Multilocus sequence analysis of root nodule isolates from Lotus arabicus (Senegal), Lotus creticus, Argyrolobium uniflorum and Medicago sativa (Tunisia) and description of Ensifer numidicus sp. nov. and Ensifer garamanticus sp. nov.

Nine isolates from Argyrolobium uniflorum, Lotus creticus , Medicago sativa (Tunisia) and Lotus arabicus (Senegal) were analysed by multilocus sequence analysis (MLSA) of five housekeeping genes (recA, atpD, glnA, gltA and thrC), the 16S rRNA gene and the nodulation gene nodA. Analysis of the individual and concatenated gene sequences demonstrated that the nine new strains constituted three stable, well-supported (bootstrap and gene sequence similarity values) monophyletic clusters, A, B and C, all belonging to the branch of the genus Ensifer, regardless of the phylogenetic reconstruction method used (maximum likelihood, maximum-parsimony, neighbour-joining). The three groups were further characterized by API 100 auxanographic tests, host specificity and nodA gene sequence analysis. On the basis of these data, clusters A and C are suggested as representing two novel species within the genus Ensifer, for which the names Ensifer numidicus sp. nov. (type strain ORS 1407(T)=LMG 24690(T)=CIP 109850(T)) and Ensifer garamanticus sp. nov. (type strain ORS 1400(T)=LMG 24692(T)=CIP 109916(T)) are proposed. The cluster B strains were assigned to Ensifer adhaerens genomovar A.

A new family of plasma membrane polypeptides differentially regulated during plant development.

Two cDNAs encoding polypeptides identified in a tobacco leaf plasma membrane fraction prepared by phase partitioning were cloned. The deduced polypeptides, P16 and P17, exhibit a striking primary structure, similar to that of P19, a previously cloned plasma membrane polypeptide. Antibodies raised to the recombinant proteins were used to probe the cellular location of P16, P17 and P19 by means of western blotting of sucrose density-gradient fractions; all three polypeptides were found to be located solely at the plasma membrane. Furthermore, P19 antigen accumulated transiently at the time of floral induction while P16 and P17 antigens accumulated towards the end of the life-cycle. These results together with sequence database searches and multiple-sequence alignments suggest that we have identified a new family of plasma membrane polypeptides that (i) are putatively plant specific and (ii) are differentially regulated during plant development. These polypeptides are termed DREPPs for developmentally regulated plasma membrane polypeptides.

Cloning of a cDNA encoding a developmentally regulated 22 kDa polypeptide from tobacco leaf plasma membrane.

A polypeptide doublet (P18-P19, ca 22 kDa, pI 4.5) has been shown to accumulate in tobacco leaf plasma membrane in a development-dependent way, under constant environmental conditions. P18 and P19 were purified by 2D-PAGE and microsequenced. Microsequences revealed only small differences between the two polypeptides. A PCR-based cloning strategy identified a cDNA displaying a 591 bp ORF. The encoded polypeptide contained P19 specific microsequences. It was expressed in E. coli and a specific rabbit antiserum was raised. Western-blots confirmed its identification as P19. The accumulation pattern of hybridizable mRNA around the floral induction period was similar to that of P18 and P19. Searching of databases revealed no significant hits except unidentified plant ESTs. P18 and P19 are proposed as the first example of plant-specific and developmentally regulated plasma membrane proteins.

Rhizobium meliloti fixGHI sequence predicts involvement of a specific cation pump in symbiotic nitrogen fixation.

We present genetic and structural analyses of a fix operon conserved among rhizobia, fixGHI from Rhizobium meliloti. The nucleotide sequence of the operon suggests it may contain a fourth gene, fixS. Adjacent open reading frames of this operon showed an overlap between TGA stop codons and ATG start codons in the form of an ATGA motif suggestive of translational coupling. All four predicted gene products contained probable transmembrane sequences. FixG contained two cysteine clusters typical of iron-sulfur centers and is predicted to be involved in a redox process. FixI was found to be homologous with P-type ATPases, particularly with K+ pumps from Escherichia coli and Streptococcus faecalis but also with eucaryotic Ca2+, Na+/K+, H+/K+, and H+ pumps, which implies that FixI is a pump of a specific cation involved in symbiotic nitrogen fixation. Since prototrophic growth of fixI mutants appeared to be unimpaired, the predicted FixI cation pump probably has a specifically symbiotic function. We suggest that the four proteins FixG, FixH, FixI, and FixS may participate in a membrane-bound complex coupling the FixI cation pump with a redox process catalyzed by FixG.

Cascade regulation of nif gene expression in Rhizobium meliloti.

We report the discovery of two genes from Rhizobium meliloti, fixL and fixJ, which are positive regulators of symbiotic expression of diverse nitrogen fixation (nif and fix) genes. nif gene regulation is shown to consist of a cascade: the fixLJ genes activate nifA, which in turn activates nifHDK and fixABCX. Like nifA, fixN can be induced in free-living microaerobic cultures of R. meliloti, indicating a major physiological role for oxygen in nif and fix gene regulation. Microaerobic expression of fixN and nifA depends on fixL and fixJ. The FixL and FixJ proteins belong to a family of two-component regulatory systems widely spread among prokaryotes and responsive to the cell environment. We propose that FixL, which has features of a transmembrane protein, senses an environmental signal and transduces it to FixJ, a transcriptional activator of nif and fix genes.

Transcription patterns of Rhizobium meliloti symbiotic plasmid pSym: identification of nifA-independent fix genes.

We performed a systematic survey of transcription of a large region of the Rhizobium meliloti symbiotic plasmid pSym. This led to the discovery of two new sequences induced during symbiosis. The first sequence was linked to the known nitrogen fixation (nif-fix) gene cluster, and its expression depended on the nifA gene product. The second sequence was a novel fix locus (M.-H. Renalier, J. Batut, J. Ghai, B. Terzaghi, M. Gherardi, M. David, A.-M. Garnerone, J. Vasse, G. Truchet, T. Huguet, and P. Boistard, J. Bacteriol. 169:2231-2238, 1987) whose expression was independent of the nifA gene product; therefore this fix locus undergoes a novel type of symbiotic regulation.