PssO, a unique extracellular protein important for exopolysaccharide synthesis in Rhizobium leguminosarum bv. trifolii.

Synthesis and secretion of polysaccharides by Gram-negative bacteria are a result of a concerted action of enzymatic and channel-forming proteins localized in different compartments of the cell. The presented work comprises functional characterization of PssO protein encoded within the previously identified, chromosomal exopolysaccharide (EPS) biosynthesis region (Pss-I) of symbiotic bacterium Rhizobium leguminosarum bv. trifolii TA1 (RtTA1). pssO gene localization between pssN and pssP genes encoding proteins engaged in exopolysaccharide synthesis and transport, suggested its role in EPS synthesis and/or secretion. RtTA1 pssO deletion mutant and the PssO protein overproducing strains were constructed. The mutant strain was EPS-deficient, however, this mutation was not complemented. The PssO-overproducing strain was characterized by increase in EPS secretion. Subcellular fractionation, pssO-phoA/lacZ translational fusion analyses and immunolocalisation of PssO on RtTA1 cell surface by electron microscopy demonstrated that PssO is secreted to the extracellular medium and remains attached to the cell. Western blotting analysis revealed the presence of immunologically related proteins within the species R. leguminosarum bv. trifolii, bv. viciae and Rhizobium etli. The secondary structure of PssO-His(6), as determined by FTIR spectroscopy, consists of at least 32% alpha-helical and 12% beta-sheet structures. A putative function of PssO in EPS synthesis and/or transport is discussed in the context of its cellular localization and the phenotypes of the deletion mutant and pssO-overexpressing strain.

Isolation and sequencing of Rhizobium leguminosarum Bv. Trifolii PssN, PssO and PssP genes encoding the proteins involved in polymerization and translocation of exopolysaccharide.

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) that plays an important role in symbiotic interaction with clover plants. The sequence of 6.0-kb DNA fragment located upstream of the previously described prsDEorf3 and pssCDE genes involved in exopolysaccharide biosynthesis revealed three new genes designated pssN, pssO and pssP. The predicted protein product of pssP gene shares a significant homology to members of the membrane-periplasmic auxiliary (MPA1) family, that are involved in polymerization of the repeating subunits of EPS. The putative pssN protein product is highly homologous to the family of the outer membrane auxiliary (OMA) proteins engaged in translocation of polysaccharides in bacteria. The PssO did not reveal homology to the known bacterial proteins, but showed characteristic features of outer membrane proteins, and with PssN and PssP, it might be a part of the system involved in polymerization and translocation of EPS across the bacterial membranes.

Expression of bovine leukemia virus protein p24 in Escherichia coli and its use in the immunoblotting assay.

The gag gene encoded protein, p24 of bovine leukemia virus (BLV), was cloned and expressed as thioredoxin-6xHis-p24 protein in Escherichia coli. The bacterial cells carrying plasmid pT7THis-p24 expressed the protein of 38 kDa that was detected by immunoblotting analysis using anti-p24 monoclonal antibodies and sera from BLV infected cattle and sheep. The purified p24 fusion protein was shown to be sensitive and specific for detection of BLV antibodies in the infected cattle.

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.

The Rhizobium leguminosarum bv. trifolii pssB gene product is an inositol monophosphatase that influences exopolysaccharide synthesis.

The pssB gene of Rhizobium leguminosarum bv. trifolii encodes a protein of 284 amino acids with sequence similarity to eukaryotic inositol monophosphatases. The gene was cloned and overexpressed in Escherichia coli. The purified gene product of pssB showed inositol monophosphatase activity with a Km of 0.23 mM, and a Vmax of 3.27 mumol Pi min-1 (mg protein)-1. Its substrate specificity, Mg+2 requirement, Li+ inhibition, and subunit association (dimerization) were studied and compared to those of other inositol monophosphatases. Western immunoblotting with anti-PssB antibodies showed the presence of PssB in R. leguminosarum bv. trifolii strain TA1 and lack of this protein in the pssB mutant strain Rt12A. The presence of PssB protein in R. leguminosarum bv. trifolii TA1 was correlated with phosphatase activity with myo-inositol 1-phosphate as a substrate. Evidence for a regulatory function of PssB protein in exopolysaccharide (EPS) synthesis is presented. The mutation in pssB caused EPS overproduction, and introduction of pssB into the wild-type TA1 strain reduced EPS synthesis. The changes in the level of EPS production were correlated with a non-nitrogen-fixing phenotype of rhizobia.

Infection of clover by plant growth promoting Pseudomonas fluorescens strain 267 and Rhizobium leguminosarum bv. trifolii studied by mTn5-gusA.

Plant growth promoting Pseudomonas fluorescens strain 267, isolated from soil, produced pseudobactin A, 7-sulfonic acid derivatives of pseudobactin A and several B group vitamins. In coinoculation with Rhizobium leguminosarum bv. trifolii strain 24.1, strain 267 promoted clover growth and enhanced symbiotic nitrogen fixation under controlled conditions. To better understand the beneficial effect of P. fluorescens 267 on clover inoculated with rhizobia, the colonization of clover roots by mTn5-gusA marked bacteria was studied in single and mixed infections under controlled conditions. Histochemical assays combined with light and electron microscopy showed that P. fluorescens 267.4 (i) efficiently colonized clover root surface; (ii) was heterogeneously distributed along the roots without the preference to defined root zone; (iii) formed microcolonies on the surface of clover root epidermis; (iv) penetrated the first layer of the primary root cortex parenchyma and (v) colonized endophytically the inner root tissues of clover.

The pssB gene product of Rhizobium leguminosarum bv. trifolii is homologous to a family of inositol monophosphatases.

The Rhizobium leguminosarum bv. trifolii region encoding pssA and pssB genes was cloned. The pssB gene located upstream of the pssA encoded a 28.36-kDa protein which displayed 97.5% identity with the PssB of R. leguminosarum bv. viciae. Inactivation of the pssB gene by insertion of the lacZ-Gmr cassette resulted in the significant increased production of exopolysaccharide in comparison to the wild-type level. A mutant strain was also defective in nitrogen fixation suggesting a regulatory role of pssB in symbiosis with clover.

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.

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.

Tn1 insertion in Col Ib plasmid.

Eight clones ColIb::Tn1 were isolated by means of transformation of E. coli 921 with DNA ColIb, RP4. Linkage between markers controlling ampicillin-resistance and synthesis of colicine was found in conjugation and transduction experiments.

Restriction enzyme analysis of the plasmid ColIb DNA.

Plasmid ColIb (61.5 Mdal) was digested with restriction enzymes EcoRI and HindIII. The DNA digestion products were separated by electrophoresis on 1.2% agarose gels. There were identified 22 fragments of ColIb DNA generated by the endonuclease EcoRI and 21 fragments produced by HindIII. Molecular weights of the fragments were estimated. The total molecular weight of the fragments generated by EcoRI was 61.42 Mdal and for HindIII fragments 62.79 Mdal.

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.