Biochemical and genetic characterization of 2-carboxybenzaldehyde dehydrogenase, an enzyme involved in phenanthrene degradation by Nocardioides sp. strain KP7

2-Carboxybenzaldehyde dehydrogenase from the phenanthrene-degrading bacterium Nocardioides sp. strain KP7 was purified and characterized. The purified enzyme had a molecular mass of 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 205 kDa by gel filtration chromatography. Thus, the homotetramer of the 53-kDa subunit constituted an active enzyme. The apparent Km and kcat values of this enzyme for 2-carboxybenzaldehyde were 100 microM and 39 s(-1), respectively, and those for NAD+ were 83 microM and 32 s(-1), respectively. The structural gene for this enzyme was cloned and sequenced. The length of the gene was 1,455 bp. The nucleotide sequence of the 10,279 bp of DNA around the gene for 2-carboxybenzaldehyde dehydrogenase was also determined, and seven open reading frames were found in this DNA region. These were the genes for 1-hydroxy-2-naphthoate dioxygenase (phdI) and trans-2'-carboxybenzalpyruvate aldolase (phdJ), orf1, the gene for 2-carboxybenzaldehyde dehydrogenase (phdK), orf2/orf3, and orf4. The amino acid sequence of the orf1 product was similar to that of the aromatic hydrocarbon transporter gene (pcaK) in Pseudomonas putida PRS2000. The amino acid sequence of the orf4 product revealed a similarity to cytochrome P-450 proteins. The region between phdK and orf4 encoded orf2 and orf3 on different strands. The amino acid sequences of the orf2 and orf3 products exhibited no significant similarity to the reported sequences in protein databases.     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactis

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.     

Regulation and physiological role of the DAS1 gene, encoding dihydroxyacetone synthase, in the methylotrophic yeast Candida boidinii

The physiological role of dihydroxyacetone synthase (DHAS) in Candida boidinii was evaluated at the molecular level. The DAS1 gene, encoding DHAS, was cloned from the host genome, and regulation of its expression by various carbon and nitrogen sources was analyzed. Western and Northern analyses revealed that DAS1 expression was regulated mainly at the mRNA level. The regulatory pattern of DHAS was similar to that of alcohol oxidase but distinct from that of two other enzymes in the formaldehyde dissimilation pathway, glutathione-dependent formaldehyde dehydrogenase and formate dehydrogenase. The DAS1 gene was disrupted in one step in the host genome (das1Delta strain), and the growth of the das1Delta strain in various carbon and nitrogen sources was compared with that of the wild-type strain. The das1Delta strain had completely lost the ability to grow on methanol, while the strain with a disruption of the formate dehydrogenase gene could survive (Y. Sakai et al., J. Bacteriol. 179:4480-4485, 1997). These and other experiments (e.g., those to determine the expression of the gene and the growth ability of the das1Delta strain on media containing methylamine or choline as a nitrogen source) suggested that DAS1 is involved in assimilation rather than dissimilation or detoxification of formaldehyde in the cells.     

Enhanced rate of intramolecular electron transfer in an engineered purple CuA azurin

The recent expression of an azurin mutant where the blue type 1 copper site is replaced by the purple CuA site of Paracoccus denitrificans cytochrome c oxidase has yielded an optimal system for examining the unique electron mediation properties of the binuclear CuA center, because both type 1 and CuA centers are placed in the same location in the protein while all other structural elements remain the same. Long-range electron transfer is induced between the disulfide radical anion, produced pulse radiolytically, and the oxidized binuclear CuA center in the purple azurin mutant. The rate constant of this intramolecular process, kET = 650 +/- 60 s-1 at 298 K and pH 5.1, is almost 3-fold faster than for the same process in the wild-type single blue copper azurin from Pseudomonas aeruginosa (250 +/- 20 s-1), in spite of a smaller driving force (0.69 eV for purple CuA azurin vs. 0.76 eV for blue copper azurin). The reorganization energy of the CuA center is calculated to be 0.4 eV, which is only 50% of that found for the wild-type azurin. These results represent a direct comparison of electron transfer properties of the blue and purple CuA sites in the same protein framework and provide support for the notion that the binuclear purple CuA center is a more efficient electron transfer agent than the blue single copper center because reactivity of the former involves a lower reorganization energy.     

Isolation and characterization of the recA gene of Xanthomonas campestris pv. campestris

A 1.8-kb NsiI-StuI fragment containing the recA gene of Xanthomonas campestris pv. campestris was cloned by a PCR-based approach and complementation of Escherichia coli HB 101. Sequence analysis of this fragment revealed an ORF (orf343) of 1,032 bp able to encode a protein of 343 amino acids with a calculated MW of 37,021 Da, a size similar to the values detected by in vitro system and Western blotting. It showed 69.6% identity to the E. coli RecA in amino acid sequence. Amino acid residues of the E coli RecA associated with functional activities are conserved in this Xc17 RecA. The recA mutant, L1, constructed by gene replacement, was sensitive to ultraviolet irradiation and methyl methanesulfonate, and deficient in homologous recombination.     

Functional expression of two glucose transporter isoforms from the parasitic protozoan Leishmania enriettii

The parasitic protozoan Leishmania enriettii contains a family of tandemly repeated genes, designated Pro-1, that encode proteins with significant sequence similarity to mammalian facilitative glucose transporters. Pro-1 mRNAs are expressed almost exclusively in the promastigote or insect stage of the parasite life cycle. The Pro-1 tandem repeat encodes two isoforms of the putative transporter, iso-1 and iso-2, which have identical predicted amino acid sequences except for their NH2-terminal hydrophilic domains. We have now expressed both iso-1 and iso-2 by microinjecting their RNAs into Xenopus oocytes and assaying these oocytes for transport of various radiolabeled ligands. Both iso-1 and iso-2 transport [3H]2-deoxy-D-glucose, confirming that each protein is a bona fide glucose transporter. Each isoform also transports fructose and, to a much lesser degree, mannose. Compounds which inhibit 2-deoxy-D-glucose transport in L. enriettii promastigotes also inhibit transport in the microinjected oocytes expressing each isoform, indicating that the substrate specificities and pharmacological properties of each isoform are similar to those measured for 2-deoxy-D-glucose transport in intact parasites. The Km for transport of 2-deoxyglucose in oocytes expressing iso-1 is similar to that for oocytes expressing iso-2. These results reveal that both transporter isoforms have closely related functional properties and that the difference in their structures may serve some other purpose such as differential subcellular localization.     

Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis

The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.     

A flavoprotein functional as NADH oxidase from Amphibacillus xylanus Ep01: purification and characterization of the enzyme and structural analysis of its gene

Amphibacillus xylanus Ep01, a facultative anaerobe we recently isolated, shows rapid aerobic growth even though it lacks a respiratory pathway. Thus, the oxidative consumption of NADH, produced during glycolysis and pyruvate oxidation, should be especially important for maintenance of intracellular redox balance in this bacterium. We purified a flavoprotein functional as NADH oxidase from aerobically growing A. xylanus Ep01. The A. xylanus enzyme is a homotetramer composed of a subunit (M(r) 56,000) containing 1 mol of flavin adenine dinucleotide. This enzyme catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH as the preferred electron donor and exhibits no activity with NADPH. The flavoprotein gene of A. xylanus Ep01 was cloned by using a specific antibody. The amino acid sequence of 509 residues, deduced from the nucleotide sequence, showed 51.2 and 72.5% identities to the amino acid sequences of alkyl hydroperoxide reductase from Salmonella typhimurium and NADH dehydrogenase from alkalophilic Bacillus sp. strain YN-1, respectively. Bacillus spp. have a respiratory chain and grow well under aerobic conditions. In contrast, Amphibacillus spp., having no respiratory chain, grow equally well under both aerobic and anaerobic conditions, which distinguishes these two genera. Salmonella spp., which are gram-negative bacteria, are taxonomically distant from gram-positive bacteria such as Bacillus spp. and Amphibacillus spp. The above findings, however, suggest that the flavoprotein functional as NADH oxidase, the alkyl hydroperoxide reductase, and the NADH dehydrogenase diverged recently, with only small changes leading to their functional differences.     

N-Acetyltransferase 2 (NAT2) and Glutathione S-Transferase μ (GSTM1) in Bladder-cancer Patients in a Highly Industrialized Area

The gene encoding the 33 kDa piroplasm surface protein of Theileria sergenti isolated in Korea was cloned and the nucleotide sequence was determined by dideoxy chain termination method. The cloned gene corresponds to 869 bp encoding an open reading frame 283 amino acids. Comparison of the sequence between Korean and Japanese isolates showed 99.4% homology rate in the nucleotide sequence and 98.9% homology rate in the amino acid sequence.     

The relationship between the trimethylation of lysine 77 and cytochrome c metabolism in Saccharomyces cerevisiae

1. Site directed mutations were constructed in the yeast iso-1-cytochrome c gene adjacent to the lysine 77 (methylation site) codon. 2. These mutant genes were then cloned and transformed into the S. cerevisiae strain B-6642 which contains a deficiency in the iso-1-cytochrome c gene. 3. The resulting transformants were screened for cytochrome c production using gel electrophoresis. 4. Amino acid analysis of the mutated cytochromes c demonstrated varying levels of trimethyllysine formation, depending on the nature of the site directed mutation. 5. The resulting transformants were then used as tools in order to investigate the relationship between trimethyllysine formation and various aspects of cytochrome c metabolism including protein stability and heme conjugation.     

Amino-terminal sequencing of sheep CD1 antigens and identification of a sheep CD1D gene

The anti-CD1 monoclonal antibodies IAH-CC14 and SBU-T6 were used to immunopurify CD1 antigens from sheep thymocytes. The amino-terminal sequence of IAH-CC14 yielded 13 amino acids, and 29 amino acids were obtained from the SBU-T6 antigen. The sequence of the IAH-CC14 antigen was 100% identical to the predicted sequence of the sheep CD1B clone, SCD1B-42. The 29 amino acid sequence of the SBU-T6 antigen did not match identically with the derived amino acid sequence of any of the previously reported sheep CD1 genes but had closest similarity to the derived sequence of human CD1E. Degenerate polymerase chain reaction primers based on this sequence identified a group 2 sheep CD1 gene. The predicted amino acid sequence of this gene shows that it is not identical to the SBU-T6 peptide, indicating that a different, CD1D-like gene was cloned.     

Primary structure of the novel bacterial rhodopsin from extremely halophilic archaeon Haloarcula japonica strain TR-1

A novel bacterial rhodopsin was identified in Haloarcula japonica strain TR-1. The gene encoding the bacterial rhodopsin was cloned and sequenced. The structural gene consisted of an open reading frame of 750 nucleotides encoding 250 amino acids. The deduced amino acid sequence of the Ha. japonica bacterial rhodopsin showed the highest homology to those of cruxrhodopsins.     

Contribution of the 65-kilodalton protein encoded by the cloned gene cry19A to the mosquitocidal activity of Bacillus thuringiensis subsp. jegathesan

Two new crystal protein genes, cry19A and orf2, isolated from Bacillus thuringiensis subsp. jegathesan were cloned and characterized. The cry19A gene encodes a 74.7-kDa protein, and the orf2 gene encodes a 60-kDa protein. Cry19A contains the five conserved blocks present in most B. thuringiensis delta-endotoxins. The ORF2 amino acid sequence is similar to that of the carboxy terminus of Cry4 proteins. The cry 19A gene was expressed independently or in combination with orf2 in a crystal-negative B. thuringiensis host. The proteins accumulated as inclusions. Purified inclusions containing either Cry19A alone or Cry19A and ORF2 together were toxic to Anopheles stephensi and Culex pipiens mosquito larvae. They were more toxic to C. pipiens than to A. stephensi. However, inclusions containing Cry19A and ORF2 together were more toxic than inclusions of Cry19A alone but less toxic than the wild-type inclusions of B. thuringiensis subsp. jegathesan.     

Cloning of the gene encoding the Actinobacillus actinomycetemcomitans serotype b OmpA-like outer membrane protein

The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of Actinobacillus actinomycetemcomitans ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure. DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa. The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4. This monoclonal antibody reacted specifically with Omp29 of A. actinomycetemcomitans (serotype b), but not with any Omp of Escherichia coli, including OmpA. This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated. The N-terminal amino acid sequence of the protein expressed in E. coli perfectly matched those deduced from the purified Omp29 of strain Y4. The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported omp34 gene described for A. actinomycetemcomitans serotype c (NCTC 9710). Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as omp34.     

Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase

The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-2-47 in lambda EMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport system of L. lactis. The inferred amino acid sequence of PepO showed that the lactococcal endopeptidase has a marked similarity to the mammalian neutral endopeptidase EC 3.4.24.11 (enkephalinase), whereas no obvious sequence similarity with any bacterial enzyme was found. By means of gene disruption, a pepO-negative mutant was constructed. Growth and acid production of the mutant strain in milk were not affected, indicating that the endopeptidase is not essential for growth of L. lactis in milk.     

Cloning and partial characterization of the Cr32 gene of Cowdria ruminantium

Cowdria organisms were purified by density gradient centrifugation. The DNA was used to construct expression libraries. The immunodominant Cr32 protein was purified and its N-terminal amino acid sequence was determined. The expression libraries were screened with Cr32-specific monoclonal antibodies, but did not yield Cr32-positive clones. Therefore a part of the Cr32-gene was amplified using primers derived from the N-terminal and an internal amino acid sequence. This DNA was used as a probe to detect the genomic DNA fragment encoding the Cr32 protein. This fragment was cloned, using genomic DNA of the Senegal strain of Cowdria ruminantium. A part of the gene comprising two third of its total length has been expressed in vector pGEX2T. This expression product is recognized by Cr32-specific monoclonal antibodies.