Characterization of the secA gene of Streptomyces lividans encoding a protein translocase which complements an Escherichia coli mutant defective in the ATPase activity of SecA

Blanco, Jorge; Coque, Juan José R.; Martı́n, Juan F.

doi:10.1016/0378-1119(96)00220-x

Cited by 9 publications

(10 citation statements)

References 16 publications

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“…S. lividans strains were grown in yeast extract/malt extract/sucrose (YEMES) medium (YEMEϩ34% sucrose to allow dispersed growth) supplemented with thiostrepton (5 µg/ml) when required [17]. All plasmid constructions used derived from pUC19, pSECA1 [2] and pIJ699 [18].…”

Section: Methodsmentioning

confidence: 99%

“…E. coli and B. subtilis SecA proteins contain two essential ATP-binding sites: nucleotide-binding site (NBS)-I has a high affinity (K d Ϸ150 nM) and NBS-II has a low affinity (K dϷ340 µM) [36,37]. Amino acid sequence analysis revealed that both NBSs are also present in the S. lividans SecA [2].…”

Section: Purification Of S Lividans Seca Protein S Lividansmentioning

confidence: 99%

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confidence: 99%

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Biochemical characterization of the SecA protein of Streptomyces lividans

Blanco¹,

Driessen²,

Coque³

et al. 1998

European Journal of Biochemistry

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The SecA protein of Streptomyces lividans was purified to near electrophoretic homogeneity by means of FPLC from an overproducing strain harbouring plasmid pULA400, in which the secA gene (Blanco, J., Coque, J. J. R. & Martín, J. F. (1996) Gene (Amst.) 176, 61Ϫ65) was expressed from the strong promoter of the Streptomyces griseus saf gene. The native form of SecA was shown to be a dimer (M r 209 kDa) by gel filtration. It crossreacted with antibodies raised against Escherichia coli or Bacillus subtilis SecA proteins. Purified S. lividans SecA showed a low endogenous ATPase activity that was stimulated by addition of a S. lividans lipid fraction. SecA contains a high-affinity and a low-affinity nucleotide-binding site (NBS). [A-32 P]ATP could be crosslinked by ultraviolet radiation at the high-affinity site. The intrinsic tryptophan fluorescence of SecA decreased on addition of increasing concentrations of ADP and reached a saturation level at about 1 µM (the range of saturation at the NBS I). The calculated K d of the high-affinity binding site for ADP was 150 nM. Millimolar concentrations of ATP or ADP did not render the S. lividans SecA protein resistant to V8 protease degradation, in contrast to what occurs with the E. coli and B. subtilis SecA proteins. SecA was found to bind to urea-washed S. lividans membrane vesicles with high-affinity, i.e. 10 nM. SecA-dependent binding of E. coli SecB to membrane vesicles was observed when E. coli SecA was used, but not with the S. lividans SecA, suggesting that this interaction may be specific for the Gram-negative bacteria. An in vitro translocation system has been developed using inverted membrane vesicles of S. lividans. SecA supported in vitro translocation of proAmy into S. lividans membrane vesicles in an ATP-dependent manner.

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Section: Methodsmentioning

confidence: 99%

Section: Purification Of S Lividans Seca Protein S Lividansmentioning

confidence: 99%

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Biochemical characterization of the SecA protein of Streptomyces lividans

Blanco¹,

Driessen²,

Coque³

et al. 1998

European Journal of Biochemistry

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“…SecA is a homodimeric protein with a subunit mass of about 100 kDa. During the last few years, genes encoding SecA homologs from many different Gram‐positive organisms have been cloned [91–95]. Strikingly, mycobacteria even contain two secA genes [18,19,95], but it is not clear if both genes are essential.…”

Section: The Secretion Machinerymentioning

confidence: 99%

Translocation of proteins across the cell envelope of Gram-positive bacteria

et al. 2001

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In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.

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“…The SecA proteins of Gram‐positive bacteria appear to be structurally and functionally similar to SecA of E. coli . Although the secA gene of Streptomyces lividans had recently been identified by two independent groups [9, 10], the regulation of secA gene expression in Streptomycetes or other Gram‐positive bacteria has not been studied as yet. In this study we report on the cloning, functional characterization and transcriptional regulation of the secA gene from S. griseus N2‐3‐11.…”

Section: Introductionmentioning

confidence: 99%

Protein secretion in Streptomyces griseus N2-3-11: characterization of the secA gene and its growth phase-dependent expression

Pöhling

Piepersberg

Wehmeier

2006

FEMS Microbiology Letters

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The chromosomal region encoding the secA gene of Streptomyces griseus N2-3-11 was cloned and analyzed. The secA gene encodes a polypeptide of 939 aa with a molecular mass of 105 kDa. The growth defect of temperature sensitive Escherichia coli secA mutants was not restored by the S. griseus SecA. The secA promoter was analyzed and the transcriptional start point of the gene was determined. Northern blot and Western blot analyses revealed a growth phase dependent secA expression. The integration of an additional copy of the S. griseus secA gene into the genome of S. lividans TK23 had no visible effect on the efficiency of protein secretion.

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Characterization of the secA gene of Streptomyces lividans encoding a protein translocase which complements an Escherichia coli mutant defective in the ATPase activity of SecA

Cited by 9 publications

References 16 publications

Biochemical characterization of the SecA protein of Streptomyces lividans

Biochemical characterization of the SecA protein of Streptomyces lividans

Translocation of proteins across the cell envelope of Gram-positive bacteria

Protein secretion in Streptomyces griseus N2-3-11: characterization of the secA gene and its growth phase-dependent expression

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