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We report the complete genome sequence of Bacillus sp. strain ABP14 isolated from lignocellulosic compost and selected by its ability in hydrolyzing carboxymethyl cellulose. This strain does not produce a Cry-like protein but showed an insecticidal activity against larvae of Anticarsia gemmatalis (Lepidoptera). Genome-based taxonomic analysis revealed that the ABP14 chromosome is genetically close to Bacillus thuringiensis serovar finitimus YBT020. ABP14 also carries one plasmid which showed no similarity with those from YBT020. Genome analysis of ABP14 identified unique genes related to cell surface structures, cell wall, metabolic competence, and virulence factors that may contribute for its survival and environmental adaptation, as well as its entomopathogenic activity. [ABSTRACT FROM AUTHOR]

Herbaspirillum rubrisubalbicans M1 causes the mottled stripe disease in sugarcane cv. B-4362. Inoculation of this cultivar with Herbaspirillum seropedicae Sm R1 does not produce disease symptoms. A comparison of the genomic sequences of these closely related species may permit a better understanding of contrasting phenotype such as endophytic association and pathogenic life style. To achieve this goal, we constructed suppressive subtractive hybridization ( SSH) libraries to identify DNA fragments present in one species and absent in the other. In a parallel approach, partial genomic sequence from H. rubrisubalbicans M1 was directly compared in silico with the H. seropedicae Sm R1 genome. The genomic differences between the two organisms revealed by SSH suggested that lipopolysaccharide and adhesins are potential molecular factors involved in the different phenotypic behavior. The cluster wss probably involved in cellulose biosynthesis was found in H. rubrisubalbicans M1. Expression of this gene cluster was increased in H. rubrisubalbicans M1 cells attached to the surface of maize root, and knockout of wssD gene led to decrease in maize root surface attachment and endophytic colonization. The production of cellulose could be responsible for the maize attachment pattern of H. rubrisubalbicans M1 that is capable of outcompeting H. seropedicae Sm R1. [ABSTRACT FROM AUTHOR]

This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The PII proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of PII might play a role in the control of nitrogen metabolism in H. seropedicae. [ABSTRACT FROM AUTHOR]

Abstract: The Azospirillum brasilense PII and PZ proteins, encoded by the glnB and glnZ genes respectively, are intracellular transducers of nitrogen levels with distinct functions. The PII protein participates in nif regulation by controlling the activity of the transcriptional regulator NifA. PII is also involved in transducing the prevailing nitrogen levels to the Fe-protein ADP-ribosylation system. PZ regulates negatively ammonium transport and is involved in nitrogenase reactivation. To further investigate the role of PII and PZ in the regulation of nitrogen fixation, broad-host-range plasmids capable of over-expressing the glnB and glnZ genes under control of the ptac promoter were constructed and introduced into A. brasilense. The nitrogenase activity and nitrate-dependent growth was impaired in A. brasilense cells over-expressing the PII protein. Using immunoblot analysis we observed that the reduction of nitrogenase activity in cells over-expressing PII was due to partial ADP-ribosylation of the Fe-protein under derepressing conditions and a reduction in the amount of Fe-protein. These results support the hypothesis that the unmodified PII protein act as a signal to the DraT enzyme to ADP-ribosylate the Fe-protein in response to ammonium shock, and that it also inhibits nif gene expression. In cells over-expressing the PZ protein the nitrogenase reactivation after an ammonium shock was delayed indicating that the PZ protein is involved in regulation of DraG activity. [Copyright &y& Elsevier]

The interactions between maize, sorghum, wheat and rice plants and Herbaspirillum seropedicae were examined microscopically following inoculation with the H. seropedicae LR15 strain, a Nif+ (Pnif::gusA) mutant obtained by the insertion of a gusA-kanamycin cassette into the nifH gene of the H. seropedicae wild-type strain. The expression of the Pnif::gusA fusion was followed during the association of the diazotroph with the gramineous species. Histochemical analysis of seedlings of maize, sorghum, wheat and rice grown in vermiculite showed that strain LR15 colonized root surfaces and inner tissues. In early steps of the endophytic association, H. seropedicae colonized root exudation sites, such as axils of secondary roots and intercellular spaces of the root cortex; it then occupied the vascular tissue and there expressed nif genes. The expression of nif genes occurred in roots, stems and leaves as detected by the GUS reporter system. The expression of nif genes was also observed in bacterial colonies located in the external mucilaginous root material, 8 days after inoculation. Moreover, the colonization of plant tissue by H. seropedicae did not depend on the nitrogen-fixing ability, since similar numbers of cells were isolated from roots or shoots of the plants inoculated with Nif+ or Nif− strains. [Copyright &y& Elsevier]

In Azospirillum brasilense the glnB and glnA genes are clustered in an operon regulated by three different promoters: two located upstream of glnB (glnBp1-σ70, and glnBp2-σN) and one as yet unidentified promoter, in the glnBA intergenic region. We have investigated the expression of the glnB gene promoter using glnB-lacZ gene fusions, mutation analysis, heterologous expression and DNA band-shift assays. Deletion of the glnB promoter region showed that NtrC-binding sequences were essential for glnB expression under nitrogen limitation. The A. brasilense NtrC protein activated transcription of glnB-lacZ fusions in the heterologous genetic background of Escherichia coli. Expression of glnB-lacZ fusions in two A. brasilense ntrC mutants differed from that in the wild-type strain. In vitro studies also indicated that the purified NtrC protein from E. coli was able to bind to the glnB promoter region of A. brasilense. Our results show that the NtrC protein activates glnBglnA expression under nitrogen limitation in A. brasilense. [Copyright &y& Elsevier]

Analysis of the expression of the Herbaspirillum seropedicae nifA promoter in Escherichia coli and Herbaspirillum seropedicae, showed that nifA expression is primarily dependent on NtrC but also required NifA for maximal expression under nitrogen-fixing conditions. Deletion of the IHF (integration host factor)-binding site produced a promoter with two-fold higher activity than the native promoter in the H. seropedicae wild-type strain but not in a nifA strain, indicating that IHF controls NifA auto-activation. IHF is apparently required to prevent overexpression of the NifA protein via auto-activation under nitrogen-fixing conditions in H. seropedicae. [Copyright &y& Elsevier]