Nucleotide sequence of the phoM region of Escherichia coli: four open reading frames may constitute an operon.

The phoM gene is one of the positive regulatory genes for the phosphate regulon of Escherichia coli. We analyzed the nucleotide sequence of a 4.7-kilobase chromosomal DNA segment that encompasses the phoM gene and its flanking regions. Four open reading frames (ORFs) were identified in the order ORF1-ORF2-ORF3 (phoM)-ORF4-dye clockwise on the standard E. coli genetic map. Since these ORFs are preceded by a putative promotor sequence upstream of ORF1 and followed by a putative terminator distal to ORF4, they seem to constitute an operon. The 157-amino-acid ORF1 protein contains highly hydrophobic amino acids in the amino-terminal portion, which is a characteristic of a signal peptide. The 229-amino-acid ORF2 protein is highly homologous to the PhoB protein, a positive regulatory protein for the phosphate regulon. The ORF3 (phoM gene) protein contains two stretches of highly hydrophobic residues in the amino-terminal and central regions and, therefore, may be a membrane protein. The 450-amino-acid ORF4 protein contains long hydrophobic regions and is likely to be a membrane protein.     

Cloning of an insecticidal cholesterol oxidase gene and its expression in bacteria and in plant protoplasts.

We cloned and sequenced structural gene choM, which encodes an insecticidally active cholesterol oxidase in Streptomyces sp. strain A19249. The primary translation product was predicted to be a 547-amino-acid protein whose first 43 amino acids constitute a secretory signal peptide. Expression of the gene with the signal sequence in Escherichia coli resulted in production of a protein that had enzymatic and insecticidal properties which were indistinguishable from those of the cholesterol oxidase secreted by Streptomyces sp. strain A19249. Expression of the gene with or without the signal sequence in tobacco protoplasts resulted in production of an enzymatically active cholesterol oxidase.     

Heterologous expression of the Mycobacterium tuberculosis gene encoding antigen 85A in Corynebacterium glutamicum.

By using appropriate Corynebacterium glutamicum-Escherichia coli shuttle plasmids, the gene encoding the fibronectin-binding protein 85A (85A) from Mycobacterium tuberculosis was expressed in C. glutamicum, also an actinomycete and nonsporulating gram-positive rod bacterium, which is widely used in industrial amino acid production. The 85A gene was weakly expressed in C. glutamicum under the control of the ptac promoter from E. coli, but it was produced efficiently under the control of the promoter of the cspB gene encoding PS2, one of the two major secreted proteins from C. glutamicum. The 85A protein was produced in various forms, with or without its own signal sequence and with or without the signal sequence and the NH2-terminal (18-amino-acid) mature sequence of PS2. Western blot analysis with monoclonal antibodies raised against the M. tuberculosis antigen 85 complex showed that recombinant 85A protein was present in the corynebacterial cell wall extract and also released in extracellular culture medium. NH2-terminal microsequencing of recombinant 85A secreted by C. glutamicum showed that signal peptide was effectively cleaved off at the predicted site. The recombinant 85A protein was biologically active in vitro, inducing significant secretion of Th1 T-cell cytokines, particularly interleukin-2 and gamma interferon, in spleen cell cultures from mice vaccinated with live Mycobacterium bovis BCG. Heterologous expression of mycobacterial antigens in C. glutamicum now offers a potent tool for further immunological characterization and large scale preparation of these recombinant proteins.     

Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region.

The membrane orientation of the NB protein of influenza B virus, a small (Mr, approximately 18,000) glycoprotein with a single internal hydrophobic domain, was investigated by biochemical and genetic means. Cell fractionation and protein solubility studies indicate NB is an integral membrane protein, and NB has been shown to be a dimer under nonreducing conditions. Treatment of infected-cell surfaces with proteinase K and endoglycosidase F and immunoprecipitation with a site-specific antibody suggests that the 18-amino-acid NH2-terminal region of NB is exposed at the cell surface. Oligonucleotide-directed mutagenesis to eliminate each of the four potential sites of N-linked glycosylation and expression of the mutant NB proteins in eucaryotic cells suggest that the two sites adjacent to the NH2 terminus are glycosylated. This provides further evidence that NB, which lacks a cleavable NH2-terminal signal sequence, has an exposed NH2 terminus at the cell surface.     

Cloning and nucleotide sequence of a heat-stable amylase gene from an anaerobic thermophile, Dictyoglomus thermophilum

A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686-amino-acid protein of relative molecular mass 81,200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2-terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH-stability and heat-stability, were examined. The amylase was found to be a highly liquefying-type.     

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.     

Genomic structure of the human mitochondrial aldehyde dehydrogenase gene

We have isolated and characterized four overlapping clones from two cosmid human genomic libraries, which span about 90 kilobase pairs (kbp) and contain the entire human mitochondrial aldehyde dehydrogenase (ALDH2) gene. Restriction maps of the genomic clones were elucidated utilizing cDNA probes and specific oligonucleotide probes. The organization of exons and introns was established by DNA sequencing of each exon and splicing junctions. The ALDH2 gene is about 44 kbp in length and contains at least 13 exons which encode 517 amino acid residues. Except for the signal NH2-terminal peptide, which is absent in the mature enzyme, the amino acid sequence deduced from the exons coincided with the reported primary structure of human liver ALDH2 (J. Hempel, R. Kaiser, and H. J?rnvall, 1985, Eur. J. Biochem. 153: 13-28). Several introns contain Alu repetitive sequences. A TATA-like sequence (TTATAAAA) and a CAAT-like sequence (GTCATCAT) are located 473 and 515 bp, respectively, upstream from the translation initiation codon. Primer extension and S1 nuclease mapping were performed to characterize the 5'-region of the gene.     

Mutations in the NH2-terminal domain of the signal peptide of preproparathyroid hormone inhibit translocation without affecting interaction with signal recognition particle

The amino-terminal domain of a eukaryotic signal peptide, from bovine parathyroid hormone, was altered by in vitro mutagenesis of the cDNA. The function of "internalized" signal sequence mutants and of deletion mutants was assayed using an in vitro translation-translocation system. The addition of 11 amino acids to the NH2 terminus of the signal peptide did not prevent normal processing of the precursor protein, whereas a 23-amino acid extension blocked processing. These data suggest that the NH2-terminal sequences of internal signal peptides must be permissive of the signal function. Deletion of 6 NH2-terminal amino acids from the signal peptide had no effect on its cleavage by microsomal membranes, but removal of 10 or 13 amino acids, including all charged residues prior to the hydrophobic core, prevented processing. For both the extension and deletion mutations, processed proteins were protected from proteolytic digestion, whereas unprocessed forms were not, which indicated that the unprocessed mutant proteins were not translocated across the microsomal membrane. Translation of both the extension and deletion translocation-deficient mutants was arrested by signal recognition particle, and salt-washed microsomal membranes reversed the translational arrest. These data demonstrate that the NH2-terminal domain is not required for the interaction of signal recognition particle with the signal peptide or with signal recognition particle receptor, but is required for formation of a maximally translocation-competent complex with the microsomal membrane.     

Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product.

The iap gene in Escherichia coli is responsible for the isozyme conversion of alkaline phosphatase. We analyzed the 1,664-nucleotide sequence of a chromosomal DNA segment that contained the iap gene and its flanking regions. The predicted iap product contained 345 amino acids with an estimated molecular weight of 37,919. The 24-amino-acid sequence at the amino terminus showed features characteristic of a signal peptide. Two proteins of different sizes were identified by the maxicell method, one corresponding to the Iap protein and the other corresponding to the processed product without the signal peptide. Neither the isozyme-converting activity nor labeled Iap proteins were detected in the osmotic-shock fluid of cells carrying a multicopy iap plasmid. The Iap protein seems to be associated with the membrane.     

Structure of yeast pGKL 128-kDa killer-toxin secretion signal sequence. Processing of the 128-kDa killer-toxin-secretion-signal-alpha-amylase fusion protein.

The linear double-stranded DNA plasmid pGKL1 in yeast encodes a killer toxin consisting of 97-kDa, 31-kDa and 28-kDa subunits. A 128-kDa protein precursor of the 97-kDa and 31-kDa subunits, was first synthesized with a 29-amino-acid extension at its NH2-terminus as a secretion signal sequence. In the present study, the property of this signal sequence was studied by the analysis of a fusion protein with mouse alpha-amylase. Using the secretion signal sequence of the killer protein, the mouse alpha-amylase was successfully secreted into the culture medium. An intracellular precursor form of alpha-amylase was identified and purified. Analysis of the NH2-terminal sequence of this precursor molecule indicated that it corresponded to the secretory intermediate (pro form) of alpha-amylase with the removal of the hydrophobic segment (Met1-Gly16) of the secretion signal. Both the secretion of alpha-amylase into the culture medium and the detection of the pro-alpha-amylase species in the cells were prohibited by a sec 11 mutation, or by the conversion of Gly to Val at the 16th position of the secretion signal. These results strongly suggest that the cleavage occurs between Gly16 and Leu17 by a signal peptidase, and that this cleavage is required for the secretion of alpha-amylase into the medium. Based on the data from the NH2-terminal amino acid sequences of secreted alpha-amylases, we conclude that the 29-amino-acid secretion signal present in the 128-kDa killer toxin precursor protein is a prepro structure.     

Alterations to the signal peptide of an outer membrane protein (OmpA) of Escherichia coli K-12 can promote either the cotranslational or the posttranslational mode of processing

The signal sequence of the precursor of the Escherichia coli outer membrane protein OmpA was altered by oligonucleotide insertions into the corresponding gene. In one case, OmpA-S1, the hydrophobic core of the signal peptide, is reduced from 12 to 10 residues, and one positive charge is added near the NH2-terminus. In another case, OmpA-P1, the hydrophobic core is extended from 12 to 16 residues. The pro-OmpA protein is normally processed partially co- and partially posttranslationally. Processing of the pro-OmpA-S1 protein was entirely posttranslational and that of the pro-OmpA-P1 protein strictly cotranslational. Evidence is presented which strongly suggests that posttranslational processing reflects posttranslational translocation across the plasma membrane. The generation times of cells expressing pro-OmpA-P1 or pro-OmpA-S1 were identical and the pro-OmpA-S1 polypeptide could be chased into the mature protein in the absence of protein synthesis. Hence, it does not matter which mode of processing, or rather translocation, is used. The same oligonucleotides were inserted into the ompA gene of plasmid pRD87; a plasmid which leads to overproduction of the protein and to massive accumulation of both the mature protein and the precursor. In the OmpA signal sequence encoded by pRD87-P2 the hydrophobic core is extended from 12 to 20 residues. This peptide was also rapidly removed. Therefore, regardless of whether the hydrophobic core contains 12, 16, or 20 lipophilic residues, not only does the signal sequence always function correctly to mediate export, but in each case, the cleavage site is always accessible to the signal peptidase.     

Determination of the gene sequence and the molecular structure of the enterococcal peptide antibiotic AS-48.

The structural gene of the enterococcal peptide antibiotic AS-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the AS-48 Glu-C peptides, designated V8-5, was composed of a 12-amino-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+59 to W+70]) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative AS-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the antibiotic peptide was a 70-residue cyclic molecule. We propose that the AS-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves off a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tail-head linkage).     

US2, a human cytomegalovirus-encoded type I membrane protein, contains a non-cleavable amino-terminal signal peptide.

The human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH(2) terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH(2)-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable -3,-1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequent N-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein.     

An artificial hydrophobic sequence functions as either an anchor or a signal sequence at only one of two positions within the Escherichia coli outer membrane protein OmpA

The 325-residue outer membrane protein, OmpA, of Escherichia coli, like most other outer membrane proteins with known sequence, contains no long stretch of hydrophobic amino acids. A synthetic oligonucleotide, encoding the sequence Leu-Ala-Leu-Val, was inserted four times between the codons for amino acid residues 153 and 154 and two, three, or four times between the codons for residues 228 and 229, resulting in the OmpA153-4, OmpA-228-2, -3, and -4 proteins, respectively. In the first case, the lipophilic sequence anchored the protein in the plasma membrane. In the OmpA228 proteins, 16 but not 12 or 8 lipophilic residues most likely also acted as an anchor. By removal of the NH2-terminal signal peptide, the function of the insert in OmpA153-4 was converted to that of a signal-anchor sequence. Possibly due to differences in amino acid sequences surrounding the insert, no signal function was observed with the insert in OmpA228-4. Production of the OmpA153-4 protein, with or without the NH2-terminal signal sequence, resulted in a block of export of chromosomally encoded OmpA. Clearly, long hydrophobic regions are not permitted within proteins destined for the bacterial outer membrane, and these proteins, therefore, have had to evolve another mechanism of membrane assembly.     

A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene

The DNA sequence of the ompC gene which encodes one of the outer membrane porins has been determined. The gene appears to encode a secretory precursor of OmpC protein consisting of a total of 367 amino acid residues with a signal peptide of 21 amino acid residues at its NH2-terminal end. The 5' end noncoding region including the promoter of the ompC gene is extremely [A-T]-rich, and the codon usage in the ompC gene is unusual as are those in genes for other abundant outer membrane proteins. The promoter sequence of the ompC gene was compared with that of the ompF gene, both of which are controlled by the osmoregulatory operon, ompB. The deduced amino acid sequence of the OmpC protein showed extensive homology with that of the other porins (OmpF and PhoE proteins). The homology in the primary amino acid sequences, as well as the coding DNA sequences among the porins, indicates that the structural genes for the three porins evolved from a common ancestral gene. Comparison of the amino acid sequences among the OmpC, OmpF, and PhoE porins will be discussed with regard to structure and function.     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

Nine amino acid residues at the NH2-terminal of lipoprotein are sufficient for its modification, processing, and localization in the outer membrane of Escherichia coli

We have examined the structural requirements at the NH2-terminal region of the lipoprotein for its assembly in the outer membrane of Escherichia coli by constructing a hybrid protein consisting of an NH2-terminal portion of the prolipoprotein, consisting of the signal peptide and 9 amino acid residues of lipoprotein, and the entire beta-lactamase sequence. The results from this study indicate that the hybrid protein is modified with glyceride, processed in a globomycin-sensitive step, and localized in the outer membrane. The translocation of the hybrid protein across the cytoplasmic membrane occurs post-translationally and is inhibited by carbonyl cyanide m-chlorophenylhydrazone. Our results, therefore, indicate that the signal peptide and 9 amino acid residues of prolipoprotein are sufficient for its modification, processing, and localization in the outer membrane.