The amino acid sequence of the blue copper protein of Alcaligenes faecalis

The complete amino acid sequence of a blue copper protein from Alcaligenes faecalis S-6 has been determined. This protein is clearly homologous to pseudoazurins in Achromobacter cycloclastes and Pseudomonas AM1, more distantly related to plant plastocyanins, and markedly different from the azurin of Pseudomonas aeruginosa. Yet all of these proteins bind copper, and analogous ligands appear to be involved.     

Cytoplasmic expression of the Achromobacter xylosoxidans blue copper nitrite reductase in Escherichia coli and characterisation of the recombinant protein

The gene of the Achromobacter xylosoxidans (DSM 2402) blue copper-containing nitrite reductase was amplified using the polymerase chain reaction. DNA sequence analysis reveals that the amino acid sequence is identical to those of the GIFU1051 and the NCIMB11015 A. xylosoxidans nitrite reductases. The gene encoding the mature coding region for DSM 2402 nitrite reductase was cloned into a pET-vector, overexpressed in the cytoplasm of Escherichia coli BL21(DE3), and the expressed holoprotein was purified to apparent homogeneity by cation-exchange chromatography. The recombinant blue copper-containing nitrite reductase was obtained in high yields of 70mgL(-1) of culture. The specific catalytic activity as well as the electronic absorption and electron paramagnetic resonance spectra agree with corresponding data for the native protein. Mass spectroscopic analysis of the recombinant nitrite reductase gave a molecular weight of 36659.1Da for the apo-protein monomer, in agreement with the expected molecular mass based on the amino acid sequence.     

Cloning and characterization of a blue fluorescent protein from Vibrio vulnificus

The blue fluorescent protein (BFPVV) gene bfpvv from Vibrio vulnificus CKM-1 was cloned and sequenced. The transformants exhibited blue fluorescence when irradiated by UV source. Nucleotide sequence analysis predicted an ORF of 717 bp encoding a 239-amino-acid polypeptide with a calculated molecular mass of 25.8 kDa. The nucleotide sequence of the bfpvv gene and its deduced amino acid sequence showed significant homology to those of the short-chain dehydrogenase/reductase (SDR) family proteins from various organisms. Some functionally important residues in SDR were strictly conserved in BFPVV, such as an active-site Tyr145, a catalytic site Lys149, and a common GlyXXXGlyXGly pattern in the N-terminal part of the molecule. By changing three amino acid residues, Tyr145, Lys149, and Gly9 to Phe, Ile, and Val, respectively, it was found that the G9V mutant did not generate blue fluorescence, while mutants Y145F and K149I have 126 and 68.5% fluorescence compared with the wild-type BFPVV.     

Molecular cloning, sequencing, and expression in Escherichia coli of the gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A

The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited by p-chloromercuribenzoic acid, EDTA, o-phenanthroline, HgCl(2), and CuSO(4). The EDTA-inactivated enzyme was completely restored by the addition of Zn(2+) or Co(2+). In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme.     

The gene sequence and some properties of protein H. A novel IgG-binding protein

The gene for protein H, a novel bacterial cell wall protein with specific affinity for human IgG Fc, was cloned from a group A Streptococcus and expressed in Escherichia coli. Recombinant E. coli cells produced two forms of a human IgG Fc-binding protein, one with an apparent Mr of 42 kDa in a periplasmic fraction and the other with an apparent Mr of 45 kDa in a mixed fraction of cytoplasms and membranes. Both 42-kDa and 45-kDa protein preparations similarly bound to human IgG1 to IgG4, human IgG Fc, and rabbit IgG, but not to IgG of mouse, rat, bovine, sheep, goat, and human IgA, IgD, IgE, and IgM. The complete nucleotide sequence of the cloned 1.8-kb DNA fragment was determined. An open reading frame encoded a hypothetical protein of 376 amino acid residues (Mr = 42,498). The N-terminal amino acid sequence, consisting of 41 residues, which was removed post-translationally had typical characteristics of Gram-positive bacterial signal peptides. Thus, the mature form of protein H was suggested to consist of 335 residues (Mr = 38,162). There were 3 repeated sequences consisting of 42 residues that were highly homologous to those of protein Arp, an IgA-binding streptococcal cell wall protein, and streptococcal M6 and M24 proteins. The C-terminal amino acid sequence consisting of 93 residues, directly following the repeated sequences, was also highly homologous to that of M6 and M24 proteins. No sequence homology was found between protein H and protein A or protein G, two other IgG-binding bacterial cell wall proteins.     

Xylose isomerase from Escherichia coli. Characterization of the protein and the structural gene

The gene that codes for xylose isomerase in Escherichia coli has been cloned by complementation of a xylose isomerase-negative E. coli mutant. The structural gene is 1320 nucleotides in length and codes for a protein of 440 amino acids. An additional 209 nucleotides 5' and 82 nucleotides 3' to the structural gene were also sequenced. To verify that the cloned gene encodes E. coli xylose isomerase, the enzyme was purified to homogeneity and the sequence of the first 25 amino acid residues was determined by a semimicromanual Edman procedure. These results establish that the NH2-terminal methionine of xylose isomerase is specified by an ATG which is 7 nucleotides downstream from a Shine-Dalgarno sequence.     

Identification and sequence determination of the host factor gene for bacteriophage Q beta.

The host factor (HF-I) required for phage Q beta RNA-directed synthesis of complementary minus-strand RNA was purified to homogeneity from phage-infected Escherichia coli cells. The hfq gene encoding HF-I was cloned using synthetic probes designed based on the partial amino acid sequence of HF-I, and mapped at 94.8 min on the E. coli chromosome downstream of the miaA gene involved in 2-methylthio-N6-(isopentyl)-adenosine (ms2i6A) tRNA modification. Sequence determination of the cloned hfq gene indicated that HF-I is a small protein of Mr 11,166 consisting of 102 amino acid residues.     

Nucleotide sequence analysis of the gene specifying the bifunctional 6''-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities.

The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.     

Gene for an immunoglobulin-binding protein from a group G streptococcus.

The gene (spg) for an immunoglobulin G (IgG)-binding protein from a Streptococcus clinical isolate of Lancefield group G was cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and 5'-flanking sequences was determined. The DNA sequence includes an open reading frame which encodes a hypothetical protein of 448 amino acid residues (Mr = 47,595). The 5' end of this open reading frame encodes a sequence resembling a typical secretion signal sequence, and the remainder of the encoded protein has features reminiscent of staphylococcal protein A and of streptococcal M6 protein, including repeated sequences and a similar C-terminal structure. Aside from this C-terminal structure, the encoded protein has little direct amino acid sequence homology to either protein A or M6 protein. In E. coli, the cloned gene directs the synthesis of a protein which binds to immunoglobulins, including rabbit immunoglobulin, goat IgG, and human IgG3(lambda). Its binding properties are similar to those of the protein G described by Bj?rck and Kronvall (L. Bj?rck and G. Kronvall, J. Immunol. 133:969-974, 1984), a type III Fc receptor from a group G streptococcus.     

Nucleotide sequence of a novel kanamycin resistance gene, aphA-7, from Campylobacter jejuni and comparison to other kanamycin phosphotransferase genes

A novel kanamycin phosphotransferase gene, aphA-7, was cloned from a 14-kb plasmid obtained from a strain of Campylobacter jejuni and the nucleotide sequence of the gene was determined. The presumed open reading frame of the aphA-7 structural gene was 753 bp in length and encoded a protein of 251 amino acids with a calculated weight of 29,691 Da. A 29-kDa protein was demonstrated in Escherichia coli maxicells containing the cloned aphA-7 gene. A ribosomal binding site corresponding to 5 of 8 bases of the 3' end of the E. coli 16S rRNA was 8 bp upstream of the start codon. Sequences corresponding to the -35 and -10 regions of the consensus promoter sequences of E. coli were upstream of the presumed initiation codon of the gene. The DNA sequence was most closely related to the aphA-3 gene from Streptococcus faecalis, showing 55.4% sequence similarity. There was 45.6% identity at the amino acid level between the aphA-3 and the aphA-7 proteins. Of the three conserved regions noted previously in phosphotransferase genes, the aphA-7 amino acid sequence was identical to the six conserved amino acids in motif 3, but differed in one of the five conserved amino acids in motif 1 (if gaps are permitted) and 3 of the 10 conserved residues in motif 2. The 32.8% G + C ratio in the open reading frame of the aphA-7 kanamycin resistance gene, which is similar to that of the C. jejuni chromosome, suggests that the aphA-7 may be indigenous to Campylobacters.     

A putative gene of tobacco chloroplast coding for ribosomal protein similar to E. coli ribosomal protein S19.

A partial sequence of a cloned 3.2 Md BamHI fragment from tobacco chloroplast DNA revealed the occurrence of a putative gene for ribosomal protein. The putative gene is located on the left margin of the large single-copy region in the chloroplast DNA. The coding region contains 276 bp (92 codons). The amino acid sequence deduced from the DNA sequence shows 55% homology with that of E. coli S19 (91 amino acid residues).     

Cloning, expression and sequencing the molybdenum-pterin binding protein (mop) gene of Clostridium pasteurianum in Escherichia coli

mop is the structural gene for the molybdenum-pterin binding protein, which is the major molybdenum binding protein in Clostridium pastuerianum. The mop gene was detected by immunoscreening genomic libraries of C. pastuerianum and identified by determining the nucleotide sequence of the cloned insert of clostridial DNA. The deduced amino acid sequence of an open reading frame proved to be identical to the first twelve residues of purified Mop. The DNA sequence flanking the mop gene contains promoter-like consensus sequences which are probably responsible for the expression of Mop in Escherichia coli. The deduced amino acid composition shows that the protein is hydrophobic, lacks aromatic and cysteine residues and has a calculated molecular weight of 7,038. The N-terminal amino acid sequence of Mop has sequence homology with DNA binding proteins. The pattern and type of residues in the N-terminal region suggest it forms the helix-turn-helix structure observed in DNA binding proteins. We propose that Mop may be a regulatory protein binding the anabolic source of molybdenum.     

Structural similarity among Escherichia coli FtsW and RodA proteins and Bacillus subtilis SpoVE protein, which function in cell division, cell elongation, and spore formation, respectively.

The Escherichia coli cell division gene ftsW (2 min) was cloned and sequenced. It encodes a hydrophobic protein(s) with 414 and/or 384 amino acid residues. The deduced amino acid sequence and the hydropathy profile of the protein showed high homology with those of the E. coli RodA protein functioning in determination of the cell shape and the Bacillus subtilis SpoVE protein functioning in spore formation. Probably similar functional membrane proteins are involved in these three cell cycle process.     

Amino acid sequence of pheromone-inducible surface protein in Enterococcus faecalis, that is encoded on the conjugative plasmid pPD1

The major pheromone-inducible protein, PD78, believed to contribute to bacterial conjugation, was purified from Enterococcus (formerly Streptococcus) faecalis cells containing the plasmid pPD1. A cloned EcoRI-BglII 3.6-kbp fragment of the plasmid pAM351(pPdl::Tn916) contained an open reading frame corresponding to 467 amino acid residues representing PD78. In a central region of the deduced protein, there is a repeated sequence of X-X-Pro that is repeated 15 times. This is analogous to the Gln-Gln-Pro repeat in the C-terminal region of TraD product encoded on the R100 plasmid in Escherichia coli.     

Molecular analysis of a Leptospira borgpetersenii gene encoding an endoflagellar subunit protein

A flagellin gene, flaB, from Leptospira borgpetersenii (formerly L. interrogans) serovar hardjo was cloned and expressed in Escherichia coli. Expression of the 32 kDa FlaB protein was dependent upon the lacZ promoter from pUC18. Nucleotide sequence data showed an open reading frame encoding 283 amino acid residues, corresponding to a protein of molecular mass 31.3 kDa. The G + C content of the flaB gene was 54.7 mol%. Comparison of the deduced FlaB amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum FlaB proteins.     

Cloning and characterization of the gene for a protein thiol-disulfide oxidoreductase in Bacillus brevis.

The gene (bdb) for protein thiol-disulfide oxidoreductase cloned from Bacillus brevis was found to encode a polypeptide consisting of 117 amino acid residues with a signal peptide of 27 residues. Bdb contains a well-conserved motif, Cys-X-X-Cys, which functions as the active center of disulfide oxidoreductases such as DsbA, protein disulfide isomerase, and thioredoxin. The deduced amino acid sequence showed significant homology with those of several bacterial thioredoxins. The bdb gene complemented the Escherichia coli dsbA mutation, restoring motility by means of flagellar and alkaline phosphatase activity. The Bdb protein overproduced in B. brevis was enzymatically active in both reduction and oxidization of disulfide bonds in vitro. Immunoblotting indicated that Bdb could function at the periphery of the cell.     

Identification and characterization of Thermus thermophilus HB8 RuvA protein, the subunit of the RuvAB protein complex that promotes branch migration of Holliday junctions

The Escherichia coli ruvA and ruvB genes constitute an SOS-regulated operon. The products of these genes form a protein complex that promotes branch migration of the Holliday junction, an intermediate of homologous recombination. RuvA protein binds specifically to the Holliday junction and recruits RuvB protein to the junction. RuvB is an ATP-driven motor protein involved in branch migration. We previously cloned the ruvB gene of the thermophilic bacterium Thermus thermophilus HB8 (Tth) and found that, in contrast to the operon structure in most mesothermic bacteria, the ruvA gene is absent from the vicinity of ruvB. In this work, we cloned the ruvA gene from T. thermophilus HB8 and analyzed its nucleotide sequence. Tth RuvA is a protein of 20,414 Da consisting of 191 amino acid residues, and is 37% identical in amino acid sequence to E. coli RuvA. Tth ruvA complemented the DNA repair defect of E. coli deltaruvA mutants. The purified Tth RuvA protein stimulated Tth RuvB activities, such as hydrolysis of ATP and promotion of branch migration of the Holliday junction, in a manner similar to the RuvA-RuvB interactions observed in E. coli. In addition, Tth RuvA stimulated the E. coli RuvB activities in vitro, which was well consistent with the results of in vivo hetero-complementation experiments.     

Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).