Gene expression studies of Thermus thermophilus promoters PdnaK, Parg and Pscs-mdh.

AIMS: To obtain data concerning gene expression in Thermus thermophilus and demonstrate the use of the beta-galactosidase gene from Thermus sp. A4 as a convenient reporter gene. METHODS AND RESULTS: Thermus thermophilus PPKU was constructed, in which the beta-gal gene was deleted from the chromosome. Two inducible promoters PdnaK (regulating the DnaK heat shock-inducible protein) and Parg (regulating expression of an arginine-inducible protein) and a carbon-regulated promoter, Pscs-mdh (regulating expression of succinyl-coA and malate dehydrogenase) were cloned upstream of the beta-gal reporter gene derived from Thermus sp. A4 to construct vectors pTEX7, pTEX8 and pTEX9, respectively. The amount of beta-galactosidase activity produced by the PdnaK promoter in pTEX7 was substantially above the background level of 0.3 U mg(-1), and increased from 5.2 to 10.4 U mg(-1) after heat-shock induction indicating that significant amounts of DnaK are produced even when T. thermophilus is grown at its optimum temperature. The Parg promoter was found to be maximally induced by 10-30 mm arginine, but was inhibited by higher concentrations. The Pscs-mdh promoter was maximally active in the presence of malate while lower levels of activity were observed in the presence of succinate, pyruvate, glutamate, glucose and the presence of yeast extract or peptone. CONCLUSIONS: These results demonstrate that several inducible and regulated promoters are available for genetic studies in Thermus and that beta-galactosidase can be used as a convenient reporter gene for studies of transcriptional regulation in Thermus. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of characterized inducible and regulated promoters will facilitate the development of improved gene expression vectors for Thermus. The demonstration that beta-galactosidase activity in T. thermophilus PPKU can be used to allow reliable screening for beta-gal-positive transformant colonies on agar plates will add to the convenience of performing genetic manipulations in T. thermophilus. Future studies of transcriptional regulation in Thermus will benefit from the beta-gal host-vector system reported here.

Isolation and characterization of a moderate thermophile, Mycobacterium phlei GTIS10, capable of dibenzothiophene desulfurization.

An organism, identified as Mycobacterium phlei GTIS10, was isolated based on its ability to use dibenzothiophene (DBT) as a sole source of sulfur for growth at 30-52 degrees C. Similar to other biodesulfurization-competent organisms, M. phlei GTIS10 converts DBT to 2-hydroxybiphenyl (2-HBP), as detected by HPLC. The specific desulfurization activity of the 50 degrees C M. phlei GTIS10 culture was determined to be 1.1+/-0.07 micromol 2-HBP min(-1) (g dry cell)(-1). M. phlei GTIS10 can also utilize benzothiophene and thiophene as sulfur sources for growth. The dszABC operon of M. phlei GTIS10 was cloned and sequenced and was found to be identical to that of Rhodococcus erythropolis IGTS8. The presence of the R. erythropolis IGTS8 120-kb plasmid pSOX, which encodes the dszABC operon, has been demonstrated in M. phlei GTIS10. Even though identical dsz genes are contained in both cultures, the temperature at which resting cells of R. erythropolisIGTS8 reach the highest rate of DBT metabolism is near 30 degrees C whereas the temperature that shows the highest activity in resting cell cultures of M. phlei GTIS10 is near 50 degrees C, and activity is detectable at temperatures as high as 57 degrees C. In M. phlei GTIS10, the rate-limiting step in vivo appears to be the conversion of DBT to dibenzothiophene sulfone catalyzed by the product of the dszC gene, DBT monooxygenase. The thermostability of individual desulfurization enzymes was determined and 2-hydroxybiphenyl-2-sulfinate sulfinolyase, encoded by dszB, was found to be the most thermolabile. These results demonstrate that the thermostability of individual enzymes determined in vitro is not necessarily a good predictor of the functional temperature range of enzymes in vivo.

Inducible and constitutive expression using new plasmid and integrative expression vectors for Thermus sp.

AIMS: To develop molecular tools and examine inducible and constitutive gene expression in Thermus thermophilus. METHODS AND RESULTS: Two plasmid promoter probe vectors and an integrative promoter probe vector were constructed using a promoterless thermostable kanamycin nucleotidyltransferase (KmR) cassette. Three expression vectors were constructed based on a constitutive promoter J17, that functions in both Thermus and Escherichia coli. An inducible expression vector was constructed using the heat-shock inducible promoter (70 to 85 degrees C) from the dnaK gene of T. flavus, and the malate dehydrogenase gene (mdh) from T. flavus was cloned and expressed in both E. coli and T. thermophilus HB27. CONCLUSION: This report describes the construction and use of improved promoter probe and expression vectors for use in Thermus species. The mdh gene can be used as a high temperature (85 degrees C) reporter gene for Thermus sp. The dnaK promoter is thermo-inducible. SIGNIFICANCE AND IMPACT OF THE STUDY: The expression vectors and molecular tools described here are significant improvements over previously reported vectors for Thermus sp. The mdh gene and the thermo-inducible dnaK promoter will facilitate high temperature studies employing Thermus species.

New host-vector system for Thermus spp. based on the malate dehydrogenase gene.

A Thermus thermophilus HB27 strain was constructed in which the malate dehydrogenase (mdh) gene was deleted. The Deltamdh colonies are recognized by a small-colony phenotype. Wild-type phenotype is restored by transformation with Thermus plasmids or integration vector containing an intact mdh gene. The wild-type phenotype provides a positive selection tool for the introduction of plasmid DNA into Thermus spp., and because mdh levels can be readily quantified, this host-vector system is a convenient tool for monitoring gene expression.

Selective removal of nitrogen from quinoline and petroleum by Pseudomonas ayucida IGTN9m.

Enrichment culture experiments employing soil and water samples obtained from petroleum-contaminated environments succeeded in the isolation of a pure culture possessing the ability to utilize quinoline as a sole nitrogen source but did not utilize quinoline as a carbon source. This culture was identified as Pseudomonas ayucida based on a partial 16S rRNA gene sequence, and the strain was given the designation IGTN9m. Examination of metabolites using thin-layer chromatography and gas chromatography-mass spectrometry suggests that P. ayucida IGTN9m converts quinoline to 2-quinolinone and subsequently to 8-hydroxycoumarin. Resting cells of P. ayucida IGTN9m were shown to be capable of selectively removing about 68% of quinoline from shale oil in a 16-h treatment time. These results suggest that P. ayucida IGTN9m may be useful in petroleum biorefining for the selective removal of organically bound nitrogen from petroleum.

Biodesulfurization of water-soluble coal-derived material by Rhodococcus rhodochrous IGTS8.

Rhodococcus rhodochrous IGTS8 was previously isolated because of its ability to use coal as its sole source of sulfur for growth. Subsequent growth studies have revealed that IGTS8 is capable of using a variety of organosulfur compounds as sources of sulfur but not carbon. In this article, the ability of IGTS8 to selectively remove organic sulfur from water-soluble coal-derived material is investigated. The microbial removal of organic sulfur from coal requires microorganisms capable of cleaving carbon-sulfur bonds and the accessibility of these bonds to microorganisms. The use of water-soluble coal-derived material effectively overcomes the problem of accessibility and allows the ability of microorganisms to cleave carbon-sulfur bonds present in coal-derived material to be assessed directly. Three coals, two coal solubilization procedures, and two methods of biodesulfurization were examined. The results of these experiments reveal that the microbial removal of significant amounts of organic sulfur from water-soluble coal-derived material with treatment times as brief as 24 h is possible. Moreover, the carbon content and calorific value of biotreated products are largely unaffected. Biotreatment does result, however, in an increased hydrogen and nitrogen content and a decreased oxygen content of the coal-derived material. The aqueous supernatant obtained from biodesulfurization experiments does not contain sulfate, sulfite, or other forms of soluble sulfur at increased concentrations in comparison with control samples. Sulfur removed from water-soluble coal-derived material appears to be incorporated into biomass.

Instantaneous gene transfer from donor to recipient microorganisms via electroporation.

Simplified electroporation methodologies have been developed that reliably yield transformants with only minutes of effort. Neither DNA purification, cells in specific phase of growth, cell washing nor chilled cuvettes are required to obtain transformants. Electroporation can be used to transfer plasmid or chromosomal DNA directly from donor to recipient cells. This simplified direct method of electroporation has been demonstrated to work for both intra- as well as interspecies transformations using a variety of microorganisms. The use of electroporation to purify plasmid DNA was also investigated and found to be inferior to conventional plasmid isolation procedures.

Molecular characterization of Pseudomonas aeruginosa bacteriophages: identification and characterization of the novel virus B86.

We have characterized a new phage, B86, of Pseudomonas aeruginosa isolated from nature. It is a temperate, uv-inducible, generalized transducing phage. To determine the relatedness of this phage to other characterized P. aeruginosa phages, DNA homology studies were carried out. P. aeruginosa phages have previously been grouped by immunological cross-reactivity. Our studies confirm this classification by demonstrating that phages of different class share little or no DNA homology. Based on homology studies as well as cross-immunity to superinfection, B86 is related to other class B phages and is most homologous with phage B39. The virion morphology of these two phages is quite different, however, as are the restriction enzyme digestion patterns of their genomes with several restriction enzymes. Wild-type B86 is subject to the host-controlled restriction-modification systems of P. aeruginosa PAO and PAT. Virulent mutants of this phage are not restricted by these same restriction-modification systems.

Cloning and complete nucleotide sequence determination of the catB gene encoding cis,cis-muconate lactonizing enzyme.

The enzyme, cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1), has been proposed to play a key role in the beta-ketoadipate pathway of benzoate degradation. A 10.2-kb EcoRI fragment isolated from a Pseudomonas putida genomic library complemented a mutant deficient in this enzyme. The MLEI coding gene, catB, was localized to a 1.6-kb fragment which was sequenced by the dideoxy chain termination method. MLEI was purified 25-fold from crude extracts of benzoate-grown P. putida PRS2015 harboring the cloned catB gene. Purified MLEI was greater than 95% homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit Mr was 40,000 which was in close agreement with the nucleotide sequence data. N-terminal sequence analysis of purified MLEI protein agreed with the N terminus predicted by the nucleotide sequence. Comparison of the nucleotide and amino acid sequences for catB with the corresponding sequences of the clcB gene (K.L. Ngai, B.F., D.K. Chatterjee, L.N. Ornston, and A.M.C., unpublished), whose gene product catalyzes the analogous reaction in 3-chlorobenzoate degradation, showed significant homology. These results suggest that catB and clcB have diverged from a common ancestral gene.

Site-specific and illegitimate recombination in the oriV1 region of the F factor. DNA sequences involved in recombination and resolution.

We have defined some of the sequences involved in high frequency recA-independent recombination at the oriV1 region of the F factor. Using a mobilization assay, we determined that plasmid pMB080, a pBR322 derivative bearing the PvuII-BamHI (F factor co-ordinates 45.43 to 46.0) fragment from the oriV1 region of F, contained all sequences necessary to undergo efficient site-specific recombination with the F derivative pOX38, which retains the oriV1 region. We constructed a series of pMB080 deletions in vitro using exonucleases S1 and Bal31. Deletions removing a ten base-pair sequence, which forms part of an inverted repeat segment located 62 base-pairs to the left of the NcoI site (45.87) within the cloned fragment, totally eliminated the recA-independent recombination reaction. Other deletions differentially affected both the frequency and stability of cointegrate molecules formed by the site-specific recombination system. The F factor oriV1 region is involved also in low-frequency recombination with several sites on pBR322 and related plasmids. We have determined the precise location of these recombination sites within oriV1 by DNA sequencing. These studies revealed that recombination always took place within an eight base-pair spacer region between the ten base-pair inverted repeats found to be important for oriV1-oriV1 interactions. We propose that the low-efficiency recombination between pBR322 and pOX38 results from the ability of the F site-specific recombination apparatus to weakly recognize and interact with sequences that bear some resemblance to the normal oriV1 recognition elements. Furthermore, we suggest, by analogy with the lambda paradigm, that the nucleotide sequences at the junctions of secondary site recombinants define at least one crossover site used during the normal site-specific recombination process.

Genetic aspects of toxic chemical degradation.

All naturally occurring molecules are continuously being recycled in nature, constantly being synthesized, and constantly being degraded. Synthetic molecules on the other hand, often are unable to enter nature's recycling scheme because organisms that have an ability to degrade these xenobiotic compounds simply do not exist. Moreover, many synthetic chemicals are not only recalcitrant to biodegradation, but also are toxic and therefore can cause significant pollution problems even at very low concentrations. The chemical industry will continue to produce an evergrowing number of molecules, even though severe environmental problems have resulted from synthetic molecules already produced. We must find a means of bringing synthetic molecules back into nature's recycling systems if we are to preserve the environment. Biotechnology, through the genetic manipulation of microorganisms, provides a means of accomplishing this goal.

Microbial biodegradation of 2,4,5-trichlorophenoxyacetic acid and chlorophenols.

We have succeeded in isolating a pure culture of Pseudomonas cepacia, AC1100, from a chemostat enrichment culture experiment that is capable of growing on 2,4,5-trichlorophenoxyacetic acid as its sole source of carbon and energy. AC1100 is not only capable of degrading 2,4,5-T but is also able to completely or partially dehalogenate a wide variety of halophenols. The regulation of the dehalogenating ability of AC1100 has been investigated which demonstrates that the enzyme(s) which allow the conversion of 2,4,5-T to 2,4,5-TCP are constitutive, while the enzymes that allow the degradation of 2,4,5-TCP are inducible by 2,4,5-TCP (or some metabolite of 2,4,5-TCP) but not by 2,4,5-T or other halophenols which can serve as substrates. Moreover, the 2,4,5-TCP degradative pathway is repressed by the presence of an abundant alternative carbon source. The detailed pathway of 2,4,5-T degradation by AC1100 is currently under study. Although field tests have yet to be conducted, laboratory experiments have demonstrated rapid and complete degradation of 2,4,5-T from contaminated soil. Soil previously contaminated with as much as 5,000 micrograms of 2,4,5-T/g of soil could be detoxified by AC1100 treatment, allowing the growth of plants sensitive to less than 10 micrograms 2,4,5-T/g of soil. Moreover soil contaminated with as much as 20,000 micrograms of 2,4,5-T/g of soil showed greater than 90% degradation after six weekly AC1100 treatments. After 2,4,5-T has been substantially degraded in contaminated soil the titer of AC1100 rapidly falls to nearly undetectable levels, which indicates that no serious ecological disturbance is likely to result from the application of AC1100. It appears possible that the treatment of contaminated areas with appropriate microorganisms may allow essentially a total restoration of the original soil condition.

Metabolism of Halophenols by 2,4,5-trichlorophenoxyacetic acid-degrading Pseudomonas cepacia.

Resting cells of 2,4,5-trichlorophenoxyacetic acid-grown Pseudomonas cepacia AC1100 were able to completely and rapidly dechlorinate several chlorine-substituted phenols, including 2,4,5-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol. Several other trichlorophenols were only partially dechlorinated. The evidence suggests that 2,4,5-trichlorophenol is an intermediate in the degradation of 2,4,5-trichlorophenoxyacetic acid by strain AC1100. Moreover, although strain AC1100 was isolated by selection for growth on a chlorinated aromatic compound, brominated and fluorinated analogs were efficiently dehalogenated by strain AC1100 resting cells, whereas an iodinated analog was poorly dehalogenated.

Detoxification of 2,4,5-trichlorophenoxyacetic acid from contaminated soil by Pseudomonas cepacia.

The strain of Pseudomonas cepacia, AC1100, capable of utilizing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy can degrade 2,4,5-T in contaminated soil, removing more than 99% of 2,4,5-T present at 1 mg/g of soil within 1 week. Repeated application of AC1100 even allowed more than 90% removal of 2,4,5-T within 6 weeks from heavily contaminated soil containing as much as 20,000 ppm 2,4,5,-T (20 mg/g of soil). Microbial removal of 2,4,5-T allowed the soil to support growth of plants sensitive to low concentrations of 2,4,5-T. After 2,4,5-T removal, the titer of AC1100 in the soil rapidly fell to undetectable levels within a few weeks.

Biodegradation of 2,4,5-trichlorophenoxyacetic acid in soil by a pure culture of Pseudomonas cepacia.

A pure culture of Pseudomonas cepacia AC1100 was able to degrade and grow in presence of 2,4,5-trichlorophenoxyacetic acid in soil. At optimum temperature (30 degrees C) and moisture content (15 to 50% [wt/vol]) strain AC1100 could degrade as much as 95% of 2,4,5-trichlorophenoxyacetic acid at high concentration (1 mg/g of soil) within 1 week.

Biodegradation of 2,4,5-trichlorophenoxyacetic acid by a pure culture of Pseudomonas cepacia.

A pure culture of Pseudomonas cepacia, designated AC1100, that can utilize 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as its sole source of carbon and energy was isolated. An actively growing culture of AC1100 was able to degrade more than 97% of 2,4,5-T, present at 1 mg/ml, within 6 days as determined by chloride release, gas chromatographic, and spectrophotometric analyses. The ability of AC1100 to oxidize a variety of chlorophenols and related compounds is also reported.