Substrate-binding lipoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942 involved in the transport of nitrate and nitrite

Of the four genes (nrtABCD) required for active transport of nitrate in the cyanobacterium Synechococcus sp. strain PCC 7942, nrtBCD encode membrane components of an ATP-binding cassette transporter involved in the transport of nitrite as well as of nitrate, whereas nrtA encodes a 45-kDa cytoplasmic membrane protein, the biochemical function of which remains unclear. Characterization of the nrtA deletional mutants showed that the 45-kDa protein is essential for the functioning of the nitrate/nitrite transporter. A truncated NrtA protein lacking the N-terminal 81 amino acids, expressed in Escherichia coli cells as a histidine-tagged soluble protein, was shown to bind nitrate and nitrite with high affinity (Kd = 0.3 microM). Immunoblotting analysis using the antibody against the 45-kDa protein revealed a 48-kDa precursor of the protein, which accumulated in the cyanobacterial cells treated with globomycin, an antibiotic that specifically inhibits cleavage of the signal peptide of lipoprotein precursors. These findings indicated that the nrtA gene product is a nitrate- and nitrite-binding lipoprotein. The N-terminal sequences of putative cyanobacterial substrate-binding proteins suggested that lipoprotein modification of substrate-binding proteins of ATP-binding cassette transporters is common in cyanobacteria.     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

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.     

Cloning and characterization of Sel-1l, a murine homolog of the C. elegans sel-1 gene

The Notch signaling pathway regulates specification and proliferation in a variety of cell lineages in invertebrates and vertebrates. We have cloned a murine homolog of SEL-1, a key negative regulator of the Notch pathway in Caenorhabditis elegans. Murine SEL-1L (mSEL-1L) protein exhibits a high degree of similarity to SEL-1, including a signal peptide and the C-terminal region required for SEL-1 function in C. elegans. This mammalian homolog of sel-1 is widely expressed in adult mouse and human tissues, with particularly high levels in the pancreas. RNA in situ analysis of developing mouse embryos indicates that mSEL-1L is moderately expressed throughout the neural tube and dorsal root ganglia, with particularly high levels in the floor plate of the neural tube beginning at E10.5 and increasing at E11.5. Expression is high at E14.5 and E17.5 in the acini of the pancreas, and moderate in the epithelial cells of the gut villi. We localized the SEL-1L protein to the cytosol, possibly in intracellular vesicles, in a beta-islet-derived tumor cell line (RinM).     

Cloning and disruption of caPLB1, a phospholipase B gene involved in the pathogenicity of Candida albicans

The Candida albicans PLB1 gene was cloned using a polymerase chain reaction-based approach relying on degenerate oligonucleotide primers designed according to the amino acid sequences of two peptide fragments obtained from a purified candidal enzyme displaying phospholipase activity (Mirbod, F., Banno, Y., Ghannoum, M. A., Ibrahim, A. S., Nakashima, S., Yasuo, K., Cole, G. T., and Nozawa, Y. (1995) Biochim. Biophys. Acta 1257, 181-188). Sequence analysis of a 6.7-kilobase pair EcoRI-ClaI genomic clone revealed a single open reading frame of 1818 base pairs that predicts for a pre-protein of 605 residues. Comparison of the putative candidal phospholipase with those of other proteins in data base revealed significant homology to known fungal phospholipase Bs from Saccharomyces cerevisiae (45%), Penicillium notatum (42%), Torulaspora delbrueckii (48%), and Schizosaccharomyces pombe (38%). Thus, we have cloned the gene encoding a C. albicans phospholipase B homolog. This gene, designated caPLB1, was mapped to chromosome 6. Disruption experiments revealed that the caplb1 null mutant is viable and displays no obvious phenotype. However, the virulence of strains deleted for caPLB1, as assessed in a murine model for hematogenously disseminated candidiasis, was significantly attenuated compared with the isogenic wild-type parental strain. Although deletion of caPLB1 did not produce any detectable effects on candidal adherence to human endothelial or epithelial cells, the ability of the caplb1 null mutant to penetrate host cells was dramatically reduced. Thus, phospholipase B may well contribute to the pathogenicity of C. albicans by abetting the fungus in damaging and traversing host cell membranes, processes which likely increase the rapidity of disseminated infection.     

Cloning of gene responsible for tributyltin chloride (TBTC1) resistance in TBTC1-resistant marine bacterium, Alteromonas sp. M-1

A genetic library of tributyltin chloride (TBTC1)-resistant marine bacterium, Alteromonas sp. M-1, was constructed using plasmid vector pUC 19. Three positive clones were obtained from E. coli JM 109 transformed with the plasmids by the method of replica plating to LB medium containing 1 mM TBTC1. These clones could grow in LB liquid medium containing 100 microM TBTC1. Plasmids harbouring genes of Alteromonas sp. M-1 were designated pTBT1, pTBT2 and pTBT3 which contain 1.8 kb Hind III-fragment, 4.8 kb Pst I-fragment and 7.8 kb Pst I-fragment, respectively. Nucleotide sequence of the shortest fragment, 1.8 kb Hind III-fragment was determined, revealing an open reading frame (ORF) was contained in the fragment. The ORF was 324 bp (108 amino acids). The 48.5% of the amino acids encoded was hydrophobic, suggesting that the product relating to TBTC1 resistance might be membrane related protein. Homology search in amino acids alignment indicated that the product has homology with transport proteins.     

Identification and characterization of a novel competence gene, comC, required for DNA binding and uptake in Acinetobacter sp. strain BD413

A gene (comC) essential for natural transformation was identified in Acinetobacter sp. strain BD413. ComC has a typical leader sequence and is similar to different type IV pilus assembly factors. A comC mutant (T308) is not able to bind or take up DNA but exhibits a piliation phenotype indistinguishable from the transformation wild type as revealed by electron microscopy.     

A glutathione S-transferase with activity towards cis-1, 2-dichloroepoxyethane is involved in isoprene utilization by Rhodococcus sp. strain AD45

Rhodococcus sp. strain AD45 was isolated from an enrichment culture on isoprene (2-methyl-1,3-butadiene). Isoprene-grown cells of strain AD45 oxidized isoprene to 3,4-epoxy-3-methyl-1-butene, cis-1, 2-dichloroethene to cis-1,2-dichloroepoxyethane, and trans-1, 2-dichloroethene to trans-1,2-dichloroepoxyethane. Isoprene-grown cells also degraded cis-1,2-dichloroepoxyethane and trans-1, 2-dichloroepoxyethane. All organic chlorine was liberated as chloride during degradation of cis-1,2-dichloroepoxyethane. A glutathione (GSH)-dependent activity towards 3, 4-epoxy-3-methyl-1-butene, epoxypropane, cis-1,2-dichloroepoxyethane, and trans-1,2-dichloroepoxyethane was detected in cell extracts of cultures grown on isoprene and 3,4-epoxy-3-methyl-1-butene. The epoxide-degrading activity of strain AD45 was irreversibly lost upon incubation of cells with 1,2-epoxyhexane. A conjugate of GSH and 1, 2-epoxyhexane was detected in cell extracts of cells exposed to 1, 2-epoxyhexane, indicating that GSH is the physiological cofactor of the epoxide-transforming activity. The results indicate that a GSH S-transferase is involved in the metabolism of isoprene and that the enzyme can detoxify reactive epoxides produced by monooxygenation of chlorinated ethenes.     

Sucrose-phosphate synthase from Synechocystis sp. strain PCC 6803: identification of the spsA gene and characterization of the enzyme expressed in Escherichia coli

The first identification and characterization of a prokaryotic gene (spsA) encoding sucrose-phosphate synthase (SPS) is reported for Synechocystis sp. strain PCC 6803, a unicellular non-nitrogen-fixing cyanobacterium. Comparisons of the deduced amino acid sequence and some relevant biochemical properties of the enzyme with those of plant SPSs revealed important differences in the N-terminal and UDP-glucose binding site regions, substrate specificities, molecular masses, subunit compositions, and regulatory properties.     

Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp. strain 7, and overproduction of the enzyme by coexpression of tRNA for arginine rare codon

The gene encoding an extremely stable inorganic pyrophosphatase from Sulfolobus sp. strain 7, a thermoacidophilic archaeon, was cloned and sequenced. An open reading frame consisted of 516 base pairs coding for a protein of 172-amino acid residues. The deduced sequence was supported by partial amino acid sequence analyses. All the catalytically important residues were conserved. A unique 17-base-pair sequence motif was found to be repeated four times in frame in the gene, encoding a cluster of acidic amino acids essential for the function. Although the codon usage of the gene was quite different from that of Escherichia coli, the gene was effectively expressed in E. coli. Coexpression of tRNA(Arg), cognate for the rare codon AGA in E. coli, however, further improved the production of the enzyme, which occupied more than 85% of the soluble proteins obtained after removal of heat denatured E. coli proteins.     

Photosynthetic electron transport involved in PxcA-dependent proton extrusion in Synechocystis sp. Strain PCC6803: effect of pxcA inactivation on CO2, HCO3-, and NO3- uptake

The product of pxcA (formerly known as cotA) is involved in light-induced Na+-dependent proton extrusion. In the presence of 2, 5-dimethyl-p-benzoquinone, net proton extrusion by Synechocystis sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO2 fixation by glyceraldehyde in a cytochrome c oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO2 fixation is inhibited. At pH 8.0, NO3- uptake activity was very low in the pxcA mutant at low [Na+] (approximately 100 microM). At pH 6.5, the pxcA strain did not take up CO2 or NO3- at low [Na+] and showed very low CO2 uptake activity even at 15 mM Na+. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO2, HCO3-, and NO3- is discussed.     

Riboflavin biosynthesis in Saccharomyces cerevisiae. Cloning, characterization, and expression of the RIB5 gene encoding riboflavin synthase

Saccharomyces cerevisiae has a monofunctional riboflavin synthase that catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine. We have isolated the gene encoding this enzyme from a yeast genomic library by functional complementation of a mutant, rib5-10, lacking riboflavin synthase activity. Deletion of the chromosomal copy of RIB5 led to riboflavin auxotrophy and loss of enzyme activity. Intragenic complementation between point and deletion mutant alleles suggested that the encoded protein (Rib5p) assembles into a multimeric complex and predicted the existence of a discrete functional domain located at the N terminus. Nucleotide sequencing revealed a 714-base pair open reading frame encoding a 25-kDa protein. Rib5p was purified to apparent homogeneity by a simple procedure. The specific activity of the enzyme was enriched 8500-fold. The N-terminal sequence of the purified enzyme was identical to the sequence predicted from the nucleotide sequence of the RIB5 gene. Initial structural characterization of riboflavin synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a trimer of identical 25-kDa subunits. The derived amino acid sequence of RIB5 shows extensive homology to the sequences of the alpha subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes. In addition, the sequence also shows internal homology between the N-terminal and the C-terminal halves of the protein. Taken together, these results suggest that the Rib5p subunit contains two structurally related (substrate-binding) but catalytically different (acceptor and donator) domains.     

Sequence of the gene encoding a highly thermostable neutral proteinase from Bacillus sp. strain EA1: expression in Escherichia coli and characterisation

Ammonium assimilation was studied by feeding [15N]ammonium to actively growing mycelium of Agaricus bisporus. Products of ammonium assimilation were analysed using 15N-NMR. Participation of glutamine synthetase, glutamate synthase and NADP-dependent glutamate dehydrogenase was determined by inhibiting glutamine synthetase with phosphinothricin and glutamate synthase with azaserine. Our results clearly indicate that, under the conditions used, ammonium assimilation is mainly catalysed by the enzymes of the glutamine synthetase/glutamate synthase pathway. No indications were found for participation of NADP-dependent glutamate dehydrogenase. Furthermore, 15N-labelling shows that transamination of glutamate with pyruvate to yield alanine is a major route in nitrogen metabolism. Another major route is the formation of N-acetylglucosamine. Compared to the formation of N-acetylglucosamine there was only a limited formation of arginine.     

Identification and characterization of human cDNAs specific to BCS1, PET112, SCO1, COX15, and COX11, five genes involved in the formation and function of the mitochondrial respiratory chain

With a growing number of economic evaluations being conducted alongside trials, there is a need to address, and if necessary, investigate the generalizability of results derived from trials. This paper explores the generalizability of costs from a trial investigating the frequency of breast cancer screening in the United Kingdom.     

Detection of two loci involved in (1-->3)-beta-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene

A 28-item questionnaire was returned by 291 psychiatrists who had completed training between 1962 and 1992. There were positive correlations between the amount of couple and family therapy training (CFTT) they received and the following: the extent to which graduate psychiatrists practice CFT; their involvement as supervisors, teachers, teaching program directors, or researchers; the extent to which they seek continuing education in CFT; their positive attitude toward CFT; and the extent to which they feel that their attitude to and interest in CFT has had a positive effect on the milieu in which they practice and on their personal lives.     

Mutation of a gene encoding a putative glycoprotease leads to reduced salt tolerance, altered pigmentation, and cyanophycin accumulation in the cyanobacterium Synechocystis sp. strain PCC 6803

The salt-sensitive mutant 549 of the cyanobacterium Synechocystis sp. strain PCC 6803 was genetically and physiologically characterized. The mutated site and corresponding wild-type site were cloned and partially sequenced. The genetic analysis revealed that during the mutation about 1.8 kb was deleted from the chromosome of mutant 549. This deletion affected four open reading frames: a gcp gene homolog, the psaFJ genes, and an unknown gene. After construction of mutants with single mutations, only the gcp mutant showed a reduction in salt tolerance comparable to that of the initial mutant, indicating that the deletion of this gene was responsible for the salt sensitivity and that the other genes were of minor importance. Besides the reduced salt tolerance, a remarkable change in pigmentation was observed that became more pronounced in salt-stressed cells. The phycobilipigment content decreased, and that of carotenoids increased. Investigations of changes in the ultrastructure revealed an increase in the amount of characteristic inclusion bodies containing the high-molecular-weight nitrogen storage polymer cyanophycin (polyaspartate and arginine). The salt-induced accumulation of cyanophycin was confirmed by chemical estimations. The putative glycoprotease encoded by the gcp gene might be responsible for the degradation of cyanophycin in Synechocystis. Mutation of this gene leads to nitrogen starvation of the cells, accompanied by characteristic changes in pigmentation, ultrastructure, and salt tolerance level.