Dataset Information

Isolation and characterization of the nifUSVW-rpoN gene cluster from Rhodobacter sphaeroides.

ABSTRACT: The rpoN gene from Rhodobacter sphaeroides was isolated from a genomic library via complementation of a Rhodobacter capsulatus rpoN mutant. The rpoN gene was located on a 7.5-kb HindIII-EcoRI fragment. A Tn5 insertion analysis of this DNA fragment showed that a minimal DNA fragment of 5.3 kb was required for complementation. Nucleotide sequencing of the complementing region revealed the presence of nifUSVW genes upstream from rpoN. The rpoN gene was mutagenized via insertion of a gene encoding kanamycin resistance. The resulting rpoN mutant was not impaired in diazotrophic growth and was in all respects indistinguishable from the wild-type strain. Southern hybridizations using the cloned rpoN gene as a probe indicated the presence of a second rpoN gene. Deletion of the nifUS genes resulted in strongly reduced diazotrophic growth. Two conserved regions were identified in a NifV LeuA amino acid sequence alignment. Similar regions were found in pyruvate carboxylase and oxaloacetate decarboxylase. It is proposed that these conserved regions represent keto acid-binding sites.

SUBMITTER: Meijer WG

PROVIDER: S-EPMC206092 | biostudies-other | 1992 Jun

REPOSITORIES: biostudies-other

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Isolation and characterization of the nifUSVW-rpoN gene cluster from Rhodobacter sphaeroides.

Meijer W G WG Tabita F R FR

Journal of bacteriology 19920601 12

The rpoN gene from Rhodobacter sphaeroides was isolated from a genomic library via complementation of a Rhodobacter capsulatus rpoN mutant. The rpoN gene was located on a 7.5-kb HindIII-EcoRI fragment. A Tn5 insertion analysis of this DNA fragment showed that a minimal DNA fragment of 5.3 kb was required for complementation. Nucleotide sequencing of the complementing region revealed the presence of nifUSVW genes upstream from rpoN. The rpoN gene was mutagenized via insertion of a gene encoding k ...[more]

PMID: 1317839

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Project description:UnlabelledRhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 μm and a diameter of 1.4 μm, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (right-handed) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional.ImportanceR. sphaeroides possesses two functional sets of flagellar genes. The fla1 genes are normally expressed in the laboratory and were acquired by horizontal transfer. The fla2 genes are endogenous and are expressed in a Fla1(-) mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand its role in the physiology of this microorganism.

Project description:The flagellar lipoprotein FlgP has been identified in several species of bacteria, and its absence provokes different phenotypes. In this study, we show that in the alphaproteobacterium Rhodobacter sphaeroides, a ΔflgP mutant is unable to assemble the hook and the filament. In contrast, the membrane/supramembrane (MS) ring and the flagellar rod appear to be assembled. In the absence of FlgP a severe defect in the transition from rod to hook polymerization occurs. In agreement with this idea, we noticed a reduction in the amount of intracellular flagellin and the chemotactic protein CheY4, both encoded by genes dependent on σ28 This suggests that in the absence of flgP the switch to export the anti-sigma factor, FlgM, does not occur. The presence of FlgP was detected by Western blot in samples of isolated wild-type filament basal bodies, indicating that FlgP is an integral part of the flagellar structure. In this regard, we show that FlgP interacts with FlgH and FlgT, indicating that FlgP should be localized closely to the L and H rings. We propose that FlgP could affect the architecture of the L ring, which has been recently identified to be responsible for the rod-hook transition.IMPORTANCE Flagellar based motility confers a selective advantage on bacteria by allowing migration to favorable environments or in pathogenic species to reach the optimal niche for colonization. The flagellar structure has been well established in Salmonella However, other accessory components have been identified in other species. Many of these have been implied in adapting the flagellar function to enable faster rotation, or higher torque. FlgP has been proposed to be the main component of the basal disk located underlying the outer membrane in Campylobacter jejuni and Vibrio fischeri Its role is still unclear, and its absence impacts motility differently in different species. The study of these new components will bring a better understanding of the evolution of this complex organelle.

Project description:BackgroundThe genus Rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments. Representative strains of two Rhodobacter species, R. capsulatus and R. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. Gene co-expression networks can be used to identify modules of genes with similar expression profiles. Functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. In this paper, we constructed an R. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigated preservation of these modules in R. capsulatus proteomics data and in R. sphaeroides transcriptomics data.ResultsThe analysis identified 40 gene co-expression modules in R. capsulatus. Investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. We identified two R. capsulatus gene modules preserved in the protein abundance data. We also identified several gene modules preserved between both Rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. Many gene modules were non-preserved, providing insight into processes that differentiate the two species. In addition, using Local Network Similarity (LNS), a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles.ConclusionsOur analyses provide new sources of information for functional annotation in R. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional annotation. We identified R. capsulatus modules enriched with genes for ribosomal proteins, porphyrin and bacteriochlorophyll anabolism, and biosynthesis of secondary metabolites to be preserved in R. sphaeroides whereas modules related to RcGTA production and signalling showed lack of preservation in R. sphaeroides. In addition, we demonstrated that network statistics may also be applied within-species to identify congruence between mRNA expression and protein abundance data for which simple correlation measurements have previously had mixed results.

Dataset Information

Isolation and characterization of the nifUSVW-rpoN gene cluster from Rhodobacter sphaeroides.

Publications

Isolation and characterization of the nifUSVW-rpoN gene cluster from Rhodobacter sphaeroides.

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