Characterization of three propane-inducible oxygenases in Mycobacterium sp. strain ENV421.

AIMS: Mycobacterium sp. strain ENV421 has the ability to cometabolize a variety of chemicals following growth on propane as a sole source of carbon and energy. In this study, we used genetic and biochemical approaches to identify and characterize multiple propane-inducible oxygenase genes in ENV421. METHODS AND RESULTS: Gene clusters encoding a CYP153-type cytochrome P450 oxygenase (P450), an AlkB-type alkane monooxygenase (AlkB) and a soluble diiron monooxygenase were identified and cloned using degenerate PCR primers. Reverse transcriptase PCR showed that all three gene clusters were induced by propane. Substrate specificity studies revealed that despite the fact that ENV421 does not grow on medium length alkanes, cloned versions of both the AlkB and P450 were capable of octane oxidation, forming n-octanol. Additionally, the P450 oxygenase had the ability to oxidize indole, medium-to-long-chain alkylbenzenes and a variety of para-substituted methylalkylbenzenes. Successful cloning and expression of the diiron monooxygenase was not achieved, so its substrate specificity could not be determined. CONCLUSIONS: Three types of short-to-medium-chain alkane oxygenases were induced by propane in ENV421, even though the cloned AlkB and P450 oxygenases did not oxidize propane. Curiously, they both oxidized octane, which is not a growth substrate for ENV421. Furthermore, the P450, typically operating as terminal alkane hydroxylase, exhibited interesting regio- and stereoselectivity, catalysing linear alkanes, alkylbenzenes and indole. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first example of a propane-inducible P450 with a broad substrate specificity, including linear alkanes, alkylbenzenes and a multiring compound. The induction of three distinct oxygenase classes by propane is also an interesting finding because it might explain why propane serves as an effective stimulant that promotes the biodegradation of a various environmental contaminants.

DNA amplification to enhance detection of genetically engineered bacteria in environmental samples.

The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.

Designing microorganisms for the treatment of toxic wastes.

The genetic design of novel metabolic routes offers exciting possibilities for biological research and biotechnology, both in the exploration of the metabolic/evolutionary potential of cells and in the development of innovative applications. In this chapter, we review recent advances in the development of genetic tools and strategies for the design of new microorganisms for elimination of environmental pollutants. These include the design of regulated gene expression circuits that provide high levels of catalytic activity, even under environmental conditions that ordinarily repress expression of catabolic genes; the rational alteration of relevant properties of proteins that qualitatively or quantitatively restrict catabolic activities; the judicious assembly of gene blocks encoding selected metabolic modules to create novel metabolic routes and combinations of routes; and the design of microorganisms exhibiting properties that contribute to better process development.

Induction of toluene oxidation activity in Pseudomonas mendocina KR1 and Pseudomonas sp. strain ENVPC5 by chlorinated solvents and alkanes.

Toluene oxidation activity in Pseudomonas mendocina KR1 and Pseudomonas sp. strain ENVPC5 was induced by trichloroethylene (TCE), and induction was followed by the degradation of TCE. Higher levels of toluene oxidation activity were achieved in the presence of a supplemental growth substrate such as glutamate, with levels of activity of up to 86% of that observed with toluene-induced cells. Activity in P. mendocina KR1 was also induced by cis-1,2-dichloroethylene, perchloroethylene, chloroethane, hexane, pentane, and octane, but not by trans-1,2-dichloroethylene. Toluene oxidation was not induced by TCE in Burkholderia (Pseudomonas) cepacia G4, P. putida F1, Pseudomonas sp. strain ENV110, or Pseudomonas sp. strain ENV113.

Effects of acidification on mercury methylation, demethylation, and volatilization in sediments from an acid-susceptible lake.

The effect of experimental acidification on mercury methylation, demethylation, and volatilization was examined in surficial sediment samples from a weakly buffered northern Wisconsin lake. All mercury transformations were measured with radioisotopic tracers. Acidification of sediment pH with H2SO4, HCl, or HNO3 significantly decreased 203Hg(II) methylation. Acidification of pH 6.1 (ambient) sediments to pH 4.5 with either H2SO4 or HCl inhibited methylation by over 65%. The decreased methylation was due to the increased hydrogen ion concentration because methylation was not affected by concentrations of Na2SO4 or NaCl equimolar to the amount of acid added. Inhibition of methylation was observed even after prolonged acidification of sediments to pH 5.0 for up to 74 days. Acidification of sediments to pH 5.5, 4.5, and 3.5 with HNO3 resulted in a near complete inhibition of methylation at each pH. Similarly, the addition of equimolar amounts of NaNO3 resulted in a near complete inhibition of methylation, indicating that the inhibition was due to the nitrate ion rather than to the acidity. Demethylation of methyl mercury was not affected by pHs between 8.0 and 4.4, but sharply decreased below pH 4.4. Volatilization of 203Hg(II) from surface sediments was less than 2% of methylation activity and was not significantly different from that in killed sediments. This study indicated that acidification of sediments inhibits mercury methylation and that the observed increase in the mercury burdens in fish from low pH lakes is not due to increased production of methylmercury in sediments.

Chloroform mineralization by toluene-oxidizing bacteria.

Seven toluene-oxidizing bacterial strains (Pseudomonas mendocina KR1, Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas pickettii PKO1, and Pseudomonas sp. strains ENVPC5, ENVBF1, and ENV113) were tested for their ability to degrade chloroform (CF). The greatest rate of CF oxidation was achieved with strain ENVBF1 (1.9 nmol/min/mg of cell protein). CF also was oxidized by P. mendocina KR1 (0.48 nmol/min/mg of cell protein), strain ENVPC5 (0.49 nmol/min/mg of cell protein), and Escherichia coli DH510B(pRS202), which contained cloned toluene 4-monooxygenase genes from P. mendocina KR1 (0.16 nmol/min/mg of cell protein). Degradation of [14C]CF and ion analysis of culture extracts revealed that CF was mineralized to CO2 (approximately 30 to 57% of the total products), soluble metabolites (approximately 15%), a total carbon fraction irreversibly bound to particulate cellular constituents (approximately 30%), and chloride ions (approximately 75% of the expected yield). CF oxidation by each strain was inhibited in the presence of trichloroethylene, and acetylene significantly inhibited trichloroethylene oxidation by P. mendocina KR1. Differences in the abilities of the CF-oxidizing strains to degrade other halogenated compounds were also identified. CF was not degraded by B. cepacia G4, P. putida F1, P. pickettii PKO1, Pseudomonas sp. strain ENV113, or P. mendocina KRMT, which contains a tmo mutation.

Toluene monooxygenase-catalyzed epoxidation of alkenes.

Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of P. mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0. 01 to 0.33 micromol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified.

Inducible cell lysis system for the study of natural transformation and environmental fate of DNA released by cell death.

Two novel conditional broad-host-range cell lysis systems have been developed for the study of natural transformation in bacteria and the environmental fate of DNA released by cell death. Plasmid pDKL02 consists of lysis genes S, R, and Rz from bacteriophage lambda under the control of the Ptac promoter. The addition of inducer to Escherichia coli, Acinetobacter calcoaceticus, or Pseudomonas stutzeri containing plasmid pDKL02 resulted in cell lysis coincident with the release of high amounts of nucleic acids into the surrounding medium. The utility of this lysis system for the study of natural transformation with DNA released from lysed cells was assessed with differentially marked but otherwise isogenic donor-recipient pairs of P. stutzeri JM300 and A. calcoaceticus BD4. Transformation frequencies obtained with lysis-released DNA and DNA purified by conventional methods and assessed by the use of antibiotic resistance (P. stutzeri) or amino acid prototrophy (A. calcoaceticus) for markers were comparable. A second cell lysis plasmid, pDKL01, contains the lysis gene E from bacteriophage phi X174 and causes lysis of E. coli and P. stutzeri bacteria by activating cellular autolysins. Whereas DNA released from pDKL02-containing bacteria persists in the culture broth for days, that from induced pDKL01-containing bacteria is degraded immediately after release. The lysis system involving pDKL02 is thus useful for the study of both the fate of DNA released naturally into the environment by dead cells and gene transfer by natural transformation in the environment in that biochemically unmanipulated DNA containing defined sequences and coding for selective phenotypes can be released into a selected environment at a specific time point. This will allow kinetic measurements that will answer some of the current ecological questions about the fate and biological potential of environmental DNA to be made.

A nonimmunosuppressive triene-modified rapamycin analog is a potent inhibitor of peptidyl prolyl cis-trans isomerase.

A triene-modified analog of the potent immunosuppressive agent rapamycin was found to be a potent inhibitor of the peptidyl prolyl cis-trans isomerase activity of human FKBP (Ki = 12.5 nM). This analog was not immunosuppressive in a thymocyte proliferation assay itself, but was able to antagonize the effect of rapamycin. This new analog should be useful as a mechanistic probe for macrocyclic immunosuppressants.

Recovery of DNA from soils and sediments.

Experiments were performed to evaluate the effectiveness of two different methodological approaches for recovering DNA from soil and sediment bacterial communities: cell extraction followed by lysis and DNA recovery (cell extraction method) versus direct cell lysis and alkaline extraction to recover DNA (direct lysis method). Efficiency of DNA recovery by each method was determined by spectrophotometric absorbance and using a tritiated thymidine tracer. With both procedures, the use of polyvinylpolypyrrolidone was important for the removal of humic compounds to improve the purity of the recovered DNA; without extensive purification, various restriction enzymes failed to cut added target DNA. Milligram quantities of high-purity DNA were recovered from 100-g samples of both soils and sediments by the direct lysis method, which was a greater than 1-order-of-magnitude-higher yield than by the cell extraction method. The ratio of labeled thymidine to total DNA, however, was higher in the DNA recovered by the cell extraction method. than by the direct lysis method, suggesting that the DNA recovered by the cell extraction method came primarily from active bacterial cells, whereas that recovered by the direct lysis method may have contained DNA from other sources.

Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase.

Pseudomonas mendocina KR1 toluene 4-monooxygenase is a multicomponent diiron enzyme. the diiron center is contained in the tmoA polypeptide of teh hydroxylase component [alphabetagamma)2,Mr approximately 212 kDa]. Product distribution studies reveal that the natural isoform is highly specific for para hydroxylation of toluene (kcat approximately 2 s-1 with respect to an alphabetagamma promoter), o-xylene (kcat approximately 0.8 s-1), m-xylene (kcat approximately 0.6 s-1), and other aromatic hydrocarbons. This degree of regioselectivity for methylbenzenes is unmatched by numerous other oxygenase enzymes. However, during the T4MO-catalyzed oxidation of p-xylene (kcat approximately 0.4 s-1), 4-methyl benzyl alcohol is the major product, showing that the enzyme could catalyze either aromatic or benzylic hydroxylation with the appropriate substrate. Site-directed mutagenesis has been used to study the contributions of tmoA active site residues Q141, I180, and F205 to the regiospecificity. Isoforms Q141C and F205I yielded shifts of regiospecificity away from p-cresol formation, with F205I giving an approximately 5-fold increase in the percentage of m-cresol formation relative to that of the natural isoform. The kcat of purified Q141C for toluene oxidation was approximately 0.2 s-1. Isoform Q141C also functioned predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene, in direct contrast to the predominant benzylic hydroxylation observed for the natural isoform, while isoform F205I gave nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation. Isoform I180F gave no substantial shift in product distributions relativeto the natural isoform for all substrates tested. Upon the basis of a proposed active site model, both Q141 anf F205 are suggested to lie in a hydrophobic region closer to the FeA iron site, while I180 will be closer to FeB. These studies reveal that changes in the hydrophobic region predicted to be nearest to FeA can influence the regiospecificity observed for toluene 4-monooxygenase.

Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria.

Several propane-oxidizing bacteria were tested for their ability to degrade gasoline oxygenates, including methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). Both a laboratory strain and natural isolates were able to degrade each compound after growth on propane. When propane-grown strain ENV425 was incubated with 20 mg of uniformly labeled [14C]MTBE per liter, the strain converted > 60% of the added MTBE to 14CO2 in < 30 h. The initial oxidation of MTBE and ETBE resulted in the production of nearly stoichiometric amounts of tert-butyl alcohol (TBA), while the initial oxidation of TAME resulted in the production of tert-amyl alcohol. The methoxy methyl group of MTBE was oxidized to formaldehyde and ultimately to CO2. TBA was further oxidized to 2-methyl-2-hydroxy-1-propanol and then 2-hydroxy isobutyric acid; however, neither of these degradation products was an effective growth substrate for the propane oxidizers. Analysis of cell extracts of ENV425 and experiments with enzyme inhibitors implicated a soluble P-450 enzyme in the oxidation of both MTBE and TBA. MTBE was oxidized to TBA by camphor-grown Pseudomonas putida CAM, which produces the well-characterized P-450cam, but not by Rhodococcus rhodochrous 116, which produces two P-450 enzymes. Rates of MTBE degradation by propane-oxidizing strains ranged from 3.9 to 9.2 nmol/min/mg of cell protein at 28 degrees C, whereas TBA was oxidized at a rate of only 1.8 to 2.4 nmol/min/mg of cell protein at the same temperature.

Adaptive response to cold temperatures in Vibrio vulnificus.

The effectiveness of rapid chilling or freezing of oysters to reduce Vibrio vulnificus levels in shellfish may be compromised by product handling procedures that permit cold adaptation. When a V. vulnificus culture was shifted from 35 degrees C to 6 degrees C conditions, it underwent transition to a non-culturable state. Cells adapted to 15 degrees C prior to change to 6 degrees C condition, however, remain viable and culturable. In addition, cultures adapted to 15 degrees C were able to survive better upon freezing at -78 degrees C compared with cultures frozen directly from 35 degrees C. Inhibition of protein synthesis by addition of chloramphenicol in a V. vulnificus culture immediately prior to the exposure to the adaptive temperature eliminated inducible cold tolerance. These results suggest that cold-adaptive "protective" proteins may enhance survival and tolerance at cold temperatures. In addition, removal of iron from the growth medium by adding 2,2'-Dipyridyl prior to cold adaptation decreased the viability by approximately 2 logarithm levels. This suggests that iron plays an important role in adaptation at cold temperatures. Analysis of total cellular proteins on an SDS polyacrylamide gel electrophoresis, labeled with 35S-methionine during exposure at 15 degrees C, showed elevated expressions of a 6-kDa and a 40-kDa protein and decreased expression of an 80-kDa protein. These results suggest that, for V. vulnificus, survival and tolerance at cold temperatures could be due to the expression of cold-adaptive proteins other than previously documented major cold shock proteins such as CS7.4 and CsdA. In this study, for the first time we have shown that exposure to an intermediate cold temperature (15 degrees C) causes a cold adaptive response, helping this pathogen remain in culturable state when exposed to a much colder temperature (6 degrees C). This adaptive nature to cold temperatures could be important for shellfish industry efforts to reduce the risk of V. vulnificus infection from consuming raw oysters.

Detection of coliform bacteria in water by polymerase chain reaction and gene probes.

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.

Adaptive response to cold temperatures and characterization of cspA in Salmonella typhimurium LT2.

Salmonella typhimurium is a major foodborne microbial pathogen which primarily contaminates poultry products causing salmonellosis in humans. S. typhimurium LT2 cultures, when transferred from 37 degrees C to 5 degrees C or 10 degrees C, showed an initial lag period in growth with an approximate generation time of 10-25 h. Western blot assay using E. coli CS7.4 antibody and analysis of radiolabeled total cellular proteins from S. typhimurium cultures after exposure to 10 degrees C or 5 degrees C showed elevated expression of a major cold shock protein, CS7.4. Identification of a decreased level of CS7.4 at 37 degrees C suggests that the expression of this protein may require a large temperature downshift. Putative regulatory protein binding segment on the 5'-untranslated region referred as 'Fragment 7' in S. typhimurium exhibited a 90.6% and a 56.25% nucleotide sequence identity when compared with the Fragment 7 of E. coli and S. enteritidis, respectively. The differences in the nucleotide sequence within the Fragment 7 between S. typhimurium and S. enteritidis may explain the differential expression of CspA at 37 degrees C. The nucleotide sequence of the open reading frame of S. typhimurium cspA gene showed a single base difference at 816 bp position from a G to a C which altered the amino acid residue from a glycine to an alanine. In addition to CspA, an elevated expression of a 105 kDa, and decreased expression of 6 proteins were evidenced when cultures of S. typhimurium were exposed to 10 degrees C or 5 degrees C. Differential expression of the CspA and other proteins in S. typhimurium following exposure to cold temperatures suggest that adaptation and continued growth and survival at cold temperatures in this pathogen may be aided by these cold-responsive proteins.

Threonine 201 in the diiron enzyme toluene 4-monooxygenase is not required for catalysis.

The diiron enzyme toluene 4-monooxygenase from Pseudomonas mendocina KR1 catalyzes the NADH- and O(2)-dependent hydroxylation of toluene. A combination of sequence alignments and spectroscopic studies indicate that T4MO has an active site structure closely related to the crystallographically characterized methane monooxygenase hydroxylase. In the methane monooxygenase hydroxylase, active site residue T213 has been proposed to participate in O(2) activation by analogy to certain proposals made for cytochrome P450. In this work, mutagenesis of the comparable residue in the toluene 4-monooxygenase hydroxylase, T201, has been used to investigate the role of an active site hydroxyl group in catalysis. Five isoforms (T201S, T201A, T201G, T201F, and T201K) that retain catalytic activity based on an in vivo indigo formation assay were identified, and detailed characterizations of the purified T201S, T201A, and T201G variants are reported. These isoforms have k(cat) values of 1.2, 1.0, and 0.6 s(-)(1), respectively, and k(cat)/K(M) values that vary by only approximately 4-fold relative to that of the native isoform. Moreover, these isoforms exhibit 80-90% coupling efficiency, which also compares favorably to the >94% coupling efficiency determined for the native isoform. For the T201S, T201A, and T201G isoforms, the regiospecificity of toluene hydroxylation was nearly identical to that of the natural isoform, with p-cresol representing 90-95% of the total product distribution. In contrast, the T201F isoform caused a substantial shift in the product distribution, and gave o- and p-cresol in a 1:1 ratio. In addition, the amount of benzyl alcohol was increased approximately 10-fold with the T201F isoform. For reaction with p-xylene, previous studies have shown that the native isoform reacted to give 4-methybenzyl alcohol and 2, 5-dimethylphenol in a 4:1 ratio [Pikus, J. D., Studts, J. M., McClay, K., Steffan, R. J., and Fox, B. G. (1997) Biochemistry 36, 9283-9289]. For comparison, the T201S, T201A, and T201F isoforms gave a slightly relaxed 3:1 ratio of these products, while the T201G isoform gave a dramatically relaxed 1:1 ratio. On the basis of these studies, we conclude that the hydroxyl group of T201 is not essential to maintaining the turnover rate or the coupling of the toluene 4-monooxygenase complex. However, changing the volume occupied by the side chain at the position of T201 can lead to alterations in the regiospecificity of the hydroxylation, presumably by producing different orientations for substrate binding during catalysis.

Optimized expression and purification of toluene 4-monooxygenase hydroxylase.

Toluene 4-monooxygenase is a four-protein complex that catalyzes the O(2)- and NADH-dependent oxidation of toluene to p-cresol. The influence of various expression systems on the host cell growth characteristics, purified protein yields, and specific activity of the hydroxylase (T4moH) component of the complex was evaluated by considering the cell mass obtained per liter of fermentation culture medium, the purified protein obtained per gram of cell mass, and the specific activity of purified T4moH. The specific activity of purified T4moH was determined to be 1200-1250 nmol of p-cresol formed per minute per milligram of T4moH in air-saturated 50 mM phosphate buffer, pH 7.5, at 25 degrees C in the presence of optimal concentrations of the other protein components of the complex, saturating toluene (5.8 mM at 25 degrees C), and saturating NADH (1 mM). This value was obtained for T4moH purified from several different expression systems and apparently represents the maximal specific activity of the enzyme complex for toluene hydroxylation. By manipulation of vectors and gene inserts to eliminate adventitious catalytic turnover of NADH, up to 60-fold increase in the volumetric yield of T4moH activity was obtained from recombinant fermentations in Escherichia coli BL21(DE3).

Biodegradation of methyl tert-butyl ether by a pure bacterial culture.

Biodegradation of methyl tert-butyl ether (MTBE) by the hydrogen-oxidizing bacterium Hydrogenophaga flava ENV735 was evaluated. ENV735 grew slowly on MTBE or tert-butyl alcohol (TBA) as sole sources of carbon and energy, but growth on these substrates was greatly enhanced by the addition of a small amount of yeast extract. The addition of H(2) did not enhance or diminish MTBE degradation by the strain, and MTBE was only poorly degraded or not degraded by type strains of Hydrogenophaga or hydrogen-oxidizing enrichment cultures, respectively. MTBE degradation activity was constitutively expressed in ENV735 and was not greatly affected by formaldehyde, carbon monoxide, allyl thiourea, or acetylene. MTBE degradation was inhibited by 1-amino benzotriazole and butadiene monoepoxide. TBA degradation was inducible by TBA and was inhibited by formaldehyde at concentrations of >0.24 mM and by acetylene but not by the other inhibitors tested. These results demonstrate that separate, independently regulated genes encode MTBE and TBA metabolism in ENV735.

Growth, survival and characterization of cspA in Salmonella enteritidis following cold shock.

Salmonella enteritidis is a major foodborne microbial pathogen that can grow and survive at low temperatures for a considerable period of time. Increased survival was evidenced from a frozen S. enteritidis culture when treated at 10 degrees C prior to freezing. Western blot analysis with Escherichia coli CspA antibody and analysis of radiolabeled proteins from S. enteritidis cultures after cold shock at 10 degrees C and 5 degrees C showed increased expression of a 7.4-kDa major cold shock protein, CS7.4, similar in size to that reported for E. coli. Cloning followed by nucleotide sequence analysis of the cspA gene from S. enteritidis showed a 100% nucleotide sequence identity in the promoter elements (-35 and -10) and the amino acid sequence encoded by the open reading frame (ORF) with the E. coli cspA gene. However, the differences in the nucleotide sequences between E. coli and S. enteritidis cspA genes in the putative repressor protein binding domain, the fragment 7, and in various segments throughout the upstream 0.642-kbp DNA may contribute to the expression of CS7.4 at less stringent temperatures in S. enteritidis. As in E. coli, the actual role of CS7.4 in protecting S. enteritidis from the damaging effects of cold or freezing temperatures is not yet understood.

Detection and classification of hyperfine-shifted 1H, 2H, and 15N resonances of the Rieske ferredoxin component of toluene 4-monooxygenase.

T4MOC is a 12.3 kDa soluble Rieske ferredoxin that is obligately required for electron transfer between the oxidoreductase and diiron hydroxylase components of toluene 4-monooxygenase from Pseudomonas mendocina KR1. Our preliminary 1H NMR studies of oxidized and reduced T4MOC [Markley, J. L., Xia, B., Chae, Y. K., Cheng, H., Westler, W. M., Pikus, J. D., and Fox, B. G. (1996) in Protein Structure Function Relationships (Zaidi, Z., and Smith, D., Eds.) pp 135-146, Plenum Press, London] revealed the presence of hyperfine-shifted 1H resonances whose short relaxation times made it impractical to use nuclear Overhauser effect (NOE) measurements for assignment purposes. We report here the use of selective isotopic labeling to analyze the hyperfine-shifted 1H, 2H, and 15N signals from T4MOC. Selective deuteration led to identification of signals from the four Hbeta atoms of cluster ligands C45 and C64 in the oxidized and reduced forms of T4MOC. In the reduced state, the Curie temperature dependence of the Hbeta protons corresponded to that predicted from the simple vector spin-coupling model for nuclei associated with the localized ferric site. The signal at 25.5 ppm in the 1H spectrum of reduced T4MOC was assigned on the basis of selective 2H labeling to the His Hepsilon1 atom of one of the cluster ligands (H47 or H67). This assignment was corroborated by a one bond 1H-13C correlation (at 25.39 ppm 1H and 136.11 ppm 13C) observed in spectra of [U-13C]T4MOC with a 1H-13C coupling constant of approximately 192 Hz. The carbon chemical shift and one bond coupling constant are those expected for 1Hepsilon1-13Cepsilon1 in the imidazolium ring of histidine and are inconsistent with values expected for cysteine 1Halpha-13Calpha. The His Hepsilon1 proton exhibited weak Curie temperature dependence from 283 to 303 K, contrary to the anti-Curie temperature dependence predicted from the spin coupling model for nuclei associated with the localized ferrous site. A 1H peak at -12.3 ppm was observed in spectra of reduced T4MOC; this signal was found to correspond to a hydrogen (probably in an H-bond to the cluster) that exchanged with solvent with a half-time of about 2 days in the oxidized state but with a much longer (undetectable) half-time in the reduced state. These results with T4MOC call into question certain 1H assignments recently reported on the basis of NOE measurements for the comparable Rieske ferredoxin component of an evolutionarily related alkene monooxygenase from Xanthobacter sp. Py2 [Holz, R. C., Small, F. J., and Ensign, S. A, (1997) Biochemistry 36, 14690-14696]. Selective 15N labeling was used to identify hyperfine-shifted 15N NMR signals from the backbone nitrogens of all four cluster ligands (C45, H47, C64, and H67), from the Nepsilon2 atoms of the two histidine ligands (H47 and H67), and from nonligand Gln and Ala residues (Q48 and A66) present in the cluster-binding motif of T4MOC in the oxidized and reduced states. The results indicate that the Ndelta1 of each of the two ligand histidines of T4MOC are ligated to an iron atom and reveal a pattern of H-bonding to the Rieske [2Fe-2S] center involving four (H47, Q48, A66, and H67 of T4MOC) of the five backbone amide H-bonds expected on the basis of comparison with the crystal structures of other related Rieske proteins; the fifth backbone amide (I50 of T4MOC) failed to exhibit a hyperfine shift. This anomaly may arise from the lack of an associated disulfide in T4MOC, a fundamental structural difference between the three types of Rieske proteins that may be related to functional diversity in this protein family.