Fluoranthene degradation in Pseudomonas alcaligenes PA-10

Pseudomonas alcaligenes strain PA-10 degrades thefour-ring polycyclic aromatic hydrocarbon fluoranthene, co-metabolically. HPLC analysisof the growth medium identified four intermediates, 9-fluorenone-1-carboxylicacid; 9-hydroxy-1-fluorene carboxylic acid; 9-fluorenone and 9-fluorenol, formedduring fluoranthene degradation. Pre-exposure of PA-10 to 9-fluorenone-1-carboxylic acidand 9-hydroxy-1-fluorene-carboxylic acid resulted inincreases in fluoranthene removal, while pre-exposure to9-fluorenone and 9-fluorenol resulted in a decrease influoranthene degradation. The rate of indole transformation was similarly affected by pre-exposureto these metabolic intermediates, indicating a link between fluoranthenedegradation and indigo formation in this strain.     

Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis.

Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anti-cancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.     

Cloning of the uvrD gene of E. coli and identification of the product

The uvrD gene has been cloned from Escherichia coli chromosomal DNA into phage lambda, cosmid, and low-copy-number plasmid vectors. Comparison of the proteins encoded by the cloned fragments with those encoded by fragments in which the uvrD gene is inactivated by transposon insertion or by deletion shows that the uvrD gene product is a protein of Mr = 73000.     

Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase.

The ada gene of Escherichia coli encodes O6-methylguanine-DNA methyltransferase, which serves as a positive regulator of the adaptive response to alkylating agents and as a DNA repair enzyme. The gene which can make an ada-deficient strain of E. coli resistant to the cell-killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the gene potentially encoded a protein with a calculated molecular weight of 39,217. Since the nucleotide sequence of the cloned gene shows 70% similarity to the ada gene of E. coli and there is an ada box-like sequence (5'-GAATTAAAACGCA-3') in the promoter region, we tentatively refer to this cloned DNA as the adaST gene. The gene encodes Cys-68 and Cys-320, which are potential acceptor sites for the methyl group from the damaged DNA. The multicopy plasmid carrying the adaST gene significantly reduced the frequency of mutation induced by MNNG both in E. coli and in S. typhimurium. The AdaST protein encoded by the plasmid increased expression of the ada'-lacZ chromosome fusion about 5-fold when an E. coli strain carrying both the fusion operon and the plasmid was exposed to a low concentration of MNNG, whereas the E. coli Ada protein encoded by a low-copy-number plasmid increased it about 40-fold under the same conditions. The low ability of AdaST to function as a positive regulator could account for the apparent lack of an adaptive response to alkylation damage in S. typhimurium.     

Effect of surfactants on fluoranthene degradation by <Emphasis Type="Italic">Pseudomonas alcaligenes</Emphasis> PA-10

Two surfactants, Tween 80 and JBR, were investigated for their effect on fluoranthene degradation by a Pseudomonad. Both surfactants enhanced fluoranthene degradation by Pseudomonas alcaligenes PA-10 in shake flask culture. This bacterium was capable of utilising the synthetic surfactant and the biosurfactant as growth substrates and the critical micelle concentration of neither compound inhibited bacterial growth. The biosurfactant JBR significantly increased polycyclic aromatic hydrocarbon (PAH) desorption from soil. Inoculation of fluoranthene-contaminated soil microcosms with P. alcaligenes PA-10 resulted in the removal of significant amounts (45 ± 5%) of the PAH after 28 days compared to an uninoculated control. Addition of the biosurfactant increased the initial rate of fluoranthene degradation in the inoculated microcosm. The presence of a lower molecular weight PAH, phenanthrene, had a similar effect on the rate of fluoranthene removal.     

The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA.

The SPS4 gene of Saccharomyces cerevisiae, a sporulation-specific gene identified previously in a differential hybridization screen of a genomic yeast DNA library, has been characterized further. The protein encoded by this gene was inferred from its nucleotide sequence to be 38,600 daltons with an isoelectric pH of 8.2. Consistent with this, two-dimensional polyacrylamide gel electrophoresis of the in vitro translation products of RNA purified by hybridization with the cloned SPS4 DNA indicated that the SPS4 gene product is a 39-kilodalton, basic protein. This protein was found to be identical in size and charge to a major, sporulation-specific protein identified in a two-dimensional polyacrylamide gel electrophoretic comparison of the in vitro translation products of total RNA from sporulating MATa/MAT alpha cells and asporogenous MAT alpha/MAT alpha cells. A MATa/MAT alpha strain homozygous for a partial deletion of the SPS4 gene appeared, however, to be unaffected in its ability to form viable ascospores.     

Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III

Escherichia coli contains two type 1 topoisomerases, topoisomerase I and III. Although topoisomerase III can be purified as a potent decatenase, its role in DNA metabolism is unclear. In order to address this issue, the gene encoding topoisomerase III from E. coli has been molecularly cloned and its DNA sequence determined. The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa. The first 20 amino acids of this open reading frame are identical to those of topoisomerase III as determined by amino-terminal gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T7 transient expression system, results in the accumulation of a 74-kDa polypeptide. Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts. We propose that this gene be designated topB.     

Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens

A newly isolated gene from Agrobacterium tumefaciens (A. tumefaciens), which encoded a decaprenyl diphosphate synthase, was cloned in Escherichia coli (E. coli), and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1077 bp capable of encoding a 358-amino-acid protein with a calculated isoelectric point of pH 5.16 and a molecular mass of 38 960 Da. The primary structure of the enzyme shared significant homology with prenyl diphosphate synthases from various sources. The deduced amino acid sequence included oligopeptide DDxxD aspartate-rich domains conserved in the majority of prenyl diphosphate synthases. High levels of the active enzyme were expressed in the soluble fraction and were readily purified to homogeneity by Ni-NTA chromatography. E. coli JM109 harboring the dps gene produced ubiquinone-10 in addition to endogenous ubiquinone-8, while E. coli JM109 harboring the dps gene mutated on the DDxxD domain lost the ability to produce ubiquinone-10, which suggests that the A. tumefaciens dps gene is functionally expressed in E. coli and that it encodes a decaprenyl diphosphate synthase.     

Overproduction and purification of the uncI gene product of the ATP synthase of Escherichia coli

The uncI gene, the first gene of the unc operon, has been cloned into an expression vector carrying the lambda PRPL promoters in tandem orientation and the gene cI857 coding for the thermolabile repressor. Linkage of the uncI gene to an efficient ribosome binding site (the translational initiation region of the uncE gene) resulted in 10-20-fold increased gene expression. The i protein has been extracted from overproducing cells using chloroform/methanol and purified to homogeneity by ion exchange chromatography. Analyzing the products of the uncI gene encoded by different plasmids, we provide evidence that, in contrast to the previously reported data (Walker, J. E., Saraste, M., and Gay, N. J. (1984) Biochim. Biophys. Acta 768, 164-200), the chromosome-encoded i protein contains the N-terminal sequence Ser-Val-Ser-Leu-Val-Ser-Arg and has a molecular weight of 13,504.     

Gene organization of the first catabolic operon of TOL plasmid pWW53: production of indigo by the xylA gene product.

The entire operon coding for the enzymes responsible for conversion of toluenes to benzoates has been cloned from TOL plasmid pWW53 and the position of the genes accurately located. The coding region was 7.4 kilobase pairs (kbp) long, and the gene order was operator-promoter region (OP1)-a small open reading frame-xylC (1.6 kbp)-xylA (2.9 kbp)-xylB (1.8 kbp). Within the coding region there was considerable homology with the isofunctional region of the archetypal TOL plasmid pWW0. A central region of 2.9 kbp complemented an xylA (for xylene oxygenase) mutant of Pseudomonas putida mt-2 and was also capable of conferring the ability to convert indole to indigo on strains of Escherichia coli and P. putida. This reaction has been reported previously only for dioxygenases involved in aromatic catabolism but not for monooxygenases. It is proposed that the region encodes xylene oxygenase activity capable of direct monohydroxylation of indole to 3-hydroxyindole (oxindole), which then spontaneously dimerizes to form indigo.     

Cloning, sequencing, and characterization of a gene cluster involved in EDTA degradation from the bacterium BNC1

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.     

Cloning the uridine phosphorylase (udp) gene of Escherichia coli and its expression in recombinant plasmids

On the basis of Escherichia coli DNA and vectors pBR322, pUC19, hybrid plasmids restoring Udp+ phenotype in the E. coli deletion (delta udp) mutant have been obtained. The udp gene is carried by a 8 kb PstI fragment (on the pUD2) and by a smaller 2.87 kb PstI-SalGI fragment from the PstI fragment (pUD7). The uridine phosphorylase level was 30 times higher in the cells containing hybrid plasmid as compared to the strain with chromosomal location of the udp gene. On the other hand, the measurements of uridine phosphorylase activity in the cytR- and cya- background indicate that expression of the cloned udp gene escapes partially negative control of the CytR repressor and positive control of cAMP--CRP complex. These data suggest that the 2.87 kb PstI--SalGI-fragment contains the intact udp gene which is transcribed from its own promoter. Increase in the activity of beta-galactosidase encoded by udp-lacZ fusion has been observed in the presence of pUD2 or pUD7, which was suggested to be the consequence of titration of CytR repressor molecules in the operator region of the cloned udp.     

Purification of an indole alkaloid biosynthetic enzyme, strictosidine synthase, from a recombinant strain of Escherichia coli.

The gene for the indole alkaloid biosynthetic enzyme, strictosidine synthase, of Catharanthus roseus has been cloned into an inducible Escherichia coli expression vector using an expression cassette polymerase chain reaction technique. Induction of the gene resulted in overexpression of the enzyme which accumulated mainly as insoluble inclusion bodies. Denaturation and refolding of the insoluble protein resulted in the ability to purify up to 6 mg of active enzyme from a single liter of cell culture. The recombinant enzyme has good activity (approximately 30 nkat/mg).