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Cloning and characterization of the sigA gene encoding the major sigma subunit of Rhizobium meliloti.

ABSTRACT: Using PCR to create a probe based on conserved region 2 of sigma factors, we have cloned the sigA gene coding for the major sigma factor of Rhizobium meliloti. The 684-residue protein encoded by the sigA gene was expressed in vitro in coupled transcription-translation experiments with R. meliloti extracts and migrated aberrantly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its deduced amino acid sequence is similar to that of RpoD of Escherichia coli and is nearly identical to that of SigA of the closely related bacterium Agrobacterium tumefaciens. Through Southern analysis, we located the gene on the R. meliloti main chromosome rather than on one of the megaplasmids. The sigA locus does not appear to be part of a macromolecular synthesis operon (MMS), as in many other bacterial species, but rather lies downstream of a partial open reading frame showing similarity to the threonine dehydrogenase gene (tdh) of E. coli.

SUBMITTER: Rushing BG

PROVIDER: S-EPMC177565 | biostudies-other | 1995 Dec

REPOSITORIES: biostudies-other

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Cloning and characterization of the sigA gene encoding the major sigma subunit of Rhizobium meliloti.

Rushing B G BG Long S R SR

Journal of bacteriology 19951201 23

Using PCR to create a probe based on conserved region 2 of sigma factors, we have cloned the sigA gene coding for the major sigma factor of Rhizobium meliloti. The 684-residue protein encoded by the sigA gene was expressed in vitro in coupled transcription-translation experiments with R. meliloti extracts and migrated aberrantly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its deduced amino acid sequence is similar to that of RpoD of Escherichia coli and is nearly identical to t ...[more]

PMID: 7592490

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Project description:Transposon Tn5-induced C4-dicarboxylate transport mutants of Rhizobium meliloti 2011 which could be complemented by cosmid pRmSC121 were subdivided into two classes. Class I mutants (RMS37 and RMS938) were defective in symbiotic C4-dicarboxylate transport and in nitrogen fixation. They were mutated in the structural gene dctA, which codes for the C4-dicarboxylate carrier. Class II mutants (RMS11, RMS16, RMS17, RMS24, and RMS31) expressed reduced activity in symbiotic C4-dicarboxylate transport and in nitrogen fixation. These mutants were mutated in regulatory dct genes which do not play an essential role in the symbiotic state. Thin sections of alfalfa nodules induced by the wild type and class I and class II mutants were analyzed by light microscopy. Class mutants induced typical Fix- nodules, showing a large senescent zone, whereas nodules induced by class II mutants only differed in an enhanced content of starch granules compared with wild-type nodules. Class I mutants could be complemented by a 2.1-kilobase SalI-HindIII subfragment of cosmid pRmSC121. DNA sequencing of this fragment resulted in the identification of an open reading frame, which was designated dctA because Tn5 insertion sites of the class I mutants mapped within this coding region. The dctA gene was preceded by a nif consensus promoter and an upstream NifA-binding element. Upstream of the dctA promoter, the 5' end of the R. meliloti dctB gene could be localized. The amino acid sequence of the N-terminal part of the R. meliloti DctB protein shared 49% homology with the corresponding part of the R. leguminosarum DctB protein. The DctA protein consisted of 441 or 453 amino acids due to two possible ATG start codons, with calculated molecular masses of 46.1 and 47.6 kilodaltons, respectively. The hydrophobicity plot suggests that DctA is a membrane protein with several membrane passages. The amino acid sequences of the R. meliloti and the R. leguminosarum DctA proteins were highly conserved (82%).

Dataset Information

Cloning and characterization of the sigA gene encoding the major sigma subunit of Rhizobium meliloti.

Publications

Cloning and characterization of the sigA gene encoding the major sigma subunit of Rhizobium meliloti.

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