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PubMed1992

The genes for ribosomal protein L15 and the protein equivalent to secY in the archaebacterium Haloarcula (Halobacterium) marismortui.

The nucleotide sequences of the L15 gene and the secY gene, which form the last two genes of the S10/spc-operon region of Haloarcula marismortui, have been determined. The HmaL15 protein sequence translated from the DNA is 164 amino acids long, revealing 10 amino acids more at the C-terminus than the published protein sequence. The deduced HmasecY protein sequence has 487 amino acids and shows significant homology to its counterparts in Methanococcus vannielii and Escherichia coli. The gene order of the halobacterial gene cluster is similar to that in the methanogens and in eubacteria.

Amino Acid Sequence↗
PubMed1995

A secY homologous gene in the crenarchaeon Sulfolobus acidocaldarius.

The nucleotide sequence of an open reading frame, located upstream of the gene for adenylate kinase, was determined in the thermoacidophile crenarchaeon Sulfolobus acidocaldarius. Data bank searches identified the sequence as a secY homologous gene. The DNA derived protein sequence of total 463 amino acids contains 10 hydrophobic domains. A sequence alignment with other prokaryotic and eukaryotic secY sequences reveals significant homology, but the secY primary sequence of S. acidocaldarius shows only a low degree of similarity with the secY counterparts of the euryarchaea Methanococcus vannielii and Haloarcula marismortui. A transcription analysis indicates, that the secY gene is cotranscribed with the gene coding for adenylate kinase.

Adenylate Kinase↗
PubMed1996

Cloning of a secA homolog from Streptomyces lividans 1326 and overexpression in both S. lividans and Escherichia coli.

We cloned a gene encoding a SecA homolog from Streptomyces lividans 1326, a Gram-positive bacterium known to produce large amounts of extracellular proteins. A protein sequence alignment with the other bacterial SecA homologs revealed that S. lividans SecA shares from 39.5 to 44% identity with them, while it shares 34.2 to 37.2% identity with SecA homologs from plastids of algae and plants. We overexpressed the secA gene in S. lividans 1326 and Escherichia coli MM52 and in both cases we observed the production of a protein with an apparent molecular mass of 117.4 kDa. Although S. lividans SecA is similar to E. coli SecA, it does not complement a thermosensitive mutation in the E. coli secA gene. However, a hybrid polypeptide consisting of the N-terminal portion (first 242 amino acids) of the S. lividans SecA and the C-terminal portion (657 a.a.) of the wild-type E. coli SecA was able to complement this mutant.

Adenosine Triphosphatases↗
PubMed1996

Different PrlA proteins increase the efficiency of periplasmic production of human interleukin-6 in Escherichia coli.

The export efficiency of a fusion of the Escherichia coli preOmpA signal peptide to human interleukin-6 can be significantly raised by coexpressing three different prlA alleles of sec Y along with wild type secE. The effect seems prlA-specific, as prlG1 (a prl allele of secE) does not affect the export of preOmpA-hIL-6. Coexpression of secD and secF also stimulates the export of the fusion protein.

Alleles↗
PubMed1991

Suppression of an Escherichia coli secA(ts) mutant by a gene cloned from Staphylococcus carnosus.

Escherichia coli mutant MM52 (secA(ts)) was transformed with a cosmid library from Staphylococcus carnosus, and a recombinant cosmid (pBO23) allowing growth at the non-permissive temperature (42 degrees C) was isolated. pBO23 also restored the growth defects of E. coli mutants IQ85 (secY(ts)) and IT41 (lep(ts)). Nucleotide sequencing revealed that the DNA fragment responsible for the suppression effect codes for a S. carnosus protein highly homologous to the ribosomal protein L13 of E. coli. The staphylococcal L13 protein was efficiently incorporated into E. coli ribosomes. Possible explanations for the effect of this polypeptide on the growth of temperature-sensitive E. coli secretion mutants are discussed.

Adenosine Triphosphatases↗
PubMed1991

Conservation of components of the Escherichia coli export machinery in prokaryotes.

The SecA and SecB proteins are involved in protein translocation in Escherichia coli. Immunoblot analysis was used to investigate the possible conservation of these proteins in prokaryotes. Cross-reacting proteins were detected in all other Enterobacteriaceae tested, but not in microorganisms more distantly related from Escherichia coli.

Adenosine Triphosphatases↗
PubMed1993

Non-specific hole formation in the Escherichia coli inner membrane by lambda S proteins in independent of cellular secY and secA functions and of the proportion of membrane acidic phospholipids.

Formation of the lesion in the Escherichia coli inner membrane caused by lambda lysis protein S was examined by electron microscopy. We also show that macromolecules exceeding the size of the lambda R transglycosylase can pass through the S-dependent hole and that assembly of the S-dependent hole is independent of the proportion of acidic phospholipids in the inner membrane and of components of the cellular transport machinery.

Adenosine Triphosphatases↗
PubMed1995

Functional characterization of the Staphylococcus carnosus SecA protein in Escherichia coli and Bacillus subtilis secA mutant strains.

The Staphylococcus carnosus secA gene was cloned using the Bacillus subtilis secA gene as a probe. The S. carnosus secA encodes a polypeptide of 844 amino acid residues which is homologous to other known SecA proteins. The S. carnosus SecA functionally complemented the growth and secretion defects of a temperature-sensitive B. subtilis secA mutant at the non-permissive temperature. In contrast, the growth defect of an Escherichia coli secA mutant could not be complemented by the S. carnosus SecA protein. Our results suggest that the interactions of SecA with precursor proteins and/or other components of bacterial preprotein translocase are optimized within each organism.

Adenosine Triphosphatases↗
PubMed1994

Cloning and sequencing of the secY homolog from Coryneform bacteria.

A conserved domain of the secY genes from Bacillus subtilis, Mycoplasma capricolum and Escherichia coli was used to design degenerate oligodeoxyribonucleotides. These synthetic DNA sequences were used to screen a lambda library of Brevibacterium flavum MJ233. A 1.5-kb KpnI fragment of a recombinant lambda phage containing the secY homology from Br. flavum MJ233 was subsequently subcloned into plasmid pUC118. The complete nucleotide (nt) sequence of the cloned fragment indicated that the deduced gene product of the Br. flavum secY homolog is composed of 440 amino acids (aa) with a deduced M(r) of 47,871. Comparison of this aa sequence to the corresponding sequences from E. coli and B. subtilis revealed a high degree of conservation, and suggested that the Br. flavum secY homolog is a membrane protein containing ten transmembrane segments. In addition, we could identify, downstream from secY, a putative coding sequence of the enzyme adenylate kinase. This gene organization is identical to that observed in the B. subtilis genome.

Actinomycetales↗
PubMed1995

Cloning and sequencing of a secY homolog from Streptomyces scabies.

The complete DNA sequence of the Streptomyces scabies (Ss) secY homolog and partial sequences of adjacent upstream and downstream open reading frames (ORFs) have been determined. The nucleotide sequence of a 2-kb region predicts a polypeptide of 437 amino acids in length with homology to the SecY protein family. The Ss secY homolog lies upstream from a sequence that has homology to the adenylate kinase gene (adk) family. The translational stop codon of the putative SecY ORF overlaps the predicted start codon for the Adk ORF. Another ORF that lies upstream from the secY homolog has sequence similarity to the genes that code for the L15 r-protein. Within the 243-bp intergenic region between the L15 and SecY coding sequences, the presence of a streptomycete-like promoter sequence and an 18-bp inverted repeat suggests that the secY homolog and the adjacent downstream sequences may be transcribed independently of the L15 coding sequence. Transcript analysis indicates that the secY homolog is expressed in both Ss and Streptomyces lividans. The proposed gene and transcript organization of the L15-SecY-Adk coding regions in the Ss clone resembles that of Micrococcus luteus which, like the streptomycetes, has a G+C-rich genome.

Adenylate Kinase↗
PubMed1996

Genetic and transcriptional organization of the Bacillus subtilis spc-alpha region.

We used chromosomal walking methods to isolate a 10.8-kb region from the major ribosomal protein (r-protein) gene cluster of Bacillus subtilis (Bs). The gene order in this region, given by gene product, was r-proteins L16-L29-S17-L14-L24-L5-S14-S8-L6-L18-S5-L30-L15-SecY-adenylate kinase (Adk)-methionine aminopeptidase (Map)-initiation factor 1 (IF1)-L36-S13-S11-alpha subunit of RNA polymerase-L17. The region cloned, therefore, contains the homologues for the last three genes of the Escherichia coli (Ec) S10 operon, together with entire spc and alpha operons. This Bs organization differs from the corresponding region in Ec by the inclusion of the genes encoding Adk, Map and IF1 between the genes encoding SecY and L36. Plasmid integration experiments indicated that all 22 genes comprise a single large transcriptional unit controlled from a major promoter which lies upstream from the gene encoding r-protein L16. Promoter probe experiments located lesser activities internal to this large transcriptional unit, the secY and map promoters. The secY promoter region (psecY) contained two activities, each principally functioning in the stationary growth phase when high protein export is required. Thus, the Bs S10-spc-alpha region differs from its Ec counterpart in both genetic and transcriptional organization. Given this difference in transcriptional organization, the mechanisms coordinating expression of the translational apparatus are also likely to differ between Ec and Bs.

Amino Acid Sequence↗
PubMed1996

Gene organization in the ada-rplL region of Streptomyces virginiae.

The gene organization of a 7.4-kb region of the Streptomyces virginiae (Sv) chromosome was determined. The predicted open reading frames (ORFs) and their predicted products, in sequence order, were (i) ada, encoding adenosine deaminase [EC 3.5.4.4], (ii) aat, encoding a protein homologous to aspartate aminotransferase [EC 2.6.1.1], (iii) secE, encoding a protein involved in protein secretion, (iv) vbrA, encoding a NusG-like protein involved in antitermination of transcription as described by Okamoto et al. [J. Biol. Chem. 267 (1992) 1093-1098], and (v) rplKAJL, encoding the large subunits of the ribosomal proteins L11, L1, L10 and L12. Six of the ORFs (secE-rplL) were oriented in the same direction, but the other two (ada and aat) had the opposite orientation. The gene organization of the secE-rplL region in Sv was identical to that in Escherichia coli.

Adenosine Deaminase↗
PubMed1996

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

The secA gene of Streptomyces lividans was cloned using as probe a 57-mer oligonucleotide based on conserved sequences of the Escherichia coli secA and the Bacillus subtilis div genes. It encodes a protein of 946 amino acids (aa) with a deduced M(r) of 106,079, with high similarity to all known SecA proteins. All the previously described conserved motifs of SecA proteins were conserved in the S. lividans protein. The secA gene of S. lividans restored sensitivity to sodium azide in E. coli SecA4 (AzR) a mutant with an azide-resistant (ATPase defective) SecA protein. However, it did not complement the temperature-sensitive mutation in E. coli MM52 (SecAts) (a conditional lethal mutant defective in protein translocation) allowing only poor growth at the nonpermissive temperature. secA homologous sequences were present in 11 different species of Streptomyces and Nocardia.

Adenosine Triphosphatases↗
PubMed1996

Cloning and sequencing of the secY homolog from Streptomyces lividans 1326.

Two conserved regions of SecY proteins from six Gram+ bacteria were exploited in a PCR-based strategy for isolating a secY homolog from Streptomyces lividans (Sl). The nucleotide sequence of part of a 3.8-kb fragment showed that the secY homolog is flanked, at the 5' end, by the gene encoding ribosomal protein L15 and, at the 3' end, by an adenylate kinase-encoding gene. The deduced gene product of secY would have 437 amino acids (aa) and an M(r) of 47,200. Sl SecY shows 89.5, 56.1, 42 and 40% identity to its homologs from Streptomyces scabies, Brevibacterium flavum, Bacillus subtilis and Escherichia coli, respectively. Promoterprobe analyses indicated that the secY gene probably contains its own promoter.

Amino Acid Sequence↗
PubMed1994

Protein secretion pathway in Escherichia coli.

The export of proteins to the Escherichia coli periplasm is a well established system for heterologous protein production. With a better understanding of the protein export (SecA, Y-dependent) process and a greater awareness of the conditions necessary for correct folding of proteins in the periplasm, serious efforts are now being made to manipulate this system to achieve substantial increases in the yield of authentically folded proteins. Further advances in the development of methods for the recovery of recombinant proteins from the culture medium have made the use of fusion proteins secreted by the protein A or haemolysin pathways a more attractive option. Recent studies of the haemolysin system indicate its ability to secrete a wide range of polypeptides, including normally cytoplasmic proteins. As their features and potential applications become much clearer, a rapidly expanding number of protein-secretion mechanisms in Gram-negative bacteria are becoming available for heterologous protein expression. Most, if not all, of these systems can be successfully transplanted into E. coli, providing a wider choice of systems for the future.

Adenosine Triphosphatases↗
PubMedDate not supplied

Electron microscopy of thin-sectioned three-dimensional crystals of SecA protein from Escherichia coli: structure in projection at 40 A resolution.

SecA is a single-chain, membrane-associated polypeptide (102 kDa) which functions as an essential component of the protein export machinery of Escherichia coli. SecA has been crystallized from ammonium sulfate as small, three-dimensional bipyramidal crystals (0.1 x 0.1 x 0.05 mm). These crystals did not demonstrate detectable diffraction of X-rays from rotating anode sources. For study by electron microscopy, individual crystals were cross-linked in glutaraldehyde and OsO4 solutions, dehydrated, embedded in epoxy resin, and sectioned normal to crystallographic axial directions inferred from the external morphology of the crystals. Fourier transformation of processed images of untilted thin sections stained with uranyl acetate and lead citrate show reflections extending to 31 A resolution. Diffraction data and reconstructed images of the projected density of the unit cell contents indicate that the bipyramidal SecA crystals belong to orthorhombic space group C222(1) with unit cell dimensions a = 414 A, b = 381 A, and c = 243 A. Filtered images and density maps of mutually orthogonal projections of the unit cell contents are consistent with a three-dimensional model in which the asymmetric unit contains eight SecA monomers. The large unit cell dimensions and packing of protein monomers suggest that SecA is crystallizing as an oligomer of either dimers or tetramers.

Adenosine Triphosphatases↗
PubMed2005

Genes coding for SecG and Leu2-tRNA form an operon to give an unusual RNA comprising mRNA and a tRNA precursor.

The secG gene encoding the SecG subunit of the SecYEG translocon and the leuU gene encoding Leu2-tRNA are very closely located on the Escherichia coli chromosome. A secG-leuU disruptant was not viable unless secG-leuU was induced from a plasmid, indicating that leuU is an essential gene since secG is dispensable at 37 degrees C. A mutant strain in which the promoter region for secG was replaced with cat revealed the same phenotype as the secG-leuU disruptant, indicating that leuU was expressed from the secG promoter. When the secG-leuU locus was placed on a high copy plasmid, an RNA comprising both mRNA for SecG and a precursor for Leu2-tRNA was detected on a Northern blot. Moreover, a secG-leuU transcript was amplified by RT-PCR using the total RNA fraction prepared from wild type E. coli cells but not from the secG-leuU and the secG promoter disruptants, indicating that secG-leuU forms an operon. Thus, the expression of Leu2-tRNA requires expression of the upstream secG gene. The gene structure of secG-leuU was conserved among Gram-negative bacteria, although the sequences separating the two genes were quite diverse. The physiological significance of this unusual gene organization is discussed.

Amino Acid Sequence↗