Crossing the Limits of Rhizobium Existence in Extreme Conditions

An ecological survey was conducted to characterize 5000 Rhizobium sp. sesbania strains of diverse geographical origin, isolated from the root nodules of Sesbania aculeata growing in neutral (pH 7) and alkaline (pH 8.5 and above) soils. The rhizobia from the alkaline soil showed significantly higher salt tolerance than those isolated from neutral soil. Upper limits of stress survival of rhizobial isolates, Rhizobium sp. NBRI0102 sesbania selected from neutral soil, and Rhizobium sp. NBRI2505 sesbania selected from alkaline soil, were studied under free living conditions. Rhizobium sp. NBRI0102 sesbania and Rhizobium sp. NBRI2505 sesbania tolerated yeast extract mannitol broth (YEB) containing 10% and 28% salt (NaCl, wt/vol) for up to 18 h of incubation at 30°C. Growth of Rhizobium sp. NBRI0102 sesbania and Rhizobium sp. NBRI2505 sesbania at pH 7, 11, and 12 was identical, except for a lag period of about 10 h in the growth of Rhizobium sp. NBRI0102 sesbania at pH 11 and 12, as compared with pH 7. Rhizobium sp. NBRI0102 sesbania and Rhizobium sp. NBRI2505 sesbania survived at 50°C and 65°C, in YEB at pH 7 for up to 4 and 2 h, respectively. To our knowledge, this is the first report of rhizobia demonstrating survival of Rhizobium sp. NBRI2505 sesbania, estimated by counting viable cells, to such extreme conditions of salt and temperature, individually. In contrast to Rhizobium sp. NBRI0102 sesbania, high temperature was tolerated efficiently by Rhizobium sp. NBRI2505 sesbania, in the presence of salt at higher pH. Our results suggest that the possession of the trait of high salt tolerance might be of some evolutionary significance for the survival of rhizobia in alkaline soils, at high pH and temperature. Received: 23 May 2000 / Accepted: 26 June 2000     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Molecular Cloning and Characterization of <Emphasis Type="Italic">nodD</Emphasis> Genes from <Emphasis Type="Italic">Rhizobium</Emphasis> sp. SIN-1, a Nitrogen-Fixing Symbiont of <Emphasis Type="Italic">Sesbania</Emphasis> and Other Tropical Legumes

Rhizobium sp. SIN-1, a nitrogen-fixing symbiont of Sesbania aculeata and other tropical legumes, carries two copies of nodD, both on a sym plasmid. We have isolated these two nodD genes by screening a genomic library of Rhizobium sp. SIN-1 with a nodD probe from Sinorhizobium meliloti. Nucleotide sequence and the deduced amino acid sequence analysis indicated that the nodD genes of Rhizobium sp. SIN-1 are most closely related to those of R. tropici and Azorhziobium caulinodans. Rhizobium sp. SIN-1 nodD1 complemented a S. meliloti nodD1D2D3 negative mutant for nodulation on alfalfa, but failed to complement a nodD1 mutant of S. fredii USDA191 for soybean nodulation. A hybrid nodD gene, containing the N-terminus of S. fredii USDA191 nodD1 and the C-terminus of Rhizobium sp. SIN-1 nodD1, complemented the nodD1 negative mutant of USDA191 for nodulation on soybean. Received: 17 January 2002 / Accepted: 18 February 2002     

Effect of High Temperature on <Emphasis Type="Italic">Pseudomonas putida</Emphasis> NBRI0987 Biofilm Formation and Expression of Stress Sigma Factor RpoS

Pseudomonas is an efficient plant growth–promoting rhizobacteria; however, among the limiting factors for its commercialization, tolerance for high temperature is the most critical one. After screening 2,500 Pseudomnas sp. strains, a high temperature tolerant–strain Pseudomonas putida NBRI0987 was isolated from the drought-exposed rhizosphere of chickpea (Cicer arietinum L. cv. Radhey), which was grown under rain-fed conditions. P. putida NBRI0987 tolerated a temperature of 40°C for ≤ 5 days. To the best of our knowledge, this is the first report of a Pseudomnas sp. demonstrating survival estimated by counting viable cells under such a high temperature. P. putida NBRI0987 colony-forming unit (CFU)/ml on day 10 in both the absence and presence of MgSO₄.7H₂O (MgSO₄) in combination with glycerol at 40°C were 0.0 and 1.7 × 10¹¹, respectively. MgSO₄ plus glycerol also enhanced the ability of P. putida NBRI0987 to tolerate high temperatures by inducing its ability to form biofilm. However, production of alginate was not critical for biofilm formation. The present study demonstrates overexpression of stress sigma factor σ ^S (RpoS) when P. putida NBRI0987 is grown under high-temperature stress at 40°C compared with 30°C. We present evidence, albeit indirect, that the adaptation of P. putida NBRI0987 to high temperatures is a complex multilevel regulatory process in which many different genes can be involved.     

Production of Exo-polysaccharide by <Emphasis Type="Italic">Rhizobium</Emphasis> sp.

Two fold increase in the yield of glucose and maltose containing exo-polysaccharide (EPS) by Rhizobium sp. was observed during its growth in modified YEMB. EPS production, plant growth promotion activity and root colonization of Rhizobium sp. studies showed enhanced EPS synthesis, more seed germination and over all improvement in plant growth over control and R. meliloti treatment. Groundnut seeds bacterized with Rhizobium sp. resulted in 69.75% more root length, 49.51% more shoot height, 13.75% more number of branches and 13.60% more number of pods over the control and R. meliloti treatment. Bacterization of wheat seeds increased the dry matter yield of roots (1.7-fold), and roots adhering soil (RAS) (1.5) and shoot mass (1.9-fold). Rhizobium sp. inoculation also increased the population density of EPS-producing bacteria on the rhizoplane. Roots of plants inoculated with Rhizobium sp. maintained a higher K⁺/Na⁺ ratio and K⁺–Na⁺ selectivity.     

Phosphate solubilization by <Emphasis Type="Italic">Rhizobium</Emphasis> strains

Forty-six Rhizobium isolates from legume root and stem nodules were examined for their phosphate-solubilizing ability on Pikovskaya’s agar medium. Rhizobium isolates from root nodules of Cassia absus, Vigna trilobata and three strains from Sesbania sesban showed zone of tricalcium phosphate (TCP) solubilization. The isolate from C. absus showed maximum solubilization (620 μg/ml) after 12 d of incubation, while the Rhizobium sp. strain 26 (from S. sesban) showed the least amount (150 μg/ml) of phosphate solubilization. Among the carbon sources tested for their ability to solubilize TCP, maximum solubilization (620 μg/ml) was observed in glucose by Rhizobium isolate from C. absus. Phosphate solubilization increased with increase in glucose concentration steeply up to 2% and slowly above this concentration in four isolates. Among the nitrogen sources tested, maximum solubilization (620 μg/ml) was observed in ammonium sulphate by Rhizobium isolate from C. absus.     

Distribution of transposons Tn<Emphasis Type="Italic">5044</Emphasis> and Tn<Emphasis Type="Italic">5070</Emphasis> with noncanonical <Emphasis Type="Italic">mer</Emphasis> operons in environmental bacterial populations

The distribution of noncanonical mercury resistance transposons, Tn5044 and Tn5070 , was examined. A characteristic feature of Tn5044 is temperature sensitivity of its mercury operon and the presence in the mer operon of the gene homologous to RNA polymerase subunit. Structural organization of mercury operon Tn5070 , containing minimum gene set (merRTPA), differs from mer operons of both Gram-negative and Gram-positive bacteria. None of more than two thousand environmental bacterial strains displaying mercury resistance and isolated from the samples selected from different geographical regions hybridized to Tn5044- and Tn5070-specific probes. A concept on the existence of cosmopolite, endemic, and rare transposons in environmental bacterial populations was formulated.Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1717–1721.Original Russian Text Copyright © 2004 by Gorlenko, Kalyaeva, Bass, Petrova, Mindlin.     

Association Between the <Emphasis Type="Italic">cfx</Emphasis>A Gene and Transposon Tn<Emphasis Type="Italic">4555</Emphasis> in <Emphasis Type="Italic">Bacteroides distasonis</Emphasis> Strains and Other <Emphasis Type="Italic">Bacteroides</Emphasis> Species

The Bacteroides genus, the most prevalent anaerobic bacteria of the intestinal tract, carries a plethora of the mobile elements, such as plasmids and conjugative and mobilizable transposons, which are probably responsible for the spreading of resistance genes. Production of β-lactamases is the most important resistance mechanism including cephalosporin resistance to β-lactam agents in species of the Bacteroides fragilis group. In our previous study, the cfxA gene was detected in B. distasonis species, which encodes a clinically significant broad-spectrum β-lactamase responsible for widespread resistance to cefoxitin and other β-lactams. Such gene has been associated with the mobilizable transposon Tn4555. Therefore, the aim of this study was to detect the association between the cfxA gene and the presence of transposon Tn4555 in 53 Bacteroides strains isolated in Rio de Janeiro, Brazil, by PCR assay. The cfxA gene was detected in 11 strains and the Tn4555 in 15. The transposon sequence revealed similarities of approximately 96% with the B. vulgatus sequence which has been deposited in GenBank. Hybridization assay was performed in attempt to detect the cfxA gene in the transposon. It was possible to associate the cfxA gene in 11 of 15 strains that harbored Tn4555. Among such strains, 9 presented the cfxA gene as well as Tn4555, but in 2 strains the cfxA gene was not detected by PCR assay. Our results confirm the involvement of Tn4555 in spreading the cfxA gene in Bacteroides species.     

The extracellular polysaccharide produced by <Emphasis Type="Italic">Rhizobium</Emphasis> sp. isolated from the root nodules of <Emphasis Type="Italic">Phaseolus mungo</Emphasis>

The symbiont isolated from root nodules of Phaseolus mungo L., a widely grown legume in India was identified as a Rhizobium sp. a Rhizobium sp. close to R. multihospitium based on a biochemical and 16S rRNA gene-based phylogenetic approach. This Rhizobium sp. was able to produce large amounts of extracellular polysaccharides (EPS) in a yeast extract mannitol (YEM) broth medium. Both growth and EPS production started simultaneously though each had different stationary phases. EPS production increased enormously with supplementation by the preferred carbon, nitrogen and vitamin sources. Attempts were made to optimize the cultural requirements for maximum growth and maximum EPS production. The EPS produced by the symbiont contained large amount of mannose together with small amounts of arabinose and xylose. The possible role of EPS production on the Rhizobium—root nodule symbiosis is briefly discussed.     

Characterisation of <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. and <Emphasis Type="Italic">Ochrobactrum</Emphasis> sp. isolated from volcanic soil

Soil bacteria may have properties of plant growth promotion but not be sufficiently beneficial for plants under stress conditions. This challenge has led researchers to extend their searches into extreme environments for potential soil bacteria with multiple plant beneficial traits as well as abiotic stress tolerance abilities. In the current study, an attempt was made to evaluate soil bacteria from an extreme environment, volcano soils, based on plant growth promoting and abiotic stress mitigating characteristics. The screening led to the isolation of eight (NBRISH4, NBRISH6, NBRISH10, NBRISH11, NBRISH13, NBRISH14, NBRISH16 and NBRISH26) bacterial isolates capable of withstanding stresses, namely temperature (up to 45 °C), salt (up to 2 M NaCl) and drought (up to 60% Poly Ethylene Glycol 6000) in vitro. Further, the selected isolates were notable for their in vitro temporal performance with regards to survival (in terms of colony count), phosphate solubilisation, biofilm formation, auxin, alginate and exo-polysaccharide production abilities under abiotic stresses i.e. 40 °C temperature; 500 mM NaCl salt and drought (PEG) conditions. In vivo seed treatments of individual selected bacteria to maize plants resulted into significant enhancement in root and shoot length, root and shoot fresh and dry weight and number of leaves per plant. Overall, the plant growth promoting and abiotic stress tolerance ability was most evident for bacterial isolate NBRISH6 which was identified as an Ochrobactrum sp. using 16S rRNA based phylogenetic analysis.     

<Emphasis Type="Italic">Rhizobium sphaerophysae</Emphasis> sp. nov., a novel species isolated from root nodules of <Emphasis Type="Italic">Sphaerophysa salsula</Emphasis> in China

Four gram-negative, aerobic, motile, non-spore, forming rods with a wide pH and temperature range for growth (pH 7.0–11.0, optimum pH 8.0; 20–45°C, optimum 28°C) strains were isolated from root nodules of Sphaerophysa salsula and characterized by means of a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the four strains formed a new lineage related to the genus Rhizobium and the sequence similarities between the isolate and the most related type strain Rhizobium giardinii was 96.5%. These strains also formed a distinctive group from the reference strains for defined Rhizobium species based on housekeeping gene sequences (atpD and recA), BOX-PCR fingerprinting, phenotypic features and symbiotic properties. The representative strain CCNWGS0238^T has DNA-DNA relatedness of less than 33.4% with the most closely related species R. giardinii. It is therefore proposed as a new species, Rhizobium sphaerophysae sp. nov., with isolate CCNWGS0238^T (=ACCC17498^T = HAMBI3074^T) as the type strain.     

An <Emphasis Type="Italic">Rhs</Emphasis>-like genetic element is involved in bacteriocin production by <Emphasis Type="Italic">Pseudomonas savastanoi</Emphasis> pv. <Emphasis Type="Italic">savastanoi</Emphasis>

The main aim of this work was the identification of genetic determinants involved in bacteriocin production by strain ITM317 of Pseudomonas savastanoi pv. savastanoi, besides bacteriocin characterization. The bacteriocin was observed to be a heat-sensitive, high molecular weight proteinaceous compound. We identified a transposon (Tn5)-induced mutant which had lost its ability to produce the bacteriocin. The Tn5 insertion’s responsibility for the above mutated phenotype was demonstrated by marker-exchange mutagenesis. An EcoRI DNA fragment, corresponding to the EcoRI Tn5-containing fragment of the mutant, was also cloned from the wild-type strain, and its introduction into the mutant complemented the mutation. Moreover, that fragment enabled bacteriocin production by P. s. pv. savastanoi ITM302, a strain not previously capable of doing so. DNA sequence analysis revealed that Tn5 insertion occurred in the mutant within a large ORF encoding a protein which showed similarity with proteins from the Rhs family. The DNA region including that ORF showed features which have been considered typical of the Rhs genetic elements previously identified in other bacteria but whose function is as yet unclear. The results of this study for the first time identify an Rhs-like element in P. s. pv. savastanoi, and for the first time indicate that an Rhs element is involved in bacteriocin production, also suggesting this possible function for Rhs genetic elements previously characterized in other bacteria.     

Studies on Endophytic Colonization Ability of Two Upland Rice Endophytes, <Emphasis Type="Italic">Rhizobium</Emphasis> sp. and <Emphasis Type="Italic">Burkholderia</Emphasis> sp., Using Green Fluorescent Protein Reporter

Colonization ability of the two endophytic bacteria, isolated from surface sterilized roots of upland cultivated rice viz., Rhizobium sp. and Burkholderia sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp/gusA). Confocal laser scanning microscopy (CLSM) of gnotobiotically grown seedlings of Narendradhan 97, inoculated with gfp/gusA-tagged endophytes, revealed that both Rhizobium sp. and Burkholderia sp. colonized the intercellular spaces in the root cortex when inoculated separately. Colonization by gfp/gusA-tagged Rhizobium sp. was severely inhibited when co-inoculated with an equal number (10⁶ cfu ml⁻¹) of wild type Burkholderia sp. Burkholderia sp. was a more aggressive endophytic colonizer of rice than Rhizobium sp. The potential of using gfp/gusA reporter and CLSM as tools in evaluating competitive ability of colonization among endophytes is demonstrated in this study.     

<Emphasis Type="Bold">Characterization of nitrile hydratation catalysed by </Emphasis><Emphasis Type="BoldItalic">Nocardia</Emphasis><Emphasis Type="Bold"> sp. 108</Emphasis>

Abstract Nocardia sp. 108 exhibited strong acrylonitrile-hydrating activity and its nitrile hydratase was Co²⁺-dependent. Nocardia sp. 108 was active within a broad pH range from 6.0 to 10.0 at 30°C and thermostable at temperatures below 35°C, but became unstable at temperatures above 45°C. Furthermore, it was found that Nocardia sp. 108 can hydrate indole-3-acetonitrile, p-chlorobenzonitrile, p-hydroxybenzylcyanide, 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, 3-cyanopyridine, o-chlorobenzonitrile to the corresponding amides and hence displayed a broad substrate specificity. The temperature and pH optima for these hydrations were 28°C and pH 7.0–7.5, respectively. At the observed concentrations, acrylonitrile was completely converted within 5 min, while 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, indole-3-acetonitrile, p-chlorobenzonitrile were approximately 21.71, 8.98, 34.44, 93.10% hydrated. p-Chlorobenzonitrile appeared to be the preferred aromatic nitrile for Nocardia sp. 108.     

Impact of Ty3/<Emphasis Type="Italic">Gypsy</Emphasis> group retrotransposon <Emphasis Type="Italic">Lila</Emphasis> on the D-Genome specificity of common wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

An analysis of the primary structure of BAC clone 112D20 T. aestivum, that contains D-genome specific Ty3-Gypsy-retrotransposon Lila is presented. PCR analysis of nulli-tetrasomic and deletion lines of T. aestivum allowed to localize this BAC clone in the distal region of the long arm of chromosome 5D. Characteristic feature of BAC clone 112D20 is a high concentration of Ty3-Gypsy-retrotransposons (61.7%), and low content of the genes (1.2%). Only a single open reading frame was revealed homologous to an unknown gene of Ae. tauschii. Specific to the D-genome Ty3-Gypsy-retrotransposon Lila in the BAC clone 112D20 is 14 kb in length and contains unequal in size long terminal repeats. The data of in situ hybridization and PCR analysis of different Triticeae species suggest that this retroelement was amplified within the ancestral species of Ae. tauschii, the donor D-genome. The suggested time of amplification based on estimation of insertion time of Lila 112D20 is 1.7 million years, which corresponds to the formation of the first allopolyploid forms of wheat. Based on comparison with the previously obtained data, it is concluded that the amplification of retroelements specific to each genome of wheat took place during formation of the diploid progenitors of these genomes.     

Biocontrol of <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> by <Emphasis Type="Italic">Rhizobium</Emphasis> and plant growth-promoting rhizobacteria on lentil

Biocontrol of the root-knot nematode Meloidogyne javanica was studied on lentil using plant growth-promoting rhizobacteria (PGPR) namely Pseudomonas putida, P. alcaligenes, Paenibacillus polymyxa and Bacillus pumilus and root nodule bacterium Rhizobium sp. Pseudomonas putida caused greater inhibitory effect on the hatching and penetration of M. javanica followed by P. alcaligenes, P. polymyxa and B. pumilus. Inoculation of any PGPR species alone or together with Rhizobium increased plant growth both in M. javanica-inoculated and -uninoculated plants. Inoculation of Rhizobum caused greater increase in plant growth than caused by any species of plant growth-promoting rhizobacteria in nematode-inoculated plants. Among PGPR, P. putida caused greater increase in plant growth and higher reduction in galling and nematode multiplication followed by P. alcaligenes, P. polymyxa and B. pumilus. Combined use of Rhizobium with any species of PGPR caused higher reduction in galling and nematode multiplication than their individual inoculation. Use of Rhizobium plus P. putida caused maximum reduction in galling and nematode multiplication followed by Rhizobium plus P. alcaligens. Pseudomonas putida caused greater root colonization and siderophore production followed by P. alcaligenes, P. polymyxa and B. pumilus. Analysis of the protein bands of these four species by SDS-PAGE revealed that P. putida had a different protein band profile compared to the protein profiles of P. alcaligenes, P. polymyxa and B. pumilus. However, the protein profiles of P. acaligenes, P. polymyxa and B. pumilus were similar.     

Effect of temperature on the life history of <Emphasis Type="Italic">Eretmocerus</Emphasis> sp. nr. <Emphasis Type="Italic">furuhashii</Emphasis>, a parasitoid of <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Eretmocerus sp. nr. furuhashii (Hymenoptera: Aphelinidae) is an indigenous parasitoid of Bemisia tabaci (Gennadius)(Hemiptera: Aleyrodidae) from southern China; the effects of constant temperatures on the life history of E. sp. nr. furuhashii were examined in the laboratory. The developmental period ranged from 39.2 days at 20°C to 12.40 days at 32°C. A total of 263.4 degree-days were required to complete development with a lower developmental threshold temperature of 11.1°C. Of the eggs produced, 59.3% completed development at 20°C with completion increasing to 71.5% at 26°C. Adult female longevity was 10.8 days at 20°C and 5.2 days at 32°C while the mean daily offspring reproduced per female was highest at 29°C with 5.9 offspring. Adult oviposition peaked three days after emergence at 26, 29 and 32°C, and four days post-emergence at 20°C and 23°C. The total numbers of offspring produced per female ranged from 25.7 individuals at 32°C to 41.1 individuals at 20°C. The sex ratio had a female bias and ranged from 0.72 at 17°C to 0.51 at 35°C. The intrinsic rate of increase was 0.1727 at 29°C followed with 0.1606 at 32°C. Results indicated that E. sp. nr. furuhashii reaches its maximum biological potential at temperatures ranging from 26°C to 32°C.