Construction of a gene library from the nitrogen-fixing aerobe Azotobacter vinelandii

We have cloned the DNA of Azotobacter vinelandii in the cosmid pHC79. Recombinant cosmids that can transform Escherichia coli leuB- to a Leu+ phenotype, as well as those having sequence homology to the nitrogenase structural genes of Klebsiella pneumoniae have been selected from this library.     

Conjugation in starforming Rhizobium lupini

Summary ARhizobium strain which exhibits high efficiency of starformation has been isolated from the root nodules ofLupinus luteus. Mutants of this strain were induced by repeated treatment of the wild type with nitrous acid. The mutants are marked with one or two auxotrophic characters and with different colors (carotenoid production). Two-, three-, and four-point crosses were performed. The maximum recombination rate is around 10%. A quantitative evaluation of the results of the crosses indicates the existence of a circular linkage map.The recombination is the result of a conjugation between cells during which most probably always the whole chromosome is transferred.The research was supported by NSF Grant. No. GB-4287 and NJH Grant No. 1 PO 1 GM 13234.     

Chemische Zusammensetzung des Lipopolysaccharids eines Stammes von Rhizobium lupini

Die chemische Zusammensetzung des Lipopolysaccharids des Stammes R. lupini 16–2 wurde untersucht. Der Lipidanteil enthält die Fettsäuren Hydroxymyristinsäure, Myristinsäure und Palmitinsäure im molaren Verhältnis 4:1: 1. Folgende Zuckerkomponenten konnten nachgewiesen werden: Glucosamin, Galaktosamin, 2-Keto-3-desoxyoctonat (KDO), Rhamnose und wahrscheinlich Heptose. Der Phosphatgehalt betrug 2% (w/w). Rhamnose stellt 37% (w/w) des Gesamt-LPS dar. Dieser Zucker bildet wahrscheinlich die O-spezifischen Seitenketten. Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft unterstützt. Herrn Prof. Dr. K. Jann und Frau Dr. B. Jann möchten wir sehr herzlich für anregende Diskussionen danken. Frau Dr. I. Fromme danken wir für die Hilfe bei der gaschromatographischen Analyse.     

Die Fimbrien von Rhizobium lupini 1/50, sta-

Zusammenfassung Die Fimbrien (oder Pili) einer nicht sternbildenden Mutante (1–50, sta^-) von Rhizobium lupini wurden elektronenmikroskopisch untersucht. Die Fimbien sind peritrich an der Bakterienzelle inseriert, und zwar während der exponentiellen Wachstumsphase meist einzeln. In der stationären Phase nehmen die Fimbrien an Zahl und Länge kontinuierlich stark zu; sie sind dann häufig büschelweise inseriert. Zusammen mit den oft zopfbildenden Geißeln verflechten sie sich zu einem ausgedehnten Netzwerk. Die Aneinanderlagerung der Fimbrien erfolgt unspezifisch durch Kohäsion; durch ebenfalls unspezifische Adhäsion haften sie auf dem Substrat.Die Fimbrien haben ca. 30 Å Durchmesser und sind röhrenförmig gebaut. Ihre lichte Weite beträgt 8 bis 10 Å. Ein allgemeines Bauschema der Fimbrien wird diskutiert. In der Diskussion über die allgemeine Funktion aller Arten von Fimbrien wird ihre Haftfähigkeit herausgestellt. Die Fimbrien der Mutante 1/50, sta^- sind wegen ihres geringen Innendurchmessers nicht als Transportröhren für doppelsträngige DNS und wahrscheinlich auch nicht für einzelsträngige DNS oder RNS geeignet.

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The fimbriae of Rhizobium lupini 1/50, sta^-

Summary The fimbriae (pili) of a non-starforming mutant (1/50, sta^-) of Rhizobium lupini were studied under the electron microscope. In the exponential phase of growth, fimbriae are singly, peritrichously inserted. During stationary growth these fimbriae increase by number and length. Together with the larger flagella they often form an extended reticulum. The fimbriae often stick together by unspecific cohesion forces; their attachment to the substrate can be explained by unspecific adhesion.The outer diameter of the fimbriae is 30 Å, the inner diameter 8 to 10 Å.The tubelike structure of fimbriae (pili) is discussed in terms of a general model.The most obvious general function of all types of fimbriae is their connecting power.The inner dimension of the fimbriae of the 1/50, sta^-—mutant exclude a model where they function as transport tubes for double-stranded DNA and probably for single stranded DNA or RNA either.

     

Synthesis and breakdown of poly-beta-hydroxybutyric acid in Rhizobium lupini]

The effect of various substrates on synthesis and decomposition of poly-beta-hydroxybutyric acid (PHBA) was studied in cell suspensions of the effective strain of Rhizobium lupini and in suspensions of the bacteroids of this strain which were isolated from lupine nodules at different growth stages of the plants. Glucose and beta-hydroxybutyrate were found to be the best substrates for synthesis of PHBA in all variants. The content of PHBA in the presence of these substrates increased in suspensions by 2.0 to 2.5 times during ten hours of incubation. In the presence of succinate, PHBA was actively synthesized only by the bacteroids isolated during the stage of active nitrogen fixation (flowering of the plants). In the absence of exogenous substrates, PHBA was decomposed, especially if ammonium ions were present in suspensions. No synthesis of PHBA was registered in the presence of ammonium and glucose, and the rate of PHBA decomposition was high in this case during the stage of active nitrogen fixation.     

Effect of cultivation conditions on the accumulation of poly-beta-hydroxy-butyric acid in Rhizobium lupini]

The influence of the age of the culture and nitrogen source on the accumulation of poly-beta-hydroxybutyric acid by different strains of Rhizobium lupini was studied. The accumulation depended on the age of the culture and reached maximum at the end of the logarithmic and at the beginning of the stationary phase of the bacterial growth (about 50-60% dry weight). The accumulation varied in relation to the nitrogen source used: it was the highest in the glutamate medium and the lowest on nitrate nitrogen; the culture grown on ammonium phosphate was intermediate.     

Corals form characteristic associations with symbiotic nitrogen-fixing bacteria

The complex symbiotic relationship between corals and their dinoflagellate partner Symbiodinium is believed to be sustained through close associations with mutualistic bacterial communities, though little is known about coral associations with bacterial groups able to fix nitrogen (diazotrophs). In this study, we investigated the diversity of diazotrophic bacterial communities associated with three common coral species (Acropora millepora, Acropora muricata, and Pocillopora damicormis) from three midshelf locations of the Great Barrier Reef (GBR) by profiling the conserved subunit of the nifH gene, which encodes the dinitrogenase iron protein. Comparisons of diazotrophic community diversity among coral tissue and mucus microenvironments and the surrounding seawater revealed that corals harbor diverse nifH phylotypes that differ between tissue and mucus microhabitats. Coral mucus nifH sequences displayed high heterogeneity, and many bacterial groups overlapped with those found in seawater. Moreover, coral mucus diazotrophs were specific neither to coral species nor to reef location, reflecting the ephemeral nature of coral mucus. In contrast, the dominant diazotrophic bacteria in tissue samples differed among coral species, with differences remaining consistent at all three reefs, indicating that coral-diazotroph associations are species specific. Notably, dominant diazotrophs for all coral species were closely related to the bacterial group rhizobia, which represented 71% of the total sequences retrieved from tissue samples. The species specificity of coral-diazotroph associations further supports the coral holobiont model that bacterial groups associated with corals are conserved. Our results suggest that, as in terrestrial plants, rhizobia have developed a mutualistic relationship with corals and may contribute fixed nitrogen to Symbiodinium.     

Localization of symbiotic mutations in Rhizobium meliloti

A total of 5 Nod- and 57 Fix- symbiotic mutants of Rhizobium meliloti strain 41 have been isolated after either nitrosoguanidine or Tn5 transposition mutagenesis. Chromosomal locations of mutations in 1 Nod- and 11 Fix- derivatives were ascertained by transferring the chromosome (mobilized by plasmid R68.45), in eight fragments, into symbiotically effective recipients and testing the recombinants for symbiotic phenotype. Alternatively, the kanamycin resistance marker of Tn5 was mapped. In five mutants the fix alleles were localized on different chromosomal regions, but six other fix mutations and one nod mutation tested did not map onto the chromosome. It was shown that the chromosome-mobilizing ability (Cma+) of R68.45 was not involved in the mobilization of genes located extrachromosomally. Moreover, Cma- derivatives of R68.45 could mobilize regions of the indigenous plasmid pRme41b but not chromosomal genes. Thus, mobilization of a marker by Cma- R68.45 indicates its extrachromosomal location. With a 32P-labeled DNA fragment carrying Tn5 as a hybridization probe, it was shown that in five extrachromosomally located Tn5-induced fix mutants and one nod mutant Tn5 was localized on plasmid pRme41b. This is in agreement with the genetic mapping data.     

Draft genome sequence of the nitrogen-fixing symbiotic bacterium Bradyrhizobium elkanii 587

The draft sequence of the genome of Bradyrhizobium elkanii 587 is presented. This was obtained using Illumina Next-Gen DNA sequencing combined with Sanger sequencing. Genes for the pathways involved in biological nitrogen fixation (the nif gene cluster), nod genes including nodABC, and genes for the type III protein secretion system (T3SS) are present.     

Chromosomal gene controlling symbiotic nitrogen fixation in Rhizobium meliloti L5-30

Auxotrophic Rhizobium meliloti strain RM 246 carries two independent mutations: in the biosynthesis of cysteine (cys) and symbiotic nitrogen fixation process (fix). These two mutations were mapped by transduction between his-240 and ade-4 markers. Cotransduction frequencies show the following order of genes: his-240 fix-1 cys-246 ade-4.