Effects of nutritional and environmental conditions on Sinorhizobium meliloti biofilm formation

Rhizobia are non-spore-forming soil bacteria that fix atmospheric nitrogen into ammonia in a symbiosis with legume roots. However, in the absence of a legume host, rhizobia manage to survive and hence must have evolved strategies to adapt to diverse environmental conditions. The capacity to respond to variations in nutrient availability enables the persistence of rhizobial species in soil, and consequently improves their ability to colonize and to survive in the host plant. Rhizobia, like many other soil bacteria, persist in nature most likely in sessile communities known as biofilms, which are most often composed of multiple microbial species. We have been employing in vitro assays to study environmental parameters that might influence biofilm formation in the Medicago symbiont Sinorhizobium meliloti. These parameters include carbon source, amount of nitrate, phosphate, calcium and magnesium as well as the effects of osmolarity and pH. The microtiter plate assay facilitates the detection of subtle differences in rhizobial biofilms in response to these parameters, thereby providing insight into how environmental stress or nutritional status influences rhizobial survival. Nutrients such as sucrose, phosphate and calcium enhance biofilm formation as their concentrations increase, whereas extreme temperatures and pH negatively affect biofilm formation.Fil: Rinaudi, Luciana Veronica. Universidad Nacional de Río Cuarto; ArgentinaFil: Fujishige, Nancy A.. University of California; Estados UnidosFil: Hirsch, Ann M.. University of California; Estados UnidosFil: Banchio, Erika. Universidad Nacional de Río Cuarto; ArgentinaFil: Zorreguieta, Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Giordano, Walter Fabian. Universidad Nacional de Río Cuarto; Argentin

Expression of MsLEC1 transgenes in alfalfa plants causes symbiotic abnormalities

Legume lectins have been proposed to have important symbiotic roles during Rhizobium-legume symbioses. To test this hypothesis, the symbiotic responses of transgenic alfalfa plants that express a portion of the putative alfalfa lectin gene MsLEC1 or MsLEC2 in either the antisense or sense orientation were analyzed following inoculation with wild-type Sinorhizobium meliloti 1021. MsLEC1-antisense (LEC1AS) plants were stunted, exhibited hypernodulation, and developed not only abnormally large nodules but also numerous small nodules, both of which senesced prematurely. MsLEC2-antisense plants were intermediate in growth and nodule number compared with LEC1AS and vector control plants. The symbiotic abnormalities of MsLEC1-sense transgene plants were similar to but milder than the responses shown by the LEC1AS plants, whereas MsLEC2-sense transgene plants exhibited symbiotic responses that were identical to those of vector and nontransgenic control plants. MsLEC1 mRNA accumulation was not detected in nodule RNA by Northern blot analysis but was localized to alfalfa nodule meristems and the adjacent cells of the invasion zone by in situ hybridization; transcripts were also detected in root meristems. A similar spatial pattern of MsLEC2 expression was found by using a whole-mount in situ hybridization procedure. Moreover, mRNAs for an orthologous lectin gene (MaLEC) were detected in white sweetclover (Melilotus alba) nodules and root tips

Dg93, a Nodule-Abundant mRNA of Datisca glomerata with Homology to a Soybean Early Nodulin Gene

We have isolated a 590-bp full-length cDNA clone designated Dg93, an mRNA that is highly expressed in symbiotic root nodules of the actinorhizal host Datisca glomerata. Dg93 mRNA encodes a deduced polypeptide of 105 amino acids with significant identity (74%) to the soybean (Glycine max) early nodulin (ENOD) gene GmENOD93 (Kouchi and Hata, 1993). Dg93 mRNA is abundant in nodules at 4 weeks post inoculation, the earliest time assayed, and steady-state mRNA levels remain elevated 11 weeks after inoculation. Spatial patterns of Dg93 mRNA expression are complex, with transcript accumulation in the nodule lobe meristem, early infection zone, periderm, and cells of the vascular cylinder, but not in the surrounding uninfected cortical cells. Dg93 is encoded by a small gene family in D. glomerata. To our knowledge, this is the first report of a gene from an actinorhizal host that is expressed in the nodule meristem and that shares sequence homology with an early nodulin gene from a legume

Investigations of Rhizobium biofilm formation

The development of nitrogen-fixing nodules of the Rhizobium-legume symbiosis, especially the early stages of root hair deformation and curling, infection thread formation, and nodule initiation, has been well studied from a genetic standpoint. In contrast, the factors important for the colonization of surfaces by rhizobia, including roots-an important prerequisite for nodule formation-have not been as thoroughly investigated. We developed conditions for analyzing the ability of two fast-growing rhizobia, Sinorhizobium meliloti and Rhizobium leguminosarum bv. viciae, to produce biofilms on abiotic surfaces such as glass, plastic microtiter plates, sand and soil as a prelude to characterizing the genes important for aggregation and attachment. Factors involved in adherence to abiotic surfaces are likely to be used in rhizobial attachment to legume root cells. In this report, we show that S. meliloti exopolysaccharide-deficient mutants as well as exopolysaccharide overproducers exhibit reduced biofilm phenotypes that show parallels with their nodulation abilities. We also investigated two flagella-less S. meliloti mutants and found them to have reduced biofilming capabilities. To investigate whether there was a symbiotic phenotype, we tested one of the Fla(-) mutants on two different S. meliloti hosts, alfalfa and white sweetclover, and found that nodule formation was significantly delayed on the latter

Rhizobium common nod genes are required for biofilm formation.

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

Bacillus simplex—A Little Known PGPB with Anti-Fungal Activity—Alters Pea Legume Root Architecture and Nodule Morphology When Coinoculated with Rhizobium leguminosarum bv. viciae

Two strains, 30N-5 and 30VD-1, identified as Bacillus simplex and B. subtilis, were isolated from the rhizospheres of two different plants, a Podocarpus and a palm, respectively, growing in the University of California, Los Angeles (UCLA) Mildred E. Mathias Botanical Garden. B. subtilis is a well-known plant-growth promoting bacterial species, but B. simplex is not. B. simplex 30N-5 was initially isolated on a nitrogen-free medium, but no evidence for nitrogen fixation was found. Nevertheless, pea plants inoculated with B. simplex showed a change in root architecture due to the emergence of more lateral roots. When Pisum sativum carrying a DR5::GUSA construct, an indicator for auxin response, was inoculated with either B. simplex 30N-5 or its symbiont Rhizobium leguminosarum bv. viciae 128C53, GUS expression in the roots was increased over the uninoculated controls. Moreover, when pea roots were coinoculated with either B. simplex 30N-5 or B. subtilis 30VD-1 and R. leguminosarum bv. viciae 128C53, the nodules were larger, clustered, and developed more highly branched vascular bundles. Besides producing siderophores and solubilizing phosphate, the two Bacillus spp., especially strain 30VD-1, exhibited anti-fungal activity towards Fusarium. Our data show that combining nodulating, nitrogen-fixing rhizobia with growth-promoting bacteria enhances plant development and strongly supports a coinoculation strategy to improve nitrogen fixation, increase biomass, and establish greater resistance to fungal disease