Production of Extracellular Polysaccharide by a Rhizobium Species from Root Nodules of the Leguminous Tree Dalbergia lanceolaria

The ability of the Rhizobium D1 10 species, which was isolated from the root nodules of the leguminous forest tree Dalbergia lanceolaria, for the production of extracellular polysaccharides (EPS) was investigated. High amounts of EPS (765 μg/mL) were produced by the bacteria (Rhizobium D1 10) in yeast extract mannitol medium. Both growth and EPS production started simultaneously, but the EPS production was at its maximum in the stationary phase of growth at 32 h. The EPS production was maximal when the medium was supplemented with mannitol (2 %), thiamine hydrochloride (1 μg/mL) and KNO₃ (0.1 %), which was accompanied by a great increase in the production compared to the control. The EPS contained xylose, rhamnose, glucose, galactose and arabinose. The possible role of rhizobial EPS production in root nodule symbiosis is discussed.     

Carbohydrate-mediated Recognition of Larval Mosquito Hosts by Romanomermis culicivorax

Proteases, glycosidases, and lectins were tested and the results supported a role in host recognition for glycoproteins containing β-glucose and α-mannose on the cuticular surface of host and parasite. Carbohydrates containing α-glucose, galactose, fucose, or N-acetylglucosamine residues apparently are not involved in nematode attachment. Chitin or a related N-acetylglucosamine polymer was found in R. culicivorax preparasites. Treatment of preparasites with neuraminidase, which hydrolyzes sialic acids, increased nematode attachment to Anopheles freeborni larvae.     

Correlation between NaCl Sensitivity of Rhizobium Bacteria and Ineffective Nodulation of Leguminous Plants

A sodium chloride (NaCl)-sensitive mutant of Rhizobium fredii USDA191, which contained a single copy of Tn5-Mob transposed into chromosomal DNA, was obtained by Tn5-Mob random insertion. The growth rate of this mutant was lower than that of the wild type in the presence of 0.2 M NaCl and it seemed to lack the inductive ATP production in response to the addition of NaCl. This mutant induced the formation of small and whitish nodules on lateral roots of soybeans, which were negative for acetylene reduction activity, indicating that the nodules were ineffective for nitrogen fixation. The mutant also reduced the weight of above-ground portions and roots to 64 and 55%, respectively, compared with the weight of the plants inoculated with the wild-type cells. These results suggest that NaCl sensitivity of Rhizobium bacteria is one of the important factors for nodule formation and nitrogen fixation.     

根瘤菌选育研究进展

生物固氮是一个全球性的战略课题,其中豆科植物与根瘤菌共生固氮一直是生物固氮研究的焦点。该文从菌株选育的角度,通过对比总结国内外根瘤菌选育方法的研究进展,详细阐述了各种育种方法在根瘤菌选育过程中的应用和优缺点,指出筛选周期过长和筛选技术低效是当前研究中的限制问题,并进一步对选育工作的前景进行了展望。     

Natural Diversity of Frankia Strains in Actinorhizal Root Nodules from Promiscuous Hosts in the Family Myricaceae

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N₂-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.     

Rhizoremediation of Dioxin-like Compounds by a Recombinant Rhizobium tropici Strain Expressing Carbazole 1,9a-Dioxygenase Constitutively

A recombinant Rhizobium strain, PBK3-IS, that constitutively expressed the oxygenase component of carbazole 1,9a-dioxygenase from Sphingomonas sp. strain KA1, was constructed. In the water-cultured siratro rhizospheres inoculated with strain PBK3-IS, 48% of the dibenzofuran was removed within 3 days (initial substrate, 25 μg). Similar results were obtained in soil-cultured siratro rhizospheres using sterile vermiculite. When non-sterile field soils were used instead of sterile vermiculite, the inoculated recombinant strain could grow on the siratro root in all soils tested, except for wet paddy field.     

Rhizobium Strain Identification and Quantification in Commercial Inoculants by Immunoblot Analysis

An immunoblot procedure for the strain-specific quantitative analysis of commercial Rhizobium inoculants was developed. The technique greatly reduced the time required for inoculant analysis. Correlation between immunoblot analysis and traditional plant nodule grow-out most-probable-number techniques was r = 0.90 for 16 commercial alfalfa inoculants tested.     

Isolation and Characterization of a Bacteriophage Tail-Like Bacteriocin from a Strain of Rhizobium

Bacterial strain 16-3 spontaneously produces a bacteriocin which inhibits the growth of closely related strain 16-2. Both strains were newly isolated from root nodules of lupines and probably belong to the species Rhizobium lupini. Production of infectious progeny of newly isolated virulent phage 16-2-4 in strain 16-2 is inhibited completely if complexes are bacteriocin-treated during the first half of the latent period. Treatment begun during the second half leads to premature lysis of complexes and inactivates only those progeny phages which were not yet fully matured at the moment of the particle-induced lysis. Examination by electron microscope of the bacteriocin enrichment revealed the presence of particles 123 nm in length which resemble the tails of T-even bacteriophages. Since the particles sediment together with the bactericidal activity in the sucrose gradient and adsorb specifically to bacteriocin-sensitive cells, it is concluded that they are identical with the bactericidal agent. The particles are not found attached to phage heads and cannot self-propagate; they are regarded as incomplete and are named INCO particles. INCO particles consist of a core enveloped by a contractile sheath. One end of the sheath is connected to a baseplate to which six fibers, each 32 nm in length, are attached. These connect the baseplate of an adsorbing particle to the cell surface. Since INCo cores are probably empty, it is concluded that specific adsorption of the particles to the bacterial surface is sufficient to inactive sensitive cells irreversibly.     

Role of Lectins in the Specific Recognition of Rhizobium by Lotononis bainesii

Fluorescein isothiocyanate (FITC)-labeled lectin purified from the root of Lotononis bainesii Baker was bound by cells of five out of seven L. bainesii-nodulating strains of Rhizobium under culture conditions. With the exception of a strain of Rhizobium leguminosarum, strains of noninfective rhizobia failed to bind the root lectin under these conditions. The two nonlectin binding L. bainesii-specific strains did not bind root lectin on the L. bainesii rhizoplane although this was observed with three other L. bainesii-nodulating strains. A single Rhizobium japonicum strain bound root lectin on the L. bainesii rhizoplane. There was no evidence of an interaction between the L. bainesii seed lectin and the Rhizobium strains tested.

Root lectin-specific FITC-labeled antibodies were bound to the tips of developing root hairs and lateral growth points of more mature root hairs of L. bainesii seedlings. The damaged edges of severed root hairs always bound FITC-labeled root lectin antibody. Seed lectin-specific FITC-labeled antibodies were not bound to the roots of L. bainesii. The preemergent root hair region of L. bainesii was most susceptible to infection by rhizobia but nodules also emerged in the developing and mature root hair regions. Lectin exposed at growth points on L. bainesii root hairs may provide a favorable site for host plant recognition of infective strains of Rhizobium.

     

A Positive Strain Identification Method for Rhizobium meliloti

About 80% of Rhizobium meliloti strains contain 1 to 11 copies of insertion sequence ISRm1 in their genomes (R. Wheatcroft and R. J. Watson, J. Gen. Microbiol. 134:113-121, 1988). Hybridization to separated genomic DNA fragments with an ISRm1-specific probe produces patterns of hybridization bands which are distinctive for each strain. These patterns can be compared between strains to prove or disprove common identity. In most cases relatedness can be inferred despite phenotypic differences or minor genomic alterations.     

Strain Identification in Rhizobium Using Intrinsic Antibiotic Resistance

The variation in intrinsic resistance to low levels of eight antibiotics was used as an identifying characteristic for 26 Rhizobium leguminosarum strains. The pattern of antibiotic resistance of each strain was a stable property by which rhizobia isolated from root nodules of inoculated Pisum sativum could be recognized. The antibiotic tests for strain identification with R. leguminosarum were applied to R. phaseoli . It was necessary to include reference cultures in tests with this species, as the tests most suitable for the R. leguminosarum strains showed some variability with R. phaseoli .     

Mimosine, a Toxin Present in Leguminous Trees (Leucaena spp.), Induces a Mimosine-Degrading Enzyme Activity in Some Rhizobium Strains

Thirty-seven Rhizobium isolates obtained from the nodules of leguminous trees (Leucaena spp.) were selected on the basis of their ability to catabolize mimosine, a toxin found in large quantities in the seeds, foliage, and roots of plants of the genera Leucaena and Mimosa. A new medium containing mimosine as the sole source of carbon and nitrogen was used for selection. The enzymes of the mimosine catabolic pathway were inducible and were present in the soluble fraction of the cell extract of induced cells. On the basis of a comparison of the growth rates of Rhizobium strains on general carbon and nitrogen sources versus mimosine, the toxin appears to be converted mostly to biomass and carbon dioxide. Most isolates able to grow on mimosine as a source of carbon and nitrogen are also able to utilize 3-hydroxy-4-pyridone, a toxic intermediate of mimosine degradation in other organisms.     

Asymbiotic Acetylene Reduction by a Fast-Growing Cowpea Rhizobium Strain with Nitrogenase Structural Genes Located on a Symbiotic Plasmid

A procedure was designed which enabled the detection of ex planta nitrogenase activity in the fast-growing cowpea Rhizobium strain IHP100. Nitrogenase activity in agar culture under air occurred at a rate similar to that found for Bradyrhizobium strain CB756 but lower than that for Rhizobium strain ORS571. Hybridization studies showed that both nod and nif genes were located on a 410-kilobase Sym plasmid in strain IHP100.     

Procedure for Obtaining Efficient Root Nodulation of a Pea Cultivar by a Desired Rhizobium Strain and Preempting Nodulation by Other Strains

The specificity between the sym-2 gene bred into certain cultivars of pea (Pisum sativum L.) and the nodX gene, present only rarely in isolates of Rhizobium leguminosarum, can be exploited to preempt competition or nodulation blocking by a Rhizobium strain indigenous to a soil environment. The principle is to isolate an R. leguminosarum strain prevalent in a locale, convert it into a strain that will nodulate a desirable pea cultivar carrying sym-2 by establishing nodX in it, and then use the resulting Rhizobium strain with the pea cultivar carrying sym-2. To accomplish this, we first constructed a transposon Tn5 derivative called Tn5nodX and an efficient delivery vehicle that is suicidal in R. leguminosarum. We tested the potential utility of the system in greenhouse experiments. The results are encouraging enough to warrant extensive experiments under field conditions.     

Rhizobium sp. Strain NGR234 Possesses a Remarkable Number of Secretion Systems

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.Diverse soil bacteria interact with plants in ways that range from symbiotic to pathogenic. Symbiotic Eubacteria (both alpha- and betaproteobacteria, collectively called rhizobia) form nitrogen-fixing associations of tremendous environmental importance (41, 66). Although some rhizobia are able to reduce atmospheric nitrogen to ammonia under saprophytic, free-living conditions, the reduced oxygen tensions found within the intracellular environment of specialized organs called nodules, maximizes this process (16). As legume roots penetrate the soil, they come in contact with rhizobia. Symbiotic interactions are initiated by the exchange of diverse molecules between the partners. Among them, plants liberate flavonoids into the rhizosphere that upregulate rhizobial genes. As a result, lipo-chito-oligo-saccharidic Nod factors are produced that trigger the nodulation pathway in susceptible legumes. Then, in many hosts, rhizobia enter the roots through root hairs, make their way to the cortex, multiply and fill the intracellular spaces of mature nodules. Centripetal progression of rhizobia into the plant and their maturation into nitrogen-fixing symbiosomes depends on the continued exchange of diverse signals. Many, but not all of these signals have been identified; one sure way to take stock of what is necessary for effective symbiosis is to sequence the partners. We began this work by assembling overlapping sets of cosmids (contigs) of the microsymbiont Rhizobium sp. strain NGR234 (hereafter NGR234) (63), which enabled us to elucidate the nucleotide sequence of the symbiotic (pNGR243a) plasmid (29). Similar techniques permitted the assembly of sections of the extremely large megaplasmid pNGR234b (86), and some snapshot genome information was made available earlier (91); however, the use of pyrosequencing methods greatly facilitated this process. We report here the genome sequence of NGR234 that is able to nodulate more than 120 genera of legumes and the nonlegume Parasponia andersonii (69). It seems likely that the vast richness of secretory systems might be a major key to the broad host range.     

Impact of Latently Infected Cells on Strain Archiving Within HIV Hosts

Latently infected cells are a barrier to HIV eradication on therapy due to long half-lives of between 6 and 44 months. The mechanism behind this long term maintenance is unclear although bystander proliferation and asymmetric division have both been put forward for consideration in mathematical models. The latently infected cell reservoir seems to act as an archive for strains of HIV no longer dominant in the blood, such as wild-type virus when the individual is on therapy. This is particularly significant when patients wish to come off medication and wild-type virus re-emerges.We use a two target cell model capable of producing low-level viral load on therapy and include latent cells and two strains of virus, wild-type and drug resistant, to investigate the impact of two possible mechanisms of latent cell reservoir maintenance on strain archiving. We find that although short term (less than a year) archiving of viral strains is possible in a model with no mechanism for reservoir maintenance, both bystander proliferation and asymmetric division of latent cells allow archiving to occur over much longer timescales (2 or more years). We suggest that regardless of the mechanism involved, latent cell reservoir maintenance allows strain archiving to occur. We interpret our results for clinical consideration.     

Strain selection for pigeon pea Rhizobium under greenhouse conditions

Pigeon pea obtains N for growth by N₂ fixation although yield generally is not improved by either the inoculation of Rhizobium or by the application of N fertilizer in Puerto Rico. Sixteen strains ofRhizobium spp., different in geographical origin, were tested for N-fixing effectiveness, determined from comparisons with uninoculated controls, N controls and the standard strain 176A22. Inoculated treatments showed significant differences in nodulation, plant dry weight and %N. Several strain x plant combinations had higher N content than the N treatment, reflecting the ubiquity of effective strains and the possible lack of response of pigeon pea to inoculation or N fertilization. Strains superior in N₂ fixation were selected for testing for symbiotic effectiveness under field conditions.     

A Note on a Highly Sensitive Modified ELISA Technique for Rhizobium Strain Identification

A modification of the ELISA technique using a fluorescent substrate is described and comparisons are made between this highly sensitive technique and the conventional ELISA method for investigations of the Rhizobium /legume symbiosis. The technique can be used to detect Rhizobium spp. both from pure culture and from nodules on Trifolium repens , at a concentration of 10⁴ cells/ml. Tests for cross-reactivity indicated that the technique will facilitate a wide range of experiments which require the identification of Rhizobium strains.     

Some Bacteriophages Active Against Rhizobium trifolii Strain W19

Fourteen soil bacteriophages active against Rhizobium trifolii W19 have been studied which fall into four structural groups. Group 1 phages have contractile tails. Some particles show double base plates to which at least three spikes are attached, and fibers are attached to the base plates. Group 2 phages also have contractile tails. At least five spikes are attached to the base plate, and there are spherical bodies adjacent to the tail, at the ends of fibers attached to the tail base. Group 3 phages have contractile tails, but are larger than phages of groups 1 and 2. The end of the tail has a complex structure. Group 4 phages have long, noncontractile tails.