Isolation of the replication DNA region from a Rhizobium plasmid and examination of its potential as a replicon for Rhizobiaceae cloning vectors

The DNA region essential for replication and stability of a native plasmid (pTM5) from Rhizobium sp. (Hedysarum) has been identified and isolated within a 5.4-kb PstI restriction fragment. The isolation of this region was accomplished by cloning endonuclease-restricted pTM5 DNA into a ColE1-type replicon and selecting the recombinant plasmids containing the pTM5 replicator (pTM5 derivative plasmids) by their ability to replicate in Rhizobium. DNA homology studies revealed that pTM5-like replicons are present in cryptic plasmids from some Rhizobium sp. (Hedysarum) strains but not in plasmids from strains of other Rhizobium species or Agrobacterium tumefaciens. The pTM5 derivative plasmids were able to replicate in Escherichia coli and A. tumefaciens and in a wide range of Rhizobium species. On the basis of stability assays in the absence of antibiotic selective pressure, the pTM5 derivative plasmids were shown to be highly stable in both free-living and symbiotic cells of Rhizobium sp. (Hedysarum). The stability of these plasmids in other species of Rhizobium and in A. tumefaciens varied depending on the host and on the plasmid. Most pTM5 derivative plasmids tested showed significantly higher symbiotic stability than RK2 derivative plasmids pRK290 and pAL618 in Rhizobium sp. (Hedysarum), R. meliloti, and R. leguminosarum by. phaseoli. Consequently, we consider that the constructed pTM5 derivative plasmids are potentially useful as cloning vectors for Rhizobiaceae.     

Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.     

A region of the broad-host-range plasmid RK2 causes stable in planta inheritance of plasmids in Rhizobium meliloti cells isolated from alfalfa root nodules.

We demonstrate for the first time that the broad-host-range stabilization loci from plasmid RK2 cause total retention of plasmids in cells of Rhizobium meliloti during symbiosis with alfalfa. Two derivatives of plasmid RK2, pRK290 and a 7.3-kb mini-RK2 plasmid, were stabilized in R. meliloti cells isolated from root nodules by the insertion of a 3.2-kb DNA fragment or a smaller 0.8-kb DNA fragment derived from the RK2 stabilization region.     

High-frequency transformation of Rhizobium meliloti.

Transformation of R factor RP4 and its derivative pRK290 from Escherichia coli to Rhizobium meliloti is reported. The efficiency of transformation was in the range of 10(-5) per viable cell. In addition, chromosomal DNA prepared from one R. meliloti strain resistant to streptomycin was transferred to the isoleucine-valine-requiring mutant susceptible to streptomycin.     

Gene transfer system for Rhodopseudomonas viridis.

A gene transfer system for Rhodopseudomonas viridis was established which uses conjugation with Escherichia coli S17-I as the donor and mobilizable plasmids as vectors. Initially, plasmids of the incompatibility group P1 (pRK290 and pRK404) were used. The more effective shuttle vectors between E. coli and R. viridis, pKV1 and pKVS1, were derived from plasmid pBR322 and showed the highest conjugation frequency (10(-2] thus far demonstrated in purple bacteria. It was also demonstrated that Rhizobium meliloti can be used as a donor for conjugation with R. viridis. From a genomic cosmid library of R. viridis constructed in the vector pHC79, clones that coded for subunits H (puh operon), L, M and cytochrome c (puf operon) of the photosynthetic reaction center were isolated and characterized. For linkage of the two operons on the genome, cosmids that overlapped with the operon-carrying clones were identified. The relative positions of the two operons could not be determined, but the operons must be more than 100 kilobase pairs apart. Thus, the genomic organization of the reaction center in R. viridis is different from that of Rhodobacter capsulatus, for which a distance of about 39 kilobase pairs was determined. From a spontaneous mutant of R. viridis that is resistant to the herbicide terbutryn, the puf operon was cloned in pKVS1 and transferred by conjugation into R. viridis wild-type cells. The resulting exconjugants were resistant to the herbicide, which demonstrated that the puf operon on pKVS1 constructions was functionally expressed in R. viridis.     

The construction of recA–deficient Rhizobium meliloti and R. leguminosarum strains marked with gusA or luc cassettes for use in risk–assessment studies

A vector system was developed employing the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar. viciae as target sequences for the stable genomic integration of foreign DNA. The plasmid vectors can be used either as integration vectors (single cross–over), or as gene replacement vectors (double cross–over). Gene replacement results in the antibiotic–marker–free integration of cloned DNA into the recA genes of R. meliloti and R. leguminosarum bv. viciae. Consequently, the recombinant strains become recombination deficient (RecA^-). The expression of integrated genes is under the control of the neomycin phosphotransferase II (nptll) promoter of transposon Tn5. The system was used to construct recA mutant strains of R. meliloti and R. leguminosarum by. viciae, carrying the Escherichia coli gusA gene encoding β–glucuronidase as well as the firefly (Photinus pyralis) luc gene encoding luciferase as marker genes. The GUS activity in the constructed strains was found to be absolutely stable over more than 100 generations of non–selective growth in liquid culture. The stability was also confirmed in root–nodule passages. In addition, the potential use of the luc gene as a stable genetic marker in the unequivocal identification of tagged strains among indigenous microbes in non–sterile soil was demonstrated. It is proposed to use bioluminescent recA mutants as model organisms in risk assessment studies with genetically engineered Rhizobium strains.     

Construction and Properties of Cloning Vectors Based on a 7.2-kb Rhizobium meliloti Cryptic Plasmid

Cloning vectors (pFD1001, pFD1192, pFD1194, and pFD1212) were constructed by extension of the host range of a 7.2-kb Rhizobium meliloti cryptic plasmid (pRm1132f) with the ColE1-based plasmids, pBR322, pACYC177, pACYC 184, pSUP301, or pHC179; mobilization was facilitated by introduction of the ori T region from pRK2, a broad-host-range plasmid. The vector plasmids transferred readily into a wide range of gram-negative bacteria and had relatively low copy number in R. meliloti; two constructs, pFD1001 and pFD1212, were completely stable in R. meliloti isolated from nodules of alfalfa (Medicago sativa). A representative of the vector constructs (pFD1001) could be maintained in R. meliloti in the presence of the broad-host-range shuttle plasmid pRK290. These two vector plasmids could be introduced into R. meliloti, either simultaneously or singly when pRK290 was the resident plasmid; however, entry of pRK290 was blocked when pFD1001 was the resident plasmid. The cloning vectors constructed in this study should prove to be useful for the genetic manipulation of Rhizobium.     

Direct selection for curing and deletion of Rhizobium plasmids using transposons carrying the Bacillus subtilis sacB gene

We have constructed derivatives of the transposon Tn5 carrying the mob site (oriT) of plasmid RP4, and an nptI-sacB-sacR cassette [Ried and Collmer, Gene 57 (1987) 239-246]. The mob site, in conjunction with the antibiotic-resistance markers carried on the transposons, allows identification of transposon inserts in cryptic plasmids by mobilisation to other strains. The sacB-sacR genes allow direct selection for the loss or curing of plasmids, because only strains which no longer contain an active sacB gene are able to grow on media containing sucrose. We have tested these transposons in four strains of Rhizobium leguminosarum and two strains of Rhizobium meliloti, and have been able to demonstrate curing of several large cryptic plasmids, and generation of large deletions in many other plasmids. This method has enabled us to show that the R. leguminosarum plasmids pRL12JI and pR1eVF39f carry auxotrophic markers, and that the plasmid pR1eVF39c carries genes which affect colony morphology.     

Isolation and characterization of the recA gene of Rhizobium meliloti.

Interspecific complementation of an Escherichia coli recA mutant with plasmids containing a gene bank of Rhizobium meliloti DNA was used to identify a clone which contains the recA gene of R. meliloti. The R. meliloti recA protein can function in recombination and in response to DNA damage when expressed in an E. coli recA host, and hybridization studies have shown that DNA sequence homology exists between the recA gene of E. coli and that of R. meliloti. The isolated R. meliloti recA DNA was used to construct a recA R. meliloti, and this bacterium was not deficient in its ability to carry out symbiotic nitrogen fixation.     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

Expression of Rhizobium trifolii early nodulation genes on maize and rice plants.

An IncQ multicopy vector (pKT230) and an IncP1 low-copy-number vector (pRK290), both carrying Rhizobium trifolii root hair curling (Hac) genes, were transferred to a Sym plasmid-cured derivative of R. trifolii ANU843. The resulting transconjugants were used to inoculate the monocotyledonous plants sorghum, maize, rice, and wheat. Transconjugants carrying the Hac genes on the multicopy vector caused a root hair curling response on maize and rice plants 14 days after inoculation.     

Expression cloning and biochemical characterization of a Rhizobium leguminosarum lipid A 1-phosphatase

Lipid A of Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, displays several significant structural differences when compared with Escherichia coli. An especially striking feature of R. leguminosarum lipid A is that it lacks both the 1- and 4'-phosphate groups. Distinct lipid A phosphatases that attack either the 1 or the 4' positions have previously been identified in extracts of R. leguminosarum and Rhizobium etli but not Sinorhizobium meliloti or E. coli. Here we describe the identification of a hybrid cosmid (pMJK-1) containing a 25-kb R. leguminosarum 3841 DNA insert that directs the overexpression of the lipid A 1-phosphatase. Transfer of pMJK-1 into S. meliloti 1021 results in heterologous expression of 1-phosphatase activity, which is normally absent in extracts of strain 1021, and confers resistance to polymyxin. Sequencing of a 7-kb DNA fragment derived from the insert of pMJK-1 revealed the presence of a lipid phosphatase ortholog (designated LpxE). Expression of lpxE in E. coli behind the T7lac promoter results in the appearance of robust 1-phosphatase activity, which is normally absent in E. coli membranes. Matrix-assisted laser-desorption/time of flight and radiochemical analysis of the product generated in vitro from the model substrate lipid IVA confirms the selective removal of the 1-phosphate group. These findings show that lpxE is the structural gene for the 1-phosphatase. The availability of lpxE may facilitate the re-engineering of lipid A structures in diverse Gram-negative bacteria and allow assessment of the role of the 1-phosphatase in R. leguminosarum symbiosis with plants. Possible orthologs of LpxE are present in some intracellular human pathogens, including Francisella tularensis, Brucella melitensis, and Legionella pneumophila.     

Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.     

Phenotypic expression of mutations in a wide-host-range R plasmid in Escherichia coli and Rhizobium meliloti.

Eight different derivatives of R plasmid RP1 with thermosensitive mutations affecting maintenance in Escherichia coli and Pseudomonas aeruginosa were introduce into Rhizobium meliloti. None of the plasmids showed a thermosensitive character in R. meliloti. On the other hand, a certain deletion mutation in RP1 was found to cause plasmid instability in rhizobia and agrobacteria, but not in E. coli.     

Reduction of adenosine-5'-phosphosulfate instead of 3'-phosphoadenosine-5'-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and other members of the family Rhizobiaceae.

We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizobium meliloti) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the Escherichia coli cysDN genes, which code for an ATP sulfurylase (EC 2.7.7.4). The third gene has homology to the E. coli cysH gene, a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase (EC 1.8.99.4), but has greater homology to a set of genes found in Arabidopsis thaliana that encode an adenosine-5'-phosphosulfate (APS) reductase. In order to determine the specificity of the R. meliloti reductase, the R. meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the A. thaliana reductases, the histidine-tagged R. meliloti cysH gene product appears to favor APS over PAPS as a substrate, with a Km for APS of 3 to 4 microM but a Km for PAPS of >100 microM. In order to determine whether this preference for APS is unique to R. meliloti among members of the family Rhizobiaceae or is more widespread, cell extracts from R. leguminosarum, Rhizobium sp. strain NGR234, Rhizobium fredii (Sinorhizobium fredii), and Agrobacterium tumefaciens were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS.     

Origin of the 2-amino-2-deoxy-gluconate unit in Rhizobium leguminosarum lipid A. Expression cloning of the outer membrane oxidase LpxQ

An unusual feature of the lipid A from the plant endosymbionts Rhizobium etli and Rhizobium leguminosarum is the presence of a proximal sugar unit consisting of a 2-amino-2-deoxy-gluconate moiety in place of glucosamine. An outer membrane oxidase that generates the 2-amino-2-deoxy-gluconate unit from a glucosamine-containing precursor is present in membranes of R. leguminosarum and R. etli but not in S. meliloti or Escherichia coli. We now report the identification of a hybrid cosmid that directs the overexpression of this activity by screening 1800 lysates of individual colonies of a R. leguminosarum 3841 genomic DNA library in the host strain R. etli CE3. Two cosmids (p1S11D and p1U12G) were identified in this manner and transferred into S. meliloti, in which they also directed the expression of oxidase activity in the absence of any chromosomal background. Subcloning and sequencing of the oxidase gene on a 6.5-kb fragment derived from the approximately 20-kb insert in p1S11D revealed that the enzyme is encoded by a gene (lpxQ) that specifies a protein of 224 amino acid residues with a putative signal sequence cleavage site at position 28. Heterologous expression of lpxQ using the T7lac promoter system in E. coli resulted in the production of catalytically active oxidase that was localized in the outer membrane. A new outer membrane protein of the size expected for LpxQ was present in this construct and was subjected to microsequencing to confirm its identity and the site of signal peptide cleavage. LpxQ expressed in E. coli generates the same products as seen in R. leguminosarum membranes. LpxQ is dependent on O(2) for activity, as demonstrated by inhibition of the reaction under strictly anaerobic conditions. An ortholog of LpxQ is present in the genome of Agrobacterium tumefaciens, as shown by heterologous expression of oxidase activity in E. coli.     

Gene fusion vehicles for the analysis of gene expression in Rhizobium meliloti.

A set of plasmid cloning vehicles was developed to facilitate the construction of gene or operon fusions in Rhizobium meliloti. The vehicles also contain a broad-host-range replicon and could be introduced into bacteria either by transformation or by transduction, using bacteriophage P2. Insertion of foreign DNA into a unique restriction endonuclease cleavage site promotes the synthesis of either the Escherichia coli lactose operon or the kanamycin phosphotransferase gene from transposon Tn5. Expression of the lactose operon could be detected by observing the color of Rhizobium colonies on medium that contained a chromogenic indicator. We also determined the growth conditions that make it possible to select either for or against the expression of the E. coli lactose operon in R. meliloti. Recombinant plasmids were constructed by inserting MboI restriction fragments of R. meliloti DNA into one of the vehicles, pMK353 . Expression of beta-galactosidase by a number of these recombinants was measured in both R. meliloti and E. coli.