This paper summarizes a multinational collaborative project to search for natural, intimate associations between rhizobia and rice (Oryza sativa L.), assess their impact on plant growth, and exploit those combinations that can enhance grain yield with less dependence on inputs of nitrogen (N) fertilizer. Diverse, indigenous populations of Rhizobium leguminosarum bv. trifolii (the clover root-nodule endosymbiont) intimately colonize rice roots in the Egyptian Nile delta where this cereal has been rotated successfully with berseem clover (Trifolium alexandrinum L.) since antiquity. Laboratory and greenhouse studies have shown with certain rhizobial strain-rice variety combinations that the association promotes root and shoot growth thereby significantly improving seedling vigour that carries over to significant increases in grain yield at maturity. Three field inoculation trials in the Nile delta indicated that a few strain-variety combinations significantly increased rice grain yield, agronomic fertilizer N-use efficiency and harvest index. The benefits of this association leading to greater production of vegetative and reproductive biomass more likely involve rhizobial modulation of the plant's root architecture for more efficient acquisition of certain soil nutrients [e.g. N, phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), zinc (Zn), sodium (Na) and molybdenum (Mo)] rather than biological N 2 fixation. Inoculation increased total protein quantity per hectare in field-grown grain, thereby increasing its nutritional value without altering the ratios of nutritionally important proteins. Studies using a selected rhizobial strain (E11)

Lipopolysaccharides (LPSs) isolated from several strains of Rhizobium, Bradyrhizobium, Agrobacterium, and Azorhizobium were screened for the presence of 27-hydroxyoctacosanoic acid. The LPSs from all strains, with the exception of Azorhizobium caulinodans, contained various amounts of this long-chain hydroxy fatty acid in the lipid A fractions. Analysis of the lipid A sugars revealed three types of backbones: those containing glucosamine (as found in Rhizobium meliloti and RhizobiumfrediO), those containing glucosamine and galacturonic acid (as found in Rhizobium leguminosarum bv. phaseoli, trifolii, and viciae), and those containing 2,3-diamino-2,3-dideoxyglucose either alone or in combination with glucosamine (as found in Bradyrhizobium japonicum and Bradyrhizobium sp.[Lupinus] strain DSM 30140). The distribution of 27-hydroxyoctacosanoic acid as well as analysis of lipid A backbone sugars revealed the taxonomic relatedness of various strains of the Rhizobiaceae.Bacteria belonging to the family Rhizobiaceae are gram negative and are able to form nitrogen-fixing symbiotic relationships with legume plants. There are three distinct genera: the symbiotic nitrogen-fixing Rhizobium and Bradyrhizobium spp. and the plant pathogenic Agrobacterium spp. Quite recently a new genus, so far comprising only the stem-nodulating nitrogen-fixing species Azorhizobium caulinodans, was defined (13). Of these genera, the species of Rhizobium are taxonomically closely related and show genetic similarities to the genus Agrobacterium as evidenced by 16S rRNA homology studies (12). On the other hand, the slowly growing species of Bradyrhizobium are rather distantly related to the other two genera as revealed by their low SAB values determined by DNA-rRNA hybridization studies (1). In addition to the nucleotide sequence homology studies, differentiation of various members of the Rhizobiaceae has been attempted by several chemotaxonomic approaches such as cellular fatty acid analysis (21, 31), polyacrylamide gel electrophoresis of cellular proteins (19), and composition analysis of extracellular gum (26). However, results of these studies were not sufficient to adequately distinguish between members of the Rhizobiaceae. More recently, the backbone sugar composition of lipid A fractions of lipopolysaccharide (LPS) has been used as a taxonomic marker for recognition and relatedness of various nonsulfur bacteria (23). Therefore, in this study, the lipid A fractions from rhizobial LPSs were examined to see whether they represented a marker for determining the relatedness of these bacteria.The surface polysaccharides, including the LPS, of strains of Rhizobium have been hypothesized to be involved in the molecular mechanisms of symbiotic infection (5). In an attempt to elucidate the structure of LPS from rhizobial strains, an unusual very-long-chain hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27-OH-28:0), was discovered to be the major fatty acid constituent of the lipid A region (15). More recently, we have also identified this long-cha...

Past studies of cold-acclimated bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study cold-acclimated physiology. Changes in membrane composition and exopolysaccharides were defined as a function of growth at 24, 4 and -2.5 degrees C in the presence and absence of 5% NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the colder temperatures and a further decrease in the degree of saturation at higher osmolarity. A shift in carbon source utilization and antibiotic resistance occurred at 4 versus 24 degrees C growth, perhaps due to changes in the membrane transport. Some carbon substrates were used uniquely at 4 degrees C and, in general, increased antibiotic sensitivity was observed at 4 degrees C. All the permafrost strains tested were resistant to long-term freezing (1 year) and were not particularly unique in their UVC tolerance. Most of the tested isolates had moderate ice nucleation activity, and particularly interesting was the fact that the Gram-positive Exiguobacterium showed some soluble ice nucleation activity. In general the features measured suggest that the Siberian organisms have adapted to the conditions of long-term freezing at least for the temperatures of the Kolyma region which are -10 to -12 degrees C where intracellular water is likely not frozen.

exo mutants of Rhizobium meliloti SU47, which fail to secrete acidic extracellular polysaccharide (EPS), induce Fix-nodules on alfalfa. However, mutants of R. meliloti Rm4l carrying the same exo lesions induce normal Fix' nodules. We show that such induction is due to a gene from strain Rm4l, which we call lpsZ+, that is missing in strain SU47. lpsZ+ does not restore EPS production but instead alters the composition and structure of lipopolysaccharide. In both SU47 and Rm4l, either lpsZ+ or exo+ is sufficient for normal nodulation. This suggests that in R. meliloti EPS and lipopolysaccharide can perform the same function in nodule development.The nitrogen-fixing alfalfa symbiont Rhizobium meliloti secretes an acidic extracellular polysaccharide (EPS) called EPS-I (or EPSa) (2, 47). EPS-I binds to fluorochrome dye calcofluor white, which causes EPS-I-producing colonies to fluoresce blue-green in UV light (calcofluor-bright phenotype). Genetic evidence indicates that EPS-I functions in bacterial invasion of developing nodules (15,25). In R. meliloti natural isolate SU47 (41), exo mutants that fail to fluoresce on calcofluor agar (calcofluor-dark phenotype) do not secrete EPS-I and are concomitantly defective in invasion of nodule cells (Inft), inducing small white nodules that are unable to fix nitrogen (Fix-).Strain SU47 can also be made to produce a second unrelated EPS, EPS-II (or EPSb), by mutation of the presumptive negative regulatory loci, expR (18) or mucR (48). Production of EPS-II by expR results in a mucoid-colony phenotype and allows invasion and Fix' nodule formation by some exo mutants on alfalfa (Medicago sativa), although not on other R. meliloti macrosymbionts (Medicago caerulea, Medicago trunculata, Melilotus alba, Trigonella foenum-graecium). Production of EPS-II by mucR also results in mucoidy and in Fix' nodule formation on alfalfa and Melilotus alba (nodulation on other hosts has not been reported).A variety of exo mutants with different characteristics have been described previously (15,25,28). Mutations in exoA, -B, -F, -L, -P, -Q, and -T are calcofluor dark and are clustered in a 20-kilobase (kb) region of the symbiotic megaplasmid pRmeSU47b, which we call pEXO (see below); all of these mutants are deficient in EPS-I and are Inf Fix-. Unlike the other pEXO exo mutants, exoB mutants do not produce EPS-II in an expR or mucR background, presumably because exoB is also required for the synthesis of EPS-II. exoB mutants are also resistant to a number of R. Fix' nodules on all of the macrosymbionts tested. lpsZ+ is involved in the synthesis of LPS in Rm4l and has no known effect on EPS. Therefore, LPS appears to be responsible for suppression of the exo defect. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids which we used are listed in Table 1.Media and growth conditions. The media and growth conditions which we used have been described previously (14)(15)(16)36). Phage assays were done in A broth or in top agar (7) containing 20 mM CaC12 and 20 mM MgSO4.G...

The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. tnifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 ,ug per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by N03 or NH4', and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-0-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (0-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.Establishment of an effective Rhizobium-legume symbiosis can be viewed as a process of cellular recognition and compatibility between bacterial and plant cells. The infection process involves bacterial attachment, root hair deformation, bacterial penetration of the root hair wall, formation and sustained development of the infection thread, bacterial release from infection threads within emerging root nodule cells, and bacterial differentiation into nitrogen-fixing bacteroids.The lipopolysaccharides (LPS) of rhizobia are likely to be involved in the infection process. They are major glycoconjugates on the surface of Rhizobium leguminosarum biovars * Corresponding author. t Present address: Laboratoire des Relationes Plantes-Microorganismes,

A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C 30 fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities).Microorganisms such as Saccharomyces or Zymomonas strains that are used for industrial ethanol production from glucose or sucrose have high alcohol tolerance for growth (i.e., Ͼ6% [vol/vol]). Other species that produce ethanol from cheaper substrates such as cellulose or starch, like Clostridium thermocellum or Thermoanaerobacter ethanolicus, generally have a low alcohol tolerance for growth (Ͻ2% [vol/vol]). In general, alcohol-producing microbes respond to increasing solvent concentrations by increasing the percentage of unsaturated versus saturated fatty acids, long-chain fatty acids, and hopanes into their cytoplasmic membranes (2,8,9). These structural changes prevent the loss of membrane function from fluidization caused by a high solvent concentration.Thermophilic ethanol fermentations offer the potential of direct degradation of cellulose or starch and direct recovery of ethanol at fermentation temperatures under reduced pressure (5,16,17,18,21,23). This potential has not been demonstrated because of low-end product concentrations caused by bacterial ethanol inhibition. Thermophilic bacteria employ two different pathways for ethanol production, using either a primary alcohol dehydrogenase (ADH), as in C. thermocellum, or primary and secondary ADHs, as in T. ethanolicus (13,15). Herrero and coworkers (3, 4) studied ethanol tolerance in C. thermocellum and concluded that the low tolerance to ethanol (Ͻ2% [vol/vol]) was a combined result of general solvent effects on membrane fluidity and a specific inhibition of enzymes involved in sugar metabolism. Work in the labs of Ljundahl, Wiegel, Demain, Zeikus, and others has showed that thermophilic anaerobic bacteria can adapt their tolerance to about 4% (vol/vol) ethanol (for a review, see reference 16).We previously demonstrated (15) that moderate ethanol tolerance (Ͻ4% [vol/vol]) of a T. ethanolicus mutant strain was related to enzymatic prevention of metabolic inhibition caused by ethanol overreducing the pyridine nucleotide pool and inhibiting glycolysis. The ethanol-tolerant mutant 39EA lacked primary ADH...

No abstract

No abstract