Analysis of pss genes of Rhizobium leguminosarum required for exopolysaccharide synthesis and nodulation of peas: their primary structure and their interaction with psi and other nodulation genes

Summary Strains of Rhizobium leguminosarum (R. l.) biovar viciae containing pss mutations fail to make the acidic exopolysaccharides (EPS) and are unable to nodulate peas. It was found that they also failed to nodulate Vicia hirsuta, another host of this biovar. When peas were co-inoculated with pss mutant derivatives of a strain of R.l. bv viciae containing a sym plasmid plus a cured strain lacking a sym plasmid (and which is thus Nod^-, but for different reasons) but which makes the acidic EPS, normal numbers of nodules were formed, the majority of which failed to fix nitrogen (the occasional Fix⁺ nodules were pressumably induced by strains that arose as a result of genetic exchange between cells of the two inoculants in the rhizosphere). Bacteria from the Fix^- nodules contained, exclusively, the strain lacking its sym plasmid. When pss mutant strains were co-inoculated with a Nod^- strain with a mutation in the regulatory gene nodD (which is on the sym plasmid pRL1JI), normal numbers of Fix⁺ nodules were formed, all of which were occupiced solely by the nodD mutant strain. Since a mutation in nodD abolishes activation of other nod genes required for early stages of infection, these nod genes appear to be dispensable for subsequent stages in nodule development. Recombinant plasmids, containing cloned pss genes, overcame the inhibitory effects of psi, a gene which when cloned in the plasmid vector pKT230, inhibits both EPS production and nodulation ability. Determination of the sequence of the pss DNA showed that one, or perhaps two, genes are required for correcting strains that either carry pss mutations or contain multi-copy psi. The predicted polypeptide product of one of the pss genes had a hydrophobic aminoterminal region, suggesting that it may be located in the membrane. Since the psi gene product may also be associated with the bacterial membrane, the products of psi and pss may interact with each other.     

Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide.

In eukaryotes, tyrosine protein phosphorylation has been studied extensively, while in bacteria, it is considered rare and is poorly defined. We demonstrate that Escherichia coli possesses a gene, etk, encoding an inner membrane protein that catalyses tyrosine autophosphorylation and phosphorylation of a synthetic co-polymer poly(Glu:Tyr). This protein tyrosine kinase (PTK) was termed Ep85 or Etk. All the E.coli strains examined possessed etk; however, only a subset of pathogenic strains expressed it. Etk is homologous to several bacterial proteins including the Ptk protein of Acinetobacter johnsonii, which is the only other known prokaryotic PTK. Other Etk homologues are AmsA of the plant pathogen Erwinia amylovora and Orf6 of the human pathogen Klebsiella pneumoniae. These proteins are involved in the production of exopolysaccharide (EPS) required for virulence. We demonstrated that like Etk, AmsA and probably also Orf6 are PTKs. Taken together, these findings suggest that tyrosine protein phosphorylation in prokaryotes is more common than was appreciated previously, and that Etk and its homologues define a distinct protein family of prokaryotic membrane-associated PTKs involved in EPS production and virulence. These prokaryotic PTKs may serve as a new target for the development of new antibiotics.     

The Rhizobium leguminosarum prsDE genes are required for secretion of several proteins, some of which influence nodulation, symbiotic nitrogen fixation and exopolysaccharide modification

NodO is a secreted protein from Rhizobium leguminosarum bv. viciae with a role in signalling during legume nodulation. A Tn5-induced mutant was identified that was defective in NodO secretion. As predicted, the secretion defect decreased pea and vetch nodulation but only when the nodE gene was also mutated. This confirms earlier observations that NodO plays a particularly important role in nodulation when Nod factors carrying C18:1 (but not C18:4) acyl groups are the primary signalling molecules. In addition to NodO secretion and nodulation, the secretion mutant had a number of other characteristics. Several additional proteins including at least three Ca2+-binding proteins were not secreted by the mutant and this is thought to have caused the pleiotropic phenotype. The nodules formed by the secretion mutant were unable to fix nitrogen efficiently; this was not due to a defect in invasion because the nodule structures appeared normal and nodule cells contained many bacteroids. The mutant formed sticky colonies and viscous liquid cultures; analysis of the acidic exopolysaccharide revealed a decrease in the ratio of reducing sugars to total sugar content, indicating a longer chain length. The use of a plate assay showed that the mutant was defective in an extracellular glycanase activity. DNA sequencing identified the prsDE genes, which are homologous to genes encoding protease export systems in Erwinia chrysanthemi and Pseudomonas aeruginosa. An endoglycanase (Egl) from Azorhizobium caulinodans may be secreted from R. leguminosarum bv. viciae in a prsD-dependent manner. We conclude that the prsDE genes encode a Type I secretion complex that is required for the secretion of NodO, a glycanase and probably a number of other proteins, at least one of which is necessary for symbiotic nitrogen fixation.     

Rhizobium meliloti exoG and exoJ mutations affect the exoX-exoY system for modulation of exopolysaccharide production.

R. meliloti Rm1021 normally produces an acidic Calcofluor-binding exopolysaccharide, called succinoglycan or EPS I, which is required for successful nodulation of alfalfa by this strain. At least 13 loci affecting production of EPS I were previously mapped to a cluster on the second of two symbiotic megaplasmids in Rm1021, pRmeSU47b. A putative regulatory region was originally defined by the exoG and exoJ mutations. exoG and exoJ mutants produced less exopolysaccharide than wild-type strains and induced nitrogen-fixing nodules on alfalfa with reduced efficiency compared with the wild type. These mutants appeared to produce only a low-molecular-weight form of EPS I. Mutations called exoX cause an increase in exopolysaccharide production and map in the same region as the exoG and exoJ mutations. The DNA sequence of this region reveals that it contains two open reading frames, called exoX and exoY, which have homologs in other Rhizobium species. Interestingly, the exoG insertion mutations fall in an intergenic region and may affect the expression of exoX or exoY. The exoJ mutation falls in the 3' portion of the exoX open reading frame and is probably an allele of exoX that results in altered function. exoG and exoJ mutations limit EPS I production in the presence of exoR95 or exoS96 mutations, which cause overproduction of EPS I. Gene regulation studies suggest that ExoX and ExoY constitute a system that modulates exopolysaccharide synthesis at a posttranslational level. The deduced sequence of ExoY is homologous to a protein required for an early step in xanthan gum biosynthesis, further suggesting that the modulatory system may affect the exopolysaccharide biosynthetic apparatus.     

The REP2 repeats of the genome of Neisseria meningitidis are associated with genes coordinately regulated during bacterial cell interaction

Interaction with host cells is essential in meningococcal pathogenesis especially at the blood-brain barrier. This step is likely to involve a common regulatory pathway allowing coordinate regulation of genes necessary for the interaction with endothelial cells. The analysis of the genomic sequence of Neisseria meningitidis Z2491 revealed the presence of many repeats. One of these, designated REP2, contains a -24/-12 type promoter and a ribosome binding site 5 to 13 bp before an ATG. In addition most of these REP2 sequences are located immediately upstream of an ORF. Among these REP2-associated genes are pilC1 and crgA, described as being involved in steps essential for the interaction of N. meningitidis with host cells. Furthermore, the REP2 sequences located upstream of pilC1 and crgA correspond to the previously identified promoters known to be induced during the initial localized adhesion of N. meningitidis with human cells. This characteristic led us to hypothesize that at least some of the REP2-associated genes were upregulated under the same circumstances as pilC1 and crgA. Quantitative PCR in real time demonstrated that the expression of 14 out of 16 REP2-associated genes were upregulated during the initial localized adhesion of N. meningitidis. Taken together, these data suggest that these repeats control a set of genes necessary for the efficient interaction of this pathogen with host cells. Subsequent mutational analysis was performed to address the role of these genes during meningococcus-cell interaction.     

The rkpGHI and -J genes are involved in capsular polysaccharide production by Rhizobium meliloti.

The first complementation unit of the fix-23 region of Rhizobium meliloti, which comprises six genes (rkpAB-CDEF) exhibiting similarity to fatty acid synthase genes, is required for the production of a novel type of capsular polysaccharide that is involved in root nodule development and structurally analogous to group II K antigens found in Escherichia coli (G. Petrovics, P. Putnoky, R. Reuhs, J. Kim, T. A. Thorp, K. D. Noel, R. W. Carlson, and A. Kondorosi, Mol. Microbiol. 8:1083-1094, 1993; B. L. Reuhs, R. W. Carlson, and J. S. Kim, J. Bacteriol. 175:3570-3580, 1993). Here we present the nucleotide sequence for the other three complementation units of the fix-23 locus, revealing the presence of four additional open reading frames assigned to genes rkpGHI and -J. The putative RkpG protein shares similarity with acyltransferases, RkpH is homologous to short-chain alcohol dehydrogenases, and RkpJ shows significant sequence identity with bacterial polysaccharide transport proteins, such as KpsS of E. coli. No significant homology was found for RkpI. Biochemical and immunological analysis of Tn5 derivatives for each gene demonstrated partial or complete loss of capsular polysaccharides from the cell surface; on this basis, we suggest that all genes in the fix-23 region are required for K-antigen synthesis or transport.     

PilC of pathogenic Neisseria is associated with the bacterial cell surface

Adherence of pathogenic Neisseria to target host cells is mediated by pili. PilC1 and PilC2 are two high-molecular-weight proteins involved in pilus assembly and cellular adherence functions of the pili. Inactivation of pilC1 or pilC2 in N. meningitidis resulted in clones that expressed the same number of pili as the parent, contained no alterations in pilE and showed no detectable differences in PilE glycosylation. However, the PilC2+ pilC1- mutant showed much reduced adherence to target cells, indicating that production of PilC1 is essential for pilus-mediated adherence. To study further the functional differences between the meningococcal pilC genes, we determined the complete nucleotide sequence of pilC1 and pilC2 of N. meningitidis. Alignment of six PilC sequences demonstrated that PilC is composed of both conserved and variable regions. By immunogold labelling of bacterial sections we showed that PilC is present in the membranes of both piliated and non-piliated bacteria. Further, we demonstrated that PilC is associated with the bacterial cell surface.     

The Rhizobium meliloti exoK gene and prsD/prsE/exsH genes are components of independent degradative pathways which contribute to production of low-molecular-weight succinoglycan

When grown on medium supplemented with the succinoglycan-binding dye, Calcofluor, and visualized under UV light, colonies of Rhizobium meliloti (Sinorhizobium meliloti) exoK mutants produce a fluorescent halo with a delayed onset relative to wild-type colonies. By conducting transposon mutagenesis of exoK mutants of R. meliloti and screening for colonies with even more severe delays in production of these fluorescent halos, we identified three genes, designated prsD, prsE, and exsH, which are required for the eventual production of fluorescent halos by exoK colonies. Nucleotide sequence indicates that the prsD and prsE genes encode homologues of ABC transporters and membrane fusion proteins of Type I secretion systems, respectively, whereas exsH encodes a homologue of endo-1,3-1,4-beta-glycanases with glycine-rich nonameric repeats typical of proteins secreted by Type I secretion systems. The exoK gene and the prsD/prsE/exsH genes were shown to be components of independent pathways for production of extracellular succinoglycan degrading activities and for production of low-molecular-weight succinoglycan by R. meliloti. Based on these results, we propose that ExsH is a succinoglycan depolymerase secreted by a Type I secretion system composed of PrsD and PrsE, and that the ExsH and ExoK glycanases contribute to production of low-molecular-weight succinoglycan.     

Wnt family proteins are secreted and associated with the cell surface.

Members of the Wnt gene family are proposed to function in both normal development and differentiation as well as in mammary tumorigenesis. To understand the function of Wnt proteins in these two processes, we present here a biochemical characterization of seven Wnt family members. For these studies, AtT-20 cells, a neuroendocrine cell line previously shown to efficiently process and secrete Wnt-1, was transfected with expression vectors encoding Wnt family members. All of the newly characterized Wnt proteins are glycosylated, secreted proteins that are tightly associated with the cell surface or extracellular matrix. We have also identified native Wnt proteins in retinoic acid-treated P19 embryonal carcinoma cells, and they exhibit the same biochemical characteristics as the recombinant proteins. These data suggest that Wnt family members function in cell to cell signaling in a fashion similar to Wnt-1.     

Polymorphisms of DNA repair genes are associated with renal cell carcinoma

DNA repair gene alterations have been shown to cause a reduction in DNA repair capacity and may influence an individual's susceptibility to carcinogenesis. Single nucleotide polymorphisms (SNPs) of DNA repair genes have been shown to cause a reduction in repair activity. We hypothesized that SNPs of DNA repair genes may be a risk factor for renal cell carcinoma (RCC). To test this hypothesis, DNA samples from 112 cases of renal cell cancer and healthy controls (n=180) were analyzed by PCR-RFLP to determine the genotypic frequency of six different polymorphic loci on five DNA repair genes (XRCC1, XPC, ERCC1, XRCC3, and XRCC7). The chi(2) test was applied to compare the genotype frequency between patients and controls. We found that the frequency of 399Gln variant at XRCC1 Arg399Gln was significantly higher in RCC cases than in controls (OR=2.83, 95%CI=1.24-6.49, P=0.01). The frequency of T-A haplotype of XRCC1 194 Trp and XRCC1 399Gln was significantly higher in RCC than controls. No differences in genotypes were observed at the other sites. This is the first report on SNPs of DNA repair genes in renal cell carcinoma that suggests XRCC1 399Gln polymorphism may be a risk factor for RCC. Our present data suggest that the XRCC1 399Gln allele may be linked to susceptibility for RCC.     

Temperature and pH affect the production of bacterial biofilm

The effect of different cultivation temperatures (30 and 37 °C) and pH of the media (5.5, 7.5, 8.5) on the biofilm production was compared in Pseudomonas aeruginosa, Klebsiella pneumoniae, and Vibrio cholerae non-O1 and O1 using the crystal-violet test for estimation of quantitative production of the biofilm. Decrease (46.4–98.4 %) in the biofilm production was observed at 37 °C in 8 of the tested strains (P. aeruginosa three strains, K pneumoniae two, V. cholerae non-O1 two, and V. cholerae O1 one strain) compared with the production at 30 °C. On the other hand, five strains (P. aeruginosa 1, K. pneumoniae 3, V. cholerae non-O1 1) exhibited under these conditions a higher biofilm production (103–143 %). However, this difference was not significant (p = 0.196). Increased pH lead to a higher biofilm production using all media tested. In P. aeruginosa the biofilm production at pH 8.5 was 139–244 %, at pH 7.5 136–164 % in comparison with pH 5.5. Similarly, in K. pneumoniae the biofilm production increased to 151–319 % at pH 8.5 while with the drop of pH to 7.5 the biofilm production was 113–177 % compared with pH 5.5. In V. cholerae non-O1 and O1 the biofilm production reached 204–329 % at pH 8.5, and 123–316 % at pH 7.5 (compared with the production at pH 5.5). An increase in biofilm production represented an average of 169 % (p = 0.001) at pH change from 5.5 to 7.5, with the rise of pH from 5.5 to 8.5 caused an average difference of 229 % (p = 0.001).     

Two distinct proteins are associated with tetrameric acetylcholinesterase on the cell surface

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.     

The Tuber borchii fruiting body-specific protein TBF-1, a novel lectin which interacts with associated Rhizobium species

Lectins, proteins that are able to bind carbohydrate structures, are typically involved in cell recognition mechanisms. We demonstrate here that TBF-1, the main soluble protein in the Tuber borchii Vittad. fruiting body, is a phase-specific lectin that is able selectively to bind the exopolysaccharides produced by ascoma-associated Rhizobium spp. Characterization of TBF-1 was performed using both the protein purified from the truffles and the recombinant protein overexpressed in Escherichia coli. The two proteins exhibit the same hemagglutination activity toward rabbit red blood cells and the same sugar binding specificity. The discovery of lectin activity for TBF-1 led us to propose revising the protein name to 'T. borchii fruiting body lectin 1' with the acronym TBFL-1.     

Extracellular glycanases of Rhizobium leguminosarum are activated on the cell surface by an exopolysaccharide-related component

Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS(+) strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn of R. leguminosarum. This indicates that the surface of A. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.     

Altered gene products are associated with activation of cellular rasK genes in human lung and colon carcinomas

Two lung and two colon carcinoma cell lines of human origin, which contained the same activated ras^K transforming gene, expressed abnormal species of p21 that were distinct from the p21 proteins expressed in normal human cells and other human carcinomas. The abnormal species of p21 expressed by three of these cell lines were indistinguishable from each other, but differed from the abnormal p21 expressed by one lung carcinoma cell line. NIH cells transformed by DNAs of these carcinomas expressed the same abnormal p21 species, indicating that these abnormal proteins were encoded by the activated ras^K genes detected by transfection. These results indicate that transforming activity of ras^K genes in human lung and colon carcinoma cell lines is activated by mutations which alter the structure of their gene products, and that activation of ras^K genes can result from different molecular alterations in different individual neoplasms.