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The rhizobia-legume, root-nodule symbiosis provides the most efficient source of biologically fixed ammonia fertilizer for agricultural crops. Its development involves pathways of specificity, infectivity, and effectivity resulting from expressed traits of the bacterium and host plant. A key event of the infection process required for development of this root-nodule symbiosis is a highly localized, complete erosion of the plant cell wall through which the bacterial symbiont penetrates to establish a nitrogen-fixing, intracellular endosymbiotic state within the host. This process of wall degradation must be delicately balanced to avoid lysis and destruction of the host cell. Here, we describe the purification, biochemical characterization, molecular genetic analysis, biological activity, and symbiotic function of a cell-bound bacterial cellulase (CelC2) enzyme from Rhizobium leguminosarum bv. trifolii, the clover-nodulating endosymbiont. The purified enzyme can erode the noncrystalline tip of the white clover host root hair wall, making a localized hole of sufficient size to allow wild-type microsymbiont penetration. This CelC2 enzyme is not active on root hairs of the nonhost legume alfalfa. Microscopy analysis of the symbiotic phenotypes of the ANU843 wild type and CelC2 knockout mutant derivative revealed that this enzyme fulfils an essential role in the primary infection process required for development of the canonical nitrogen-fixing R. leguminosarum bv. trifolii-white clover symbiosis.nitrogen fixation ͉ nodulation ͉ clover ͉ root hair ͉ cellulose A central event in development of the Rhizobium-legume root-nodule symbiosis is the localized erosion of a cellulosic plant wall through which the bacterial symbiont passes to establish a nitrogen-fixing, intracellular endosymbiotic state within its legume host. Plant cell wall-degrading enzymes are predicted to participate in two steps of this infection process: during primary infection of host root hairs leading to infection thread formation (Inf) and later during bacterial release (Bar) from infection threads within host nodule cells. This process of plant cell wall degradation must be delicately balanced to allow the localized penetration of the bacterial symbiont into the host cell without its overt lysis and destruction.Several studies indicate that rhizobia produce enzymes capable of degrading plant cell wall polymers (1-14), but little is known about their molecular properties, none have previously been purified to homogeneity, and their specific role (if any) in symbiosis is undefined. The relatively low activities of these rhizobial enzymes have hampered research progress in this area. Using improved assays with increased sensitivity that reliably detect these enzyme activities, we established that cellulases are produced by wild-type strains of Rhizobium leguminosarum (biovars trifolii, phaseoli, and viciae), Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti (7, 9). Further studies using R. leguminosarum bv. trifolii ANU843 indic...

Bacteria belonging to the family Rhizobiaceae may establish beneficial or harmful relationships with plants. The legume endosymbionts contain nod and nif genes responsible for nodule formation and nitrogen fixation, respectively, whereas the pathogenic strains carry vir genes responsible for the formation of tumors or hairy roots. The symbiotic and pathogenic strains currently belong to different species of the genus Rhizobium and, until now, no strains able to establish symbiosis with legumes and also to induce tumors or hairy roots in plants have been reported. Here, we report for the first time the occurrence of two rhizobial strains (163C and ATCC11325T) belonging to Rhizobium rhizogenes able to induce hairy roots or tumors in plants and also to nodulate Phaseolus vulgaris under natural environmental conditions. Symbiotic plasmids (pSym) containing nod and nif genes and pTi- or pRi-type plasmids containing vir genes were found in these strains. The nodD and nifH genes of the strains from this study are phylogenetically related to those of Sinorhizobium strains nodulating P. vulgaris. The virA and virB4 genes from strain 163C are phylogenetically related to those of R. tumefaciens C58, whereas the same genes from strain ATCC 11325T are related to those of hairy root-inducing strains. These findings may be of high relevance for the better understanding of plant-microbe interactions and knowledge of rhizobial phylogenetic history.

A bacterial strain designated GR21 T was isolated from apoplastic fluid of Saccharum officinarum (sugar cane). Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate forms a separate branch within the family 'Paenibacillaceae', with Paenibacillus as the closest related genus. Within this genus, the closest related species is Paenibacillus xylanilyticus, with 93.4 % similarity to the sequence of the type strain. The isolate has Gram-variable, facultatively anaerobic, rod-shaped cells, motile by polar and subpolar flagella. Round, non-ornamented, central or subterminal spores are formed in unswollen sporangia. The strain is catalase-positive and oxidase-negative on nutrient agar medium. Cellulose and aesculin were hydrolysed, whereas xylan, starch and gelatin were not. Growth was supported by many carbohydrates as carbon sources. Strain GR21 T displayed a lipid profile consisting of diphosphatidylglycerol, phosphatidylglycerol, an unknown aminophospholipid, two unknown glycolipids and an unknown phosphoglycolipid. MK-7 was the predominant menaquinone and anteiso-C 15 : 0 was the major fatty acid. The DNA G+C content was 57.8 mol%. Phylogenetic and phenotypic analyses, including assimilation of carbon sources and exoenzyme production commonly used for classification within the family 'Paenibacillaceae', showed that strain GR21 T belongs to a new genus within this family, for which the name Saccharibacillus sacchari gen. nov., sp. nov. is proposed. The type strain of Saccharibacillus sacchari is GR21 T (5LMG 24085 T 5DSM 19268 T ).The family 'Paenibacillaceae' currently contains eight genera of Gram-positive, -variable or -negative sporulating bacilli. The description of the most recently described genus of the family, Cohnella (Kämpfer et al., 2006), was based on its phylogenetic relationship with respect to the remaining genera of the family as well as on its polar lipid profile. Some species of this family, mainly belonging to the genus Paenibacillus, have been isolated from the plant rhizosphere, but only Paenibacillus polymyxa has been reported as a plant endophyte (Cho et al., 2006). In the present work, we describe the isolation and identification of a novel bacterium isolated from apoplastic sap of sugar cane (Saccharum officinarum) in Granada (Spain).For the isolation of strain GR21 T , stem pieces were washed with sterile tap water and soaked in 5 % chloramine T solution for 2 min. After removal of the rind with a sterile sharp knife, the cylinder of parenchyma tissue was rinsed with 70 % ethanol and flame-sterilized. Apoplastic sap from the internodes was collected by centrifugation according to Dong et al. (1994), and serial dilutions were streaked on tryptone soybean agar (TSA) and incubated at 30 u C for 7-10 days. After flaming, no contamination was observed when several stem pieces were incubated on TSA plates.Strain GR21 T was grown on nutrient agar for 48 h at 22 u C to check for motility by phase-contrast microscopy using the hanging drop method. Gram staining was carried o...

The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial isolates of Phaseolus vulgaris grown in the soil of Lanzarote, an island of the Canary Islands, were identical to a less-common pattern found within Sinorhizobium meliloti (assigned to group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northern Spain. The P. vulgaris isolates and the group II LMW RNA S. meliloti isolates also were distinguishable in that both had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed spacer region that were not present in other strains of S. meliloti. The isolates from P. vulgaris nodulated bean but not Medicago sativa, while those recovered from Medicago, Melilotus, and Trigonella spp. nodulated both host legumes. The bean isolates also were distinguished from those of Medicago, Melilotus, and Trigonella spp. by nodC sequence analysis. The nodC sequences of the bean isolates were most similar to those reported for S. meliloti bv. mediterranense and Sinorhizobium fredii bv. mediterranense (GenBank accession numbers DQ333891 and AF217267, respectively). None of the evidence placed the bean isolates from Lanzarote in the genus Rhizobium, which perhaps is inconsistent with seed-borne transmission of Rhizobium etli from the Americas to the Canaries as an explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote.