Lectin-like glycoprotein PsNLEC-1 is not correctly glycosylated and targeted in boron-deficient pea nodules.

Symbiosome development was studied in pea root nodules from plants growing in the absence of boron (B). Rhizobia released into the host cells of nodules from B-deficient plants developed to abnormal endophytic forms with an altered electrophoretic lipopolysaccharide pattern. Immunostaining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting of nodule homogenates with antibodies that recognize glycoprotein components showed that two previously described lectin-like glycoproteins (PsNLEC-1A and PsNLEC-1B) did not harbor the carbohydrate epitope normally recognized by specific monoclonal antibodies. Material derived from B-deficient nodules, however, still contained three antigenic isoforms with similar electrophoretic mobilities to PsNLEC-1 isoforms A, B, and C. These could be detected following immunoblotting and immunostaining with a specific antiserum originating from the purified PsNLEC protein that had been heterologously expressed in Escherichia coli. Immunogold localization of PsNLEC-1 sugar epitopes in B-deficient nodules showed that they were associated mostly with cytoplasmic vesicles rather than normal localization in the symbiosome compartment of mature infected cells. These results suggest that a modification of the glycosyl-moieties of PsNLEC-1 and an alteration of vesicle targeting occur during the development of pea nodules in the absence of B, and that these changes are associated with the development of aberrant nonfunctional symbiosomes.

PCR use of highly conserved DNA regions for identification of Sinorhizobium meliloti.

A PCR identification method in which four primers that recognize homologous conserved regions in the Sinorhizobium meliloti genome are used was developed and tested. The regions used for identification were the nodbox 4 locus, which is located in one of the symbiotic megaplasmids, and the mucR gene, which is located in the chromosome. The new method was used to establish a collection of S. meliloti strains from polluted soils.

MucR is necessary for galactoglucan production in Sinorhizobium meliloti EFB1.

Sinorhizobium meliloti can produce two types of acidic exopolysaccharides, succinoglycan and galactoglucan, that are interchangeable for infection of alfalfa nodules. Strain SU47 and derivatives produce only succinoglycan, unless it grows under phosphate limitation or carries a mutation in either of two regulatory loci, mucR or expR. It has been proposed that MucR acts as a transcriptional repressor that blocks the expression of the exp genes responsible for galactoglucan production. Strain EFB1 simultaneously produces both exopolysaccharides. Heterologous expression of lacZ transcriptional fusions of the expE promoters has shown that genetic background is more important that promoter sequence for exp gene expression, since expE promoters from both strains are expressed at high level in EFB1 and not in SU47. We have found that mucR is present in mucoid and nonmucoid strains, and in EFB1 differs from SU47 in only one conservative amino acid change. MucR proteins from both strains are interchangeable. An mucR mutant of EFB1 cannot produce galactoglucan and does not express mucS.

Exopolysaccharide II production is regulated by salt in the halotolerant strain Rhizobium meliloti EFB1.

The halotolerant strain Rhizobium meliloti EFB1 modifies the production of extracellular polysaccharides in response to salt. EFB1 colonies grown in the presence of 0.3 M NaCl show a decrease in mucoidy, and in salt-supplemented liquid medium this organism produces 40% less exopolysaccharides. We isolated transposon-induced mutant that, when grown in the absence of salt, had a colony morphology (nonmucoid) similar to the colony morphology of the wild type grown in the presence of salt. Calcofluor fluorescence, proton nuclear magnetic resonance spectroscopy, and genetic analysis of the mutant indicated that galactoglucan, which is not produced under normal conditions by other R. meliloti strains, is produced by strain EFB1 and that production of this compound decreases when the organism is grown in the presence of salt. The mutant was found to be affected in a genetic region highly homologous to genes for galactoglucan production in R. meliloti Rm2011 (expE genes). However, sequence divergence occurs in a putative expE promoter region. A transcriptional fusion of the promoter with lacZ demonstrated that, unlike R. meliloti Rm2011, galactoglucan is produced constitutively by EFB1 and that its expression is reduced 10-fold during exponential growth in the presence of salt.

Ionic Stress and Osmotic Pressure Induce Different Alterations in the Lipopolysaccharide of a Rhizobium meliloti Strain.

A halotolerant strain of Rhizobium meliloti was isolated from nodules of a Melilotus plant growing in a salt marsh in Donana National Park (southwest Spain). This strain, EFB1, is able to grow at NaCl concentrations of up to 500 mM, and no effect on growth is produced by 300 mM NaCl. EFB1 showed alterations on its lipopolysaccharide (LPS) structure that can be related to salt stress: (i) silver-stained electrophoretic profiles showed a different mobility that was dependent on ionic stress but not on osmotic pressure, and (ii) a monoclonal antibody, JIM 40, recognized changes in LPS that were dependent on osmotic stress. Both modifications on LPS may form part of the adaptive mechanism of this bacterium for saline environments.

Rhizobium leguminosarum NodT is related to a family of outer-membrane transport proteins that includes TolC, PrtF, CyaE and AprF.

Cells containing a protein fusion consisting of the Rhizobium leguminosarum bv. viciae nodulation protein, NodT, fused to PhoA, produced alkaline phosphatase activity, indicating that the N terminus of NodT could translocate PhoA across the inner membrane. Cellular fractionation suggested that the NodT::PhoA fusion is targetted to the outer membrane. NodT resembles a family of bacterial outer membrane proteins including TolC, PrtF, CyaE and AprF, which are involved in secretion. By analogy, NodT (together with the inner membrane putative transport proteins NodI and NodJ) is proposed to be involved in the secretion of nodulation factors.

Identification of a Rhizobium leguminosarum gene homologous to nodT but located outside the symbiotic plasmid.

An open reading frame (ORF471), homologous to nodT from Rhizobium leguminosarum, has been found outside the symbiotic plasmid. The deduced amino-acid sequence indicates that the gene product is an outer membrane lipoprotein similar to the NodT proteins. This ORF seems to be widespread among R. leguminosarum bv. viciae strains and its presence in the genetic background of different strains may explain the lack of a nodulation-deficient phenotype found in such strains carrying a nod mutation.

A note on the isolation of psychrotrophic coliform organisms from faecal-polluted environments.

The Simmons citrate salicin (SCS) medium was developed for the enrichment and isolation of presumptive psychrotrophic coliforms from polluted environments. The selectivity and resolution of the medium were tested with pure and mixed laboratory cultures. A procedure for the isolation of psychrotrophic coliforms from polluted environments using a lactose-bile broth and SCS medium is presented.

Seasonal variations of pollution indicators in a wildfowl reserve (Doñana National Park, Spain).

Pollution indicators in Doñana National Park show a cyclic seasonal pattern with maximum level in the autumn and minimum in the spring. While faecal streptococci proved to be the best indicator micro-organisms in this type of salty, alkaline environment, total coliforms gave an overestimation of pollution because of the presence of hydroteluric coliforms.

Simplified methods for the microbiological evaluation of bottled natural mineral waters.

The conventional methods for the microbiological examination of natural mineral water were compared with a simplified procedure. The results indicate that when indicator micro-organisms are present in water, they may not be detected in the simplified method. An alternative procedure, including the determination of Pseudomonas aeruginosa, is suggested.