Identification of the sym plasmid of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other Rhizobia and Agrobacterium tumefaciens

A plasmid of 150 Mdal from Rhizobium leguminosarum RCC1001 was found to be a Sym plasmid (pSym1) carrying genes for root nodulation and nitrogen fixation on plants of the pea vetch cross-inoculation group. The plasmid was expressed not only in different R. leguminosarum and R. trifolii hosts, but also in Agrobacterium tumefaciens and R. meliloti, although in root nodules induced by A. tumefaciens and R. meliloti hosts no nitrogen was fixed. The host range for root nodule induction appeared to be determined by pSym1 and only included plants of the pea vetch cross-inoculation group; in contrast, the host range for the induction of root hair deformations, which was found also to be determined by pSym1 was less restricted and included besides plants of the pea vetch group in addition plants of the clover group. This corroborates previous findings that host specificity for nodulation and nitrogen fixation is exerted at a stage after the induction of root hair deformations.     

Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment

Abstract An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II ( npt II) promotor of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.     

Analysis of three nodD genes in Rhizobium leguminosarum biovar phaseoli; nodD 1 is preceded by nolE, a gene whose product is secreted from the cytoplasm

In a strain of Rhizobium leguminosarum biovar phaseoli, three copies of the regulatory nodulation gene nodD were identified on the Sym plasmid and sequenced. Two were closely linked to each other and the third was near, but not adjacent, to the nodABC genes. Each of these nodD genes could correct the Nod- defect of a nodD mutant strain of R. leguminosarum biovar viciae on peas. A truncated form of nodD2 could also correct this mutant, indicating that the C-terminus of NodD2 is not needed for inducing activity. Upstream of nodD1 and in the same operon is a newly described gene, noIE, whose product appears to be exported into the periplasm. Close to nodD2 is another gene, noIP, with no known counterpart in other rhizobia. Both noIP and noIE-nodD1 are preceded by 'nod-box' sequences and, in the former case, there appear to be two tandemly repeated nod-box sequences. Mutations in each of the nodD genes and in the noIE and noIP genes did not abolish nodulation or nitrogen fixation on beans.     

Direct amplification of nodD from community DNA reveals the genetic diversity of Rhizobium leguminosarum in soil

Sequences of nodD , a gene found only in rhizobia, were amplified from total community DNA isolated from a pasture soil. The polymerase chain reaction (PCR) primers used, Y5 and Y6, match nodD from Rhizobium leguminosarum biovar trifolii , R. leguminosarum biovar viciae and Sinorhizobium meliloti . The PCR product was cloned and yielded 68 clones that were identified by restriction pattern as derived from biovar trifolii [11 restriction fragment length polymorphism (RFLP) types] and 15 clones identified as viciae (seven RFLP types). These identifications were confirmed by sequencing. There were no clones related to S. meliloti nodD . For comparison, 122 strains were isolated from nodules of white clover ( Trifolium repens ) growing at the field site, and 134 from nodules on trap plants of T. repens inoculated with the soil. The nodule isolates were of four nodD RFLP types, with 77% being of a single type. All four of these patterns were also found among the clones from soil DNA, and the same type was the most abundant, although it made up only 34% of the trifolii -like clones. We conclude that clover selects specific genotypes from the available soil population, and that R. leguminosarum biovar trifolii was approximately five times more abundant than biovar viciae in this pasture soil, whereas S. meliloti was rare.     

Growth requirements of Rhizobium leguminosarum,strain PRE

Summary Rhizobium leguminosarum, strain PRE, is unable to use sulphate as the sulphur source. Sulfhydryl compounds must be added to achieve growth.Omission of FeCl₃ from the synthetic growth medium resulted in a sharp decrease in growth of this Rhizobium strain as contrasted to other strains of R. leguminosarum. The pyrimidine bases uracil and cytosine could replace FeCl₃. Thymine almost completely inhibited bacterial growth. Adenine and guanine showed no effect. re]19760809     

Ammonium uptake by cowpea Rhizobium strain MNF 2030 and Rhizobium trifolii MNF 1001

Free-living Rhizobium trifolii MNF 1001 and cowpea Rhizobium MNF 2030 grown in chemostat culture under nitrogen limitation had high activities of an ammonium permease. In phosphate-limited, nitrogen-excess conditions, strains MNF 1001 and MNF 2030 retained 20% and 50%, respectively, of the ammonium uptake activity found in nitrogen-limited cells. Uptake in both strains was sensitive to azide, cyanide, carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol. A gradient of ammonium concentration greater than 150-fold developed across the membrane within 20 min in cells of strain MNF 1001 grown under ammonia limitation. The pH optimum for ammonium uptake by N-limited cells of both MNF 1001 and MNF 2030 was around pH 7. The apparent K _m values for the ammonium permease in strains MNF 2030 and MNF 1001 were 3.9±1.6 M and 2.0±1.6 M respectively, and the V _max was 47±2.6 nmol min^-1 (mg protein)^-1 for MNF 2030 and 101±5.1 nmol min^-1 (mg protein)^-1 for MNF 1001. Isolated snake bean bacteroids of strain MNF 2030 capable of transporting succinate and l-glutamate had no detectable ammonium uptake activity. It therefore appears that the ammonium permeases in cells of these two strains are not as tightly regulated as in R. leguminosarum MNF 3841.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrzone - HEPES N-Hydroxyethylpiperazine-N-2-ethanesulphonic acid     

katG基因在豌豆根瘤菌抗氧化中的功能

摘要:【目的】细菌中过氧化物/过氧化氢酶KatG参与活性氧(ROS)的解毒过程,从而防止其对细菌生长伤害,本文研究根瘤菌中katG基因的抗氧化功能对豌豆根瘤菌3841生长及共生固氮的影响。【方法】通过基因敲除、遗传互补和对菌株的抗氧化和共生能力分析,系统地探究了根瘤菌中katG基因的功能。【结果】katG基因突变不影响菌株在自生培养条件下生长状况,但H2O2短时间处理导致突变株的存活率显著下降。实时荧光定量RT-PCR结果显示,H2O2不能诱导豌豆根瘤菌3841katG基因的表达。进一步研究发现突变体中katG基因缺失能显著提高抗氧化基因ohrB的表达,而降低grxC基因的表达。植物盆栽实验发现,katG突变虽然对根瘤菌共生固氮能力和竞争结瘤能力均无影响,但katG在类菌体中表达显著下调。同时,katG突变显著影响了根瘤菌在植物根圈中的定殖能力。【结论】研究表明katG虽对豌豆根瘤菌自生和共生固氮无明显影响,但在抗氧化和根圈定殖中起重要作用,外源H2O2对katG的表达无诱导作用,但katG调节ohrB和grxC等抗氧化基因的表达,从而在抗氧化和共生中发挥作用。     

Identification and DNA sequence of fixZ, a nifB-like gene from Rhizobium leguminosarum.

Previously, several mutants which nodulated peas but which failed to fix nitrogen were isolated following Tn5 mutagenesis of pRL 1JI, a symbiotic plasmid of Rhizobium leguminosarum. Two of these alleles, fix52::Tn5 and fix137::Tn5 were in a region of pRL 1JI which hybridized to a probe that contained the nifA gene and the amino-terminal region of the nifB gene of Klebsiella pneumoniae. The nitrogen fixation defect of the fix52::Tn5 mutant strain was corrected by a 2.0kb fragment of the corresponding wild-type DNA cloned in a wide host-range plasmid. The DNA sequence of this region revealed an open reading frame corresponding to the gene within which the fix52::Tn5 allele was located. The polypeptide corresponding to this open reading frame had a deduced molecular weight of 39,936 and the gene was termed fixZ. The deduced amino acid sequence of the fixZ gene product contained two clusters of cysteine residues, suggesting that the protein may contain an iron-sulphur cluster. The sequence of the fixZ polypeptide was very similar to the sequence of the K. pneumoniae nifB gene (provided by W. Arnold and A. Pühler) which is required for the synthesis of the FeMo-cofactor of nitrogenase. It was shown that the previously observed hybridization was due to homology between the amino terminal regions of fixZ and nifB. Upstream from fixZ was found another open reading frame whose 5' terminus was not established, but within which was located the fix137::Tn5 allele. This gene was termed fixY. The deduced amino acid sequence of the sequenced part of fixY showed similarity to that of the regulatory nifA gene of K. pneumoniae (provided by W. J. Buikema and F. M. Ausubel). Thus in R. leguminoarum the fix genes that correspond to the nifA and nifB genes are in the same relative orientation as in K. pneumoniae.     

The expression of the nodD and nodABC genes of Rhizobium leguminosarum is not regulated in response to combined nitrogen

Abstract Using a Rhizobium leguminosarum bv. viciae strain harboring nodD :: lacZ or nodC :: lacZ translational fusions, grown in minimal media containing different concentrations of nitrate and/or ammonium salts, lacZ expression was monitored. Based on these experiments it is shown that the induction of Rhizobium leguminosarum bv. viciae nodD and nodABC operons by the flavanone naringenin is not regulated in response to nitrate and/or ammonium salts.     

Identification of a NodD repressible gene adjacent to nodM in Rhizobium leguminosarum biovar viciae

The nodFEL and nodMNT operons in Rhizobium leguminosarum biovar viciae are transcribed in the same orientation and induced by NodD in response to flavonoids secreted by legumes. In the narrow intergenic region between nodFEL and nodMNT, we identified a small gene divergently transcribed from nodM to the 3' end of nodL. Unlike the promoters upstream of nodF and nodM, the promoter of this gene is constitutively expressed. It appeared that its promoter might partially overlap with that of nodM and its expression was repressed by nodD. A deletion mutation was made and proteins produced by the mutant were compared with those by wild-type using 2D gel electrophoresis. Several protein differences were identified suggesting that this small gene influences the expression or stability of these proteins. However, the mutant nodulated its host plant (pea) normally.     

The glnB gene of Rhizobium leguminosarum biovar viceae

Abstract An open-reading frame (ORF111) upstream of the glutamine synthetase I structural gene ( glnA ) in Rhizobium leguminosarum biovar viceae encodes a protein which is highly homologous to the P_II protein (encoded by glnB ) of enteric bacteria. ORF111 was cloned in a number of different plasmid vectors and shown to complement a K. pneumoniae glnB mutant. We propose that ORF111 encodes the P_II protein of R. leguminosarum and that it should be designated glnB .     

Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum

DNA sequencing of the nodIJ region from Rhizobium leguminosarum biovar trifolii revealed the nodT gene immediately downstream of nodJ. DNA hybridizations using a nodT-specific probe showed that nodT is present in several R. leguminosarum strains. Interestingly, a flavonoid-inducible nodT gene homologue in R. leguminosarum bv. viciae is not in the nodABCIJ operon but is located downstream of nodMN. The sequence of the nodT gene from bv. viciae was determined and a comparison of the predicted amino-acid sequences of the two nodT genes shows them to be conserved; the predicted protein sequences appear to have a potential transit sequence typical of outer-membrane proteins. Mutations affecting nodT in either biovar had no observed effect on nodulation of the legumes tested.     

Identification and characterization of a functional nodD gene in Azorhizobium caulinodans ORS571.

Azorhizobium caulinodans ORS571, a bacterium capable of nodulating roots and stems of the tropical legume Sesbania rostrata, has been shown to have no nodD-like gene located immediately upstream from its common nodABC locus. A clone carrying a functional nodD gene of strain ORS571 has now been isolated from a pLAFR1 gene library by screening for naringenin-induced expression of the common nod genes in an Agrobacterium background. Tn5 mutagenesis of the cloned insert DNA delimited the inducing activity to a +/- 0.8-kilobase-pair fragment. One of the Tn5 insertions in the activator locus was homogenotized in the ORS571 genome. This resulted in a mutant strain (ORS571-3) that was unable to induce common nod gene expression in the presence of host plant exudate or the flavanone naringenin and that had lost the capacity to nodulate the roots and stems of S. rostrata. Complementation of both mutant phenotypes was achieved upon introduction of the cloned nodD gene. Sequencing of the nodD locus indicated the presence of a single, 942-base-pair-long open reading frame (ORFD) with significant homology to the nodD gene of (brady)rhizobia. The level of homology, however, is the lowest thus far reported for this kind of gene. ORFD most likely initiates translation with a TTG start codon. Upstream from ORFD, a divergently oriented nod box-like sequence is present, the function of which remains to be determined.