Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or Rhizobium. The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.Neptunia natans (L.f.) Druce is an aquatic legume native to several continents of the humid tropics and is used for both human consumption and as green manure for rice cultivation in Asiatic countries. This legume is unusual in that it normally develops buoyant floating stems that grow profusely on the surface of freshwater ponds, and in this aquatic environment it develops many stem-associated nitrogen-fixing nodules.The developmental morphology of the infection process leading to formation of nitrogen-fixing nodules in N. natans has been examined under strict gnotobiotically controlled conditions, and several unique aspects of this specialized, aquatic plant-bacterium symbiosis distinguish it from other legume root-nodule symbioses (22). After colonizing the root and floating stem surfaces, the aquatic bacterium symbiont enters the primary root cortex and stem interior through natural wounds caused by splitting of the epidermis and emergence of young lateral and adventitious roots, respectively, and then stimulates early development of nodules in the cortex at the base of these roots primordia, but not in the stem itself (22). Following crack entry through the nodule periphery, the bacteria penetrate internal nodule host cells; induce formation of bona fide tubular infection threads that disseminate them further intracellularly; and then release the bacteria into infection droplets, where they multiply. The endosymbiotic bacteria transform into nitrogen-fixing bacteroids within symbiosomes that eventually become filled with an unusual fibrillar matrix towards the end of their active nitrogen-fixing cycle in the aquatic e...

The genes coding for the five outer membrane proteins (OMPs) of the Omp25/Omp31 family expected to be located in the outer membrane (OM) of rough virulent Brucella ovis PA were inactivated to evaluate their role in virulence and OM properties. The OM properties of the mutant strains and of the mutants complemented with the corresponding wild-type genes were analyzed, in comparison with the parental strain and rough B. abortus RB51, in several tests: (i) binding of anti-Omp25 and anti-Omp31 monoclonal antibodies, (ii) autoagglutination of bacterial suspensions, and (iii) assessment of susceptibility to polymyxin B, sodium deoxycholate, hydrogen peroxide, and nonimmune ram serum. A tight balance of the members of the Omp25/Omp31 family was seen to be essential for the stability of the B. ovis OM, and important differences between the OMs of B. ovis PA and B. abortus RB51 rough strains were observed. Regarding virulence, the absence of Omp25d and Omp22 from the OM of B. ovis PA led to a drastic reduction in spleen colonization in mice. While the greater susceptibility of the ⌬omp22 mutant to nonimmune serum and its difficulty in surviving in the stationary phase might be on the basis of its dramatic attenuation, no defects in the OM able to explain the attenuation of the ⌬omp25d mutant were found, especially considering that the fully virulent ⌬omp25c mutant displayed more important OM defects. Accordingly, Omp25d, and perhaps Omp22, could be directly involved in the penetration and/or survival of B. ovis inside host cells. This aspect, together with the role of Omp25d and Omp22 in the virulence both of B. ovis in rams and of other Brucella species, should be thoroughly evaluated in future studies.The genus Brucella comprises six classical species (Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae) that infect terrestrial mammals (41), although in recent years an increasing number of Brucella strains have been isolated from marine mammals and proposed to be included in two new species, B. cetaceae and B. pinnipediae (9).Brucella spp. may have smooth or rough lipopolysaccharide (S-LPS or R-LPS) depending on the presence or absence, respectively, of O-polysaccharide chains. Rough mutants derived from smooth Brucella strains show an important reduction in virulence (1,26,30,40,45). Accordingly, the O-polysaccharide chains of LPS are thought to be necessary for the pathogenicity of Brucella strains bearing S-LPS. However, B. ovis and B. canis lack O chains in the LPS but are pathogenic for rams and dogs, respectively, and induce long-lasting infections with high levels of splenic colonization in laboratory animals (34). Since O chains mask other components of the Brucella spp. outer membrane (OM), OM proteins (OMPs) are more exposed at the bacterial surface of rough B. ovis and B. canis (4, 7) and their involvement in virulence in rough Brucella strains may be more relevant than in smooth Brucella strains.The Brucella spp. Omp25/Omp31 family comprises seven homologous OMPs. Omp25 and Omp31...

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Bacterial strain PETP02 T was isolated from nodules of Trifolium pratense growing in a Spanish soil. Phylogenetic analysis of the 16S rRNA gene sequence showed that this strain represents a member of the genus Phyllobacterium. However, divergence found with the 16S rRNA gene sequence of the single recognized species of this genus, Phyllobacterium myrsinacearum, indicated that strain PETP02 T belongs to a different species. The results of DNA-DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain represents a novel species of the genus Phyllobacterium, for which the name Phyllobacterium trifolii sp. nov. is proposed. The type strain is PETP02 T (=LMG 22712 T =CECT 7015 T). This strain was strictly aerobic and used several carbohydrates as carbon source. It was not able to reduce nitrate. Aesculin hydrolysis was negative. It did not produce urease, arginine dihydrolase, gelatinase or b-galactosidase. The DNA G+C content was 56?4 mol%. The nodD gene of this strain showed a sequence closely related to those of strains able to nodulate Lupinus. Infectivity tests showed that this strain is able to produce nodules in both Trifolium repens and Lupinus albus.