Receptor-mediated exopolysaccharide perception controls bacterial infection.

Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume-rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3 regulates bacterial passage through the plant's epidermal cell layer. Finally, we show that Epr3 expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant-bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.

Serratia entomophila bet gene induction and the impact of glycine betaine accumulation on desiccation tolerance.

AIMS: The genes involved in choline transport and oxidation to glycine betaine in the biopesticidal bacterium Serratia entomophila were characterized, and the potential of osmoprotectants, coupled with increased NaCl concentrations, to improve the desiccation tolerance of this species was investigated. METHODS AND RESULTS: Serratia entomophila carries sequences similar to the Escherichia coli betTIBA genes encoding a choline transporter and dehydrogenase, a betaine aldehyde dehydrogenase and a regulatory protein. Disruption of betA abolished the ability of Ser. entomophila to utilize choline as a carbon source. Quantitative reverse-transcriptase PCR analysis revealed that betA transcription was reduced compared to that of the upstream genes in the operon, and that NaCl and choline induced bet gene expression. Glycine betaine and choline increased the NaCl tolerance of Ser. entomophila, and osmotically preconditioned cultures survived better than control cultures following desiccation and immediately after application to agricultural soil. CONCLUSIONS: Addition of glycine betaine and NaCl to growth medium can greatly enhance the desiccation survival of Ser. entomophila, and its initial survival in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Serratia entomophila is sensitive to desiccation and does not persist under low soil moisture conditions. Techniques described here for enhancing the desiccation survival of Ser. entomophila can be used to improve formulations of this bacterium, and allow its application under a wider range of environmental conditions.

Plasmid-located pathogenicity determinants of Serratia entomophila, the causal agent of amber disease of grass grub, show similarity to the insecticidal toxins of Photorhabdus luminescens.

Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in S. entomophila is required for disease causation and, when introduced into Escherichia coli, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on E. coli and a pADAP-cured strain of S. entomophila was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (sepA, sepB, and sepC) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium Photorhabdus luminescens. Transposon insertions in sepA, sepB, or sepC completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by S. entomophila and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP.

Cloning and overexpression of glycosyltransferases that generate the lipopolysaccharide core of Rhizobium leguminosarum.

The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizobium leguminosarum is more amenable to enzymatic study than that of Escherichia coli because much of it is synthesized from readily available sugar nucleotides. The inner portion of the R. leguminosarum core contains mannose, galactose, and three 3-deoxy-D-manno-octulosonate (Kdo) residues, arranged in the order: lipid A-(Kdo)2-Man-Gal-Kdo-[O antigen]. A mannosyltransferase that uses GDP-mannose and the conserved precursor Kdo2-[4'-32P]lipid IVA (Kadrmas, J. L., Brozek, K. A., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32119-32125) is proposed to represent a key early enzyme in R. leguminosarum core assembly. Conditions for demonstrating efficient galactosyl- and distal Kdo-transferase activities are now described using a coupled assay system that starts with GDP-mannose and Kdo2-[4'-32P]lipid IVA. As predicted, mannose incorporation precedes galactose addition, which in turn precedes distal Kdo transfer. LPS core mutants with Tn5 insertions in the genes encoding the putative galactosyltransferase (lpcA) and the distal Kdo-transferase (lpcB) are shown to be defective in the corresponding in vitro glycosylation of Kdo2-[4'-32P]lipid IVA. We have also discovered the new gene (lpcC) that encodes the mannosyltransferase. The gene is separated by several kilobase pairs from the lpcAB cluster. All three glycosyltransferases are carried on cosmid pIJ1848, which contains at least 20 kilobase pairs of R. leguminosarum DNA. Transfer of pIJ1848 into R. meliloti 1021 results in heterologous expression of all three enzymes, which are not normally present in strain 1021. Expression of the lpc genes individually behind the T7 promoter results in the production of each R. leguminosarum glycosyltransferase in E. coli membranes in a catalytically active form, demonstrating that lpcA, lpcB, and lpcC are structural genes.

Evolution of rhizobia by acquisition of a 500-kb symbiosis island that integrates into a phe-tRNA gene.

Nodulation and nitrogen fixation genes of Mesorhizobium loti are encoded on the chromosome of the bacterium. Nevertheless, there is strong evidence that these genes can be transferred from an inoculant strain to nonsymbiotic mesorhizobia in the field environment. Here we report that the chromosomal symbiotic element of M. loti strain ICMP3153 is transmissible in laboratory matings to at least three genomic species of nonsymbiotic mesorhizobia. The element is 500 kb in size, integrates into a phe-tRNA gene, and encodes an integrase of the phage P4 family just within its left end. The entire phe-tRNA gene is reconstructed at the left end of the element upon integration, whereas the 3' 17 nucleotides of the tRNA gene are present as a direct repeat at the right end. We termed the element a symbiosis island on the basis of its many similarities to pathogenicity islands. It may represent a class of genetic element that contributes to microbial evolution by acquisition.

Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus.

Previously, we found that genetically diverse rhizobia nodulating Lotus corniculatus at a field site devoid of naturalized rhizobia had symbiotic DNA regions identical to those of ICMP3153, the inoculant strain used at the site (J. T. Sullivan, H. N. Patrick, W. L. Lowther, D. B. Scott, and C. W. Ronson, Proc. Natl. Acad. Sci. USA 92:8985-8989, 1995). In this study, we characterized seven nonsymbiotic rhizobial isolates from the rhizosphere of L. corniculatus. These included two from plants at the field site sampled by Sullivan et al. and five from plants at a new field plot adjacent to that site. The isolates did not nodulate Lotus species or hybridize to symbiotic gene probes but did hybridize to genomic DNA probes from Rhizobium loti. Their genetic relationships with symbiotic isolates obtained from the same sites, with inoculant strain ICMP3153, and with R. loti NZP2213T were determined by three methods. Genetic distance estimates based on genomic DNA-DNA hybridization and multilocus enzyme electrophoresis were correlated but were not consistently reflected by 16S rRNA nucleotide sequence divergence. The nonsymbiotic isolates represented four genomic species that were related to R. loti; the diverse symbiotic isolates from the site belonged to one of these species. The inoculant strain ICMP3153 belonged to a fifth genomic species that was more closely related to Rhizobium huakuii. These results support the proposal that nonsymbiotic rhizobia persist in soils in the absence of legumes and acquire symbiotic genes from inoculant strains upon introduction of host legumes.

Nodulating strains of Rhizobium loti arise through chromosomal symbiotic gene transfer in the environment.

Rhizobia were isolated from nodules off a stand of Lotus corniculatus established with a single inoculant strain, ICMP3153, 7 years earlier in an area devoid of naturalized Rhizobium loti. The isolates showed diversity in growth rate, Spe I fingerprint of genomic DNA, and hybridization pattern to genomic DNA probes. The 19% of isolates that grew at the same rate as strain ICMP3153 were the only isolates that had the same fingerprint as strain ICMP3153. Sequencing of part of the 16S rRNA gene of several diverse isolates confirmed that they were not derived from the inoculant strain. Nevertheless, all non-ICMP3153 strains gave EcoRI and Spe I hybridization patterns identical to ICMP3153 when hybridized to nodulation gene cosmids. Hybridization of digests generated by the very rare cutting enzyme Swa I revealed that the symbiotic DNA region (at least 105 kb) was chromosomally integrated in the strains. The results suggest that the diverse strains arose by transfer of chromosomal symbiotic genes from ICMP3153 to nonsymbiotic rhizobia in the environment.

Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression.

The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported. However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions. This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field. The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nif regulon and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule. These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass. The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control. Each treatment was replicated eight times and was conducted at four field sites in Wisconsin. Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9% compared with the yield with the wild-type strain RMBPC and 17.9% over that in the uninoculated control plot at the site where soil nitrogen and organic matter content was lowest. These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season. Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site. At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. We conclude that RMBPC -2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient moisture.

Isolation and characterization of the lantibiotic salivaricin A and its structural gene salA from Streptococcus salivarius 20P3.

A bacteriocin-like inhibitory substance, salivaricin A, was purified from cultures of Streptococcus salivarius 20P3 and was shown by ion spray mass spectrometry to have a molecular mass of 2,315 +/- 1.1 Da. Amino acid composition analysis demonstrated the presence of lanthionine, indicating that salivaricin A may be a member of the lantibiotic class of antibiotic substances. The sequence of eight amino acids at the N terminus of the molecule was determined by Edman degradation, and mixed oligonucleotide probes based on part of this sequence (GSGWIA) were used to detect the salivaricin A structural gene. A 6.2-kb EcoRI fragment of chromosomal DNA from strain 20P3 that hybridized with the probes was cloned, and the hybridizing region was further localized to a 379-bp DraI-AluI fragment. Analysis of the nucleotide sequence of this fragment indicated that salivaricin A is synthesized as a 51-amino-acid prepeptide that is posttranslationally modified and cleaved to give a biologically active 22-residue peptide containing one lanthionine and two beta-methyllanthionine residues. The secondary structure of presalivaricin A was predicted to be similar to that of type A lantibiotics, with a hydrophilic alpha-helical leader sequence and a propeptide region with potential for beta-turn formation and a lack of alpha-helicity. The sequence around the cleavage site of presalivaricin A differed from that of other type A lantibiotics but was similar to that of several bacteriocin-like inhibitory substances produced by lactic acid bacteria.

Identification of a gene linked to Rhizobium meliloti ntrA whose product is homologous to a family to ATP-binding proteins.

The ntrA gene of Rhizobium meliloti has recently been identified and shown to be required for a diverse set of metabolic functions (C. W. Ronson, B. T. Nixon, L. M. Albright, and F. M. Ausubel, J. Bacteriol. 169:2424-2431, 1987). As a result of sequencing the ntrA gene and its flanking regions from R. meliloti, we identified an open reading frame directly upstream of ntrA, ORF1, whose predicted product is homologous to a superfamily of ATP-binding proteins involved in transport, cell division, nodulation, and DNA repair. The homology of ORF1 to this superfamily and its proximity to ntrA led us to investigate its role in symbiosis by mutagenesis and expression studies. We were unable to isolate an insertion mutation in ORF1, suggesting that ORF1 may code for an essential function. We identified the start of transcription for the ntrA gene in vegetative cells and bacteroids and showed that ORF1 and ntrA are transcriptionally unlinked. ORF1 appears to be in an operon with one or more upstream genes.

Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products.

We have sequenced two genes dctB and dctD required for the activation of the C4-dicarboxylate transport structural gene dctA in free-living Rhizobium leguminosarum. The hydropathic profile of the dctB gene product (DctB) suggested that its N-terminal region may be located in the periplasm and its C-terminal region in the cytoplasm. The C-terminal region of DctB was strongly conserved with similar regions of the products of several regulatory genes that may act as environmental sensors, including ntrB, envZ, virA, phoR, cpxA, and phoM. The N-terminal domains of the products of several regulatory genes thought to be transcriptional activators, including ntrC, ompR, virG, phoB and sfrA. In addition, the central and C-terminal regions of DctD were strongly conserved with the products of ntrC and nifA, transcriptional activators that require the alternate sigma factor rpoN (ntrA) as co-activator. The central region of DctD also contained a potential ATP-binding domain. These results are consistent with recent results that show that rpoN product is required for dctA activation, and suggest that DctB plus DctD-mediated transcriptional activation of dctA may be mechanistically similar to NtrB plus NtrC-mediated activation of glnA in E. coli.

Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions.

We report the identification and cloning of an ntrA-like (glnF rpoN) gene of Rhizobium meliloti and show that the R. meliloti ntrA product (NtrA) is required for C4-dicarboxylate transport as well as for nitrate assimilation and symbiotic nitrogen fixation. DNA sequence analysis showed that R. meliloti NtrA is 38% homologous with Klebsiella pneumoniae NtrA. Subcloning and complementation analysis suggested that the R. meliloti ntrA promoter lies within 125 base pairs of the initiation codon and may be constitutively expressed.

Identification and characterization of the Rhizobium meliloti ntrC gene: R. meliloti has separate regulatory pathways for activation of nitrogen fixation genes in free-living and symbiotic cells.

We show here that Rhizobium meliloti, the nitrogen-fixing endosymbiont of alfalfa (Medicago sativa), has a regulatory gene that is structurally homologous to previously characterized ntrC genes in enteric bacteria. DNA sequence analysis showed that R. meliloti ntrC is homologous to previously sequenced ntrC genes from Klebsiella pneumoniae and Bradyrhizobium sp. (Parasponia) and that an ntrB-like gene is situated directly upstream from R. meliloti ntrC. Similar to its counterparts in K. pneumoniae and Escherichia coli, R. meliloti ntrC is expressed when the cells are grown in nitrogen-limiting media. In addition, R. meliloti ntrC is required for growth on media containing nitrate as the sole nitrogen source and for the ex planta transcription of several R. meliloti nif genes. On the other hand, root nodules elicited by R. meliloti ntrC mutants fix nitrogen as well as nodules elicited by wild-type R. meliloti. These latter results indicate that R. meliloti has separate regulatory pathways for activating nif gene expression ex planta and during symbiotic nitrogen fixation.

Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes.

The fixA, fixB, fixC, and fixX genes of Rhizobium meliloti 1021 constitute an operon and are required for nitrogen fixation in alfalfa nodules. DNA homologous to the R. meliloti fixABC genes is present in all other Rhizobium and Bradyrhizobium species examined, but fixABC-homologous sequences were found in only one free-living diazotroph, Azotobacter vinelandii. To determine whether the fixABCX genes share sequence homology with any of the 17 Klebsiella pneumoniae nif genes, we determined the entire nucleotide sequence of the fixA, fixB, fixC, and fixX genes and defined four open reading frames that code for polypeptides of molecular weights 31,146, 37,786, 47,288, and 10,937, respectively. Neither DNA nor amino acid sequence homology to the R. meliloti fixA, -B, -C, and -X genes was found in the K. pneumoniae nif operon. The fixX gene contains a cluster of cysteine residues characteristic of ferredoxins and is highly homologous to an Azotobacter ferredoxin which has been shown to donate electrons to nitrogenase. The fixABC operon contains a promoter region that is highly homologous to other nifA-activated promoters. We also found a duplication of the 5' end of the fixABCX operon; a 250-bp region located 520 bp upstream of the fixABCX promoter bears more than 65% homology to the 5' end of the transcribed region, including the first 32 codons of fixA.

Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC.

We report that the ntrB and ntrC proteins of Bradyrhizobium sp. [Parasponia] strain RP501 share homology with other regulatory proteins. There is extensive conservation of C-terminal regions between products of RP501 ntrB; Klebsiella pneumoniae ntrB; Escherichia coli envZ, cpxA, and phoR; Agrobacterium tumefaciens virA; and, to a lesser extent, E. coli cheA. There is also extensive conservation of N-terminal regions between products of RP501 ntrC; K. pneumoniae ntrC; E. coli ompR, sfrA, phoB, cheY and cheB; Salmonella typhimurium cheB and cheY; Bacillus subtilis spoOA and spoOF; and A. tumefaciens virG. We propose that these regulatory genes comprise two-component regulatory systems that evolved from a common ancestral system that involved transduction of information about the status of the environment by one protein domain (the C-terminal regions conserved among ntrB, envZ, etc.) to a second one (the N-terminal region conserved among ntrC, ompR, etc.). The ntrC-set protein then acts upon a specific responding mechanism, typically as a transcriptional activator but also as an effector of the maturation of outer membrane proteins or as a modulator of the direction of flagella rotation.

Molecular cloning and genetic organization of C4-dicarboxylate transport genes from Rhizobium leguminosarum.

Cosmids containing C4-dicarboxylate transport (dct) genes were identified from a gene bank of Rhizobium leguminosarum DNA made in the broad-host-range vector pLAFR1 by their ability to complement R. trifolii dct mutants. The dct genes were further characterized by subcloning, restriction site mapping, and transposon Tn5 and Tn7 mutageneses. Three dct loci were identified within a 5.5-kilobase region of DNA, in the order dctA-dctB-dctC. The results suggested that dctA encoded a structural component necessary for C4-dicarboxylate transport, whereas dctB and dctC encoded positive regulatory elements, and that dctA was transcribed divergently from dctB and dctC. Expression of dctA and dctC was obtained from vector promoters in some pLAFR1- and pSUP106-based plasmids.

Identification and mobilization by cointegrate formation of a nodulation plasmid in Rhizobium trifolii.

A nodulation plasmid, pRtr-514a, of molecular size 180 megadaltons (Mdal) was identified in Rhizobium trifolii strain NZP514. This plasmid was absent in both spontaneous and heat-cured Nod- derivatives of NZP514, and these strains were unable to induce root hair curling. The ability to nodulate clover was transferred from the wild-type strain to a Nod- derivatives, PN104, with the broad-host-range plasmid R68.45 (39 megadaltons) at a cotransfer frequency of about 4 X 10(-3). Most of the Nod+ transconjugants were resistant to kanamycin, tetracycline, and carbenicillin and had received a plasmid approximately 36 or 70 Mdal larger than pRtr514a but did not contain a plasmid of the size of R68.45, indicating that pRtr-514a was mobilized as a cointegrate plasmid containing either one or possibly two copies of R68.45. Use of these cointegrate-containing strains as donors in further crosses with the Nod- derivative strain PN118 resulted in high-frequency transfer of Nod+ (10(-3) to 10(-4), with cotransfer frequencies with kanamycin of up to 100%. Introduction of R68.45 into a derivative of NZP514 containing the broad-host-range plasmid pJP4 (52 Mdal) resulted in a high frequency of transconjugants carrying a cointegrate plasmid composed of pRtr-514a and pJP4. When used as donors to Nod- derivatives, such strains cotransferred Nod+ with kanamycin plus mercury at a frequency of 67%. The identification of stable cointegrates between pRtr-514a and the broad-host-range plasmids R68.45 and pJP4 should enable several genetic manipulations to be carried out with this nodulation plasmid, including the transfer of the plasmid to most gram-negative bacterial genera.