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.

Bacterial strain PETP02^Twas isolated from nodules ofTrifolium pratensegrowing in a Spanish soil. Phylogenetic analysis of the 16S rRNA gene sequence showed that this strain represents a member of the genusPhyllobacterium. However, divergence found with the 16S rRNA gene sequence of the single recognized species of this genus,Phyllobacterium myrsinacearum, indicated that strain PETP02^Tbelongs 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 genusPhyllobacterium, for which the namePhyllobacterium trifoliisp. 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 orβ-galactosidase. The DNA G+C content was 56·4 mol%. ThenodDgene of this strain showed a sequence closely related to those of strains able to nodulateLupinus. Infectivity tests showed that this strain is able to produce nodules in bothTrifolium repensandLupinus albus.

Two strains named ESC1^T and ESC5 were isolated from nodules of Cytisus scoparius growing in a Spanish soil. Phylogenetic analysis of the 16S rRNA gene showed that these strains belong to the genus Ochrobactrum, their closest relatives being Ochrobactrum anthropi and Ochrobactrum lupini, with 100 and 99.9 % similarity to the respective type strains. Despite this high similarity, the results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses showed that these strains represent a novel species of genus Ochrobactrum. The DNA–DNA hybridization values were respectively 70, 66 and 55 % with respect to O. lupini LUP21^T, O. anthropi DSM 6882^T and Ochrobactrum tritici DSM 13340^T. The predominant fatty acids were C_18 : 1 ω7c and C_18 : 1 2-OH. Strains ESC1^T and ESC5 were strictly aerobic and were able to reduce nitrate and to hydrolyse aesculin. They produced β-galactosidase and β-glucosidase and did not produce urease after 48 h incubation. The G+C content of strain ESC1^T was 56.4 mol%. Both strains ESC1^T and ESC5 contained nodD and nifH genes on megaplasmids that were related phylogenetically to those of rhizobial strains nodulating Phaseolus, Leucaena, Trifolium and Lupinus. From the results of this work, we propose that the strains isolated in this study be included in a novel species named Ochrobactrum cytisi sp. nov. The type strain is ESC1^T (=LMG 22713^T=CECT 7172^T).

Summary: The omp-31 gene, encoding a major outer-membrane protein in Brucella melitensis, was PCR-amplified from Brucella strains representing all species and known biovars by using primers selected according to the B. melitensis 16M omp-31 published sequence. Amplification of omp-31 was achieved from DNA of all Brucella species with the exception of Brucella abortus, the only Brucella species where expression of omp-31 was not detected by reactivity with an mAb specific for an epitope located in Omp-31. Southern blot hybridization of plasmid probes, bearing inserts (4.4-17 kb) containing B. melitensis 16M omp-31 and adjacent DNA of different sizes, with HindIII-digested total DNA showed that a large fragment, comprising the entire omp-31 gene and flanking DNA, was actually absent in B. abortus strains. The size of this DNA fragment has been determined to be about 10 kb. Southern blot hybridization with the different plasmid probes identified species-specific markers for B. abortus and B. melitensis. At the biovar level, a specific marker for B. melitensis bv. 1 was also identified. Additionally, PCR-RFLP studies of omp-31 revealed specific markers for Brucella ovis, Brucella canis and Brucella suis bv. 2. Using a combination of omp-31 PCR-RFLP patterns and Southern blot hybridization profiles Brucella species were differentiated with the sole exception of Brucella neotomae which was not differentiated from B. suis bv. 1, 3, 4 and 5. Results presented in this paper demonstrate the potential of omp-31 for differentiating the brucellae and show that B. abortus lacks a large DNA fragment of about 10 kb containing omp-31 and flanking DNA. In such a large deletion, other genes in addition to omp-31 are probably involved. Sequencing of this DNA fragment will help to identify the missing genes in B. abortus which could possibly be involved in the differences of pathogenicity and host preference seen in Brucella species.

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 virulentBrucella ovisPA 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 roughB. abortusRB51, 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 theB. ovisOM, and important differences between the OMs ofB. ovisPA andB. abortusRB51 rough strains were observed. Regarding virulence, the absence of Omp25d and Omp22 from the OM ofB. ovisPA led to a drastic reduction in spleen colonization in mice. While the greater susceptibility of the Δomp22mutant 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 Δomp25dmutant were found, especially considering that the fully virulent Δomp25cmutant displayed more important OM defects. Accordingly, Omp25d, and perhaps Omp22, could be directly involved in the penetration and/or survival ofB. ovisinside host cells. This aspect, together with the role of Omp25d and Omp22 in the virulence both ofB. ovisin rams and of otherBrucellaspecies, should be thoroughly evaluated in future studies.