SecA proteins of Bacillus subtilis and Escherichia coli possess homologous amino-terminal ATP-binding domains regulating integration into the plasma membrane.

The Bacillus subtilis secA homolog, div, was cloned and expressed at a variety of different levels in wild-type and secA mutant strains of Escherichia coli. Analysis of Div function showed that it could not substitute for SecA despite being present at a wide range of concentrations at or above the physiological level. Location of regions of functional similarity between the two proteins using div-secA chimeras revealed that only the amino-terminal ATP-binding domain of Div could functionally substitute for the corresponding region of SecA. The role of this domain was revealed by subcellular localization experiments that demonstrated that in both B. subtilis and E. coli Div had cytoplasmic, peripheral, and integral membrane distributions similar to those of its SecA homolog and that an intact ATP-binding domain was essential for regulating integration of this protein into the plasma membrane. These results suggest strongly that the previously observed cycle of membrane binding, insertion, and deinsertion of SecA protein (A. Economou and W. Wickner, Cell 78:835-843, 1994) is common to these two bacteria, and they demonstrate the importance of the conserved ATP-binding domain in promoting this cycle.     

Isolation and characterization of a Bacillus subtilis secA mutant allele conferring resistance to sodium azide

Abstract A mutation has been isolated in the Bacillus subtilis secA gene ( secA10 ) which allows cell growth and residual protein translocation in the presence of 1.5 mM sodium azide. Besides conferring resistance to sodium azide, the corresponding SecA10 mutant protein, in which glutamic acid at position 338 has been changed to glycine, seems to possess a secretion defect even in the absence of azide. In addition, the secA10 mutant protein was found to be recessive to wild-type secA with regard to azide resistance. Our results strongly suggest that, like the situation in Escherichia coli , the B. subtilis SecA protein is a main target for the lethal action of sodium azide.     

Molecular characterization and functional analysis of a secA gene homolog in Actinobacillus actinomycetemcomitans

Results of Southern blot analyses and polymerase chain reaction revealed that the Gram-negative pathogen, Actinobacillus actinomycetemcomitans, harbored DNA homologous to the secA gene of Escherichia coli. In E. coli, the secA gene product is essential for translocation of proteins across the inner membrane via the Sec system. This A. actinomycetemcomitans secA homolog was cloned and its nucleotide sequence determined. Amino acid sequence analysis of the cloned gene revealed significant homology to the SecA proteins of Haemophilus influenzae, E. coli, Caulobacter crescentus and Bacillus subtilis. Although the cloned gene did not complement a temperature sensitive mutation in the E. coli secA gene, strains harboring the cloned gene did produce a protein that cross-reacted with anti-SecA antibody. In addition, the cloned gene did restore sensitivity to sodium azide in an E. coli azide mutant. These data support the hypothesis that A. actinomycetemcomitans may use a system similar to the Sec system of E. coli to transport proteins across the cytoplasmic membrane, but suggest that the A. actinomycetemcomitans gene product may require genera-specific Sec proteins to complement some Sec mutations in E. coli.     

Molecular cloning of a Bacillus subtilis gene involved in cell division, sporulation, and exoenzyme secretion

The wild type div-341+ gene of Bacillus subtilis was cloned in a temperate phage rho 11, and was recloned in a smaller temperate phage phi 105. The resulting Div+ transducing phage carried a 3 kilobase Cfr13I digested chromosomal fragment which showed Div+ transforming activity and contained the whole div-341+ gene which is involved in cell division, sporulation, exoenzyme secretion, competent cell formation, and autolysis. A partial restriction map of the fragment was established. The merodiploid system of the div-341+ gene, wild type gene on the phage genome and mutant gene on the chromosome, resulted in the suppression of mutant phenotypes and indicated that the wild type div-341+ gene is dominant over mutant gene.     

Cloning,expression, and functional characterization of the Mycobacterium tuberculosis secA gene

To better understand the protein secretion mechanisms involved in the growth and pathogenesis of Mycobacterium tuberculosis, we examined the secA gene from M. tuberculosis (tbsecA; cosmid sequence accession No. z95121.gb_ba). We generated plasmids containing the full-length tbsecA gene or a fusion containing the 5' sequence from the M. tuberculosis secA gene and the remainder from the Escherichia coli secA gene and evaluated the ability of each construct to complement the defective SecA protein in E. coli MM52ts when grown at the non-permissive temperature. The full-length tbsecA gene was unable to compensate for the temperature-sensitive defect, whereas E. coli MM52ts that has been transformed with plasmid pMF8TB226 containing a chimeric secA gene was able to grow at 42 degrees C. This work confirms that the topography of SecA and its ATP binding sites are highly conserved, whereas its membrane insertion domains are species specific.     

Cloning and expression analysis of Phytoplasma protein translocation genes

Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.     

Isolation and analysis of dominant secA mutations in Escherichia coli.

The secA gene product is an autoregulated, membrane-associated ATPase which catalyzes protein export across the Escherichia coli plasma membrane. Previous genetic selective strategies have yielded secA mutations at a limited number of sites. In order to define additional regions of the SecA protein that are important in its biological function, we mutagenized a plasmid-encoded copy of the secA gene to create small internal deletions or duplications marked by an oligonucleotide linker. The mutagenized plasmids were screened in an E. coli strain that allowed the ready detection of dominant secA mutations by their ability to derepress a secA-lacZ protein fusion when protein export is compromised. Twelve new secA mutations were found to cluster into four regions corresponding to amino acid residues 196 to 252, 352 to 367, 626 to 653, and 783 to 808. Analysis of these alleles in wild-type and secA mutant strains indicated that three of them still maintained the essential functions of SecA, albeit at a reduced level, while the remainder abolished SecA translocation activity and caused dominant protein export defects accompanied by secA depression. Three secA alleles caused dominant, conditional-lethal, cold-sensitive phenotypes and resulted in some of the strongest defects in protein export characterized to date. The abundance of dominant secA mutations strongly favors certain biochemical models defining the function of SecA in protein translocation. These new dominant secA mutants should be useful in biochemical studies designed to elucidate SecA protein's functional sites and its precise role in catalyzing protein export across the plasma membrane.     

Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export.

Export of the outer membrane lipoprotein in Escherichia coli was examined in conditionally lethal mutants that were defective in protein export in general, including secA, secB, secC, and secD. Lipoprotein export was affected in a secA(Ts) mutant of E. coli at the nonpermissive temperature; it was also affected in a secA(Am) mutant of E. coli at the permissive temperature, but not at the nonpermissive temperature. The export of lipoprotein occurred normally in E. coli carrying a null secB::Tn5 mutation; on the other hand, the export of an OmpF::Lpp hybrid protein, consisting of the signal sequence plus 11 amino acid residues of mature OmpF and mature lipoprotein, was affected by the secB mutation. The synthesis of lipoprotein was reduced in the secC mutant at the nonpermissive temperature, as was the case for synthesis of the maltose-binding protein, while the synthesis of OmpA was not affected. Lipoprotein export was found to be slightly affected in secD(Cs) mutants at the nonpermissive temperature. These results taken together indicate that the export of lipoprotein shares the common requirements for functional SecA and SecD proteins with other exported proteins, but does not require a functional SecB protein. SecC protein (ribosomal protein S15) is required for the optimal synthesis of lipoprotein.     

SecA protein is directly involved in protein secretion in Escherichia coli

A high-expression plasmid for the secA gene was constructed. The SecA protein was then overproduced in E. coli and purified. The purified SecA stimulated the in vitro translocation of a model secretory protein into inverted membrane vesicles pretreated with 4 M urea. Membrane vesicles from a secAts mutant exhibited lower translocation activity, which was enhanced by SecA. These results indicate that SecA is directly involved in protein secretion across the cytoplasmic membrane.     

Autogenous translation regulation by Escherichia coli ATPase SecA may be mediated by an intrinsic RNA helicase activity of this protein.

The seven conserved motifs typical of the helicase superfamily II have been identified in the sequences of Escherichia coli protein SecA, an ATPase mediating protein translocation across the inner membrane of the bacterium, and its Bacillus subtilis homolog Div. It is hypothesized that SecA and Div possess an RNA helicase activity and may couple ATP hydrolysis both to membrane translocation of proteins, and to hairpin unwinding in their own mRNAs, leading to the known autogenous regulation of translation.     

Regulation of a membrane component required for protein secretion in Escherichia coli

We have previously described a gene, secA, which may code for a component of the secretion machinery of E. coli. Temperature-sensitive mutations in this gene lead to the cytoplasmic accumulation of precursors to a number of secreted proteins. In this paper, we describe the use of antibody to the SecA protein to characterize the cellular location and regulation of the protein. The antibody was elicited in response to a SecA-LacZ hybrid protein, produced by a strain carrying a secA-lacZ gene fusion. The secA gene product is a 92 kd polypeptide that is present in small amounts in the cell and that fractionates as a peripheral cytoplasmic membrane protein. The synthesis of the SecA protein is greatly derepressed (at least tenfold) when secretion in E. coli is blocked either in a secAts mutant or in the presence of a MalE-LacZ hybrid protein. We suggest that components of the secretion machinery of E. coli, such as the SecA protein, may be regulated in response to the secretion needs of the cell. When suppression of a secAam mutant is eliminated, leading to the absence of SecA protein, the synthesis of maltose-binding protein is greatly reduced. These results support a mechanism in which secretion and translation are coupled.     

Preprotein translocation by a hybrid translocase composed of Escherichia coli and Bacillus subtilis subunits

Bacterial protein translocation is mediated by translocase, a multisubunit membrane protein complex that consists of a peripheral ATPase SecA and a preprotein-conducting channel with SecY, SecE, and SecG as subunits. Like Escherichia coli SecG, the Bacillus subtilis homologue, YvaL, dramatically stimulated the ATP-dependent translocation of precursor PhoB (prePhoB) by the B. subtilis SecA-SecYE complex. To systematically determine the functional exchangeability of translocase subunits, all of the relevant combinations of the E. coli and B. subtilis secY, secE, and secG genes were expressed in E. coli. Hybrid SecYEG complexes were overexpressed at high levels. Since SecY could not be overproduced without SecE, these data indicate a stable interaction between the heterologous SecY and SecE subunits. E. coli SecA, but not B. subtilis SecA, supported efficient ATP-dependent translocation of the E. coli precursor OmpA (proOmpA) into inner membrane vesicles containing the hybrid SecYEG complexes, if E. coli SecY and either E. coli SecE or E. coli SecG were present. Translocation of B. subtilis prePhoB, on the other hand, showed a strict dependence on the translocase subunit composition and occurred efficiently only with the homologous translocase. In contrast to E. coli SecA, B. subtilis SecA binds the SecYEG complexes only with low affinity. These results suggest that each translocase subunit contributes in an exclusive manner to the specificity and functionality of the complex.     

Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation

Summary A DNA fragment that codes for the 364 amino-terminal amino acid residues of a putative Bacillus subtilis SecA homologue has been cloned using the Escherichia coli SecA gene as a probe. The deduced amino acid sequence showed 58% identity to the aminoterminus of the E. coli SecA protein. A DNA fragment which codes for 275 amino-terminal amino acid residues of the B. subtilis SecA homologue was expressed in E. coli and the corresponding gene product was shown to be recognized by anti-E. coli SecA antibodies. This polypeptide, although only about 30% the size of the E. coli SecA protein, also restored growth of E. coli MM52 (secA ^ts) at the non-permissive temperature and the translocation defect of proOmpA in this mutant was relieved to a substantial extent.     

SecA protein is required for secretory protein translocation into E. coli membrane vesicles

The soluble and membrane components of an E. coli in vitro protein translocation system prepared from a secA amber mutant, secA13[Am], contain reduced levels of SecA and are markedly defective in both the cotranslational and posttranslational translocation of OmpA and alkaline phosphatase into membrane vesicles. Moreover, the removal of SecA from soluble components prepared from a wild-type strain by passage through an anti-SecA antibody column similarly abolishes protein translocation. Translocation activity is completely restored by addition of submicrogram amounts of purified SecA protein, implying that the observed defects are solely related to loss of SecA function. Interestingly, the translocation defect can be overcome by reconstitution of SecA into SecA-depleted membranes, suggesting that SecA is an essential, membrane-associated translocation factor.     

Two Nonredundant SecA Homologues Function in Mycobacteria

The proper extracytoplasmic localization of proteins is an important aspect of mycobacterial physiology and the pathogenesis of Mycobacterium tuberculosis. The protein export systems of mycobacteria have remained unexplored. The Sec-dependent protein export pathway has been well characterized in Escherichia coli and is responsible for transport across the cytoplasmic membrane of proteins containing signal sequences at their amino termini. SecA is a central component of this pathway, and it is highly conserved throughout bacteria. Here we report on an unusual property of mycobacterial protein export--the presence of two homologues of SecA (SecA1 and SecA2). Using an allelic-exchange strategy in Mycobacterium smegmatis, we demonstrate that secA1 is an essential gene. In contrast, secA2 can be deleted and is the first example of a nonessential secA homologue. The essential nature of secA1, which is consistent with the conserved Sec pathway, leads us to believe that secA1 represents the equivalent of E. coli secA. The results of a phenotypic analysis of a Delta secA2 mutant of M. smegmatis are presented here and also indicate a role for SecA2 in protein export. Based on our study, it appears that SecA2 can assist SecA1 in the export of some proteins via the Sec pathway. However, SecA2 is not the functional equivalent of SecA1. This finding, in combination with the fact that SecA2 is highly conserved throughout mycobacteria, suggests a second role for SecA2. The possibility exists that another role for SecA2 is to export a specific subset of proteins.     

A flower-specific cDNA encoding a novel thionin in tobacco

Summary A gene library of Bacillus subtilis chromosomal DNA was screened for genes capable of reverting the growth defects of the Escherichia coli secA51(Ts) mutant at 42° C. A B. subtilis gene, designated csaA, was found to phenotypically suppress not only the growth defects of the E. coli mutant, but also to relieve the detrimental accumulation of precursors of exported proteins. The csaA gene encoded a protein of 15 kDa (137 amino acids) and was likely to be the distalmost member of an operon. No similarity to csaA was found among DNA or protein sequences deposited in databases. In contrast to other homologous or heterologous suppressors of the E. coli secA51(Ts) mutation, the csaA gene did not exert pleiotropic effects on either the E. coli sec Y24(Ts) or lep9(Ts) mutations. However, it restored the ability of a SecB-deficient mutant to grow on complex medium. It is proposed that CsaA serves as a molecular chaperone for exported proteins or alternatively acts by stabilizing the SecA protein.     

Effects of secA mutations on the synthesis and secretion of proteins in Escherichia coli. Evidence for a major export system for cell envelope proteins

We have followed the synthesis and secretion of a number of periplasmic and outer membrane proteins in three strains of Escherichia coli, a secA amber mutant, a secA temperature-sensitive mutant, and a strain that blocks protein secretion due to a high level of expression of an export-defective hybrid protein between maltose-binding protein and beta-galactosidase (MalE-LacZ). Our results show that after several hours under nonpermissive conditions the specificity and extent of the export blocks in the secA temperature-sensitive mutant and the strain producing the MalE-LacZ hybrid protein are identical, affecting at least four major outer membrane proteins and most but not all periplasmic proteins. The secA gene product, therefore, appears to be an essential component of the major export pathway in E. coli which is used by many envelope proteins independent of whether they are cotranslationally or post-translationally secreted. In contrast, the synthesis of only a subset of these envelope proteins is reduced in the secA amber mutant after shift to the nonpermissive condition. These results indicate that the SecA protein serves roles both in the synthesis and the secretion of certain cell envelope proteins.     

Membrane biogenesis in Escherichia coli: effects of a secA mutation

In Escherichia coli K-12, temperature-sensitive mutations in the secA gene have been shown to interfere with protein export. Here we show that the effect of a secA mutation is strongly pleiotropic on membrane biogenesis. Freeze-fracture experiments as well as cryosections of the cells revealed the appearance of intracytoplasmic membranes upon induction of the SecA phenotype. The permeability barrier of the outer membrane to detergents was lost. Two alterations in the outer membrane may be responsible for this effect, namely the reduced amounts of outer membrane proteins, or the reduction of the length of the core oligosaccharide of the lipopolysaccharide, which was observed in phage-sensitivity experiments and by SDS-polyacrylamide gel electrophoresis. Phospholipid analysis of the secA mutant, grown under restrictive conditions, revealed a lower content of the negatively charged phospholipid cardiolipin and of 18:1 fatty acid compared to those of the parental strain grown under identical conditions. These results are in line with the hypothesis that protein export and lipid metabolism are coupled.