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Presence of Megaplasmids in Rhizobium tropici and Further Evidence of Differences between the Two R. tropici Subtypes

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Using a modified Eckhardt method, we visualized replicons larger than 1,000 kb in Rhizobium tropici strains belonging to both subgroup A and subgroup B. The megaplasmid of R. tropici CFN299 was characterized. This megaplasmid is different from a cointegrate of various plasmids and from the chromosome. Hybridization of Eckhardt blots of 15 R. tropici strains with fragments derived from the megaplasmids of the type strains of subgroups A and B revealed that the megaplasmids are subgroup specific.

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... Originally R. tropici were recovered from bean nodules from plants in acid soils in Colombia and Brazil (Martínez-Romero et al., 1991). Two types of R. tropici have been clearly distinguished (Table 2) that seem to be diverging lineages that share a common symbiotic plasmid (Geniaux et al., 1995; Martínez-Romero, 1996) but there are also R. tropici strains with intermediate characteristics which do not fall into either type A or type B (Hungria et al., 2000; Martínez-Romero, 1996; Mostasso et al., 2002). Type B strains are more diverse than type A strains when a similar number of strains were analyzed from each and a wide spectrum of symbiotic efficiency with bean has been found in R. tropici (Mostasso et al., 2002; Oliveira and Graham, 1990; Oliveira et al., 1998) with highly effective strains belonging either to type B (Aguilar et al., 2001; Montealegre et al., 1995), type A (S M Tsai, personal communication) or intermediate types (Mostasso et al., 2002). ...
... A B Colony morphology in YM medium wet, translucent opaque Colony morphology in PY medium flat, pearly white creamy Motility - + Growth in LB - + Growth at 37 • C - + Acid tolerance (pH 4.5) + + Tolerance to heavy metals and high salt concentration - + Induction of Nod factor by genistein + - Presence of a 200 kb plasmid (pb) + - Presence of teu genes (bean exudate uptake genes) in pSym + - Presence of teu genes in plasmid a + + Presence of exo genes in megaplasmid + + nifH gene hybridization band in EcoR1 digests 8 kb 8 kb Citrate synthase gene in pSym + + Glutamine synthetase II electromorphs a b a Data taken from Martínez-Romero et al. (1991); Pardo et al. (1994); Geniaux et al. (1995); Rosenblueth et al. (1998); Priefer et al. (2001); Taboada et al. 1996; ...
Article
Common bean (Phaseolus vulgaris) has become a cosmopolitan crop, but was originally domesticated in the Americas and has been grown in Latin America for several thousand years. Consequently an enormous diversity of bean nodulating bacteria have developed and in the centers of origin the predominant species in bean nodules is R. etli. In some areas of Latin America, inoculation, which normally promotes nodulation and nitrogen fixation is hampered by the prevalence of native strains. Many other species in addition to R. etli have been found in bean nodules in regions where bean has been introduced. Some of these species such as R. leguminosarum bv. phaseoli, R. gallicum bv. phaseoli and R. giardinii bv. phaseoli might have arisen by acquiring the phaseoli plasmid from R. etli. Others, like R. tropici, are well adapted to acid soils and high temperatures and are good inoculants for bean under these conditions. The large number of rhizobia species capable of nodulating bean supports that bean is a promiscuous host and a diversity of bean-rhizobia interactions exists. Large ranges of dinitrogen fixing capabilities have been documented among bean cultivars and commercial beans have the lowest values among legume crops. Knowledge on bean symbiosis is still incipient but could help to improve bean biological nitrogen fixation.
... (Martínez et al., 1985; Piñero et al., 1988), but that two distinct groups could be distinguished (Martínez et al., 1988). Further characterization led us to propose that these two groups be classified as novel Rhizobium species: R. etli (Segovia et al., 1993), and R. tropici (Martínez-Romero et al., 1991) with two sub-types (Geniaux et al., 1995; Martínez-Romero, 1994). R. etli was the Rhizobium most commonly isolated from bean nodules in Mesoamerica, while R. tropici was obtained from South American acid-soil regions. ...
... ( Martínez et al., 1985;Piñero et al., 1988), but that two distinct groups could be distinguished ( Martínez et al., 1988). Further characterization led us to propose that these two groups be classified as novel Rhizobium species: R. etli , and R. tropici (Martínez-Romero et al., 1991) with two sub-types (Geniaux et al., 1995;Martínez-Romero, 1994). R. etli was the Rhizobium most commonly isolated from bean nodules in Mesoamerica, while R. tropici was obtained from South American acid-soil regions. ...
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Rhizobium etli and R. tropici form nitrogen-fixing nodules on Phaseolus vulgaris (common bean). In the hope that R. etli strains with additional citrate synthase genes have better carbon economies, merodiploid strains were constructed. Previously, one such construct was shown to have an increased nodulation capacity in the standard bean cultivar Negro Xamapa. In the present work, derivatives from different R. etli strains carrying the R. tropici plasmid-borne or chromosomal citrate synthase gene were constructed and tested for nodulation in bean cultivars selected for their high capacity to fix nitrogen. Nodule numbers were dependent on the strain and the cultivar used. Differences in nodule number were not reflected in plant biomass.
... When we tried to clone the R. tropici CFN299 symbiotic plasmid (pSym) of approximately 600 kb (8), only fragments, in the range of 200-500 kb, were obtained (Figure 2A, lanes 7-9, 11, and 12). The regions obtained were overlapping and covered most of the pSym of R. tropici CFN299, as tested by PCR and sequence analyses of several genetic markers. ...
... Genome-wide expression profiling using cDNA microarrays or oligonucleotide microarrays (GeneChips; Affymetrix, Santa Clara, CA, USA) has become an important tool in biomedical studies (13,18,23). Expression data have been obtained from a wide variety of samples including bacteria, fungi, mammalian cells, and tissues (8,(19)(20)(21)(22)24). The application of array technology generally requires the isolation of RNA from millions of cells containing micrograms to milligrams of total RNA. ...
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We have developed a simple system to clone indigenous Rhizobium plasmids into E. coli. The strategy consists of three matings: the first is to insert Tn5 in the plasmid to be cloned, the second incorporates the integrative vector into the inserted Tn5 in the native Rhizobium plasmid, and the last mating transfers the target plasmid directly into E. coli. This mating-based system was successfully used to clone plasmids of Rhizobium species with sizes ranging from 150 to 270 kb. In addition, a 500-kb fragment of a 600-kb megaplasmid was also cloned. This strategy could be used for cloning indigenous replicons of other gram-negative bacteria into a different host.
... Rhizobium strains from Mimosa caesalpiniaefolia were isolated by Campelo & Dobereiner (1969). Extensive isolations were made by $ Plasmid patterns were detected in Eckhardt gels (0.75 YO agarose) and the sizes of the plasmids were estimated from their migration distances using the computer program SEQAID 11 version 3.5 (Rhoads & Roufa, 1989) and the plasmids of R. etli CFN42 (Romero et al., 1991) and R. tropici CFN299 (Geniaux et al., 1995; Martinez et al., 1987) as molecular size standards. The symbiotic plasmids identified by nqgene probing are marked in bold. ...
... Linkage disequilibrium was calculated as described previously (Souza et al., 1992).Hynes & McGregor, 1990). Plasmids of R. etli CFN42 (Romero et al., 1991) and R. tropici CFN299 (Geniaux et al., 1995; Martinez et at., 1987) were used as molecular size standards and as positive controls for hybridization analyses. The electrophoretic plasmid patterns were hybridized as described previously (Wang et al., 1998) to the following probes labelled with [32P] dCTP (Amersham ) : a 600 bp internal SalI fragment of n f H from pEM 15 (Morett et al., 1988), a 5.6 kbp Hind111 fragment from pH3 containing the replicator region of CFN42 plasmid d (Romero et al., 1997), a PCR-amplified internal lipopolysaccharide gene (Lpspl) fragment (250 bp) from pAGSlO (Garcia-de 10s Santos & Brom, 1997) and pAGS4, in which a 0.718 kbp internal fragment of IpsP2 was cloned (Garcia-de 10s Santos & Brom, 1997) (Table 2). ...
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Fifty rhizobial isolates from root nodules of Mimosa affinis, a small leguminous plant native to Mexico, were identified as Rhizobium etli on the basis of the results of PCR-RFLP and RFLP analyses of small-subunit rRNA genes, multilocus enzyme electrophoresis and DNA-DNA homology. They are, however, a restricted group of lineages with low genetic diversity within the species. The isolates from M. affinis differed-from the R. etli strains that orginated from bean plants (Phaseolus vulgaris) in the size and replicator region of the symbiotic plasmid and in symbiotic-plasmid-borne traits such as nifH gene sequence and organization, melanin production and host specificity. A new biovar, bv. mimosae, is proposed within R. etli to encompass Rhizobium isolates obtained from M. affinis. The strains from common bean plants have been designated previously as R. etli bv. phaseoli. Strains of both R. etli biovars could nodulate P. vulgaris, but only those of bv. mimosae could form nitrogen-fixing nodules on Leucaena leucocephala.
... También, R. leguminosarum bv. phaseoli tipo II ha sido reclasificado como Rhizobium tropici tipos A y B, que son portadores de una única copia del gen nifH, y que se distinguen entre sí por los distintos valores de hibridación ADN- ADN, características fenotípicas y la presencia de megaplásmidos específicos (Martínez-Romero et al., 1991; Geniaux et al., 1995). De este modo se establecen dos nuevas especies, R. tropici y R. etli, y se mantiene la biovariedad phaseoli, que incluye a las cepas no americanas que nodulan Phaseolus vulgaris. ...
... Sin embargo, un análisis más pormenorizado pone de manifiesto que tanto la secuencia de R. tropici tipo A (X67234), como la de la cepa P2-13 poseen un " inserto " de 75 nucleótidos (Martínez-Romero et al., 1991; Willems y Collins, 1993) (Fig. IV.5) que las hace muy diferentes a las otras cuatro secuencias aquí comparadas. Por ésta y otras razones, como son el bajo porcentaje de hibridación ADN-ADN, alrededor de un 36% (Martínez-Romero y Caballero-Mellado, 1991), y las diferencias en las secuencias de los megaplásmidos, se ha propuesto que R. tropici tipos A y B sean incluidas en especies diferentes (Geniaux et al., 1995). Sin embargo, la existencia de aislados con características intermedias entre los tipos A y B de R. tropici (Martínez-Romero, 1996; Mostasso et al., 2002) parece ir en contra de esta división (Acosta-Durán y Romero, 2002). ...
Article
La alubia, Phaseolus vulgaris, es un cultivo de gran importancia en la Península Ibérica, lo que la convierte en un modelo muy adecuado para el estudio de poblaciones endófitas en distintos suelos. En este estudio se han elegido una serie de suelos en el Noroeste de Portugal, que presentan la particularidad de ser suelos de montaña que han sido sometidos a técnicas de cultivo tradicional, sin abonos químicos, durante muchísimos años, y sobre los que se ha cultivado alubias de una variedad local de generación en generación. Se han aislado microorganismos endosimbiontes a partir de nódulos de plantas de Phaseolus vulgaris, y se ha realizado la caracterización polifásica de las cepas aisladas mediante la utilización de métodos fenotípicos y moleculares. Los resultados más sobresaliente fueron: Se aislaron 186 cepas de microorganismos endófitos de Phaseolus vulgaris, de las cuales 180 fueron capaces de producir nódulos efectivos y el resto fueron capaces de penetrar en sus raíces sin originar estructuras diferenciadas. La caracterización genotípica y fenotípica permitió la identificación de todas las cepas aisladas a nivel de especie y/o subespecie. De acuerdo con los resultados obtenidos, el 82% de las cepas fueron identificadas como Rhizobium rhizogenes, el 15% como Rhizobium tropici tipo A y el 3% como cepas del género Herbaspirillum. Por primera vez se aislaron cepas de R. rhizogenes (Agrobacterium rhizogenes) capaces de formar nódulos en plantas de Phaseolus vulgaris. De acuerdo con los resultados de los perfiles de TP-RAPD dentro de la especie Rhizobium rhizogenes puede haber más de una subespecie, hecho que será necesario comprobar en posteriores estudios. Se ha demostrado que existen cepas de Rhizobium tropici tipo A en la Península Ibérica y que, además, estas cepas portan en el gen 16S el mismo “inserto” que las cepas americanas. Se ha caracterizado e identificado una nueva especie del género Herbaspirillum que se ha denominado Herbaspirillum lusitanum. La especie Herbaspirillum lusitanum es la primera que se describe como endófito de leguminosas. The common bean, Phaseolus vulgaris, is a staple grain-legume in the Iberian Peninsula and, therefore, a very appropriate model for studying the soil bacterial endophytic populations. In this study, different mountain soils from the north-west of Portugal were chosen because they have been cultivated following traditional agronomical practices from the antiquity, avoiding the use of chemical fertilizers, and where a local genotype of of Phaseolus has been recurrently planted. Endosymbiont microorganisms were isolated from nodules of Phaseolus vulgaris plants, and their polyphasic characterization performed by using phenotypic and genotypic methods. The main results were: From a total of 186 isolates, 180 formed effective root nodules in Phaseolus vulgaris plants. The remainder six strains were able to infect Phaseolus vulgaris roots but they did not form any differentiated structure. Their genotypic and phenotypic characterization allowed to identify all these isolates at a taxonomic level of species and/or subspecies. From the 186 isolates, the 82% was identified as Rhizobium rhizogenes, 15 % as Rhizobium tropici type A, and the remaining 3 % as members of the genus Herbaspirillum. To the best of our knowledge, strains of R. rhizogenes (Agrobacterium rhizogenes) able to nodulate Phaseolus vulgaris plants were isolated by the first time. The TP-RAPD profiles indicated that there could be more than one subespecies of Rhizobium rhizogenes, although more detailled studies should be done to verify that result. It has been demonstrated that Rhizobium tropici type A strains can be found in soils from the Iberian Peninsula. These strains showed a “insert” in its 16S rRNA gen that is identical to that found in strains from American origen. It has been characterized and identified a new species of the genus Herbaspirillum designated as Herbaspirillum lusitanum. To our knowledge, this is the first report on the undoubted association of a bacterium of the genus Herbaspirillum with a leguminous plant. Peer reviewed
... One, which contains ribosomal gene sequences, corresponds to the chromosome; another corresponds to the well-characterized symbiotic plasmid; and the other is a megaplasmid with a very high molecular mass. Megaplasmids (Ͼ1,000 kb) have been observed in several Rhizobium species, such as R. meliloti (1,8,16), R. fredii (11), R. galegae (13,19), and R. tropici (7). Such replicons have been classified as plasmids due to the absence of rDNA; however, they might contain genes essential for the survival of the organism. ...
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Rhizobium sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234a, a megaplasmid (pNGR234b), and the chromosome. Symbiotic gene sequences not present in pNGR234a were analyzed by hybridization. DNA sequences homologous to the genes fixLJKNOPQGHIS were found on the chromosome, while sequences homologous to nodPQ and exoBDFLK were found on pNGR234b.
... Asimismo, se detectó un plásmido de aproximadamente 279Kb en la cepa tipo B (R. tropici CiAT 899), mediante la técnica de Casse et al. (1979) (Fig. 3), la cual nos permite identificar plásmidos cercanos a los 300 Kb. Sin embargo, en los 2 subgrupos A y B de R. tropici, CFN299 y CiAT 899 respectivamente, ha sido visualizado mediante el método de Eckhardt (1978), un megaplásmido mayor de 1000Kb no homólogo (subgrupo específico) (Geniaux et al. 1995). De igual manera se ha evidenciado un plásmido de 200Kb en la cepa tipo A (CFN 299), pero no en la tipo B (CiAT 899) (Martínez-Romero 2003). ...
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Phenotypic and genotypic characterization of twelve rhizobial isolates from different regions of Venezuela. Rhizobial taxonomy and systematics have progressed substantially, nevertheless, few studies have been developed on venezuelan species. This study evaluated the phenotypic and genetic variation between 12 venezuelan indigenous rhizobial isolates and 10 international referential strains, by phenotypical traits and DNA molecular markers. in this regard, a PCR-RFLP of the 16S rDNA gene, the presence of large plasmids, metabolic assays in solid media, salinity resistance, pH and temperature growth conditions, and intrinsic antibiotic resis-tance were assayed. in reference to the phenotypic attributes, we recognized three main groups: A group i, which comprised all the strains metabolizing between 67.5%-90% of the C and N sources. They were also acid-tolerant, as well as acid producers, capable of growing at 40ºC and in high salinity conditions (2-2.5% NaCl). With regard to the antibiotic sensitivity, this group was susceptible to a 30% of the antibiotic assayed. Strains belonging to Group ii exhibited a lower salt tolerance (0.1-1.5%NaCl), as well as a lower acid tolerance, since they grew well at pH values equal or higher than 5.0. This group appeared to be resistant to all of the antibiotics assayed and only metabolized between 52.5%-82.5% of the C and N sources. Group iii was represented by a single bacte-rial strain: it has a extremely low salt tolerance (0.1% NaCl). This strain grew at a pH equal or higher than 5.6, was susceptible to 50% of the antibiotics assayed and metabolized 72% of the C and N sources. On the basis of a PCR-RFLP of the 16S rDNA, three groups were also obtained. Members of the group A showed a close resemblance to Rhizobium tropici CiAT 899 and Sinorhizobium americanum CFN-Ei 156, while Group B was closely related to Bradyrhizobium spp. Group C, was also represented by only one isolate. The Trebol isolate, was the only one strain able to form nodules and does not appear to be related to any of the referential rhizobial strains, suggesting a possible symbiotic horizontal gene transfer. Finally, in this work, there are evidences of a genetic diversity in the venezuelan rhizobial strains. A different geographical origin is perhaps an important factor affecting the diversity of the indigenous rhizobia in this study. Rev. Biol. Trop. 59 (3): 1017-1036. Epub 2011 September 01. Las bacterias denominadas comúnmen-te rizobios presentan varias formas de vida; pueden comportarse como saprófitos en el suelo establecer una asociación simbiótica y formar nódulos con las raíces y tallos de las leguminosas, donde tiene lugar la reducción del nitrógeno atmosférico en amonio, el cual es transportado a la planta y convertido en biomoléculas esenciales, o bien estar presentes como endófito en raíces de diferentes especies vegetales, donde ejercen efectos promotores del crecimiento (Wang et al. 2001). La taxonomía rizobiana basada tanto en técnicas moleculares como en las aproxima-ciones polifásicas ha permitido reconocer seis géneros hasta la actualidad, los cuales son: Allorhizobium, Mesorhizobium, Azorhizobium, Sinorhizobium, Bradyrhizobium y Rhizobium
... The plasmids may carry genes encoding traits beneficial for the organism's survival, for example, resistance to antibiotics or to heavy metals. Usually the plasmid size is relatively small but in some cases plasmids are comparable in size to the chromosome, like the megaplasmid in Rhizobium tropici (Geniaux et al., 1995). ...
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Background: The genus Burkholderia consists of species that occupy remarkably diverse ecological niches. Its best known members are important pathogens, B. mallei and B. pseudomallei, which cause glanders and melioidosis, respectively. Burkholderia genomes are unusual due to their multichromosomal organization. Results: We performed pan-genome analysis of 127 Burkholderia strains. The pan-genome is open with the saturation to be reached between 86,000 and 88,000 genes. The reconstructed rearrangements indicate a strong avoidance of intra-replichore inversions that is likely caused by selection against the transfer of large groups of genes between the leading and the lagging strands. Translocated genes also tend to retain their position in the leading or the lagging strand, and this selection is stronger for large syntenies. We detected parallel inversions in the second chromosomes of seven B. pseudomallei. Breakpoints of these inversions are formed by genes encoding components of multidrug resistance complex. The membrane components of this system are exposed to the host's immune system, and hence these inversions may be linked to a phase variation mechanism. We identified 197 genes evolving under positive selection. We found seventeen genes evolving under positive selection on individual branches; most of the positive selection periods map to the branches that are ancestral to species clades. This might indicate rapid adaptation to new ecological niches during species formation. Conclusions: This study demonstrates the power of integrated analysis of pan-genomes, chromosome rearrangements, and selection regimes. Non-random inversion patterns indicate selective pressure, inversions are particularly frequent in a recent pathogen B. mallei, and, together with periods of positive selection at other branches, may indicate adaptation to new niches. One such adaptation could be possible phase variation mechanism in B. pseudomallei.
... . Strains of R. tropici have been divided into two groups, A and B, based on multilocus enzyme electro-Rhisobium tropici induces the formation of nitrogen-phoresis, DNA-DNA hybridization, 16s rRNA gene fixing nodules on the roots of plants belonging to several sequences, plasmid profiles and an analysis of phenogenera of tropical legumes, including Phaseolus, types (Geniaux et al., 1995 ;Martinez-Romero et al., Leucaena and Macroptilium (Martinez-Romero et al., 1991). CIAT899 (the type strain of R. tropici) can form colonies on acidic solid medium (pH 4.3, tolerates 50p.p.m. ...
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Type B strains of Rhizobium tropici induce severe foliar chlorosis when applied at planting to seeds of symbiotic host and non-host dicotyledonous plants. A Tn5-induced mutant, designated CT4812, or R. tropici strain CIAT899 that was unable to induce chlorosis was isolated. Cloning and sequencing of the DNA flanking the transposon in CT4812 revealed that the Tn5 insertion is located in a gene similar to glnD, which encodes uridylyltransferase/uridylyl-removing enzyme in enteric bacteria. Two marker-exchange mutants with insertions in glnD also failed to induce chlorosis in bean (Phaseolus vulgaris) plants. The 5'-most insertion in glnD (in mutant strain ME330) abolished the ability of R. tropici to utilize nitrate as a sole carbon source, whereas a mutation in glnD further downstream (in mutant strain ME245) did not have an obvious effect on nitrate utilization. A gene similar to the Salmonella typhimurium virulence gene mviN overlaps the 3' end of the R. tropici glnD homologue. A mutation in mviN had no effect on the ability of CIAT899 to induce chlorosis in bean plants. Therefore the glnD homologue, but not mviN, appears to be required for induction of chlorosis in plants by R. tropici strain CIAT899. A high nitrogen: carbon ratio in the rhizosphere of bean plants also prevented R. tropici from inducing chlorosis in bean plants. Mutations in either the glnD homologue or mviN had no significant effect on root nodule formation or acetylene reduction activity. A mutation in mviN eliminated motility in R. tropici. The sequence data, the inability of the glnD mutant to utilize nitrate, and the role of the R. tropici glnD gene in chlorosis induction in plants, a process that is nitrogen regulated, suggest that glnD plays a role in nitrogen sensing in R. tropici as its homologues do in other organisms.
... Plasmid mobilities determined in 0.7% agarose gels were used for estimating the approximate molecular sizes of plasmids with the computer program Seqaid I1 version 3.5 (Rhoads & Roufa, 1989). The plasmids of R. etli CFN 42T (Romero et al., 1991) and of R. tropici CFN 299 (Geniaux et al., 1995; Martinez et al., 1987) were used as reference molecular size markers. ...
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Fifty-five Chinese isolates from nodules of Amorpha fruticosa were characterized and compared with the type strains of the species and genera of bacteria which form nitrogen-fixing symbioses with leguminous host plants. A polyphasic approach, which included RFLP of PCR-amplified 16S rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 16S rRNA gene sequencing, electrophoretic plasmid profiles, cross-nodulation and a phenotypic study, was used in the comparative analysis. The isolates originated from several different sites in China and they varied in their phenotypic and genetic characteristics. The majority of the isolates had moderate to slow growth rates, produced acid on YMA and harboured a 930 kb symbiotic plasmid (pSym). Five different RFLP patterns were identified among the 16S rRNA genes of all the isolates. Isolates grouped by PCR-RFLP of the 16S rRNA genes were also separated into groups by variation in MLEE profiles and by DNA-DNA hybridization. A representative isolate from each of these DNA homology groups had a separate position in a phylogenetic tree as determined from sequencing analysis of the 16S rRNA genes. A new species, Mesorhizobium amorphae, is proposed for the majority of the isolates, which belonged to a moderately slow- to slow-growing, acid-producing group based upon their distinct phylogenetic position, their unique electrophoretic type, their low DNA homology with reference strains representing the species within the genus Mesorhizobium and their distinct phenotypic features. Strain ACCC 19665 was chosen as the type strain for M. amorphae sp. nov.
... No hybridization to a 16S rDNA probe was observed with the plasmids or megaplasmids of the isolates analysed (not shown). This is similar to other megaplasmids in Rhizobium (Geniaux et al. 1995), but not in Brucella (Michaux et al. 1993;Humas-Bilak et al. 1998). Symbiotic plasmids were identified in some isolates by hybridization to the nifH and nodDAB genes (Table 1). ...
Article
Leucaena species are leguminous plants native to Mexico. Using two L. leucocephala cultivars grown in different soils, we obtained 150 isolates from the nodules. Twelve rDNA types were identified which clustered into groups corresponding to Mesorhizobium, Rhizobium, and Sinorhizobium by restriction fragment length polymorphism (RFLP) of amplified 16S rRNA genes. Types 2, 4, 5, 6, 10, 11, and 12 were distinct from all the defined species. Others had patterns indistinguishable from some recognized species. Most of the isolates corresponded to Sinorhizobium. Forty-one electrophoretic types (ETs) were identified among the isolates based on the different combinations of electrophoretic patterns of 13 metabolic enzymes. ETs were clustered into groups in general agreement with the rDNA types. Diverse plasmid patterns were obtained among the isolates, but common plasmids were observed among most isolates within rDNA types 5, 10, and 11. The symbiotic plasmids were identified among most of the isolates, except for the Mesorhizobium isolates. The affinities of host cultivars for different rhizobial groups and the impact of soil cultivation on the soil populations of rhizobia were analysed from the estimation of isolation frequencies and diversity. The results showed differences in rhizobial populations in cultivated and uncultivated soils and also differences in rhizobia trapped by L. leucocephala cv. Cunningham or Peruvian.
... Plasmid patterns, visualized with a modified Eckhard procedure (Hynes & McGregor 1990), were different among different strains even within the same novel species (data not shown) and this further confirmed that the strains within each novel species were not siblings. All strains tested showed megaplasmids of~1700 bp (not shown), as do other strains from the 'tropici' group (Geniaux et al., 1995). ...
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Calliandra grandiflora has been used as a medicinal plant for thousands of years in Mexico. Rhizobial strains were obtained from root nodules of C. grandiflora collected from different geographical regions in Chiapas and characterized by BOX-PCR, ARDRA and 16S rRNA gene sequences. Most isolates corresponded to Rhizobium and those not related to described species were further characterized by recA, atpD, rpoB and nifH gene phylogenies, phenotypic and DNA-DNA hybridization analyses. Three new, related Rhizobium species within the 'Rhizobium tropici group' share the same symbiovar that may be named sv. calliandrae. The type strains are for Rhizobium calliandrae CCGE524T (=ATCC BAA-2435T =CIP110456T =LBP2-1T), Rhizobium mayense CCGE526T (=ATCC BAA-2446T =CIP110454T =NSJP1-1T) and Rhizobium jaguaris CCGE525T (=ATCC BAA-2445T =CIP110453T =SJP1-2T).
... The 3,837,060-bp chromosome contained all three ribosomal operons. The plasmid sizes were in general agreement with previously reported estimates based on Eckhardt gels [31], except for the 2,083,197-bp megaplasmid (pRtrCIAT899c) which was somewhat larger than expected. The second largest replicon (549,467 bp) was the symbiotic plasmid (pSym). ...
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Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean and other legume hosts.
... Asimismo, se detectó un plásmido de aproximadamente 279Kb en la cepa tipo B (R. tropici CiAT 899), mediante la técnica de Casse et al. (1979) (Fig. 3), la cual nos permite identificar plásmidos cercanos a los 300 Kb. Sin embargo, en los 2 subgrupos A y B de R. tropici, CFN299 y CiAT 899 respectivamente, ha sido visualizado mediante el método de Eckhardt (1978), un megaplásmido mayor de 1000Kb no homólogo (subgrupo específico) (Geniaux et al. 1995). De igual manera se ha evidenciado un plásmido de 200Kb en la cepa tipo A (CFN 299), pero no en la tipo B (CiAT 899) (Martínez-Romero 2003). ...
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Rhizobial taxonomy and systematics have progressed substantially, nevertheless, few studies have been developed on venezuelan species. This study evaluated the phenotypic and genetic variation between 12 venezuelan indigenous rhizobial isolates and 10 international referential strains, by phenotypical traits and DNA molecular markers. In this regard, a PCR-RFLP of the 16S rDNA gene, the presence of large plasmids, metabolic assays in solid media, salinity resistance, pH and temperature growth conditions, and intrinsic antibiotic resistance were assayed. In reference to the phenotypic attributes, we recognized three main groups: A group I, which comprised all the strains metabolizing between 67.5%-90% of the C and N sources. They were also acid-tolerant, as well as acid producers, capable of growing at 40 degrees C and in high salinity conditions (2-2.5% NaCl). With regard to the antibiotic sensitivity, this group was susceptible to a 30% of the antibiotic assayed. Strains belonging to Group II exhibited a lower salt tolerance (0.1-1.5%NaCl), as well as a lower acid tolerance, since they grew well at pH values equal or higher than 5.0. This group appeared to be resistant to all of the antibiotics assayed and only metabolized between 52.5%-82.5% of the C and N sources. Group III was represented by a single bacterial strain: it has a extremely low salt tolerance (0.1% NaCl). This strain grew at a pH equal or higher than 5.6, was susceptible to 50% of the antibiotics assayed and metabolized 72% of the C and N sources. On the basis of a PCR- RFLP of the 16S rDNA, three groups were also obtained. Members of the group A showed a close resemblance to Rhizobium tropici CIAT 899 and Sinorhizobium americanum CFN-EI 156, while Group B was closely related to Bradyrhizobium spp. Group C, was also represented by only one isolate. The Trebol isolate, was the only one strain able to form nodules and does not appear to be related to any of the referential rhizobial strains, suggesting a possible symbiotic horizontal gene transfer. Finally, in this work, there are evidences of a genetic diversity in the venezuelan rhizobial strains. A different geographical origin is perhaps an important factor affecting the diversity of the indigenous rhizobia in this study.
... Both chromosomes from each Brucella strain hybridized to the 16S rRNA gene probe, while only the larger one hybridized to the citrate synthase gene (not shown). The megaplasmid of R. tropici, which is similar in size to the smaller chromosome of B. suis bv. 2 and 4, did not hybridize to the homologous 16S rRNA DNA gene probe as was reported previously (11). ...
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Multilocus enzyme electrophoresis (MLEE) of 99 Brucellaisolates, including the type strains from all recognized species, revealed a very limited genetic diversity and supports the proposal of a monospecific genus. In MLEE-derived dendrograms, Brucella abortus and a marine Brucella sp. grouped into a single electrophoretic type related to Brucella neotomaeand Brucella ovis. Brucella suis andBrucella canis formed another cluster linked toBrucella melitensis and related to Rhizobium tropici. The Brucella strains tested that were representatives of the six electrophoretic types had the same rRNA gene restriction fragment length polymorphism patterns and identical ribotypes. All 99 isolates had similar chromosome profiles as revealed by the Eckhardt procedure.
... GenBank accession numbers are given in Section 2. The tree was generated using MEGA version 3.1 with default parameters, K2P distance model and the Neighbor-Joining algorithm. (Martı´nez-Romero et al, 1991;Laguerre et al. 1994;Geniaux et al., 1995). Among several intrinsic characteristics of type A group, Willems and Collins (1993) and van Berkum et al. (1994) reported an insertion of 72 nucleotides in the 16S rRNA genes, although later Laguerre et al. (1994) found that not all strains shared this characteristic. ...
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Common bean (Phaseolus vulgaris) is native to the Americas, and Rhizobium etli is the dominant microsymbiont in both the Mesoamerican and the Andean centers of genetic diversification. Wild common beans are not found in Brazil, although the legume has been cropped in the country throughout time and all but one of the rhizobial species that nodulate it (Rhizobium gallicum) have been broadly detected in Brazilian soils. However, the majority of the effective rhizobial strains isolated so far from field-grown plants belong to R. tropici. This study describes the analysis of symbiotic and non-symbiotic genes of 15 effective R. tropici strains, isolated from four geographically distant regions in Brazil. With RFLP-PCR of the 16S and 23S rRNA genes and sequence analysis of 16S rRNA, two clusters were observed, one related to R. tropici type A and another to type B strains. Diversity in ribosomal genes was high, indicating that type A strains might represent a new species. High intraspecies diversity was also observed in the rep-PCR analysis with BOX, ERIC and REP primers. However, in the RFLP-PCR analysis of nifH and nodC genes, all R. tropici showed unique combinations of profiles, which might reflect an evolutionary strategy to maximize N2 fixation.
... It is worth mentioning that Rhizobium tropici type A and type B strains have DNA-DNA hybridization values of around 36% (Martínez-Romero et al. 1991). For this reason and due to their differences in megaplasmid sequences, it has been proposed that type A and type B strains are different species (Geniaux et al. 1995), but the existence of strains Table 1 Patterns of 16S rDNA of selected isolates from Gliricidia sepium nodules and reference strains. The different letters represent the patterns of the PCR products obtained respectively with restriction enzymes (MspI, HinfI, HhaI, RsaI, DdeI), as described by Wang et al. (1999) following the techniques of Laguerre et al. (1994) 16S rDNA patterns Mostasso et al. 2002) argues against this proposal. ...
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The Rhizobium species that nodulate the legume tree Gliricidia sepium were analyzed by phenotypic characteristics (including nodule formation in different hosts), PCR-RFLP patterns and sequences of 16S rRNA genes, multilocus enzyme electrophoresis, and plasmid patterns. Strains of Rhizobium tropici type A and B, Sinorhizobium spp., and Rhizobium etli bv. phaseoli were encountered in G. sepium nodules and their presence depended on the site sampled.
... strain OR191 and R. etli could not be distinguished by the results of isoelectric focusing (Fig. 1C). It has been observed previously that the differences between the two R. tropici types could be enough to reclassify them as different subspecies or even as different species (23). Our results, which show that the type A strain GSII is different from the GSII found in type B strains, support the subdivision of R. tropici. ...
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Different Rhizobium species may be identified by using polymorphisms in their glutamine synthetases (GSII) but not by their GSI profiles. We analyzed the GSs of various Rhizobium tropici and Rhizobium etli strains (which are capable of nodulating and fixing nitrogen in Phaseolus vulgaris beans), as well as strains of other species included for comparison. The GS polymorphisms were determined by identifying variations in native enzyme mobility (revealed by GS activity staining) and in the isoelectric points of the monomers (revealed by immunodetection with antibodies against the GS proteins) by using gel electrophoresis. Restriction fragment length polymorphism patterns obtained by hybridizing an internal fragment of the GSII gene obtained from R. etli with total fragmented DNAs from different strains clearly distinguished the different groups. GSII is a novel and useful marker for Rhizobium groups and species, and GSII data support R. tropici and R. etli as bona fide species.
... Thus, it is possible that the over-production of EPS by the nrcR mutant could contribute to its increased motility. Remarkably, nrcR gene is located in the CIAT 899 megaplasmid that also carries the exo genes, which are implied in the biosynthesis of EPS [53]. Thus, the relative position of all these genes is another clue that could be relating EPS production with the NrcR protein. ...
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The establishment of nitrogen-fixing rhizobium-legume symbioses requires a highly complex cascade of events. In this molecular dialogue the bacterial NodD transcriptional regulators in conjunction with plant inducers, mostly flavonoids, are responsible for the biosynthesis and secretion of Nod factors which are key molecules for successful nodulation. Other transcriptional regulators related to the symbiotic process have been identified in rhizobial genomes, including negative regulators such as NolR. Rhizobium tropici CIAT 899 is an important symbiont of common bean (Phaseolus vulgaris L.), and its genome encompasses intriguing features such as five copies of nodD genes, as well as other possible transcriptional regulators including the NolR protein. Here we describe and characterize a new regulatory gene located in the non-symbiotic plasmid pRtrCIAT899c, that shows homology (46% identity) with the nolR gene located in the chromosome of CIAT 899. The mutation of this gene, named nrcR (nolR-like plasmid c Regulator), enhanced motility and exopolysaccharide production in comparison to the wild-type strain. Interestingly, the number and decoration of Nod Factors produced by this mutant were higher than those detected in the wild-type strain, especially under salinity stress. The nrcR mutant showed delayed nodulation and reduced competitiveness with P. vulgaris, and reduction in nodule number and shoot dry weight in both P. vulgaris and Leucaena leucocephala. Moreover, the mutant exhibited reduced capacity to induce the nodC gene in comparison to the wild-type CIAT 899. The finding of a new nod-gene regulator located in a non-symbiotic plasmid may reveal the existence of even more complex mechanisms of regulation of nodulation genes in R. tropici CIAT 899 that may be applicable to other rhizobial species.
... The large plasmids (mega-plasmids), with a molecular size up to 1 Mb or more, harbor, in certain circumstances, some essential genes that are required for bacterial viability and survival; these genes are not easily cured, eliminated, or transferred from one cell to another. These mega-plasmids have been observed in several rhizobia species; referring to, as Bsecondary chromosomes or chromids^ (Geniaux et al. 1995;Flores et al. 1998;Landeta et al. 2011). ...
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Rhizobia are a well-known group of soil bacteria that establish symbiotic relationship with leguminous plants, fix atmospheric nitrogen, and improve soil fertility. To fulfill multiple duties in soil, rhizobia are elaborated with a large and complex multipartite genome composed of several replicons. The genetic material is divided among various replicons, in a way to cope with, and satisfy the diverse functions of rhizobia. In addition to the main chromosome, which is carrying the essential (core) genes required for sustaining cell life, the rhizobia genomes contain several extra-chromosomal plasmids, carrying the nonessential (accessory) genes. Occasionally, some mega-plasmids, denoted as secondary chromosomes or chromids, carry some essential (core) genes. Furthermore, specific accessory gene sequences (the symbiotic chromosomal islands) are incorporated in the main chromosome of some rhizobia species in Bradyrhizobium and Mesorhizobium genera. Plasmids in rhizobia are of variable sizes. All of the plasmids in a Rhizobium cell constitute about 30–50% of the genome. Rhizobia plasmids have specific characters such as miscellaneous genes, independent replication system, self-transmissibility, and instability. The plasmids regulate several cellular metabolic functions and enable the host rhizobia to survive in diverse habitats and even under stress conditions. Symbiotic plasmids in rhizobia are receiving increased attention because of their significance in the symbiotic nitrogen fixation process. They carry the symbiotic (nod, nif and fix) genes, and some non-symbiotic genes. Symbiotic plasmids are conjugally-transferred by the aid of the non-symbiotic, self-transmissible plasmids, and hence, brings about major changes in the symbiotic interactions and host specificity of rhizobia. Besides, the rhizobia cells harbor one or more accessory, non-symbiotic plasmids, carrying genes regulating various metabolic functions, rhizosphere colonization, and nodulation competitiveness. The entire rhizobia-plasmid pool interacting in harmony and provides rhizobia with substantial abilities to fulfill their complex symbiotic and non-symbiotic functions in variable environments. The above concepts are extensively reviewed and fairly discussed.
... Rhizobium tropici Type A CFN 299, recently renamed as Rhizobium leucaenae CFN 299 , is a soil bacterium that establishes symbiosis with at least 22 legume species, including Leucaena leucocephala and Phaseolus vulgaris (common bean) (Martínez-Romero et al. 1991;Hernández-Lucas et al. 1995b; Acosta-Durán and . The CFN 299 genome comprises an approximately 3-MB chromosome, a 1.5-MB megaplasmid with genes for exopolysaccharide synthesis, plasmid A (185 kb) containing genes for the transport of compounds present in bean root exudates, plasmid B (240 kb) that participates in nodulation competitiveness and the 500-kb symbiotic plasmid (pSym) (Martínez et al. 1987;Geniaux et al. 1995;Rosenblueth et al. 1998;Ormeño-Orrillo et al. 2016). The pSym contains genes required for nodulation, nodulation competitiveness, nitrogen fixation, phytohormone biosynthesis and multiple functional insertion sequence (IS) elements (Mavingui et al. 1997;Hernández-Lucas et al. 2006;Ormeño-Orrillo et al. 2012). ...
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The alpha-protobacterium Rhizobium leucaenae CFN 299 is able to nodulate and fix nitrogen in symbiosis with a wide range of legumes, including Phaseolus vulgaris (common bean). Strain CFN 299 contains a 500-kb symbiotic plasmid (pSym) that encodes genes required for nodulation and nitrogen fixation as well as many genes whose function is unknown. In this work, we characterized the transcriptional expression of 16 pSym genes in common bean nodules and in free-living cells grown in culture. A functionally diverse group of genes were expressed during discrete stages of the symbiosis or in free-living cells. These included genes whose products are involved in nodulation and nitrogen fixation, carbon metabolism, vitamin synthesis, sulfur utilization, conjugation, transposition and DNA replication. We also examined the functionality of two replication systems encoded on pSym and found that repABC, but not repC2, is required for pSym replication.
... In these areas, R. tropici has been proved to be far more competitive for nodulation than R. etli, blocking the nodulation of R. etli [67]. Two types of R. tropici, A and B have been clearly distinguished that seem to be diverging lineages sharing a common symbiotic plasmid [56,68], although there are some R. tropici strains with intermediate characteristics between A and B that do not belong to any of them [56,66,69]. Recently, accumulated phylogenetic data supported the reclassification of R. tropici strains belonging to type A into the new species R. leucaneae [43]. ...
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Common bean is one of the most important crops for human feed, and the most important legume for direct consumption by millions of people, especially in developing countries. It is a promiscuous host legume in terms of nodulation, able to associate with a broad and diverse range of rhizobia, although the competitiveness for nodulation and the nitrogen fixation capacity of most of these strains is generally low. As a result, common bean is very inefficient for symbiotic nitrogen fixation, and nitrogen has to be supplied with chemical fertilizers. In the last years, symbiotic nitrogen fixation has received increasing attention as a sustainable alternative to nitrogen fertilizers, and also as a more economic and available one in poor countries. Therefore, optimization of nitrogen fixation of bean-rhizobia symbioses and selection of efficient rhizobial strains should be a priority, which begins with the study of the natural diversity of the symbioses and the rhizobial populations associated. Natural rhizobia biodiversity that nodulates common bean may be a source of adaptive alleles acting through phenotypic plasticity. Crosses between accessions differing for nitrogen fixation may combine alleles that never meet in nature. Another way to discover adaptive genes is to use association genetics to identify loci that common bean plants use for enhanced biological nitrogen fixation and, in consequence, for marker assisted selection for genetic improvement of symbiotic nitrogen fixation. In this review, rhizobial biodiversity resources will be discussed, together with what is known about the loci that underlie such genetic variation, and the potential candidate genes that may influence the symbiosis’ fitness benefits, thus achieving an optimal nitrogen fixation capacity in order to help reduce reliance on nitrogen fertilizers in common bean.
... Further analysis with glutamine synthetase II isoenzymes clearly distinguished type A and type B strains (Taboada et al., 1996). Megaplasmids of similar size (over 1700 kb) were observed in both type A and type B strains but they were found to be subgroup-specific, indicating that type A and B strains belonged to different taxa (Geniaux et al., 1995). Such megaplasmids may correspond to chromids (E. ...
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Rhizobium tropici is a well-studied legume symbiont characterized by high genetic stability of the symbiotic plasmid and tolerance to tropical environmental stresses such as high temperature and low soil pH. However, high phenetic and genetic variabilities among R. tropici strains have been largely reported, with two subgroups, designated type A and B, already defined within the species. A polyphasic study comprising multilocus sequence analysis, phenotypic and genotypic characterizations, including DNA-DNA hybridization, strongly supported the reclassification of R. tropici type A strains as a novel species. Type A strains formed a well-differentiated clade that grouped with R. tropici, Rhizobium multihospitium, Rhizobium miluonense, Rhizobium lusitanum and Rhizobium rhizogenes in the phylogenies of the 16S rRNA, recA, gltA, rpoA, glnII and rpoB genes. Several phenotypic traits differentiated type A strains from all related taxa. The novel species, for which the name Rhizobium leucaenae sp. nov. is proposed, is a broad host range rhizobium being able to establish effective root-nodule symbioses with Leucaena leucocephala, Leucaena esculenta, common beans (Phaseolus vulgaris) and Gliricidia sepium. Strain CFN 299(T) ( = USDA 9039(T) = LMG 9517(T) = CECT 4844(T) = JCM 21088(T) = IAM 14230(T) = SEMIA 4083(T) = CENA 183(T) = UMR1026(T) = CNPSo 141(T)) is designated the type strain of Rhizobium leucaenae sp. nov.
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The broad-host-range, heat-tolerant Rhizobium strain BR816 produces sulphated Nod metabolites. Two ORFs highly homologous to the Sinorhizobium meliloti nodPQ genes were isolated and sequenced. It was found that Rhizobium sp. BR816 contained two copies of these genes; one copy was localized on the symbiotic plasmid, the other on the megaplasmid. Both nodP genes were interrupted by insertion of antibiotic resistance cassettes, thus constructing a double nodP1P2 mutant strain. However, no detectable differences in Nod factor TLC profile from this mutant were observed as compared to the wild-type strain. Additionally, plant inoculation experiments did not reveal differences between the mutant strain and the wild-type. It is proposed that a third, functionally homologous locus complements mutations in the Nod factor sulphation genes. Southern blot analysis suggested that this locus contains genes necessary for the sulphation of amino acids.
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The nitrogen-fixing rhizobial symbionts of Sesbania herbacea growing in the nature reserve at the Sierra de Huautla, Mexico, were isolated and characterized. All 104 isolates together with the type strain for Rhizobium galegae, HAMBI 540T, had similar 16S rRNA genes as revealed by PCR-RFLP analysis. Similarity in the sequences of the 16S rRNA genes placed the isolates on a phylogenetic branch shared with R. galegae. Among 66 randomly selected isolates, three closely related electrophoretic alloenzyme types (ETs) were identified, which were distinct from 10 ETs distinguished among 23 strains of R. galegae. A new species Rhizobium huautlense, represented by the Sesbania isolate SO2T, is proposed based upon low estimates of DNA relatedness between our chosen type strain and the type strains for the other species, the dissimilarity of the nucleotide sequence of the 16S rRNA genes, and their distinct ETs compared with R. galegae. The description of R. huautlense is significant because in the reconstruction of the phylogeny at R. huautlense there was a shift in the node of the branch of Agrobacterium vitis relative to that of R. galegae. The revised phylogenetic tree would tend to indicate common ancestry between R. galegae and Rhizobium leguminosarum.
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Fifty-eight new isolates were obtained from root nodules of common bean (Phaseolus vulgaris) cultivated in soils originating from different agroecological areas in Senegal and Gambia (West Africa). A polyphasic approach including both phenotypic and genotypic techniques was used to study the diversity of the 58 Rhizobium isolates and to determine their taxonomic relationships with reference strains. All the techniques performed, analysis of multilocus enzyme electrophoretic patterns, SDS-PAGE profiles of total cell proteins, PCR-RFLP analysis of the genes encoding 16S rRNA and of the 16S-23S RNA intergenic spacer region (ITS-PCR-RFLP), auxanographic tests using API galleries and nodulation tests lead to the consensus conclusion that the new rhizobial isolates formed two main distinct groups, I and II, belonging to Rhizobium tropici type B and Rhizobium etli, respectively. By MLEE R. etli and group II strains showed several related electrophoretic types, evidencing some extent of internal heterogeneity among them. This heterogeneity was confirmed by other techniques (ITS-PCR-RFLP, SDS-PAGE and host-plant-specificity) with the same nine distinct strains of group II showing some differences from the core of group II (54 strains).
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The phylogenetic relationships among Rhizobium species that nodulate Phaseolus vulgaris (common bean) were determined by directly sequencing the amplified 16S ribosomal DNA genes of these organisms. The bean strains formed four separate clusters. One cluster was composed of Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, and R. leguminosarum bv. phaseoli. Two other clusters comprised Rhizobium etli and Rhizobium tropici, and the fourth cluster contained a single bean-nodulating strain. Data for species identification were obtained from DNA-DNA reassociation experiments. The levels of DNA relatedness among strains belonging to the three biovars of R. leguminosarum ranged from 58 to 67%. The levels of DNA relatedness between R. leguminosarum bv. phaseoli and R. etli and R. tropici ranged from 43 to 45% and 13 to 16%, respectively. The levels of DNA relatedness between the strain belonging to the fourth cluster and strains of the other three Rhizobium species that nodulate beans were less than 10%.
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The Leguminosae is one of the largest families of plants. It has a broad geographical distribution. The principal legume species have defined sites of origin and these coincide with the diversification centers for their “specific” symbiotic bacteria. These nitrogen-fixing bacteria, which form nodules in the roots or stems of the plants, belong to different bacterial lineages (Rhizobium, Bradyrhizobium, and Azorhizobium) related to other nonsymbiotic bacteria. A remarkable characteristic of these bacteria is their large genetic diversity. The genetic relationships among the different bacterial groups are being defined based mainly on the analysis of the sequences of the ribosomal genes. Recent results point out the need to have a broader genomic scope. Gene maps, genome sizes, and sequence of metabolic genes would serve to validate the present Rhizobium and Bradyrhizobium phylogenies. More realistic phylogenies should perhaps consider lateral transfer between clusters of bacteria.A compilation of records of bacterial genetic diversity, including enterobacteria and pathogens, is presented and compared with Rhizobium diversity. It is proposed that human activities are having important effects on microbe diversity.
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Two oligonucleotide primer pairs, based on the nifH gene sequence of Rhizobium etli, were used in the polymerase chain reaction to amplify genomic DNA from a collection of bean-nodulating Rhizobium strains that were isolated from the Americas. The primers that were derived from a poorly conserved region among the nifH sequences of other rhizobial species, generated a positive and specific reaction with all the R. etli strains assayed. By contrast, strains belonging to type II R. tropici and to unclassified bean strains did not yield an amplification product. Since the nifH primers appeared to be universal for a collection of R. etli strains representing a diversity of genotypes, the nifH-PCR method provides a tool for the rapid typing of bean nodule isolates.
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Functional analyses of rhizobial plasmids have concentrated mainly on symbiotic plasmids (pSym) that carry genes required for nitrogen fixation and nodule formation. However, information on the other plasmids, termed cryptic or non-symbiotic, is still scarce. In this work, a collection of sequential cured derivatives with different combinations of plasmids was generated from the Rhizobium tropici strain CIAT 899 to study their functions in symbiosis with common bean plants. PCR analysis of the nif genes from all cured derivatives indicated that plasmid b is the symbiotic plasmid (pSym) while a and c are the cryptic plasmids. This genotype was confirmed by the presence of nodules in common bean plants inoculated with derivatives containing plasmid b. However, when plasmid a was missing in the derivative containing the pSym, nodules were formed but in lower numbers and were smaller in size. The derivative cured of plasmid a formed nodules with a large quantity of starch and crystals and showed a significant decrease in nitrogenase activity. In addition, the presence of bacteria in the intercellular spaces of the nodules was observed. A co-inoculation experiment involving both the wild-type CIAT 899 strain and derivative CIAT 899a suggested that the cryptic plasmid a contains genes involved in the competition for nodulation of Phaseolus vulgaris.
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SUMMARY Large plasmids were detected in Rhizobium species by sedimentation analysis of lysates on alkaline sucrose gradients and by dye buoyant density centrifugation. Mitomycin C treatment did not increase the amount of plasmid DNA in the bacteria. Renaturation kinetics were used to confirm that these large DNA molecules had the molecular complexity of plasmids and to estimate their molecular weights. These ranged from 0.7 x roS to 4.0~ IO* daltons. The maximum yield of isolated plasmid DNA relative to chromosomal DNA was 3-8 %.
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Recent developments inRhizobium taxonomy are presented from a molecular and evolutionary point of view. Analyses of ribosomal RNA gene sequences provide a solid basis to infer phylogenies in the Rhizobiaceae family. These studies confirmed thatRhizobium andBradyrhizobium are only distantly related and showed thatRhizobium andBradyrhizobium are related to other groups of bacteria that are not plant symbionts.Rhizobium andAgrobacterium species are intermixed. Differences in plasmid content may explain to a good extent the different behavior ofRhizobium andAgrobacterium as symbionts or pathogens. Other approaches to identify and classify bacteria such as DNA-DNA hybridization, fatty acid analysis, RFLP and RPD-PCR techniques and phylogenies derived from other genes are in general agreement to the groupings derived by ribosomal sequences. Only a small proportion of nodulated legumes have been sampled for their symbionts and more knowledge is required on the systematics and taxonomy ofRhizobium andBradyrhizobium species.
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The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes. Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots. All the isolates clustered with R. leguminosarum bv. phaseoli reference strains and did not encompass any other Rhizobium taxa. Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R. leguminosarum bv. phaseoli reference strains. When complemented with an R. leguminosarum bv. phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain. The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R. leguminosarum strains.
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A new Rhizobium species that nodulates Phaseolus vulgaris L. and Leucaena spp. is proposed on the basis of the results of multilocus enzyme electrophoresis, DNA-DNA hybridization, an analysis of ribosomal DNA organization, a sequence analysis of 16S rDNA, and an analysis of phenotypic characteristics. This taxon, Rhizobium tropici sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type II strains) and was recognized by its host range (which includes Leucaena spp.) and nif gene organization. In contrast to R. leguminosarum biovar phaseoli, R. tropici strains tolerate high temperatures and high levels of acidity in culture and are symbiotically more stable. We identified two subgroups within R. tropici and describe them in this paper.
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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.
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Eight symbiotic mutants defective in lipopolysaccharide (LPS) synthesis were isolated from Rhizobium leguminosarum biovar phaseoli CFN42. These eight strains elicited small white nodules lacking infected cells when inoculated onto bean plants. The mutants had undetectable or greatly diminished amounts of the complete LPS (LPS I), whereas amounts of an LPS lacking the O antigen (LPS II) greatly increased. Apparent LPS bands that migrated between LPS I and LPS II on sodium dodecyl sulfate-polyacrylamide gels were detected in extracts of some of the mutants. The mutant strains were complemented to wild-type LPS I content and antigenicity by DNA from a cosmid library of the wild-type genome. Most of the mutations were clustered in two genetic regions; one mutation was located in a third region. Strains complemented by DNA from two of these regions produced healthy nitrogen-fixing nodules. Strains complemented to wild-type LPS content by the other genetic region induced nodules that exhibited little or no nitrogenase activity, although nodule development was obviously enhanced by the presence of this DNA. The results support the idea that complete LPS structures, in normal amounts, are necessary for infection thread development in bean plants.
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Mutants of Rhizobium meliloti which are deficient in exopolysaccharide synthesis have been classified into six different genetic groups (A through F) (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). Using physical and genetic techniques, we have demonstrated that the group E Exo- mutants carry deletions in the exoA-exoB region of the megaplasmid pRmeSU47b. We have constructed strains carrying defined deletions which remove up to 200 kilobases of pRmeSU47b, including the exoA-exoB region. These derivatives have the same phenotypes as do the group E mutants.
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We characterized mutants of Rhizobium meliloti SU47 that were unable to grow on succinate as the carbon source. The mutants fell into five groups based on complementation of the succinate mutations by individual recombinant plasmids isolated from a R. meliloti clone bank. Enzyme analysis showed that mutants in the following groups lacked the indicated common enzyme activities: group II, enolase (Eno); group III, phosphoenolpyruvate carboxykinase (Pck); group IV, glyceraldehyde-3-phosphate dehydrogenase (Gap), and 3-phosphoglycerate kinase (Pgk). Mutants in groups I and V lacked C4-dicarboxylate transport (Dct-) activity. Wild-type cells grown on succinate as the carbon source had high Pck activity, whereas no Pck activity was detected in cells that were grown on glucose as the carbon source. It was found that in free-living cells, Pck is required for the synthesis of phosphoenolpyruvate during gluconeogenesis. In addition, the enzymes of the lower half of the Embden-Meyerhoff-Parnas pathway were absolutely required for gluconeogenesis. Eno, Gap, Pck, and one of the Dct loci (ntrA) mapped to different regions of the chromosome; the other Dct locus was tightly linked to a previously mapped thi locus, which was located on the megaplasmid pRmeSU47b.
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Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.
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Experience from different laboratories indicates that Rhizobium strains can generate variability in regard to some phenotypic characteristics such as colony morphology or symbiotic properties. On the other hand, several reports suggest that under certain stress conditions or genetic manipulations Rhizobium cells can present genomic rearrangements. In search of frequent genomic rearrangements, we analyzed three Rhizobium strains under laboratory conditions that are not considered to cause stress in bacterial populations. DNAs from direct descendants of a single cell were analyzed in regard to the hybridization patterns obtained, using as probes different recombinant plasmids or cosmids; while most of the probes utilized did not show differences in the hybridization patterns, some of them revealed the occurrence of frequent genomic rearrangements. The implications and possible biological significance of these observations are discussed.
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Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained.
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The taxonomic status of 16 collection strains of chickpea (Cicer arietinum L.) rhizobia which were previously determined to belong to two groups (groups A and B) were compared with reference strains belonging to different genera and species of the family Rhizobiaceae. We used the following taxonomic, phylogenetic, and phenotypic characteristics and approaches to study these organisms: DNA homology, guanine-plus-cytosine content, restriction fragment length polymorphism of the amplified 16S-intergenic spacer rRNA gene, partial 16S rRNA sequencing, and auxanographic tests performed with 147 carbon sources. Similar groups of chickpea strains were identified by the different approaches. The chickpea strains were found to belong to the genus Rhizobium regardless of the phylogenetic group to which they belonged (group A or B). All strains fell into a tight cluster which included Rhizobium loti and Rhizobium galegae, and the group B strains were closely related to R. loti. An analysis of partial 16S ribosomal DNA sequences revealed identical nucleotide sequences for the slowly growing strains and fast-growing strains that were used as representatives of groups A and B, respectively, and these organisms fell into the Rhizobium-Agrobacterium lineage. When the sequences of these organisms were compared with the partial sequences of Rhizobium huakuii and R. loti, one- and two-nucleotide mismatches were observed, respectively, indicating that the chickpea rhizobia are closely related to these two species. The DNA-DNA hybridization data revealed that the chickpea rhizobia exhibited low levels of homology (less than 17%) to previously described Rhizobium and Bradyrhizobium species. Moreover, when we compared chickpea strains to R. loti and R. huakuii, the most closely related species as determined by the partial 16S rRNA sequence analysis, the homology values ranged from 21 to 52% and the delta Tm values were greater than 5 degrees C (delta Tm is the difference between the denaturation temperatures of the heterologous and homologous duplexes). These results confirmed that the rhizobia that nodulate chickpeas cannot be assigned to a previously described species. Within the chickpea rhizobia, the DNA homology values obtained when members of groups A and B were compared were less than 38%, indicating that the group A and group B organisms belong to different species. Furthermore, these organisms can be distinguished from each other by the results of phenotypic tests. We propose that the group B chickpea rhizobia should be assigned to a new species, Rhizobium ciceri; UPM-Ca7 is the type strain of R. ciceri.
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Levels of DNA relatedness between strains isolated from root nodules of Phaseolus vulgaris and reference strains of different Rhizobium species were determined by performing DNA-DNA hybridization experiments (S1 nuclease method). The nine strains examined were members of three genomic groups previously delineated by a restriction fragment length polymorphism analysis among strains isolated from P. vulgaris at different sites in France. In agreement with the results of the restriction fragment length polymorphism analysis, three genomic species were found. We confirmed that one of these species corresponded to Rhizobium leguminosarum since the strain examined was 100% related to the type strain of this species. The other two species were new genomic species which were less than 21% related to reference strains belonging to other Rhizobium species, including Rhizobium etli and Rhizobium tropici, and were 18% related to each other. As determined by an analysis of partial 16S ribosomal DNA sequences, each of the genomic species was found to belong to a lineage independent from the lineages of previously described Rhizobium species. Nevertheless, they were included in the group formed by the fast-growing Rhizobium species. Both genomic species 1 and genomic species 2 contained a majority of strains which were capable of nodulating both P. vulgaris and Leucaena leucocephala, like R. tropici. However, they also contained strains with a nodulation phenotype restricted to P. vulgaris, like R. leguminosarum bv. phaseoli and R. etli bv. phaseoli. Our data are the first evidence that in Europe species other than R. leguminosarum nodulate P. vulgaris.
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Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.
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Plant specificities and deoxyribonucleic acid homologies were studied among 122 strains of Rhizobium. Some strains were assigned to species on the basis of their source of isolation and present nodulation capabilities, but many did not fit into one of the six currently recognized species of the genus Rhizobium. Among those strains assigned to species were many which also nodulated plants outside their species-specific, cross-inoculation group. Conversely, isolates from a wide variety of plants could be designated Rhizobium phaseoli since they were capable of nodulating Phaseolus vulgaris. Acid production and growth rate on yeastmannitol agar were tested for all strains. Some strains grew rapidly but did not produce an acid reaction; these were grouped with the fast growing acid producers. Deoxyribonucleic acid homology was used to identify four genetic groups of fastgrowing, acid-producing rhizobia. Group 1 included strains of Rhizobium trifolii (except strains obtained from Trifolium lupinaster, Rhizobium leguminosarum, Rhizobium phaseoli (obtained from Phaseolus vulgaris), and two strains obtained from Neptunia gracilis. Group 2 comprised six American strains obtained from crown vetch (Coronilla varia), sainfoin (Onobrychis vicifolia), and Sophora spp. Species status for this group should remain tentative until further strains have been studied. Group 3 corresponded with Rhizobium meliloti as presently defined. Group 4 included fast-growing Lotus rhizobia, two strains obtained from T. lupinaster, and a wide variety of previously unclassified strains. Nine fastgrowing strains could not be included in any of these groups. The nine slowgrowing, non-acid producing strains included in this study showed < 10% homology with DNAs from seven fast-growing reference strains. The relationships between subgroups in group 1 are discussed, and the genetic diversity of strains obtained from Phaseolus vulgaris is examined. It is proposed that fast-growing rhizobia comprise at least four species corresponding with the four genetic groups described.
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Bacteria of the genus Rhizobium form symbiotic nitrogen-fixing relationships with legumes. To investigate the organization of the nitrogen-fixation (nif) genes in Rhizobium phaseoli, which nodulates Phaseolus vulgaris, we have taken advantage here of the sequence homology between the nif D and nif H genes of Klebsiella pneumoniae and those of other nitrogen-fixing species1–3. Strains from different geographical origins were studied, and a common pattern of organization of nif-homologous sequences was found. Initial characterization of nif sequences in R. phaseoli has revealed the presence of stable reiterations of DNA sequences including nif sequences, and suggests that at least some of these sequences are located on a large plasmid.
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Evidence is presented which indicates that genetic information required for nodulation is carried on a plasmid. The ability to nodulate peas was transferred by conjugation at a high frequency from a strain of Rhizobium leguminosarum to a nonnodulating strain of the same species and to three other species of Rhizobium nodulating legumes other than peas.
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This chapter discusses the plasmids of Rhizobium and their role in symbiotic nitrogen fixation. Rhizobia are gram-negative soil bacteria which fix nitrogen in a symbiotic association with plants of the family Leguminosae; however, this classical definition must be extended now to include nodulation and nitrogen fixation on a nonlegume, Parasponia. Rhizobium parasponia can infect this tropical tree and fix nitrogen in a manner very similar to that observed in legumes. In both the cases, establishment of the symbiosis starts with invasion of plant root or stem by free-living rhizobia followed by a series of steps that result in the formation of a nodule. It is in these nodules that nitrogen fixation takes place. Both the plant and the bacteria undergo differentiation that is regulated by gene expression. Members of the genus Rhizobium are of great economic importance because of their ability to fix nitrogen. The genus has somewhat informally been separated into those species that are fast growers and those that are slow growers. An understanding of the genes of Rhizobium involved in plant symbiosis and nitrogen fixation has moved very rapidly with the fast-growing strains (Rhizobium leguminosarum, Rhizobium trifolii, Rhizobium meliloti, and R. parasponia and R.fredii).
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We studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production. The mutants produced non-mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture. A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified. EcoRI restriction digests of the cosmids revealed nine unique inserts. Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups. On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899. On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS-deficient mutants induced effective nodules. Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS-deficient mutant contained infected cells. These data indicate that EPS is not required for normal nodulation of bean by R tropici, that it may contribute to competitiveness of R. tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro.
Article
Response of bean (Phaseolus vulgaris L.) cultivars to inoculation with Rhizobium phaseoli under field conditions in Latin America has been inconsistent, one of the contributory factors being the low soil pH in many regions of bean production. In the study the tolerance of 55 strains of Rhizobium phaseoli to acid pH, aluminium (Al) and manganese (Mn) in nutrient medium was examined and the tolerance of these stresses related to the survival and nodulating ability of strains of R. phaseoli in acid soils.At pH 4.5 only six of the 55 strains tested produced isolate colonies on modified Keyser-Munns medium. Eight strains grew well on pH 4.7 medium to which 6 ppm Al had been added, but only six of these grew when the Al was replaced with 50 ppm Mn. When two strains differing in tolerance to pH in nutrient medium were introduced into an Ultisol from Santander de Quilichao, Colombia, which had been limed to differing pH values, the tolerant strain CIAT 899 survived better from pH 4.15 to 4.90. In a field trial with soil limed to pH values from 3.8 to 4.4, the percentage of plants nodulated and the number of nodules per plant was greater when CIAT 899 was used as inoculant, instead of the strain CIAT 632, shown sensitive to pH in both nutrient media and soil tests. In this trial granular, soil applied inoculants gave better results than those which were seed applied. In a second field trial, on a Typic Distrandept limed from pH 4.45 to 5.20, yields of P. vulgaris were enhanced by inoculation, but again CIAT 899 performed better than did CIAT 632. In this trial there was no significant difference between inoculation methods. The possible value of granular inoculation methods to the production of P. vulgaris under small-farm conditions is discussed.
Article
A fast and very sensitive procedure is described for detecting plasmids in bacterial strains. The size of plasmids is determined by agarose gel electrophoresis. Plasmids present in one or more copies per cell with a molecular mass ranging from 2 to over 150 megadaltons may be identified.
Article
It is known that the Rhizobium galegae genomes contain megaplasmids. The suicide vector pSUP2111 with nifH gene of R. meliloti was introduced into the strains CIAM 0703 and CIAM 0711 of R. galegae inducing effective nodules on Galega orientalis plants. The formation of self-transmissible megaplasmids was observed. The megaplasmid transfer into non-nodulating R. meliloti mutants resulted in partial complementation of the nodulation defect in recipient strains though only one transconjugant showed the nitrogen-fixing activity in symbiosis with alfalfa and another one in symbiosis with G. orientalis plants. Among the Agrobacterium strains harbouring R. galegae megaplasmids there were four classes of transconjugants: (1) Nod+ Fix- in symbiosis with goat's rue plants (three strains); (2) Nod+ Fix- on Medicago sativa (two strains); (3) Nod+ Fix+ on M. sativa (five strains); (4) Nod- with both plant hosts (11 strains).
Article
Rhizobium phaseoli CFN299 forms nitrogen-fixing nodules in Phaseolus vulgaris (bean) and in Leucaena esculenta. It has three plasmids of 185, 225, and 410 kilobases. The 410-kilobase plasmid contains the nitrogenase structural genes. We have transferred these plasmids to the plasmid-free strain Agrobacterium tumefaciens GMI9023. Transconjugants containing different combinations of the R. phaseoli plasmids were obtained, and they were exhaustively purified before nodulation was assayed. Only transconjugants harboring the 410-kilobase plasmid nodulate P. vulgaris and L. esculenta. Nodules formed by all such transconjugants are able to reduce acetylene. Transconjugants containing the whole set of plasmids from CFN299 nodulate better and fix more nitrogen than the transconjugants carrying only the Sym plasmid. Microscopic analysis of nodules induced by A. tumefaciens transconjugants reveals infected cells and vascular bundles. None of the A. tumefaciens transconjugants, not even the one with the whole set of plasmids from CFN299, behaves in symbiosis like the original R. phaseoli strain; the transconjugants produce fewer nodules and have lower acetylene reduction (25% as compared to the original R. phaseoli strain) and more amyloplasts per nodule. More than 2,000 bacterial isolates from nodules of P. vulgaris and L. esculenta formed by the transconjugants were analyzed by different criteria. Not a single rhizobium could be detected. Our results show that R. phaseoli plasmids may be expressed in the A. tumefaciens background and direct the formation of effective, differentiated nodules.
Article
Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.
Article
A DNA fragment of the broad host range plasmid RP4 carrying the cis-acting DNA recognition site for conjugative DNA transfer between bacterial cells (Mobsite) was cloned into the kanamycin-neomycin resistance transposon Tn5. Using conventional transposon mutagenesis techniques the new transposon, called Tn5-Mob, can easily be inserted into the host DNA of gram-negative bacteria. A host replicon carrying Tn5-Mob is then mobilizable into any other gram-negative species if the transfer functions of plasmid RP4 are provided in trans. The potential of Tn5-Mob was demonstrated by mobilizing Rhizobium meliloti plasmids as well as the E. coli chromosome at high frequencies.
Article
Large plasmids of molecular weight varying from 90 to around 200 x 10(6) have earlier been detected in most Rhizobium meliloti strains using an alkaline denaturation - phenol extraction procedure. With a less destructive method (Eckardt 1978) it was possible additionally to detect one plasmid of molecular weight clearly greater than 300 x 10(6) (= megaplasmid) in all of twenty-seven R. meliloti strains of various geographical origins and nodulation groupings investigated. Four strains (RCR 2011, A145, S26 and CC2013) were found to carry one megaplasmid and no smaller plasmids. Hybridization experiments with Klebsiella pneumoniae and R. meliloti cloned nitrogenase structural genes D and H showed that these genes are located on the megaplasmid and not on the smaller plasmids. All of the ten independent spontaneous non-nodulating derivatives of three strains of R. meliloti were shown to have suffered a deletion in the nifDH region of the megaplasmid. These results indicate that a gene controlling an early step in nodule formation is located in the nifDH region of the megaplasmid. This indicates that the same replicon carries genes controlling early and late functions in symbiosis.
Article
Phenotypic and genetic characterization indicated that Hup+ bean rhizobial strains are type IIA and type IIB Rhizobium tropici. The Hup+ strain USDA 2840, which did not cluster with either of the two types of R. tropici in a restriction fragment length polymorphism analysis, had electrophoretic patterns of PCR products generated with primers for repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences similar to those of three reference strains of R. tropici type IIA. The Hup+ strain USDA 2738, which clustered with the reference strain of R. tropici IIB in a restriction fragment length polymorphism analysis, had electrophoretic patterns of PCR products generated with primers for repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences more closely resembling those of the reference strains of R. tropici type IIA than those of type IIB. DNA amplification with the Y1 and Y2 primers to generate a portion of the 16S rDNA operon was useful to distinguish R. tropici type IIA strains from other bean rhizobial strains. The phylogenetic position of the type IIA strain of R. tropici USDA 2840, determined from the partial 16S rDNA sequence, indicated a more distant relationship with the type IIB strain of R. tropici CIAT899 than with the as yet unnamed rhizobial species of Leucaena leucocephala, TAL 1145. Therefore, we suggest that it may be appropriate either to separate R. tropici types IIA and IIB into two different species or to identify TAL 1145 to the species level as a third type of R. tropici.
Article
Rhizobium species elicit the formation of nitrogen-fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen-fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen-fixing nodules on the roots of beans (Phaseolus vulgaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl-CoA and oxaloacetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5-mob insertion causes the bacteria to form fewer nodules (30-50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented all the phenotypic alterations of the pcsA mutant, and conferred Rhizobium leguminosarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.
Article
Rhizobium tropici CIAT899 induced chlorosis in the leaves of its symbiotic hosts, common bean (Phaseolus vulgaris L.), siratro (Macroptilium atropurpureum Urb.), and Leucaena leucocephala (Lam.) de Wit. Chlorosis induction by strains CIAT899 and CT9005, an exopolysaccharide-deficient mutant of CIAT899, required carbon substrate. When the bacteria were added at planting in a solution of mannitol (50 g/liter), as few as 10 cells of CIAT899 were sufficient to induce chlorosis in bean plants. All carbon sources tested, including organic acids and mono- and disaccharides, supported chlorosis induction. The addition of a carbon source did not affect the growth rate or the population density of CT9005 in the bean plant rhizosphere. Cell-free filtrates of cultures of CT9005 did not induce detectable chlorosis. All type B strains of R. tropici tested also induced chlorosis in common bean. Type A strains of R. tropici and all other species of bacteria tested did not induce chlorosis. Several lines of evidence indicated that nodulation was not required for chlorosis induction. Strain RSP900, a pSym-cured derivative of CIAT899, induced chlorosis in wild-type P. vulgaris. In addition, NOD125, a nodulation-defective line of common bean, developed chlorosis when inoculated with CIAT899, but did not develop nodules. CIAT899 consistently induced severe chlorosis in the leaves of the nonhost legumes alfalfa (Medicago sativa L.) and Berseem clover (Trifolium alexandrinum L.), and induced chlorosis in 29 to 58% of the plants tested of sunflower, cucumber, and tomato seedlings, but it did not induce chlorosis in the leaves of corn or wheat. Chlorosis induction in nonhost plants also required carbon substrate. The data are consistent with the hypothesis that R. tropici type B strains produce a chlorosis-inducing factor that affects a wide range of plant species.
Article
Fast-growing rhizobia have been isolated from soybean root nodules collected in China. These new isolates are physiologically distinct from slow-growing soybean rhizobia. They formed effective nitrogen-fixing associations with wild soybean and an unbred soybean cultivar from China, but were largely ineffective as nitrogen-fixing symbionts with common commercial cultivars of soybeans.
Genetic relatedness and taxonomic considerations of Rhizobium strains that nodulate Phaseolus vulgaris Nitrogen fixation: achievements and objectives
  • E Martinez
  • M A Pardo
  • F Martins
  • P Graham
  • A Franco
  • R Palacios
  • L Segovia
Martinez, E., M. A. Pardo, F. Martins, P. Graham, A. Franco, R. Palacios, and L. Segovia. 1990. Genetic relatedness and taxonomic considerations of Rhizobium strains that nodulate Phaseolus vulgaris (L.), p. 831. In P. M. Gresshoff, L. E. Roth, G. Stacey, and W. E. Newton (ed.), Nitrogen fixation: achievements and objectives. Chapman and Hall, New York.