Identification and solubilization of a signal peptidase from the phototrophic bacterium <i>Rhodobacter capsulatus</i>

Wieseler, Beate; Schiltz, Emile; Müller, Matthias

doi:10.1016/0014-5793(92)80075-r

Cited by 20 publications

(9 citation statements)

References 28 publications

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“…Positively charged amino acids in the N-terminal part are marked (+), and hydrophobic stretches of amino acid residues are shown in lowercase letters. The E. coli Skp protein and cytochrome c2 of R. sphaeroides were shown to be correctly cleaved by a signal peptidase of R. capsulatus (52). The cleavage site is indicated by an arrow.…”

Section: Resultsmentioning

confidence: 99%

“…The R. capsulatus ModA protein contained a leader sequence of 27 amino acid residues, which conformed to signal sequences of other proteins that are cleaved during translocation to the periplasm. A signal peptidase able to cleave the precursors of the E. coli Skp protein and Rhodobacter sphaeroides cytochrome c2 at the authentic cleavage site was identified recently in R capsulatus (52). From the high degree of structural and sequence similarities of the putative leader peptide of R. capsulatus ModA to these proteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins

1993

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The alternative, heterometal-free nitrogenase of Rhodobacter capsulatus is repressed by traces of molybdenum in the medium. Strains carrying mutations located downstream of nipB copy II were able to express the alternative nitrogenase even in the presence of high molybdate concentrations. DNA sequence analysis of a 5.5-kb fragment of this region revealed six open reading frames, designated modABCD, mopA, and mopB. The gene products of modB and modC are homologous to ChU and ChID of Escherichia coil and represent an integral membrane protein and an ATP-binding protein typical of high-affinity transport systems, respectively.ModA and ModD exhibited no homology to known proteins, but a leader peptide characteristic of proteins cleaved during export to the periplasm is present in ModA, indicating that ModA might be a periplasmic molybdate-binding protein. The MopA and MopB proteins showed a high degree of amino acid sequence homology to each other. Both proteins contained a tandem repeat of a domain encompassing 70 amino acid residues, which had significant sequence similarity to low-molecular-weight molybdenum-pterin-binding proteins from Clostridium pasteurianum. Compared with that for the parental nijfDK deletion strain, the molybdenum concentrations necessary to repress the alternative nitrogenase were increased 4-fold in a modD mutant and 500-fold in modA, modB, and modC mutants. No significant inhibition of the heterometal-free nitrogenase by molybdate was observed for mopA mopB double mutants. The uptake of molybdenum by mod and mop mutants was estimated by measuring the activity of the conventional molybdenum-containing nitrogenase. Molybdenum transport was not affected in a mopA mopB double mutant, whereas strains carrying lesions in the binding-protein-dependent transport system were impaired in molybdenum uptake.The process of N2 fixation has been studied for many years in a variety of different diazotrophs, and the conventional molybdenum-containing nitrogenase has been well characterized. However, it was not realized until recently that some organisms harbor, in addition to the molybdenum nitrogenase, other genetically distinct nitrogenase enzyme complexes. These alternative nitrogenases have been best characterized in the obligate aerobic soil bacterium Azotobacter vinelandii, which contains three distinct nitrogenase systems. The conventional nitrogenase (nitrogenase 1) includes a molybdenum cofactor, nitrogenase 2 is a vanadium enzyme, and nitrogenase 3 is a heterometal-free enzyme complex (for a review, see reference 5). The phototrophic purple bacterium Rhodobacter capsulatus harbors two nitrogenase systems, corresponding to nitrogenase 1 and nitrogenase 3, whereas a vanadium-containing enzyme is apparently not present (36). The expression of alternative nitrogenases in both A. vinelandii and R capsulatus is repressed by extremely low molybdenum concentrations, indicating that high-affinity systems are involved in gene regulation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins

1993

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“…Only a few experiments have been performed on protein translocation in photosynthetic bacteria (10,41,42,45 (1). Thus, once C2Asig associates with the membrane, attachment of this hydrophobic ligand might aid its export across the CM in the absence of a signal peptide.…”

Section: Discussionmentioning

confidence: 99%

The Rhodobacter sphaeroides cytochrome c2 signal peptide is not necessary for export and heme attachment

Brandner

Donohue

1994

J Bacteriol

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Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a member of the heme-containing cytochrome c protein family that is found in the periplasmic space of this gram-negative bacterium. This exported polypeptide is made as a higher-molecular-weight precursor with a typical procaryotic signal peptide. Therefore, cyt c2 maturation is normally expected to involve precursor translocation across the cytoplasmic membrane, cleavage of the signal peptide, and covalent heme attachment. Surprisingly, synthesis as a precursor polypeptide is not a prerequisite for cyt c2 maturation because deleting the entire signal peptide does not prevent export, heme attachment, or function. Although cytochrome levels were reduced about threefold in cells containing this mutant protein, steady-state cyt c2 levels were significantly higher than those of other exported bacterial polypeptides which contain analogous signal peptide deletions. Thus, this mutant protein has the unique ability to be translocated across the cytoplasmic membrane in the absence of a signal peptide. The covalent association of heme with this mutant protein also suggests that the signal peptide is not required for ligand attachment to the polypeptide chain. These results have uncovered some novel aspects of bacterial c-type cytochrome biosynthesis.

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“…Separation of precursor and mature forms of cytochrome c 2 required SDS-urea gels (2). Immunoblotting has been detailed elsewhere (67). Antibodies against Bacillus subtilis SecA were raised in female rabbits (chinchilla bastard) by applying 300 g of pure SecA initially and 200 g for each booster injection.…”

Section: Molecular Cloning Techniquesmentioning

confidence: 99%

“…We have started to analyze protein export in Rhodobacter capsulatus (57,58,66,67), which belongs to the group of ␣-subclass purple bacteria. Based on a 16S rRNA sequence analysis (68), these bacteria are closely related to the ancestral prokaryote that, according to the endosymbiont theory, gave rise to mitochondria.…”

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confidence: 99%

Comparative characterization of SecA from the alpha-subclass purple bacterium Rhodobacter capsulatus and Escherichia coli reveals differences in membrane and precursor specificity

et al. 1997

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We have cloned the secA gene of the ␣-subclass purple bacterium Rhodobacter capsulatus, a close relative to the mitochondrial ancestor, and purified the protein after expression in Escherichia coli. R. capsulatus SecA contains 904 amino acids with 53% identity to E. coli and 54% identity to Caulobacter crescentus SecA. In contrast to the nearly equal partitioning of E. coli SecA between the cytosol and plasma membrane, R. capsulatus SecA is recovered predominantly from the membrane fraction. A SecA-deficient, cell-free synthesistranslocation system prepared from R. capsulatus is used to demonstrate translocation activity of the purified R. capsulatus SecA. This translocation activity is then compared to that of the E. coli counterpart by using various precursor proteins and inside-out membrane vesicles prepared from both bacteria. We find a preference of the R. capsulatus SecA for the homologous membrane vesicles whereas E. coli SecA is active with either type of membrane. Furthermore, the two SecA proteins clearly select between distinct precursor proteins. In addition, we show here for the first time that a bacterial c-type cytochrome utilizes the canonical, Sec-dependent export pathway.

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Identification and solubilization of a signal peptidase from the phototrophic bacterium Rhodobacter capsulatus

Cited by 20 publications

References 28 publications

Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins

Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins

The Rhodobacter sphaeroides cytochrome c2 signal peptide is not necessary for export and heme attachment

Comparative characterization of SecA from the alpha-subclass purple bacterium Rhodobacter capsulatus and Escherichia coli reveals differences in membrane and precursor specificity

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