Application of subtraction hybridization for the development of a Rhizobium leguminosarum biovar phaseoli and Rhizobium tropici group-specific DNA probe

Abstract A combined subtraction hybridization and polymerase chain reaction/amplification technique was used to develop a DNA probe which was specific for the Rhizobium leguminosarum biovar phaseoli and the Rhizobium tropici group. Total genomic DNA preparations from Rhizobium leguminosarum biovar viciae, Rhizobium leguminosarum biovar trifolii, Rhizobium sp., Agrobacterium tumefaciens, Rhizobium fredii, Bradyrhizobium japonicum, Bradyrhizobium ssp. and Rhizobium meliloti were pooled and used as subtracter DNA against total genomic DNA from the Rhizobium leguminosarum biovar phaseolo strain KIM5s. Only one round of subtraction hybridization at 65°C was necessary to remove all cross-hybridizing sequences. Dot blot hybridizations with total genomic DNA of the eight subtracter organisms and 29 bacteria of different groups confirmed the high specificity of the isolated DNA sequences. Dot blot hybridizations and total genomic DNA from ten different R. Leguminosarum biovar phaseoli and R. tropici strains resulted in strong hybridization signals for all strains tested. The DNA probe for the R. tropici and R. leguminosarum biovar phaseoli group was used for dot blot hybridization with DNA extracts from three tropical and one boreal soil. When correlated with data from Most Probable Number analyses the probe was capable of detecting as low as 3 × 10⁴ homologous indigenous rhizobia per g soil. The technique offers great benefits for the development of DNA probes for monitoring bacterial populations in environmental samples.     

Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species.

Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp. strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI. A second gene product, 500 bp downstream of the chvI-like gene in R. meliloti, was homologous to the A. tumefaciens ChvG protein. The homology between the R. meliloti and A. tumefaciens genes was confirmed, because the R. meliloti chvI and chvG genes complemented A. tumefaciens chvI and chvG mutants for growth on complex media. We were unable to construct chvI or chvG insertion mutants of R. meliloti, whereas mutants carrying insertions outside of these genes were readily obtained. A 108-bp repeat element characterized by two large palindromes was identified in the chvI and chvG intergenic regions of both Rhizobium species. This element was duplicated in Rhizobium sp. strain NGR234. Another structurally similar element with a size of 109 bp was present in R. meliloti but not in Rhizobium sp. strain NGR234. These elements were named rhizobium-specific intergenic mosaic elements (RIMEs), because their distribution seems to be limited to members of the family Rhizobiaceae. A homology search in GenBank detected six more copies of the first element (RIME1), all in Rhizobium species, and three extra copies of the second element (RIME2), only in R. meliloti. Southern blot analysis with a probe specific to RIME1 showed the presence of several copies of the element in the genome of R. meliloti, Rhizobium sp. strain NGR234, Rhizobium leguminosarum, and Agrobacterium rhizogenes, but none was present in A. tumefaciens and Bradyrhizobium japonicum.     

Occurrence of lipid A variants with 27-hydroxyoctacosanoic acid in lipopolysaccharides from members of the family Rhizobiaceae.

Lipopolysaccharides (LPSs) isolated from several strains of Rhizobium, Bradyrhizobium, Agrobacterium, and Azorhizobium were screened for the presence of 27-hydroxyoctacosanoic acid. The LPSs from all strains, with the exception of Azorhizobium caulinodans, contained various amounts of this long-chain hydroxy fatty acid in the lipid A fractions. Analysis of the lipid A sugars revealed three types of backbones: those containing glucosamine (as found in Rhizobium meliloti and Rhizobium fredii), those containing glucosamine and galacturonic acid (as found in Rhizobium leguminosarum bv. phaseoli, trifolii, and viciae), and those containing 2,3-diamino-2,3-dideoxyglucose either alone or in combination with glucosamine (as found in Bradyrhizobium japonicum and Bradyrhizobium sp. [Lupinus] strain DSM 30140). The distribution of 27-hydroxyoctacosanoic acid as well as analysis of lipid A backbone sugars revealed the taxonomic relatedness of various strains of the Rhizobiaceae.     

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.     

A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum.

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).     

Regulation of Predation by Prey Density: the Protozoan-Rhizobium Relationship

Tetramitus rostratus and strains of Hartmanella, Naegleria, and Vahlkampfia consumed large numbers of Rhizobium meliloti cells in a salt solution, but protozoan multiplication and the bacterial decline stopped when the prey density fell to about 10-6 to 10-7 cells/ml. At higher prey densities, the maximum numbers of Hartmanella sp. and Naegleria sp. were proportional to the quantity of R. meliloti initially provided to the amoebas. When supplemental rhizobia were supplied to Hartmanella sp. or Naegleria sp. after their active feeding had terminated, presumably because the remaining 10-6 or 10-7 bacteria/ml could not be captured, replication of the protozoa was initiated. The rate of elimination of rhizobia present in large populations was proportional to the initial abundance of Naegleria sp., but the final numbers of amoebas and surviving R. meliloti cells were independent of initial numbers of predators. The surviving bacteria were not intrinsically resistant to attack because 98% of the survivors, when concentrated, were consumed. It is suggested that large populations of bacteria in nature may be reduced in size by predatory protozoa, but many of the prey cells will not be eliminated.     

Rhizobium meliloti nodD genes mediate host-specific activation of nodABC.

To differentiate among the roles of the three nodD genes of Rhizobium meliloti 1021, we studied the activation of a nodC-lacZ fusion by each of the three nodD genes in response to root exudates from several R. meliloti host plants and in response to the flavone luteolin. We found (i) that the nodD1 and nodD2 products (NodD1 and NodD2) responded differently to root exudates from a variety of hosts, (ii) that NodD1 but not NodD2 responded to luteolin, (iii) that NodD2 functioned synergistically with NodD1 or NodD3, (iv) that NodD2 interfered with NodD1-mediated activation of nodC-lacZ in response to luteolin, and (v) that a region adjacent to and upstream of nodD2 was required for NodD2-mediated activation of nodC-lacZ. We also studied the ability of each of the three R. meliloti nodD genes to complement nodD mutations in R. trifolii and Rhizobium sp. strain NGR234. We found (i) that nodD1 complemented an R. trifolii nodD mutation but not a Rhizobium sp. strain NGR234 nodD1 mutation and (ii) that R. meliloti nodD2 or nodD3 plus R. meliloti syrM complemented the nodD mutations in both R. trifolii and Rhizobium sp. strain NGR234. Finally, we determined the nucleotide sequence of the R. meliloti nodD2 gene and found that R. meliloti NodD1 and NodD2 are highly homologous except in the C-terminal region. Our results support the hypothesis that R. meliloti utilizes the three copies of nodD to optimize the interaction with each of its legume hosts.     

The genomes of the family Rhizobiaceae: size, stability, and rarely cutting restriction endonucleases.

The lack of high-resolution genetic or physical maps for the family Rhizobiaceae limits our understanding of this agronomically important bacterial family. On the basis of statistical analyses of DNA sequences of the Rhizobiaceae and direct evaluation by pulsed-field agarose gel electrophoresis (PFE), five restriction endonucleases with AT-rich target sites were identified as the most rarely cutting: AseI (5'-ATTAAT-3'), DraI (5'-TTTAAA-3'), SpeI (5'-ACTAGT-3'), SspI (5'-AATAAT-3'), and XbaI (5'-TCTAGA-3'). We computed the sizes of the genomes of Bradyrhizobium japonicum USDA 424 and Rhizobium meliloti 1021 by adding the sizes of DNA fragments generated by SpeI digests. The genome sizes of R. meliloti 1021 and B. japonicum USDA 424 were 5,379 +/- 282.5 kb and 6,195 +/- 192.4 kb, respectively. We also compared the organization of the genomes of free-living and bacteroid forms of B. japonicum. No differences between the PFE-resolved genomic fingerprints of free-living and mature (35 days after inoculation) bacteroids of B. japonicum USDA 123 and USDA 122 were observed. Also, B. japonicum USDA 123 genomic fingerprints were unchanged after passage through nodules and after maintenance on a rich growth medium for 100 generations. We conclude that large-scale DNA rearrangements are not seen in mature bacteroids or during free-living growth on rich growth media under laboratory conditions.     

Isolation and characterization of an ndvB locus from Rhizobium fredii

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in beta-1,2-glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of beta-1,2-glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment-specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for beta-1,2-glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).     

Hyperreiterated DNA regions are conserved among Bradyrhizobium japonicum serocluster 123 strains.

We have identified and cloned two DNA regions which are highly reiterated in Bradyrhizobium japonicum serocluster 123 strains. While one of the reiterated DNA regions, pFR2503, is closely linked to the B. japonicum common and genotype-specific nodulation genes in strain USDA 424, the other, pMAP9, is located next to a Tn5 insertion site in a host-range extension mutant of B. japonicum USDA 438. The DNA cloned in pFR2503 and pMAP9 are reiterated 18 to 21 times, respectively, in the genomes of B. japonicum serocluster 123 strains. Gene probes from the reiterated regions share sequence homology, failed to hybridize (or hybridized poorly) to genomic DNA from other B. japonicum and Bradyrhizobium spp. strains, and did not hybridize to DNA from Rhizobium meliloti, Rhizobium fredii, Rhizobium leguminosarum biovars trifolii, phaseoli, and viceae, or Agrobacterium tumefacians. The restriction fragment length polymorphism hybridization profiles obtained by using these gene probes are useful for discriminating among serologically related B. japonicum serocluster 123 strains.     

Hyperreiterated DNA regions are conserved among Bradyrhizobium japonicum serocluster 123 strains.

We have identified and cloned two DNA regions which are highly reiterated in Bradyrhizobium japonicum serocluster 123 strains. While one of the reiterated DNA regions, pFR2503, is closely linked to the B. japonicum common and genotype-specific nodulation genes in strain USDA 424, the other, pMAP9, is located next to a Tn5 insertion site in a host-range extension mutant of B. japonicum USDA 438. The DNA cloned in pFR2503 and pMAP9 are reiterated 18 to 21 times, respectively, in the genomes of B. japonicum serocluster 123 strains. Gene probes from the reiterated regions share sequence homology, failed to hybridize (or hybridized poorly) to genomic DNA from other B. japonicum and Bradyrhizobium spp. strains, and did not hybridize to DNA from Rhizobium meliloti, Rhizobium fredii, Rhizobium leguminosarum biovars trifolii, phaseoli, and viceae, or Agrobacterium tumefacians. The restriction fragment length polymorphism hybridization profiles obtained by using these gene probes are useful for discriminating among serologically related B. japonicum serocluster 123 strains.     

B12 Coenzyme-dependent Ribonucleotide Reductase in Rhizobium Species and the Effects of Cobalt Deficiency on the Activity of the Enzyme

This investigation revealed that the ribonucleotide reductases in extracts of Rhizobium leguminosarum, R. trifolii, R. phaseoli, R. japonicum, and R. meliloti 3DOal (ineffective in nitrogen fixation) are dependent upon B(12) coenzyme for activity. Rhizobium and certain Lactobacillus species are the only two groups of organisms known to contain B(12) coenzyme-dependent ribonucleotide reductases. Extracts of cobalt-deficient R. meliloti cells assayed in the presence of optimum B(12) coenzyme showed a 5- to 10-fold greater ribonucleotide reductase activity than comparable extracts from cells grown on a complete medium. Furthermore, cobalt-deficient cells were abnormally elongated and contained reduced contents of deoxyribonucleic acid. The addition of purified deoxyribonucleosides to cobalt-deficient cultures of R. meliloti failed to alleviate deficiency symptoms.     

Phylogenetic analysis reveals gene conversions in multigene families of rhizobia

Gene families are an important and intrinsic trait of rhizobial species. These gene copies can participate in non-reciprocal recombination events, also called gene conversions. Gene conversion has diverse roles, but it is usually implicated in the evolution of multigene families. Here, we searched for gene conversions in multigene families of six representative rhizobial genomes. We identified 11 gene families with different numbers of copies, genome location and function in CFN42 and CIAT652 strains of Rhizobium etli, Rhizobium sp NGR234, Mesorhizobium loti MAFF303099, Sinorhizobium meliloti 1021, and Bradyrhizobium japonicum USDA110. Gene conversions were detected by phylogenetic inference in the nifD and nifK gene families in R. etli. Sequence analysis confirmed multiple gene conversions in these two gene families. We suggest that gene conversion events have an important role in homogenizing multigene families in rhizobia.     

Resistance of Rhizobium strains to phygon, spergon, and thiram.

Strains of Rhizobium meliloti, Rhizobium sp. nodulating cowpeas, and R. phaseoli derived from cultures susceptible to tetramethylthiuram disulfide (thiram), 2,3-dichloro-1,4-naphthoquinone (phygon), and 2,3,5,6-tetrachloro-p-benzoquinone (spergon), respectively, grew in the presence of high concentrations of the fungicides and converted them to products not toxic to the sensitive rhizobia. The results of chemical assays demonstrated that the pesticides were destroyed by the resistant bacteria but not by the susceptible parent rhizobia. Resting cells of thiram-metabolizing R. meliloti formed large quantities of dimethyldithiocarbamate, dimethylamine, and CS2 from the pesticide. The products were characterized by gas and thin-layer chromatography, colorimetric reactions, and ultraviolet spectrometry. Dimethylamine and CS2 were formed spontaneously from dimethyldithiocarbamate, but the yield was higher in the presence of R. meliloti. The phygon-resistant bacterium converted the fungicide to five metabolites and thereby rendered the chemical nontoxic to a test fungus. The resistant strain of R. phaseoli generated at least one organic product and released about one-third of the chlorine during its detoxication of spergon.     

Expression of the adenyl cyclase-encoding gene (cya) of Rhizobium meliloti F34: existence of two cya genes?

To gain insight into the role of cyclic AMP (cAMP) in Gram-negative soil bacteria, we have studied the expression of an adenyl cyclase-encoding gene 'cya' of Rhizobium meliloti F34. In both Escherichia coli and Bradyrhizobium japonicum, the gene is expressed from a promoter(s) contained on a 2.6-kb fragment of the cloned insert, which may indicate the presence of a functional 'cya' promoter or the coincidental presence of sequences which can function as promoters in these two species. The study of 'cya'-lac fusion activity in R. meliloti indicated that the 'cya' gene is not expressed at detectable levels and, thus, may not contribute to the modulation of cAMP levels under the growth conditions employed. R. meliloti strains carrying defined genomic mutations at the 'cya' locus were still capable of the synthesis of near wild-type levels of cAMP. These results suggest that the cloned 'cya' gene is not the key determinant responsible for cAMP synthesis under the culture conditions employed.     

Variation of clonal, mesquite-associated rhizobial and bradyrhizobial populations from surface and deep soils by symbiotic gene region restriction fragment length polymorphism and plasmid profile analysis

Genetic characteristics of 14 Rhizobium and 9 Bradyrhizobium mesquite (Prosopis glandulosa)-nodulating strains isolated from surface (0- to 0.5-m) and deep (4- to 6-m) rooting zones were determined in order to examine the hypothesis that surface- and deep-soil symbiont populations were related but had become genetically distinct during adaptation to contrasting soil conditions. To examine genetic diversity, Southern blots of PstI-digested genomic DNA were sequentially hybridized with the nodDABC region of Rhizobium meliloti, the Klebsiella pneumoniae nifHDK region encoding nitrogenase structural genes, and the chromosome-localized ndvB region of R. meliloti. Plasmid profile and host plant nodulation assays were also made. Isolates from mesquite nodulated beans and cowpeas but not alfalfa, clover, or soybeans. Mesquite was nodulated by diverse species of symbionts (R. meliloti, Rhizobium leguminosarum bv. phaseoli, and Parasponia bradyrhizobia). There were no differences within the groups of mesquite-associated rhizobia or bradyrhizobia in cross-inoculation response. The ndvB hybridization results showed the greatest genetic diversity among rhizobial strains. The pattern of ndvB-hybridizing fragments suggested that surface and deep strains were clonally related, but groups of related strains from each soil depth could be distinguished. Less variation was found with nifHDK and nodDABC probes. Large plasmids (>1,500 kb) were observed in all rhizobia and some bradyrhizobia. Profiles of plasmids of less than 1,000 kb were related to the soil depth and the genus of the symbiont. We suggest that interacting selection pressures for symbiotic competence and free-living survival, coupled with soil conditions that restrict genetic exchange between surface and deep-soil populations, led to the observed patterns of genetic diversity.