The cpx proteins of Escherichia coli K12. Structure of the cpxA polypeptide as an inner membrane component

Gene cpxA of Escherichia coli K12 encodes the 52,000 Mr CpxA polypeptide. The complete cpxA nucleotide sequence, reported here, predicted that CpxA contains two extended, hydrophobic segments in its amino-terminal half and could therefore be a membrane protein. Using a lac-cpxA operon fusion plasmid to overproduce CpxA and an immunochemical assay to detect the polypeptide, we show that CpxA fractionated with the bacterial inner membrane during differential and isopycnic sedimentation. Moreover, the protein could be solubilized by extraction of crude membranes with non-ionic detergents but not with KCl or NaOH, indicating that Cpx is an intrinsic membrane component. Analysis of TnphoA insertions in cpxA indicated that the region between the hydrophobic segments of CpxA is periplasmic, whereas the region carboxy-terminal to the second such segment is cytoplasmic. Based on these structural data, we propose that CpxA functions as a trans-membrane sensory protein. The DNA sequence data also indicate that cpxA is the 3' gene of an operon.     

Nuclear-encoded chloroplast ribosomal protein L27 of Nicotiana tabacum: cDNA sequence and analysis of mRNA and genes.

A tobacco (Nicotiana tabacum cv. Petite Havana) leaf cDNA library was constructed in the expression vector lambda gt11. Immunological and nucleic acid hybridization screening yielded several cDNAs encoding an M(r) 19,641 precursor to an M(r) 14,420 mature protein which is homologous to Escherichia coli ribosomal protein L27. One cDNA (L27-1; 882 nucleotides long) contains 104 bp of 5'-noncoding sequence, 51 codons for a transit peptide, 128 codons for the predicted mature L27 polypeptide, and 241 bp of 3'-noncoding sequence, including the poly(A)29 tail. A beta-galactosidase-L27 fusion protein was bound to nitrocellulose filters, expressed, and used as an affinity matrix to purify monospecific antibody to L27 protein from an antiserum of rabbits immunized with 50S chloroplast ribosomal proteins. Using this monospecific antibody, protein L27 was identified among HPLC-purified tobacco chloroplast ribosome 50S subunit proteins. The predicted amino terminus of the mature L27 protein was confirmed by partial sequencing of the HPLC-purified L27 protein. The mature L27 protein has 66%, 61%, 56%, and 48% amino acid sequence identity with the L27-type ribosomal proteins of Bacillus subtilis, E. coli, Bacillus stearo-thermophilus, and yeast mitochondria (MRP7), respectively, in the homologous overlapping regions. The transit peptide of tobacco chloroplast ribosomal protein L27 has 41% amino acid sequence similarity with the MRP7 mitochondrial targeting sequence. Tobacco chloroplast L27 protein also has a 40 amino acid long carboxyl-terminal extension (compared to its bacterial counterparts) which is similar to the corresponding portion of yeast MRP7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity.

The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain. Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells. This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E.     

Identification of the Escherichia coli K-12 cpxA locus as a single gene: construction and analysis of biologically-active cpxA gene fusions

In the accompanying communication we showed that a 2 kb EcoRI-BamHI restriction fragment from the pfkA-rha interval of the Escherichia coli K-12 chromosome fully complemented a chromosomal cpxA mutation when the fragment was cloned in pBR325. The same fragment cloned in pBR322 lacked any complementing activity. We show here that minicells containing the pBR325 derivative (pRA310) synthesized a 33 kDa polypeptide, designated phi 33, that was not synthesized in minicells containing the pBR322 derivative (pRA311) or either of the parent plasmids. Synthesis of the phi 33 polypeptide did not occur in minicells containing Tn5 insertion alleles of pRA310 that inactivated its cpxA complementing activity. These insertions mapped within the vector cat (chloramphenicol acetyltransferase gene) sequence immediately adjacent to the EcoRI site of pRA310 and within the 700-800 bp of the cloned EcoRI-BamHI fragment immediately adjacent to the EcoRI site. Tn5 insertions located within the fragment but closer to the BamHI terminus affected neither the cpxA complementing activity of pRA310 nor synthesis of the phi 33 polypeptide in minicells. Plasmid pRA311 could be converted to a plasmid with cpxA complementing activity by cloning into its EcoRI site a restriction fragment containing a hybrid trp-lacUV5 promoter, the lacZ ribosome binding site, and the first eight lacZ codons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates

Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of the herpes simplex virus type 1 virion protein encoded by the UL35 open reading frame.

The UL35 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) has been predicted from DNA sequence analysis to encode a small polypeptide with a molecular weight of 12,095. We have investigated the protein product of the UL35 ORF by using a trpE-UL35 gene fusion to produce a corresponding fusion protein in Escherichia coli. The TrpE-UL35 chimeric protein was subsequently isolated and used as a source of immunogen for the production of rabbit polyclonal antiserum directed against the UL35 gene product. The TrpE-UL35 antiserum was found to recognize a 12-kDa protein which was specifically present in HSV-1-infected cells. By utilizing the TrpE-UL35 antiserum, the kinetics of synthesis of the UL35 gene product was examined, and these studies indicate that UL35 is expressed as a gamma 2 (true late) gene. The 12-kDa protein recognized by the TrpE-UL35 antiserum was associated with purified HSV-1 virions and type A and B capsids, suggesting that the UL35 ORF may encode the 12-kDa capsid protein variably designated p12, NC7, or VP26. To confirm this assignment, immunoprecipitation and immunoblotting studies were performed to demonstrate that the TrpE-UL35 antiserum reacts with the same polypeptide as an antiserum directed against the purified p12 capsid protein (anti-NC7) (G.H. Cohen, M. Ponce de Leon, H. Diggelmann, W.C. Lawrence, S.K. Vernon, and R.J. Eisenberg, J. Virol. 34:521-531, 1980). Furthermore, the anti-NC7 serum was also found to react with the TrpE-UL35 chimeric protein isolated from E. coli, providing additional evidence that the UL35 gene encodes p12. On the basis of these studies, we conclude that UL35 represents a true late gene which encodes the 12-kDa capsid protein of HSV-1.     

Tight linkage of glnA and a putative regulatory gene in Rhizobium leguminosarum.

Rhizobium leguminosarum, biovar viceae, strain RCC1001 contains two glutamine synthetase activities, GSI and GSII. We report here the identification of glnA, the structural gene for GSI. A 2 kb fragment of DNA was shown to complement the Gln- phenotype of Klebsiella pneumoniae glnA mutant strains. DNA sequence analysis revealed an open reading frame (ORF) of 469 codons specifying a polypeptide of 52,040 daltons. Its deduced amino acid sequence was found to be highly homologous to other glutamine synthetase sequences. This ORF was expressed in Escherichia coli minicells and the corresponding polypeptide reacted with an antiserum raised against GSI. Upstream of glnA we found an ORF of 111 codons (ORF111) preceded by the consensus sequence for an ntrA-dependent promoter. Minicells experiments showed a protein band, with a molecular weight in good agreement with that (10,469) deduced from the nucleotide sequence. On the basis of homology studies we discuss the possibility that the product of ORF111 is equivalent to the PII protein of E. coli and plays a similar role in regulation of nitrogen metabolism.     

Molecular cloning of the cyanobacterial adenylate cyclase gene from the filamentous cyanobacterium Anabaena cylindrica.

Molecular cloning of the structural gene for adenylate cyclase (cya) of the cyanobacterium Anabaena cylindrica was carried out by complementation of an Escherichia coli strain defective in the cya gene. The cya-defective strain produced significant amounts of cyclic AMP when it was transformed with the cya gene isolated from A. cylindrica. This gene encodes a polypeptide consisting of 502 amino acid residues (molecular weight, 55,300). The deduced primary protein structure showed that the carboxyl-terminal region of the adenylate cyclase of A. cylindrica shows strong structural similarity to the conserved regions of the adenylate cyclases of various eukaryotes. No similarity was found between the amino acid sequences of the cya gene of A. cylindrica and that of E. coli. A hydropathy plot suggests that this protein has two hydrophobic regions, a transmembrane span and a signal peptide. An antiserum specific to this adenylate cyclase was prepared by immunizing a rabbit with a glutathione S-transferase-adenylate cyclase fusion protein expressed in E. coli. This antiserum recognized a 55-kDa protein in Anabaena cell lysates. Subcellular fractionation analysis showed that A. cylindrica adenylate cyclase localized in the thylakoid membrane.     

The signal for the termination of protein synthesis in procaryotes.

The sequences around the stop codons of 862 Escherichia coli genes have been analysed to identify any additional features which contribute to the signal for the termination of protein synthesis. Highly significant deviations from the expected nucleotide distribution were observed, both before and after the stop codon. Immediately prior to UAA stop codons in E. coli there is a preference for codons of the form NAR (any base, adenine, purine), and in particular those that code for glutamine or the basic amino acids. In contrast, codons for threonine or branched nonpolar amino acids were under-represented. Uridine was over-represented in the nucleotide position immediately following all three stop codons, whereas adenine and cytosine were under-represented. This pattern is accentuated in highly expressed genes, but is not as marked in either lowly expressed genes or those that terminate in UAG, the codon specifically recognised by polypeptide chain release factor-1. These observations suggest that for the efficient termination of protein synthesis in E. coli, the 'stop signal' may be a tetranucleotide, rather than simply a tri-nucleotide codon, and that polypeptide chain release factor-2 recognises this extended signal. The sequence following stop codons was analysed in genes from several other procaryotes and bacteriophages. Salmonella typhimurium, Bacillus subtilis, bacteriophages and the methanogenic archaebacteria showed a similar bias to E. coli.     

The molecular cloning of the gene encoding the Escherichia coli 75-kDa helicase and the determination of its nucleotide sequence and gentic map position

A previously unreported DNA unwinding enzyme, referred to as the 75-kDa helicase, was recently purified from Escherichia coli cell extracts and biochemically characterized (Wood, E. R., and Matson, S. W. (1987) J. Biol. Chem. 262, 15269-15276). In order to initiate the genetic analysis of the 75-kDa helicase, the gene encoding this enzyme was cloned. DNA sequencing confirmed the identity of the gene since the predicted amino acid sequence of the encoded polypeptide precisely matched the sequence of the first 27 NH2-terminal amino acid residues of the 75-kDa helicase as determined by peptide sequencing. The predicted amino acid sequence of the 75-kDa helicase is similar in several regions to the amino acid sequences of two other E. coli helicases, Rep protein and helicase II. The gene encoding the 75-kDa helicase was mapped to 22 min on the E. coli chromosome. We propose that this newly defined locus be referred to as helD, and, to avoid confusion with other E. coli helicases with a similar molecular size, we propose that the 75-kDa helicase be referred to as helicase IV.     

Unorthodox expression of an enzyme: evidence for an untranslated region within carA from Pseudomonas aeruginosa.

The genes encoding carbamoylphosphate synthetase from Pseudomonas aeruginosa PAO1 were cloned in Escherichia coli. Deletion and transposition analysis determined the locations of carA, encoding the small subunit, and carB, encoding the large subunit, on the chromosomal insert. The nucleotide sequence of carA and the flanking regions was determined. The derived amino acid sequence for the small subunit of carbamoylphosphate synthetase from P. aeruginosa exhibited 68% homology with its counterparts in E. coli and Salmonella typhimurium. The derived sequences in the three organisms were essentially identical in the three polypeptide segments that are conserved in glutamine amidotransferases but showed low homology at the amino- and carboxy-terminal regions. The amino-terminal amino acid sequences were determined for the large and small subunits. The first 15 amino acids of the large subunit were identical to those derived from the carB sequence. However, comparison of the derived sequence for carA with the amino-terminal amino acid sequence for the small subunit suggested that codons 5 to 8 are not translated. The DNA sequence for the region encompassing these four codons was confirmed by direct sequencing of chromosomal DNA after amplification by the polymerase chain reaction. The mRNA sequence was also deduced by in vitro synthesis of cDNA, enzymatic amplification, and sequencing, confirming that 12 nucleotides in the 5' terminal of carA are transcribed but are not translated.     

Cloning and sequencing of the gltX gene, encoding the glutamyl-tRNA synthetase of Rhizobium meliloti A2.

The gltX gene, coding for the glutamyl-tRNA synthetase of Rhizobium meliloti A2, was cloned by using as probe a synthetic oligonucleotide corresponding to the amino acid sequence of a segment of the glutamyl-tRNA synthetase. The codons chosen for this 42-mer were those most frequently used in a set of R. meliloti genes. DNA sequence analysis revealed an open reading frame of 484 codons, encoding a polypeptide of Mr 54,166 containing the amino acid sequences of an NH2-terminal and various internal fragments of the enzyme. Compared with the amino acid sequence of the glutamyl-tRNA synthetase of Escherichia coli, the N-terminal third of the R. meliloti enzyme was strongly conserved (52% identity); the second third was moderately conserved (38% identity) and included a few highly conserved segments, whereas no significant similarity was found in the C-terminal third. These results suggest that the C-terminal part of the protein is probably not involved in the recognition of substrates, a feature shared with other aminoacyl-tRNA synthetases.     

In vivo and in vitro characterization of the secA gene product of Bacillus subtilis.

The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.