Evidence for plasmid- and chromosome-borne multiple nif genes in Rhizobium fredii

Rhizobium fredii is a fast-growing rhizobium isolated from the primitive Chinese soybean cultivar Peking and from the wild soybean Glycine soja. This rhizobium harbors nif genes on 150- to 200-megadalton plasmids. By passage on acridine orange plates, we obtained a mutant of R. fredii USDA 206 cured of the 197-megadalton plasmid (USDA 206C) which carries both nif and nod genes. This strain, however, has retained its symbiotic effectiveness. Probing EcoRI digests of wild-type and cured plasmid DNA with a 2.2-kilobase nif DH fragment from Rhizobium meliloti has shown four homologous fragments in the wild-type strain (4.2, 4.9, 10, and 11 kilobases) and two fragments in the cured strain (4.2 and 10 kilobases). EcoRI digests of total DNA show four major bands of homology (4.2, 4.9, 5.8, and 13 kilobases) in both the wild-type and cured strains. The presence of major bands of homology in the total DNA not present in the plasmid DNA indicated chromosomal nif genes. Probing of HindIII digests of total and plasmid DNA led to the same conclusion. Hybridization to the smaller plasmids of USDA 206 and USDA 206C showed the presence of nif genes on at least one of these plasmids, explaining the nif homology in the USDA 206C plasmid digests.     

Evidence for plasmid- and chromosome-borne multiple nif genes in Rhizobium fredii.

Rhizobium fredii is a fast-growing rhizobium isolated from the primitive Chinese soybean cultivar Peking and from the wild soybean Glycine soja. This rhizobium harbors nif genes on 150- to 200-megadalton plasmids. By passage on acridine orange plates, we obtained a mutant of R. fredii USDA 206 cured of the 197-megadalton plasmid (USDA 206C) which carries both nif and nod genes. This strain, however, has retained its symbiotic effectiveness. Probing EcoRI digests of wild-type and cured plasmid DNA with a 2.2-kilobase nif DH fragment from Rhizobium meliloti has shown four homologous fragments in the wild-type strain (4.2, 4.9, 10, and 11 kilobases) and two fragments in the cured strain (4.2 and 10 kilobases). EcoRI digests of total DNA show four major bands of homology (4.2, 4.9, 5.8, and 13 kilobases) in both the wild-type and cured strains. The presence of major bands of homology in the total DNA not present in the plasmid DNA indicated chromosomal nif genes. Probing of HindIII digests of total and plasmid DNA led to the same conclusion. Hybridization to the smaller plasmids of USDA 206 and USDA 206C showed the presence of nif genes on at least one of these plasmids, explaining the nif homology in the USDA 206C plasmid digests.     

Evidence for the late origin of introns in chloroplast genes from an evolutionary analysis of the genus Euglena.

The origin of present day introns is a subject of spirited debate. Any intron evolution theory must account for not only nuclear spliceosomal introns but also their antecedents. The evolution of group II introns is fundamental to this debate, since group II introns are the proposed progenitors of nuclear spliceosomal introns and are found in ancient genes from modern organisms. We have studied the evolution of chloroplast introns and twintrons (introns within introns) in the genus Euglena. Our hypothesis is that Euglena chloroplast introns arose late in the evolution of this lineage and that twintrons were formed by the insertion of one or more introns into existing introns. In the present study we find that 22 out of 26 introns surveyed in six different photosynthesis-related genes from the plastid DNA of Euglena gracilis are not present in one or more basally branching Euglena spp. These results are supportive of a late origin for Euglena chloroplast group II introns. The psbT gene in Euglena viridis, a basally branching Euglena species, contains a single intron in the identical position to a psbT twintron from E.gracilis, a derived species. The E.viridis intron, when compared with 99 other Euglena group II introns, is most similar to the external intron of the E.gracilis psbT twintron. Based on these data, the addition of introns to the ancestral psbT intron in the common ancester of E.viridis and E.gracilis gave rise to the psbT twintron in E.gracilis.     

Effects of disease resistance genes on Rhizobium symbiosis in an annual legume

Summary The evolution of disease resistance in plants may be constrained if genes conferring resistance to pathogens interfere with plant responses toward other, nonpathogenic organisms. To test for such effects, we compared symbiotic nitrogen fixation in Amphicarpaea bracteata plants that differed at a major locus controlling resistance to the pathogen Synchytrium decipiens. Both resistant and susceptible plant genotypes nodulated successfully and grew significantly better in the presence of Rhizobium, although growth enhancement by Rhizobium was altered by different levels of nitrate fertilization. Plants homozygous for disease resistance achieved 2% higher growth than susceptible homozygotes across all treatments, but this difference was not significant. Resistant and susceptible plant genotypes did not differ in the mean number of nodules formed per plant or in nodule diameter. However, there was highly significant variation among replicate families within each disease resistance category for both nodulation characteristics. These results imply that genetic variation exists among A. bracteata plants both for diease resistance and for traits affecting symbiotic nitrogen fixation. However, there were no evident pleiotropic effects of disease resistance genes on the plant-Rhizobium symbiosis.     

Evidence for organelle origin

A selection of evaluations from Faculty of 1000 covering the Methanococcus maripaludis genome; recombination in animal mitochondria; modulating cell cycle progression; gene silencing by microRNA directed methylation; yeast DNA breakage and repair.     

Flavone-enhanced accumulation and symbiosis-related biological activity of a diglycosyl diacylglycerol membrane glycolipid from Rhizobium leguminosarum biovar trifolii.

Rhizobium leguminosarum bv. trifolii is the bacterial symbiont which induces nitrogen-fixing root nodules on the leguminous host, white clover (Trifolium repens L.). In this plant-microbe interaction, the host plant excretes a flavone, 4',7-dihydroxyflavone (DHF), which activates expression of modulation genes, enabling the bacterial symbiont to elicit various symbiosis-related morphological changes in its roots. We have investigated the accumulation of a diglycosyl diacylglycerol (BF-7) in wild-type R. leguminosarum bv. trifolii ANU843 when grown with DHF and the biological activities of this glycolipid bacterial factor on host and nonhost legumes. In vivo labeling studies indicated that wild-type ANU843 cells accumulate BF-7 in response to DHF, and this flavone-enhanced alteration in membrane glycolipid composition was suppressed in isogenic nodA::Tn5 and nodD::Tn5 mutant derivatives. Seedling bioassays performed under microbiologically controlled conditions indicated that subnanomolar concentrations of purified BF-7 elicit various symbiosis-related morphological responses on white clover roots, including thick short roots, root hair deformation, and foci of cortical cell divisions. Roots of the nonhost legumes alfalfa and vetch were much less responsive to BF-7 at these low concentrations. A structurally distinct diglycosyl diacylglycerol did not induce these responses on white clover, indicating structural constraints in the biological activity of BF-7 on this legume host. In bioassays using aminoethoxyvinylglycine to suppress plant production of ethylene, BF-7 elicited a meristematic rather than collaroid type of mitogenic response in the root cortex of white clover. These results indicate an involvement of flavone-activated nod expression in membrane accumulation of BF-7 and a potent ability of this diglycosyl diacylglycerol glycolipid to perform as a bacterial factor enabling R. leguminosarum bv. trifolii to activate segments of its host's symbiotic program during early development of the root nodule symbiosis.     

Expression of Rhizobium meliloti nod genes in Rhizobium and Agrobacterium backgrounds.

Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.     

Evidence for an extra-renal origin of urinary prostaglandin E2 in healthy men

In order to verify the validity of the assumption that male urinary Prostaglandin (PG) E2 reflects its renal production, PGE2 and PGF2 alpha concentrations were measured by radioimmunoassay in the renal venous plasma (RVP) and urine (U) of 12 male and 4 female healthy volunteers. While women had a similar PGE2/PFG2 alpha ratio in RVP (0.59 +/- 0.18) and U (0.41 + 0.06), men and a significantly (P less than 0.05) higher ratio in U (1.43 +/- 1.72) as compared to RVP (0.54 +/- 0.16). This was largely due to considerably higher and more variable U-PGE1 concentrations (roughly 6 times higher than female values), despite almost identical RVP levels. The possibility of an increased U excretion of a cross-reacting member of the PG-system, as a cause of such apparently high PGE2-like immunoreactivity (LI), was ruled out by TLC characterization of PGE2-LI with three different anti-PGE1 sera. Thus, male U-PGE2 may variably reflect an extra-renal source, such as contamination with trace amounts of seminal fluid. It is concluded that, unless such a contamination can be monitored and corrected for, measurement of male U-PGE1 should be considered of questionable relevance to renal PG-synthesis.     

Galega orientalis is more diverse than Galega officinalis in Caucasus--whole-genome AFLP analysis and phylogenetics of symbiosis-related genes

Legume plants can obtain combined nitrogen for their growth in an efficient way through symbiosis with specific bacteria. The symbiosis between Rhizobium galegae and its host plant Galega is an interesting case where the plant species G. orientalis and G. officinalis form effective, nitrogen‐fixing, symbioses only with the appropriate rhizobial counterpart, R. galegae bv. orientalis and R. galegae bv. officinalis, respectively. The symbiotic properties of nitrogen‐fixing rhizobia are well studied, but more information is needed on the properties of the host plants. The Caucasus region in Eurasia has been identified as the gene centre (centre of origin) of G. orientalis, although both G. orientalis and G. officinalis can be found in this region. In this study, the diversity of these two Galega species in Caucasus was investigated to test the hypothesis that in this region G. orientalis is more diverse than G. officinalis. The amplified fragment length polymorphism fingerprinting performed here showed that the populations of G. orientalis and R. galegae bv. orientalis are more diverse than those of G. officinalis and R. galegae bv. officinalis, respectively. These results support the centre of origin status of Caucasus for G. orientalis at a genetic level. Analysis of the symbiosis‐related plant genes NORK and Nfr5 reveals remarkable diversity within the Nfr5 sequence, although no evidence of adaptive evolution could be found.     

Regulation of tryptophan genes in Rhizobium leguminosarum.

Twelve tryptophan auxotrophs of Rhizobium leguminosarum were characterized biochemically. They were grown in complex and minimal media with several carbon sources, in both limiting and excess tryptophan. Missing enzyme activities allowed assignment of all mutant to the trpE, trpD, trpB, or trpA gene, confirming earlier results with the same mutants (Johnston et al., Mol. Gen. Genet. 165:323-330, 1978). In regulatory experiments, only the first enzyme of the pathway, anthranilate synthase, responded (about 15-fold) to tryptophan excess or limitation.     

Rhizobium common nod genes are required for biofilm formation

In legume nitrogen-fixing symbioses, rhizobial nod genes are obligatory for initiating infection thread formation and root nodule development. Here we show that the common nod genes, nodD1ABC , whose products synthesize core Nod factor, a chitin-like oligomer, are also required for the establishment of the three-dimensional architecture of the biofilm of Sinorhizobium meliloti . Common nod gene mutants form a biofilm that is a monolayer. Moreover, adding Nod Factor antibody to S. meliloti cells inhibits biofilm formation, while chitinase treatment disrupts pre-formed biofilms. These results attest to the involvement of core Nod factor in rhizobial biofilm establishment. However, luteolin, the plant-derived inducer of S. meliloti 's nod genes, is not required for mature biofilm formation, although biofilm establishment is enhanced in the presence of this flavonoid inducer. Because biofilm formation is plant-inducer-independent and because all nodulating rhizobia, both alpha- and beta-proteobacteria have common nod genes, the role of core Nod factor in biofilm formation is likely to be an ancestral and evolutionarily conserved function of these genes.     

The origin of endosymbiotic nitrogen fixation by Rhizobium

It is proposed, that current Rhizobia, able to fix nitrogen symbolically, developed from a bacterium, that was the common ancestor of Rhizobium and Agrobacterium tumefaciens, by the acquisition of a small plasmid or a small part of a bacterial genome, carrying a large part (but not all) of the genes necessary for the expression of nitrogen fixation.     

Evidence for asthma susceptibility genes on chromosome 11 in an African-American population

Initial genome-wide scan data provided suggestive evidence for linkage of the asthma phenotype in African-American (AA), but not Caucasian, families to chromosome 11q markers (peak at D11S1985; LOD=2). To refine this region, mapping analysis of 91 AA families (51 multiplex families and 40 asthmatic case-parent trios) was performed with an additional 17 markers flanking the initial peak linkage marker. Multipoint analyses of the 51 multiplex families yielded significant evidence of linkage with a peak non-parametric linkage score of 4.38 at marker D11S1337 (map position 68.6 cM). Furthermore, family-based association and transmission disequilibrium tests conducted on all 91 families showed significant evidence of linkage in the presence of disequilibrium for several individual markers in this region. A putative susceptibility locus was estimated to be at map position 70.8 cM with a confidence interval spanning the linkage peak. Evidence from both linkage and association analyses suggest that this region of chromosome 11 contains one or more susceptibility genes for asthma in these AA families.     

Nucleotide sequence of Rhizobium meliloti nodulation genes

A Rhizobium meliloti DNA region, determining nodulation functions common in different Rhizobium species, has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicates three large open reading frames with the same polarity coding for three proteins of 196, 217 and 402 (or 426) amino acid residues, respectively. We suggest the existence of three nod genes on this region, which were designated as nodA, B and C, respectively. Comparison of the R. meliloti nodA, B, C nucleotide and amino acid sequences with those from R. leguminosarum, as reported in the accompanying paper, shows 69-72% homology, clearly demonstrating the high degree of conservation of common nod genes in these Rhizobium species.     

Multiresistance genes of Rhizobium etli CFN42

Multidrug efflux pumps of bacteria are involved in the resistance to various antibiotics and toxic compounds. In Rhizobium etli, a mutualistic symbiont of Phaseolus vulgaris (bean), genes resembling multidrug efflux pump genes were identified and designated rmrA and rmrB. rmrA was obtained after the screening of transposon-generated fusions that are inducible by bean-root released flavonoids. The predicted gene products of rmrAB shared significant homology to membrane fusion and major facilitator proteins, respectively. Mutants of rmrA formed on average 40% less nodules in bean, while mutants of rmrA and rmrB had enhanced sensitivity to phytoalexins, flavonoids, and salicylic acid, compared with the wild-type strain. Multidrug resistance genes emrAB from Escherichia coli complemented an rmrA mutant from R. etli for resistance to high concentrations of naringenin.     

Use of marker genes in competition studies of Rhizobium

Use of marker genes has several advantages in studying rhizobial competition compared to traditional approaches. Reporter genes such as the ß-glucuronidase gene (gusA) or a thermostable ß-glucosidase gene (celB) allow detection of rhizobial strains in nodules when they are still attached to the root system. Analysis is extremely simple, fast and permits a high data throughput. This detection technique is therefore highly suitable for the study of rhizobial competition and studies using gusA-marked strains of Rhizobium are presented. By making use of gusA and celB, differentially marked strains can be produced and distinguished easily on roots. The availability of two marker genes permits competition studies of two or more than two strains and analysis of dual nodule occupancy. As this methodology does not require sophisticated equipment, a GUS Gene Marking Kit was developed.     

Subnanomolar concentrations of membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii are fully capable of eliciting symbiosis-related responses on white clover

Axenic seedling bioassays were performed on white clover, vetch, and alfalfa to assess the variety and dose responses of biological activities exhibited by membrane chitolipooligosaccharides (CLOSs) from wild type Rhizobium leguminosarum bv. trifolii ANU843. Subnanomolar concentrations of CLOSs induced deformation of root hairs (Had) and increased the number of foci of cortical cell divisions (Ccd) in white clover, some of which developed into nodule meristems. In contrast, ANU843 CLOSs were unable to induce Had in alfalfa and required a 10⁴-fold higher threshold concentration to induce this response in vetch. Also, ANU843 CLOSs were not mitogenic on either of these non-host legumes. In addition, CLOS action also increased chitinase activity in white clover root exudate. Thus, the membrane CLOSs from wild type R. leguminosarum bv. trifolii are fully capable of eliciting various symbiosis-related responses in white clover in the same concentration range as extracellular CLOSs of other rhizobia on their respective legume hosts. These results and our earlier studies indicate that membrane CLOSs represent one of many different classes of bioactive metabolites made by R. leguminosarum bv. trifolii which elicit more intense symbiosis-related responses in white clover than in other legumes. Therefore, CLOSs evidently play an important role in symbiotic development, but they may not be the sole determinant of host-range in the Rhizobium-clover symbiosis.Abbreviations Ccd cortical cell division - CLOS chitolipooligosaccharide - Had root hair deformation