Cloning of the Pseudomonas aeruginosa gene encoding CDP-diglyceride synthetase

The CDP-diglyceride synthetase (CDS)-encoding gene (cds) from Pseudomonas aeruginosa PAO1 was cloned and sequenced. The gene possessed an open reading frame of 813 bp capable of encoding a putative polypeptide of 271 amino acids (aa) (28 699 Da). The deduced aa sequence of CDS revealed a 67% similarity (45% identity) to Escherichia coli CDS.     

Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain.

The structural gene hap for the extracellular hemagglutinin/protease (HA/protease) of Vibrio cholerae was cloned and sequenced. The cloned DNA fragment contained a 1,827-bp open reading frame potentially encoding a 609-amino-acid polypeptide. The deduced protein contains a putative signal sequence followed by a large propeptide. The extracellular HA/protease consists of 414 amino acids with a computed molecular weight of 46,700. In the absence of protease inhibitors, this is processed to the 32-kDa form which is usually isolated. The deduced amino acid sequence of the mature HA/protease showed 61.5% identity with the Pseudomonas aeruginosa elastase. The cloned hap gene was inactivated and introduced into the chromosome of V. cholerae by recombination to construct the HA/protease-negative strain HAP-1. The cloned fragment containing the hap gene was then shown to complement the mutant strain.     

Homology between the photoreactivation genes of Saccharomyces cerevisiae and Escherichia coli

A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced. The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa). The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data. The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E. coli gene. Despite the difference in G + C content there is a 35% homology between the deduced aa sequences. This homology suggests that both genes have originated from a common ancestral gene.     

Revised nucleotide sequence of the gltP gene, which encodes the proton-glutamate-aspartate transport protein of Escherichia coli K-12.

The gene encoding the proton-glutamate carrier (GltP) of Escherichia coli K-12 was sequenced, and the primary structure of the protein was analyzed. The nucleotide sequence was found to differ in several aspects from the previously published sequence (B. Wallace, Y. Yang, J. Hong, and D. Lum, J. Bacteriol. 172:3214-3220, 1990). The corrected open reading frame encodes a protein of 437 (instead of 395) amino acids. Hydropathy analysis predicts 12 membrane-spanning alpha-helical regions. The complementary strand does contain an open reading frame possibly encoding a highly hydrophilic polypeptide of 272 amino acids.     

Cloning and expression in Escherichia coli of the gene encoding an extracellular deoxyribonuclease (DNase) from Aeromonas hydrophila.

The gene encoding an extracellular DNase from Aeromonas hydrophila CHC-1 has been cloned and sequenced. Following expression of the dns in Escherichia coli, it was revealed that some of the cloned enzyme was present in the cell-free extracellular supernatant fluid, and there was no cell lysis and concurrent release of cytoplasmic or periplasmic proteins. Therefore, results suggest that E. coli cells were capable of secreting the DNase extracellularly, albeit very inefficiently. The dns is transcribed from its own promoter in E. coli, and expressed as a 25-kDa product, as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of the culture supernatant preparations followed by a DNA-hydrolysis assay. Nucleotide sequence analysis predicted a single open reading frame of 690 bp encoding a 230-amino acid (aa) polypeptide, with a potential 20-aa signal peptide located at the N terminus of the predicted protein. The deduced aa sequence of the entire protein is highly homologous with that of the DNase of Vibrio cholerae.     

Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis

The nucleotide sequence of the Synechococcus sp. PCC7942 glgB gene has been determined. The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206. Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp. glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa). In contrast, the N-terminal portions shared little homology. The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase. This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp. is different from that described for E. coli. A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp. glgB gene. The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.     

Mutational analysis of the phage T4 morphogenetic 31 gene, whose product interacts with the Escherichia coli GroEL protein

The phage T4 morphogenetic gene 31 has been sequenced. Its deduced gene product is a polypeptide of 111 aa, with a predicted Mr of 12064 and a pI of 4.88. The proof that the assigned open reading frame (ORF) encodes Gp31 rests on the sequencing of two known gene 31 amber mutations, amN54 and NG71, demonstrating that these mutations result in translational termination within the assigned ORF. Furthermore, the sequencing of four different T4 epsilon mutations, isolated on the basis of allowing the phage to propagate on Escherichia coli groEL- hosts, showed that they are either missense mutations or 3-bp deletions in the gene 31 reading frame. The sequencing of neighboring DNA revealed the presence of five other ORFs, one of which overlaps gene 31 substantially, but in the opposite orientation.     

Investigation of the regulation of the Escherichia coli btuB gene using operon fusions

Operon fusions were isolated between Mu dX (lac CmR ApR) and btuB, the gene encoding the multivalent vitamin B12 outer membrane receptor. Using these fusions, vitamin B12-mediated repression of btuB in Escherichia coli was demonstrated. Mutations in metH, metE and ompR as well as exogenous methionine, membrane pertubants, high osmolar conditions and temperature had no major effect on the expression of the btuB gene.     

Cloning and sequencing of the locus encoding the large and small subunit genes of the periplasmic [NiFe]hydrogenase from Desulfovibrio fructosovorans

The genetic locus encoding the periplasmic [NiFe]hydrogenase (Hyd) from Desulfovibrio fructosovorans was cloned and sequenced. The genes of this two-subunit enzyme have an operon organization in which the 0.94-kb gene encoding the small subunit precedes the 1.69-kb gene encoding the large subunit. A Shine-Dalgarno sequence is centered at -9 bp from the ATG of both subunits. The possible presence of another open reading frame downstream from the large-subunit-encoding gene is considered. The N-terminal sequence of the large 61-kDa subunit deduced from the nucleotide sequence is in perfect agreement with the results of the amino acid (aa) sequence determined by Edman degradation. A 50-aa leader peptide precedes the small 28-kDa subunit. The aa sequence of the enzyme shows nearly 65% homology with the [NiFe]Hyd aa sequence of Desulfovibrio gigas. Comparisons with a large range of Hyds from various bacterial species indicate the presence of highly conserved Cys residues, the implications of which are discussed from the point of view of nickel atom and cluster accommodation.     

Bacillus subtilis DNA polymerase III: complete sequence, overexpression, and characterization of the polC gene

Genomic DNA encompassing polC, the structural gene specifying Bacillus subtilis DNA polymerase III (PolIII), was sequenced and found to contain a 4311-bp open reading frame (ORF) encoding a 162.4-kDa polypeptide of 1437 amino acids (aa). The ORF was engineered into an Escherichia coli expression plasmid under the control of the coliphage lambda repressor. Derepression of E. coli transformants carrying the recombinant vector resulted in the high-level synthesis of a recombinant DNA polymerase indistinguishable from native PolIII. N-terminal aa sequence analysis of the recombinant polymerase unequivocally identified the 4311-bp ORF as that of polC. Comparative aa sequence analysis indicated significant homology of the B. subtilis enzyme with the catalytic alpha subunit of the E. coli PolIII and, with the exception of an exonuclease domain, little homology with other DNA polymerases. The respective sequences of the mutant polC alleles, dnaF and ts-6, were identified, and the expression of specifically truncated forms of polC was exploited to assess the dependence of polymerase activity on the structure of the enzyme's C terminus.     

A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium.

We isolated an Escherichia coli methionine auxotroph that displays a growth phenotype similar to that of known metF mutants but has elevated levels of 5,10-methylenetetrahydrofolate reductase, the metF gene product. Transduction analysis indicates that the mutant carries normal metE, metH, and metF genes; the phenotype is due to a single mutation, eliminating the possibility that the strain is a metE metH double mutant; and the new mutation is linked to the metE gene by P1 transduction. Plasmids carrying the Salmonella typhimurium metE gene and flanking regions complement the mutation, even when the plasmid-borne metE gene is inactivated. Enzyme assays show that the mutation results in a dramatic decrease in metE gene expression, a moderate decrease in metH gene expression, and a disruption of the metH-mediated vitamin B12 repression of the metE and metF genes. Our evidence suggests that the methionine auxotrophy caused by the new mutation is a result of insufficient production of both the vitamin B12-independent (metE) and vitamin B12-dependent (metH) transmethylase enzymes that are necessary for the synthesis of methionine from homocysteine. We propose that this mutation defines a positive regulatory gene, designated metR, whose product acts in trans to activate the metE and metH genes.     

Cloning and sequencing of an epoxide hydrolase gene from Rhodosporidium paludigenum.

Epoxide hydrolase (EH) activity was recently described in yeasts and highly selective hydrolysis of epoxides was observed during whole cell biotransformations. To expand the available molecular data regarding yeast EHs, the EH encoding gene from Rhodosporidium paludigenum (CBS 6565) was isolated, cloned and sequenced. The genomic EH sequence revealed a 1600 bp sequence interrupted by six introns. cDNA sequence analysis revealed an open reading frame of 1236 bp with a deduced polypeptide length of 411 amino acids. The deduced amino acid sequence revealed a relative high degree of sequence homology compared to the amino acid sequence of the EH from Rhodotorula glutinis.     

Expression and Export of Pseudomonas putida NTU-8 Creatinase by Escherichia coli Using the Chitinase Signal Sequence of Aeromonas hydrophila

The gene for the creatinase from Pseudomonas putida NTU-8 was sequenced and revealed an open reading frame (ORF) of 1209 base pairs encoding a polypeptide of 403 amino acids with a calculated molecular weight (M(r)) of 45,691. The deduced amino acid sequence is very similar to that of the creatinase of Pseudomonas putida and Flavobacterium sp. An overproduction system for the chitinase signal peptide--creatinase hybrid gene was constructed by using the pQE-51 expression vector in E. coli JM109. The amount of this fusion enzyme was about 50% exported into the periplasmic space of E. coli.     

Nucleotide sequence of the gene presumably encoding the adsorption protein of filamentous phage φLf

A 1170-nucleotide fragment of φLf DNA was sequenced. This fragment contains an open reading frame, ORF367, encoding a protein of 367 amino acids (aa) (36710 Da). ORF367 is located downstream from the gene encoding the major coat protein (gVIIIp) and a Rho-independent termination signal. Sequence analysis revealed that the gene product has a Gly-rich domain (70 aa) at the center and a hydrophobic region (26 aa) at the C terminus. These structural features suggest that ORF367 may encode the adsorption protein of φLf.