The degradation of alpha-quaternary nonylphenol isomers by Sphingomonas sp. strain TTNP3 involves a type II ipso-substitution mechanism.

The degradation of radiolabeled 4(3',5'-dimethyl-3'-heptyl)-phenol [nonylphenol (NP)] was tested with resting cells of Sphingomonas sp. strain TTNP3. Concomitantly to the degradation of NP, a metabolite identified as hydroquinone transiently accumulated and short-chain organic acids were then produced at the expense of hydroquinone. Two other radiolabeled isomers of NP, 4(2',6'-dimethyl-2'-heptyl)-phenol and 4(3',6'-dimethyl-3'-heptyl)-phenol, were synthesized. In parallel experiments, the 4(2',6'-dimethyl-2'-heptyl)-phenol was degraded more slowly than the other isomers of NP by strain TTNP3, possibly because of effects of the side-chain structure on the kinetics of degradation. Alkylbenzenediol and alkoxyphenol derivatives identified as metabolites during previous studies were synthesized and tested as substrates. The derivatives were not degraded, which indicated that the mineralization of NP does not proceed via alkoxyphenol as the principal intermediate. The results obtained led to the elucidation of the degradation pathway of NP isomers with a quaternary alpha-carbon. The proposed mechanism is a type II ipso substitution, leading to hydroquinone and nonanol as the main metabolites and to the dead-end metabolites alkylbenzenediol or alkoxyphenol, depending on the substitution at the alpha-carbon of the carbocationic intermediate formed.

Developmental downregulation of rhizobial genes as a function of symbiosome differentiation in symbiotic root nodules of Pisum sativum.

• The expression of nodA and dctA genes of Rhizobium leguminosarum bv. viciae has been studied in mutant nodules of pea (Pisum sativum L.), blocked at the following developmental stages: infection thread development inside the nodule (Itn); infection droplet differentiation (Idd); bacteroid differentiation after endocytosis (Bad); and nodule persistence (Nop). • With the use of reporter fusions to these symbiotic bacterial genes it was shown that both nodA and dctA were expressed at all developmental stages, with a pattern similar to that of constitutive, symbiosis-unrelated genes. • As well as two constitutively expressed genes, both nodA and dctA genes seemed to be subjected to gradual downregulation in nodule bacteria, correlating with the stage of bacteroid differentiation reached. No such effect was observed for the symbiotic, oxygen-regulated fixN gene. The bacteroid development stage also appeared to be related to the ability of bacteria that have been subjected to endocytosis to resume free-living vegetative growth. • The results support the suggestion that bacteroid differentiation into a nitrogen-fixing, organelle-like form, is a gradual process involving several stages, each controlled by different plant genes.

Genetic dissection of the initiation of the infection process and nodule tissue development in the Rhizobium-pea (Pisum sativum L.) symbiosis.

Twelve non-nodulating pea (Pisum sativum L.) mutants were studied to identify the blocks in nodule tissue development. In nine, the reason for the lack of infection thread (IT) development was studied; this had been characterized previously in the other three mutants. With respect to IT development, mutants in gene sym7 are interrupted at the stage of colonization of the pocket in the curled root hair (Crh- phenotype), mutants in genes sym37 and sym38 are blocked at the stage of IT growth in the root hair cell (Ith- phenotype) and mutants in gene sym34 at the stage of IT growth inside root cortex cells (Itr- phenotype). With respect to nodule tissue development, mutants in genes sym7, sym14 and sym35 were shown to be blocked at the stage of cortical cell divisions (Ccd- phenotype), mutants in gene sym34 are halted at the stage of nodule primordium (NP) development (Npd- phenotype) and mutants in genes sym37 and sym38 are arrested at the stage of nodule meristem development (Nmd- phenotype). Thus, the sequential functioning of the genes Sym37, Sym38 and the gene Sym34 apparently differs in the infection process and during nodule tissue development. Based on these data, a scheme is suggested for the sequential functioning of early pea symbiotic genes in the two developmental processes: infection and nodule tissue formation.

Genetic diversity of indigenous Rhizobium leguminosarum bv. viciae isolates nodulating two different host plants during soil restoration with alfalfa.

A total of 360 Rhizobium leguminosarum bv. viciae strains was isolated from three brown-coal mining restoration fields of different age and plant cover (without and in the first and second year of alfalfa, Medicago sativa, cultivation) using two host species (Vicia hirsuta and Pisum sativum) as capture plants. The strains were genetically typed by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-generated 16S-23S ribosomal DNA intergenic spacer regions (IGS-RFLP) and characterized by plasmid profiles and RFLP analysis of amplified nodABC genes. The R. leguminosarum bv. viciae population was dominated by the same group of strains (irrespective of the trap plant used). According to type richness, the genetic diversity of indigenous R. leguminosarum in the second year of restoration was lower than in the first year and it resembled that of the fallow field, except for plasmid types, in which it was higher than that of the fallow field. Some of the less frequent nodABC genotypes were associated with distinct chromosomal IGS genotypes and symbiotic plasmids (pSyms) of different sizes, indicating that horizontal transfer and rearrangements of pSym can occur in natural environments. However, the dominant pSym and chromosomal genotypes were strictly correlated suggesting a genetically stable persistence of the prevailing R. leguminosarum bv. viciae genotypes in the absence of its host plant.

Characterisation of Phaseolus symbionts isolated from Mediterranean soils and analysis of genetic factors related to pH tolerance.

The ultimate objective of PhIMED, in which two European (Germany, Italy) and two Mediterranean (Morocco, Egypt) countries collaborate, is to improve the cultivation of French bean (Phaseolus vulgaris) under arid and semi-arid conditions by analysing and enhancing stress tolerance of the nitrogen fixing rhizobial microsymbionts. Rhizobial strains nodulating P. vulgaris (RP strains) isolated from areas in Morocco frequently subjected to drought were analysed for their salt and pH tolerance and their phylogenetic relationship. Strain RP163, exhibiting high nodulation efficiency and a broad pH tolerance was mutagenised by Tn5 and mutants unable to grow on extreme pH media were isolated. Some of the mutants affected in low pH tolerance were found to be mutated in genes related to cobalmin biosynthesis and in succinate dehydrogenase (sdhA). In a parallel approach, promoters and genes inducible under extreme pH values were identified in Rhizobium leguminosarum bv. viciae VF39, among them gabT, which encodes the GABA transaminase and which is induced under acidic conditions. The same gene is present and similarly regulated in RP163. The actSR gene region was cloned from VF39, sequenced and mutants generated in this region were found to be impaired in growth at low pH, but also under neutral conditions. The Agrobacterium rhizogenes 'promintron' promoter, reported to be activated in stationary phase, was found to be also strongly induced under acidic conditions in rhizobia and it is currently being characterised to construct a system allowing the expression of stress tolerance genes in bacteroids and free-living bacteria.

Effect of mutations in Pisum sativum L. genes blocking different stages of nodule development on the expression of late symbiotic genes in Rhizobium leguminosarum bv. viciae.

In this report, the expression of late symbiotic genes (fnrN, fixN, and nifA) of Rhizobium leguminosarum bv. viciae was studied in nodules of mutant pea lines blocked at four successive stages of nodule development. Bacterial gene expression was analyzed in situ with transcriptional gusA reporter gene fusions. As a control, a constitutively expressed gusA gene was included. In the nodules of Nop(nodule persistence) mutants (mutant in gene sym13), which had not yet exhibited signs of premature senescence, the expression patterns observed were identical to those in wild-type nodules. Normal expression of fusions also occurred in nodules defective at the infection droplet differentiation stage (mutant in gene sym40) in which bacteria are endocytosed, but infection threads and infection droplets are hypertrophied. In contrast, in Itn- (infection thread formation inside the nodule tissue) mutants (mutant gene sym33), in which there is no endocytosis of bacteria, expression of the constitutive fusion was only in infection threads and no activity was shown for the other fusions. From this it can be concluded that functionality of the plant gene Sym33, i.e., bacterial endocytosis, is a prerequisite for the expression of late symbiotic genes in the microsymbiont. No morphologically distinct interzone II-III could be detected in nodules blocked at the bacteroid differentiation stage (mutants in gene sym31). The constitutive fusion was expressed equally throughout the nodule tissue (except for the meristem), and the activity of fusions to late symbiotic genes increased gradually with a maximal expression level at the base of the nodule. This is consistent with an altered oxygen barrier previously reported for these nodules. By including double mutants, earlier results on sequential functioning of gene pairs sym33-sym40 and sym31-sym13 could be confirmed and it could be demonstrated that the developmental epistasis found at the morphological level also is reflected in the expression pattern of late symbiotic genes in the microsymbiont.

Symbiotic plasmid rearrangement in Rhizobium leguminosarum bv. viciae VF39SM.

A rearrangement between the symbiotic plasmid (pRleVF39d) and a nonsymbiotic plasmid (pRleVF39b) in Rhizobium leguminosarum bv. viciae VF39 was observed. The rearranged derivative showed the same plasmid profile as its parent strain, but hybridization to nod, fix, and nif genes indicated that most of the symbiotic genes were now present on a plasmid corresponding in size to pRleVF39b instead of pRleVF39d. On the other hand, some DNA fragments originating from pRleVF39b now hybridized to the plasmid band at the position of pRleVF39d. These results suggest that a reciprocal but unequal DNA exchange between the two plasmids had occurred.

Unusual structure of the tonB-exb DNA region of Xanthomonas campestris pv. campestris: tonB, exbB, and exbD1 are essential for ferric iron uptake, but exbD2 is not.

The nucleotide sequence of a 3.6-kb HindIII-SmaI DNA fragment of Xanthomonas campestris pv. campestris revealed four open reading frames which, based on sequence homologies, were designated tonB, exbB, exbD1, and exbD2. Analysis of translational fusions to alkaline phosphatase and beta-galactosidase confirmed that the TonB, ExbB, ExbD1, and ExbD2 proteins are anchored in the cytoplasmic membrane. The TonB protein of X. campestris pv. campestris lacks the conserved (Glu-Pro)n and (Lys-Pro)m repeats but harbors a 13-fold repeat of proline residues. By mutational analysis, the tonB, exbB, and exbD1 genes were shown to be essential for ferric iron import in X. campestris pv. campestris. In contrast, the exbD2 gene is not involved in the uptake of ferric iron.

Functional and regulatory analysis of the two copies of the fixNOQP operon of Rhizobium leguminosarum strain VF39.

DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the heme-copper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixN-gusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation. Residual expression of fixN was observed in an fnrN/fixK double mutant.

Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue.

Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK-fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R, leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr-like gene, designated fixK, whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix+ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN-deletion mutant, whereas no nitrogen-fixation activity was detectable for a flxK/fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified down-stream of fixK-fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.

A homolog of the Rhizobium meliloti nitrogen fixation gene fixN is involved in the production of a microaerobically induced oxidase activity in the phytopathogenic bacterium Agrobacterium tumefaciens.

Hybridization analysis using the Rhizobium meliloti nitrogen fixation gene fixN as a probe revealed the presence of a homologous DNA region in the phytopathogenic bacterium Agrobacterium tumefaciens. Hybridization signals were also detected with total DNAs of Rhizobium leguminosarum bv. phaseoli, Rhodobacter capsulatus and Escherichia coli, but not those of Xanthomonas campestris pv. campestris and Pseudomonas putida. The hybridizing fragment from A. tumefaciens was cloned and sequenced. The predicted gene product of one of the two open reading frames identified on the sequenced fragment shows homology to FixN of different Rhizobiaceae as well as a low but significant similarity to subunit I of heme copper oxidases from various bacteria. The presence of five strictly conserved histidine residues previously implicated in forming ligands to heme and CuB in oxidases and the predicted membrane topology provide evidence that the A. tumefaciens fixN-like gene product is a component of the heme copper oxidase superfamily. The incomplete open reading frame starting only 8 nucleotides downstream of the fixN-like gene exhibits homology to Rhizobium fixO. Using an uidA (GUS) gene fusion it could be shown that the A. tumefaciens fixN-like gene is preferentially expressed under microaerobic conditions. Expression of the uidA fusion is abolished in R. meliloti fixJ and fixK mutants, indicating that an Fnr-like protein is involved in transcriptional regulation of the fixN-like gene in A. tumefaciens. The presence of an upstream DNA sequence motif identical to the Fnr-consensus binding site (anaerobox) further supports this hypothesis. A. tumefaciens mutated in the fixN-like gene shows decreased TMPD-specific oxidase activity under microaerobic conditions, indicating that the fixN-like gene or operon codes for proteins involved in respiration under reduced oxygen availability.

Structure of the unusual trisaccharide lipopolysaccharide component produced by a symbiotically defective mutant of Rhizobium leguminosarum biovar viciae.

The structure of an unusual trisaccharide component isolated from the lipopolysaccharide (LPS) of a Tn5 mutant of Rhizobium leguminosarum biovar viciae VF39 which is defective in infection of its host plant has been elucidated. This mutant also appears to be defective in the synthesis of a tetrasaccharide component normally synthesized by the wild-type organism. The three glycosyl components are galactose, mannose, and 3-deoxy-D-manno-2-octulosonic acid (Kdo). Mannose is linked to the 5-position and galactose to the 7-position of the 3-deoxy-2-octulosonic acid residue (Kdo). Both hexosyl components are in the alpha-pyranosyl form. In the isolated molecule the octulosonic acid appears to be present as its gamma-lactone. However, in the lipopolysaccharide molecule, it is most likely present in the pyranosyl form. The structure was determined by 1H NMR spectroscopy and methylation analysis as well as by fast-atom-bombardment mass spectrometry of the peracetylated and per(trideuterio) acetylated oligosaccharides. Small amounts of the methylation analysis product of another tetrasaccharide different to normal tetrasaccharide component made by the wild-type organisms were detected. This indicates that in this mutant, there is a block in the synthesis of the normal tetrasaccharide component in addition to a switch in the synthesis of the LPS type.

A 4.6 kb DNA region of Rhizobium meliloti involved in determining urease and hydrogenase activities carries the structural genes for urease (ureA, ureB, ureC) interrupted by other open reading frames.

A 4.6 kb DNA region of the Rhizobium meliloti strain AK631 was found to contain seven open reading frames (ORFs), all oriented in the same direction. The putative gene products of four of these ORFs were highly homologous to UreA, UreB and UreC of Klebsiella aerogenes, Proteus mirabilis, Proteus vulgaris and Canavalia ensiformis. The overall organisation of the DNA region analysed was ORF1, ureA (ORF2), ORF3, ureB (ORF4), ORF5, ORF6 and ureC (ORF7), indicating that the organisation of the urease structural genes in R. meliloti differs from that of other urease genes so far characterized. ORF1 was incomplete; only the 3' end of the coding region was present. The six complete ORFs coded for polypeptides of 11.1 (UreA), 8.9 (ORF3), 10.8 (UreB), 15.0 (ORF5), 13.8 (ORF6) and 60.7 kDa (UreC). No sequence homology to known polypeptides could be detected for the gene products of ORF1, ORF3, ORF5 and ORF6. Using a lacZ fusion and insertional mutagenesis it was shown that the seven ORFs identified were all located in the same transcription unit. For mutational analysis a resistance gene cassette was introduced into each of the complete ORFs resulting in apolar mutations. Mutations in ureA, ureB and ureC, but not in ORF3, ORF5 and ORF6, abolished urease activity in R. meliloti. The determination of hydrogen uptake in these R. meliloti mutants revealed that only ORF6 and ureB are necessary for hydrogen uptake.

A 3.9-kb DNA region of Xanthomonas campestris pv. campestris that is necessary for lipopolysaccharide production encodes a set of enzymes involved in the synthesis of dTDP-rhamnose.

By mutational analysis it was found that a 3.9-kb SmaI-XhoII DNA fragment of Xanthomonas campestris pv. campestris is involved in lipopolysaccharide (LPS) biosynthesis. LPS samples isolated from different mutants carrying mutations in the 3.9-kb SmaI-XhoII DNA fragment exhibited banding patterns in silver-stained sodium dodecyl sulfate-polyacrylamide gels markedly different from that of the wild-type LPS. Moreover, comparison of the monosaccharide composition obtained by high-performance anion-exchange chromatography with pulsed amperometric detection of LPS purified from wild-type Xanthomonas campestris pv. campestris B100 and from mutants with mutations in the 3.9-kb SmaI-XhoII DNA fragment revealed a lack of rhamnose moieties in the mutant LPS. Sequence analysis of this DNA fragment revealed four open reading frames (ORFs), designated ORF302, ORF183, ORF295, and ORF351. The deduced amino acid sequences of these ORFs showed a high degree of homology to the deduced amino acid sequences of the rfbC, rfbD, rfbA, and rfbB genes of Salmonella typhimurium LT2, which have been shown to encode a set of enzymes responsible for conversion of glucose 1-phosphate to dTDP-rhamnose.

Saturation mutagenesis in Escherichia coli of a cloned Xanthomonas campestris DNA fragment with the lux transposon Tn4431 using the delivery plasmid pDS1, thermosensitive in replication.

A system allowing transposon mutagenesis of cloned DNA fragments in Escherichia coli with Tn4431, which carries the promotorless luciferase (lux) operon of Vibrio fischeri, has been developed. The transposon delivery plasmid, pDS1, based on an IncF replicon, is thermosensitive in replication and mobilizable to many Gram-negative bacteria. We used pDS1 for Tn4431-saturation mutagenesis of a 10-kb DNA fragment of Xanthomonas campestris pv. campestris (X.c.c.) in E. coli and showed that the expression of the lux operon was dependent on orientation and location of the transposon. Transfer of a specific Tn4431 insertion to X.c.c. allowed the determination of the bioluminescence phenotype in planta.

The Rhizobium leguminosarum FnrN protein is functionally similar to Escherichia coli Fnr and promotes heterologous oxygen-dependent activation of transcription.

An open reading frame from Rhizobium leguminosarum bv. viciae strain VF39, previously identified and found to be similar to Escherichia coli fnr and Rhizobium meliloti fixK (orf240, thereafter called fnrN), was further analysed. Analysis of the expression of an fnrN-lacZ transcriptional fusion revealed that fnrN is preferentially expressed under oxygen limitation. Using R. meliloti fixN-lacZ fusions it was shown that the fnrN gene product only mediates transcriptional activation under microaerobiosis, indicating that the FnrN protein responds, directly or indirectly, to oxygen. Plasmids which expressed fnrN under the control of an E. coli promoter were able to complement an E. coli fnr mutant with respect to anaerobic growth on nitrate but not fumarate, and to promote anaerobic but not aerobic activation of the Fnr-dependent E. coli genes narGHJI, nirB and fdnGHI coding for nitrate reductase, NADH-dependent nitrite reductase and formate dehydrogenase-N, respectively. Fumarate and DMSO reductase activities were not induced by FnrN. The E. coli fnr gene substituted for fnrN in oxygen-regulated transcription of nirB- and fixN-lacZ fusions in R. leguminosarum. The results indicate that Fnr and FnrN are functionally very similar and share a common mode of oxygen-dependent transcriptional activation. From hybridization studies, it appeared that fnrN-like genes are present in a number of different R. leguminosarum strains.

Characterization of structural defects in the lipopolysaccharides of symbiotically impaired Rhizobium leguminosarum biovar viciae VF-39 mutants.

The lipopolysaccharides (LPS) of a wild type strain of Rhizobium leguminosarum biovar viciae (strain VF-39) and two symbiotically defective Tn5 mutants (VF-39-32 and VF-39-86) have been studied. The LPS of the mutants reflected impaired synthesis of the O-antigen. In the LPS of one mutant, the core tetrasaccharide was lacking and in that of the other it was truncated to a disaccharide containing mannose and 3-deoxy-D-manno-oct-2-ulosonic acid (KdO). The latter mutant also synthesized an unusual carbohydrate component containing mannose, galactose, and an unidentified saccharide. The lipid A composition was similar to that found in other strains of R. leguminosarum biovar viciae. The O-antigen of the wild-type bacterium contained 2-O-methylfucose, fucose, 3,6-dideoxy-3-(methylamino)hexose, glucose, 2-amino-2,6-dideoxyhexose, and heptose. This study clearly defines a role for the bacterial LPS in the proper functioning of the Rhizobium legume symbiosis.

Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae.

DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA- Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 bp coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA- mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.

An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK.

A 1.9 kb DNA region of Rhizobium leguminosarum biovar viciae strain VF39 capable of promoting microaerobic and symbiotic induction of the Rhizobium meliloti fixN gene was identified by heterologous complementation. Sequence analysis of this DNA region revealed the presence of two complete open reading frames, orf240 and orf114. The deduced amino acid sequence of orf240 showed significant homology to Escherichia coli Fnr and R. meliloti FixK. The major difference between ORF240 and FixK is the presence of 21 N-terminal amino acids in ORF240 that have no counterpart in FixK. A similar protein domain is also present in E. coli Fnr and is essential for the oxygen-regulated activity of this protein. Analysis of the nucleotide sequence upstream of orf240 revealed a motif similar to the NtrA-dependent promoter consensus sequence, as well as two DNA regions resembling the Fnr consensus binding sequence. A Tn5-generated mutant in orf240 lost the ability to induce the R. meliloti fixN-lacZ fusion. Interestingly, this mutant was still capable of nitrogen fixation but showed reduced nitrogenase activity.

Genes involved in lipopolysaccharide production and symbiosis are clustered on the chromosome of Rhizobium leguminosarum biovar viciae VF39.

Four mutants of Rhizobium leguminosarum biovar viciae VF39 altered in lipopolysaccharide (LPS) synthesis were isolated upon random Tn5 mutagenesis. These mutants produced matt colonies on TY medium and showed autoagglutination and loss of motility. On sodium dodecyl sulfate-polyacrylamide gels, they lacked a slow-migrating carbohydrate band, corresponding to the complete LPS (LPSI). All four mutants formed small white nodules on Vicia hirsuta. These nodules were infected but showed no nitrogen-fixing activity and senesced prematurely. Three of the mutants were complemented by a wild-type cosmid to synthesis of normal LPS and induction of nitrogen-fixing nodules. By hybridization and in vivo cloning experiments, the mutations were mapped within different EcoRI fragments which could be localized on the VF39 chromosome. Cross-complementation analyses revealed that the three mutants were affected in different transcriptional units. The results indicate that a cluster of genes necessary for LPSI production and symbiotic efficiency is located within a defined region of 20 kilobases on the R. leguminosarum bv. viciae chromosome.