Identification and Sequencing of β-Myrcene Catabolism Genes from Pseudomonas sp. Strain M1

The M1 strain, able to grow on β-myrcene as the sole carbon and energy source, was isolated by an enrichment culture and identified as a Pseudomonas sp. One β-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol) as a unique β-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and sequenced the DNA regions flanking the transposon and used these fragments to identify the M1 genomic library clones containing the wild-type copy of the interrupted gene. One of the selected cosmids, containing a 22-kb genomic insert, was able to complement the N22 mutant for growth on β-myrcene. A 5,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named myrA, myrB, myrC, and myrD, which can potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively. myrA, myrB, and myrC are likely organized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. The myrD gene starts 224 nt upstream of myrA and is divergently transcribed. The myrB sequence was found to be completely identical to the one flanking the transposon in the mutant. Therefore, we could ascertain that the transposon had been inserted inside the myrB gene, in complete agreement with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose a pathway for β-myrcene catabolism in Pseudomonas sp. strain M1.     

假单胞菌DLL-E4尿黑酸降解基因的克隆与功能的初步分析

通过接合转移将质粒pSC123上的转座子Tn5随机插入到DLL-E4基因组DNA中,从大约8,000个突变子中筛选到1株在LB培养基上积累红褐色物质的突变株M18,该突变株不能以L-苯丙氨酸(L-Phenylalanine, Phe)为唯一碳源生长。SEFA-PCR扩增转座子侧翼序列发现其与已报道的尿黑酸1,2-双加氧酶基因hmgA的同源性为92%。将hmgA定向克隆至表达载体pET-29a中,转化至Escherichia coli BL21,经IPTG诱导后可表达分子量约为48kD的蛋白;诱导后转化子粗酶液对尿黑酸有很好的降解效果。将hmgA连入自杀性载体pEX19Gm,通过同源重组整合至M18染色体中,使其恢复了DLL-E4利用Phe的能力,证实了HmgA是尿黑酸苯环裂解酶。     

Use of a promoterless lacZ gene insertion to investigate chitinase gene expression in the marine bacterium Pseudoalteromonas sp. strain S9.

Sequence data for genes encoding 16S rRNA indicated that the marine strain previously named Pseudomonas sp. strain S9 would be better identified as a Pseudoalteromonas sp. By use of transposon mutagenesis, a chitinase-negative mutant of S9 with a lacZ reporter gene insertion was isolated. Part of the interrupted gene was cloned and sequenced. The deduced amino acid sequence had homology to sequences of bacterial chitinases. Expression of the chitinase gene promoter was quantified by measuring the lacZ reporter gene product, beta-galactosidase, beta-Galactosidase production was induced 10-fold by N-acetylglucosamine and 3-fold by chitin in minimal medium. Repression of beta-galactosidase synthesis was observed in rich medium either with or without chitin but was not observed in minimal medium containing glucose. The chitinase gene promoter was induced by starvation and higher-than-ambient levels of carbon dioxide but not by cadmium ion, heat or cold shock, or UV exposure.     

Oxidation products of terpenes identified from Dendroctonus and Ips bark beetles

Exposure of adult males and females of Dendroctonus brevicomis and D. frontalis to camphene vapor resulted in oxidation of the terpene to a prominent product, which was identified as 6-hydroxy-camphene (camphenol). Exposure of D. brevicomis adults to myrcene vapor resulted in sex-specific oxidation of the hydrocarbon. A major product in both sexes was identified as 2-methyl-6-methylene-2,7-octadien-1-ol (myrcenol), whereas ipsdienol, a major product in males, was not detected in females. A compound detected in hindguts of feeding males of Ips pini and I. paraconfusus was attributed to the presence of 3-carene in the host (Pinus spp.) and subsequently identified as 1-methyl-5-(α-hydroxy-isopropyl)-cyclohexa-1,3-diene.     

Microbial transformation of geraniol and nerol by Botrytis cinerea

Summary Biotransformation of geraniol 1A and nerol 1B was studied with four strains of Botrytis cinerea and three growth media. Using grape must predominant conversion of 1A/1B to E-3,7-dimethyl-2-octen-1,8-diol 5 and 2Z,6E-3,7-dimethyl-2,6-octadien-1,8-diol 16B was observed. However, with one strain and 1A, E-2-methyl-2-hepten-6-one-1-ol 2B, 7-hydroxy-6-methyl-2-heptanone 3 and p-menth-1-ene-9-ol 7 were identified as major metabolites. As further fungal bioconversion products of 1A/1B were detected: Z-2-methyl-2-hepten-6-one-1-ol 2A, 2E,6Z-, 2E,6E-and 2Z,6Z-3,7-dimethyl-2,6-octadien-1,8-diol 4A/4B/16A, Z-3,7-dimethyl-2-octen-1,8-diol 17, 3,7-dimethyl-1,8-octandiol 6, 2E,6E-8-hydroxy-2,6-dimethyl-2,6-octadienal 8, geranial and neral 9, 18, citronellol 10, Z- and E-2,6-dimethyl-2,7-octadien-1,6-diol 13A/13B, 6-hydroxy-2,6-dimethyl-2,7-octadienal 14 as well as 2,6-dimethyl-7-octen-1,6-diol 15. Using synthetic growth medium again -hydroxylation reactions were observed, but 2-methyl-2-hepten-6-one 11 and 7 were also identified as major bioconversion products of 1A and 1B, respectively. Additionally, 2-methyl-2-hepten-6-ol 12 was detected and, using 1B, also traces of 2Z,6E-8-hydroxy-2,6-dimethyl-2,6-octadienal 19 and two 3,9-epoxy-p-menth-1-ene isomers 20A/20B were found. Addition of small amounts of grape must to the synthetic medium (1:700 to 5:700) influenced both the yields of metabolites and their qualitative and quantitative distribution. Identifications of biotransformation products of 1A/1B were performed by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.2. on-line-mass spectrometry (HRGC-MS) and-Fourier transform infrared spectroscopy (HRGC-FTIR) after extractive sample preparation.     

An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions

We have developed an alternative method to amplify DNA sequences flanking Tn5 transposon insertions. This method relies on the identical sequences of inverted terminal repeats, located at the 5' and 3' ends of Tn5, to determine the location and orientation of a transposon insertion within a restriction endonuclease fragment. From this information, PCR primers can be designed to selectively amplify by inverse PCR the DNA flanking one side of the transposon. This method avoids the problem of amplifying or cloning long sequences flanking Tn5. To demonstrate the applicability of this method, we generated Tn5 transposon mutants of Pseudomonas abietaniphila BKME-9 which no longer grew on dehydroabietic acid (DhA). The flanking sequence of one of the mutant (strain BKME-941) which accumulated 7-oxoDhA, was amplified.     

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Summary Fructose was shown to be phosphorylated by a specific phosphoenolpyruvatc-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose Ell. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.     

Aggregation pheromone of the beetle Ips confusus: Isolation and identification

The terpene alcohols 2-methyl-6-methylene-7-octen-4-ol (1), 2-methyl-6-methylene-2,7-octadien-4-ol (2), cis-verbenol (5), and trans-verbenol (6) and the ketone verbenone (7) have been isolated from frass and volatiles produced by males of Ips confusus boring in piñon pine. Females respond to naturally occurring and synthetic 1 in the laboratory bioassay; the response is intensified by the addition of 2 and 5.     

Studies on anabolic steroids--4. Identification of new urinary metabolites of methenolone acetate (Primobolan) in human by gas chromatography/mass spectrometry

The metabolism of methenolone acetate (17 beta-acetoxy-1-methyl-5 alpha-androst-1-en-3-one), a synthetic anabolic steroid, has been investigated in man. After oral administration of a 50 mg dose of the steroid to two male volunteers, twelve metabolites were detected in urine either in the glucuronide, sulfate or free steroid fractions. Methenolone, the parent steroid was detected in urine until 90 h after administration. Its cumulative urinary excretion accounted for 1.63% of the ingested dose. With the exception of 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, the major biotransformation product of methonolone acetate, metabolites were excreted in urine at lower levels, through minor metabolic routes. Most of methenolone acetate metabolites were isolated from the glucuronic acid fraction, namely methenolone, 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, 3 alpha-hydroxy-1 alpha-methyl-5 alpha-androstan-17-one, 17-epimethenolone, 3 alpha,6 beta-dihydroxy-1-methylen-5 alpha-androstan-17-one, 2 xi-hydroxy-1-methylen-5 alpha-androstan-3,17-dione, 6 beta-hydroxy-1-methyl-5 alpha-androst-1-en-3,17-dione, 16 alpha-hydroxy-1-methyl-5 alpha-androst-1-en-3,17-dione and 3 alpha,16 alpha-dihydroxy-1-methyl-5 alpha-androst-1-en-17-one. Interestingly, the metabolites detected in the sulfate fraction were isomeric steroids bearing a 16 alpha- or a 16 beta-hydroxyl group, whereas 1-methyl-5 alpha-androst-1-en-3,17-dione was the sole metabolite isolated from the free steroid fraction. Steroids identity was assigned on the basis of the mass spectral features of their TMS ether, TMS enol-TMS ether, MO-TMS, and d9-TMS ether derivatives and by comparison with reference and structurally related steroids. The data indicated that methenolone acetate was metabolized into several compounds resulting from oxidation of the 17-hydroxyl group and reduction of A-ring substituents, with or without concomitant hydroxylation at the C6 and C16 positions.     

Inactivation of a glycyl-tRNA synthetase leads to an arrest in plant embryo development.

Embryo formation is the first patterning process during vegetative plant growth. Using transposons as insertional mutagens in Arabidopsis, we identified the mutant edd1 that shows embryo-defective development. The insertion mutation is lethal, arresting embryo growth between the globular and heart stages of embryonic development. The mutant phenotype cosegregates with a transposed Dissociation element. Sequences flanking the transposed element were isolated and used to isolate a full-length cDNA clone representing the wild-type EDD1 gene. Complementation of the mutant through Agrobacterium-mediated gene transfer of an EDD1 wild-type copy as well as loss of the transposon concomitant with phenotypic reversion demonstrated that the transposon had caused the mutation. Based on homology to Escherichia coli, the EDD1 gene is predicted to encode a novel glycyl-tRNA synthetase (GlyRS) that has not been identified previously in higher plants. An N-terminal portion of the plant protein is able to direct a marker protein into pea chloroplasts. Thus, the gene identified by the embryo-defective insertion mutation encodes a GlyRS homolog, probably acting within the plastidic compartment.     

Siderophore production of a periplasmic transport binding protein kinase gene defective mutant of Magnetospirillum magneticum AMB-1

A non-magnetic mutant, NMA61, of the magnetic bacterium Magnetospirillum magneticum AMB-1 was generated by transposon mutagenesis to identify genes involved in magnetosome synthesis. The genomic region of NMA61 interrupted by a Mini-Tn5 transposon was analyzed. The transposon was inserted in an open reading frame (ORF) coding for a periplasmic transport binding protein kinase gene homologue. Three adjacent ORFs and a promoter were identified upstream, indicating that the sequences comprised an operon. Phenotype characterizations showed that the growth inhibition imposed by the exogenous non-assimilable iron chelator nitrilotriacetate was relieved in wild type but not in NMA61, by the addition of the isolated wild type siderophore. Higher concentration of siderophores accumulated in the culture medium of NMA61 than in wild type. These data suggest that the interrupted periplasmic transport binding protein kinase gene homologue is required for siderophore transport into M. magneticum AMB-1.     

A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation

The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation. The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. Co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation. Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR. The amplified fragment flanking the 3' end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST). The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium , as well as Neurospora crassa mitochondria and higher plant plastids. Thus the gene identified by the embryo-defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.     

Plant lectin-like bacteriocin from a rhizosphere-colonizing Pseudomonas isolate

Rhizosphere isolate Pseudomonas sp. strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P. putida GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a TnMod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in E. coli. This gene was designated llpA (lectin-like putidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.     

Detection and mass spectrometric characterization of novel long-term dehydrochloromethyltestosterone metabolites in human urine

The biotransformation of dehydrochloromethyltestosterone (DHCMT, 4-chloro-17β-hydroxy,17α-methylandrosta-1,4-dien-3-one) in man was studied with the aim to discover long-term metabolites valuable for the antidoping analysis. Having applied a high performance liquid chromatography for the fractionation of urinary extract obtained from the pool of several DHCMT positive urines, about 50 metabolites were found. Most of these metabolites were included in the GC-MS/MS screening method, which was subsequently applied to analyze the post-administration and routine doping control samples. As a result of this study, 6 new long-term metabolites were identified tentatively characterized using GC-MS and GC-MS/MS as 4-chloro-17α-methyl-5β-androstan-3α,16,17β-triol (M1), 4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5β-androsta-1,13-dien-3α-ol (M2), 4-chloro-18-nor-17β-hydroxymethyl,17α-methyl-5β-androst-13-en-3α-ol (M3), its epimer 4-chloro-18-nor-17α-hydroxymethyl,17β-methyl-5β-androst-13-en-3α-ol, 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-4,13-dien-3α-ol (M4) and its epimer 4-chloro-18-nor-17α-hydroxymethyl,17β-methylandrosta-4,13-dien-3α-ol. The most long-term metabolite M3 was shown to be superior in the majority of cases to the other known DHCMT metabolites, such as 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-1,4,13-trien-3-one and 4-chloro-3α,6β,17β-trihydroxy-17α-methyl-5β-androst-1-en-16-one.