from Lignobacter to other bacterial species and the transposon Tn9 was inserted into it. The molecular weight of this plasmid designated pUCS101, is of 19.8 Mdal. In this study we constructed in vitro a hybrid plasmid (pUCS110) by ligating HindIII digests of pUCS101 nif:: Tn9 and of RP4. Next it was proved that pUCS110 is able to complement the total deletion of the nif region in Klebsiella pneumoniae. The 50 Mdal plasmid pUCS110 was not maintained stably in Escherichia coli recA+ as in E. coli recA-. After being transferred to K. pneumoniae, pUCS110 showed a tendency to generate plasmids of various size from 2.8 to 78 Mdal. Bacteria harbouring plasmids of various size classes were more resistant to chloramphenicol than K. pneumoniae (pUCS110). Altered cleavage patterns were found in derivatives of pUCS110. The obtained results suggest that translocation of the transposon Tn9 can be responsible for the instability of pUCS110.

Structural studies of the lipopolysaccharide from Salmonella typhimurium 902 (ColIb drd2).

Salmonella typhimurium infected with the plasmid ColIb drd2 gave rise to changes in the composition of the bacterial lipopolysaccharide. Bacteria carrying the wild-type ColIb, the revertant of drd2 to the wild type, or the noncolicinogenic strain resulting from the elimination of ColIb drd2, showed no changes in the sugar composition of the lipopolysaccharide. The structure of the O-specific side chains of the lipopolysaccharide produced by S. typhimurium 902, infected with derepressed ColIb mutants has been investigated. As a result of these studies, it is proposed that the O-specific side chains are composed of chemical repeating units with the following structure: (formula: see text).

Detection of small cryptic plasmids in Salmonella typhimurium strain LT2.

Small cryptic plasmids of molecular weights ranging from 1 to 3 Mdal were detected by electron microscopy in Salmonella typhimurium strain LT2 (ColIb). They were divided into different size classes. Two of the cryptic plasmids were transferred simultaneously with ColIb to Escherichia coli.

Cell wall lipopolysaccharide response to the ColIb plasmid mutants.

Mutants of ColIb plasmid affected the synthesis of O-side chains of lipopolysaccharides (LPS) in Salmonella. The plasmid srd 25 (defective in colicin synthesis) caused a significant decline of rhamnose and mannose content and lack of abequose in LPS of S. typhimurium. The number of repeating units in O-side chains was decreased after the indroduction of srd 25. Cultures of S. typhimurium and S. enteritidis harboring drd2 (derepressed in colicin production) polymerised dideoxyhexose-defective O-side chains i.e. deprived of abequose and tyvelose, respectively. In dideoxyhexoseless S. meleagridis the content of rhamnose and mannose were reduced. The information for the alterations of Salmonella LPS was contained in the plasmid genome. In the wild-type plasmids the genes controlling the O-antigen changes were not expressed.

Plasmid-determined alterations of Salmonella typhimurium lipopolysaccharides.

Salmonella typhimurium Rc902 infected with derepressed ColIb mutants gave rise to changes in the composition of bacterial lipopolysaccharides (LPS). Bacteria carrying ColIbdrd7, derepressed in transfer, exhibited a marked decrease in the content of all 0-side-chain sugars of LPS. Similar effect were found upon the introduction of R64-11, also derepressed in transfer. In LPS of S. typhimurium containing ColIbdrd2, derepressed in colicin synthesis, a decrease of abequose content associated with an increase of glucose level was observed. Bacteria carrying the wild-type ColIb, the revertant of a drd mutant to the wild type, or the non colicinogenic strain resulting from the elimination of ColIbdrd2, showed no changes in the sugar composition of LPS.