Pseudomonas Butanovora Research Articles

Elucidation of Sequence–Function Relationships for an Improved Biobutanol In Vivo Biosensor in E. coli

Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ54-dependent, butanol-responsive TF–promoter pair, BmoR-PBMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized PBMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in PBMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ54-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.

Efficient Removal of Butachlor and Change in Microbial Community Structure in Single-Chamber Microbial Fuel Cells.

Microbial electrochemical technology provides an inexhaustible supply of electron acceptors, allowing electroactive microorganisms to generate biocurrent and accelerate the removal of organics. The treatment of wastewater contaminated by butachlor, which is a commonly used chloroacetamide herbicide in paddy fields, is a problem in agricultural production. In this study, butachlor was found to be removed efficiently (90 ± 1%) and rapidly (one day) in constructed single-chamber microbial fuel cells (MFCs). After the addition of sodium acetate to MFCs with butachlor as the sole carbon source, electricity generation was recovered instead of increasing the degradation efficiency of butachlor. Meanwhile, the microbial community structure was changed in anodic and cathodic biofilms after the addition of butachlor, following the bioelectrochemical degradation of butachlor. High-throughput sequencing showed the proliferation of Paracoccus and Geobacter in MFCs with butachlor as the sole carbon source and of Thauera butanivorans in MFCs with butachlor and sodium acetate as concomitant carbon sources. These species possess the ability to oxidize different substituents of butachlor and have important potential use for the bioremediation of wastewater, sediments, and soils.

Biotransformation of 6:2 polyfluoroalkyl phosphates (6:2 PAPs): Effects of degradative bacteria and co-substrates

Polyfluoroalkyl phosphates (PAPs), a group of fluorotelomer alcohol (FTOH)-based surfactants commonly used in water- and grease-proof food contact paper, have been suggested as a direct source of human exposure to health-concerned perfluoroalkyl carboxylic acids (PFCAs). This study investigated factors affecting biotranformation of 6:2 polyfluoroalkyl phosphates (6:2 PAPs) by three known FTOH-degrading Pseudomonas strains (Pseudomonas butanovora, P. oleovorans, and P. fluorescens DSM 8341) under different co-substrate conditions and compared to that by activated sludge samples. The three pure strains transformed 6:2 PAPs into eight different per- and poly-fluoroalkyl carboxylic acids (PFCAs) and/or PFCA precursors. P. fluorescens DSM 8341 produced 5:2 sFTOH [CF3(CF2)4CH(OH)CH3] and P. oleovorans produced 5:2 ketone [CF3(CF2)4C(O)CH3] as the primary transformation product, respectively, with citrate having a minimal impact on the transformation. P. butanovora with lactate produced more diverse transformation products than those by any two strains. Activated sludge was more efficient at transforming 6:2 PAPs and produced more transformation products including PFHpA [CF3(CF2)5COOH] and PFPeA [CF3(CF2)3COOH], with 5:2 sFTOH as the most abundant product on day 30. The abundance of the alkane hydroxylase (alkB) gene related to alkane oxidation, the changes of total microbial population as well as their community structure in activated sludge during 6:2 PAPs biotransformation were also investigated.

6:2 Fluorotelomer alcohol (6:2 FTOH) biodegradation by multiple microbial species under different physiological conditions

Factors affecting microbial aerobic biodegradation of 6:2 fluorotelomer alcohol [6:2 FTOH, F(CF₂)₆CH₂CH₂OH] were investigated using three alkane-degrading bacteria (Mycobacterium vaccae JOB5, Pseudomonas oleovorans, and Pseudomonas butanovora) and one fluoroacetate-degrading bacterium (Pseudomonas fluorescens DSM 8341). In the presence of formate (an external reducing energy source), P. fluorescens DSM 8341 produced perfluorobutanoic acid by removing three -CF₂- groups from 6:2 FTOH. Only P. fluorescens DSM 8341 transformed 5:3 acid to 4:3 acid and perfluoropentanoic acid. However, formate showed no effects on the degradation rates, patterns, or transformation products of 6:2 FTOH by M. vaccae JOB5. When dicyclopropylketone (an alkane hydroxylase inducer) or formate was added, P. oleovorans rapidly degraded 6:2 FTOH and produced PFPeA. In the presence of lactate, P. butanovora degraded 6:2 FTOH slowly but produced diverse metabolites. Our results demonstrate that the extent and mechanisms of 6:2 FTOH biotransformation are affected by strain types, enzyme inducers, and levels of reducing energy.

Transcription factor-based screens and synthetic selections for microbial small-molecule biosynthesis.

Continued advances in metabolic engineering are increasing the number of small molecules being targeted for microbial production. Pathway yields and productivities, however, are often suboptimal, and strain improvement remains a persistent challenge given that the majority of small molecules are difficult to screen for and their biosynthesis does not improve host fitness. In this work, we have developed a generalized approach to screen or select for improved small-molecule biosynthesis using transcription factor-based biosensors. Using a tetracycline resistance gene 3' of a small-molecule inducible promoter, host antibiotic resistance, and hence growth rate, was coupled to either small-molecule concentration in the growth medium or a small-molecule production phenotype. Biosensors were constructed for two important chemical classes, dicarboxylic acids and alcohols, using transcription factor-promoter pairs derived from Pseudomonas putida, Thauera butanivorans, or E. coli. Transcription factors were selected for specific activation by either succinate, adipate, or 1-butanol, and we demonstrate product-dependent growth in E. coli using all three compounds. The 1-butanol biosensor was applied in a proof-of-principle liquid culture screen to optimize 1-butanol biosynthesis in engineered E. coli, identifying a pathway variant yielding a 35% increase in 1-butanol specific productivity through optimization of enzyme expression levels. Lastly, to demonstrate the capacity to select for enzymatic activity, the 1-butanol biosensor was applied as synthetic selection, coupling in vivo 1-butanol biosynthesis to E. coli fitness, and an 120-fold enrichment for a 1-butanol production phenotype was observed following a single round of positive selection.

Biodefluorination and biotransformation of fluorotelomer alcohols by two alkane‐degrading Pseudomonas strains

Fluorotelomer alcohols [FTOHs, F(CF(2))(n) CH(2)CH(2)OH, n = 4, 6, and 8] are emerging environmental contaminants. Biotransformation of FTOHs by mixed bacterial cultures has been reported; however, little is known about the microorganisms responsible for the biotransformation. Here we reported biotransformation of FTOHs by two well-studied Pseudomonas strains: Pseudomonas butanovora (butane oxidizer) and Pseudomonas oleovorans (octane oxidizer). Both strains could defluorinate 4:2, 6:2, and 8:2 FTOHs, with a higher degree of defluorination for 4:2 FTOH. According to the identified metabolites, P. oleovorans transformed FTOHs via two pathways I and II. The pathway I led to the production of x:2 ketone [dominant metabolite, F(CF(2))(x)C(O)CH(3); x = n - 1, n = 6 or 8], x:2 sFTOH [F(CF(2))(x)CH(OH)CH(3)], and perfluorinated carboxylic acids (PFCAs, perfluorohexanoic, or perfluorooctanoic acid). The pathway II resulted in the formation of x:3 polyfluorinated acid [F(CF(2))(x) C(2)CH(2) COOH] and relatively minor shorter-chain PFCAs (perfluorobutyric or perfluorohexanoic acid). Conversely, P. butanovora transformed FTOHs by using the pathway I, leading to the production of x:2 ketone, x:2 sFTOH, and PFCAs. This is the first study to show that individual bacterium can bio-transform FTOHs via different or preferred transformation pathways to remove multiple --CF(2) -- groups from FTOHs to form shorter-chain PFCAs.

Growth of a non‐methanotroph on natural gas: ignoring the obvious to focus on the obscure

Methanotrophs are well known for their ability to grow on methane in natural gas environments; however, these environments also contain low concentrations of longer-chain-length gaseous alkanes. This mixture of alkanes poses a problem for organisms that might otherwise grow on alkanes ≥ C2 because methane could inhibit oxidation of growth substrates and lead to an accumulation of toxic C1 metabolites. Here, we have characterized the growth of a C2 -C9 alkane-utilizing bacterium, Thauera butanivorans, in conditions containing high concentrations of methane and small amounts (< 3% of total alkane) of C2 -C4 . During such growth, methanol accumulates transiently before being consumed in an O2 -dependent process that leads to the formation of a proton gradient and subsequent ATP generation. In contrast, formaldehyde-dependent O2 consumption is insensitive to uncouplers and does not lead to significant ATP production. This efficient C1 oxidation process that regains much of the energy loss inflicted by oxidizing methane, coupled with an alkane monooxygenase effective at limiting methane oxidation, allows T. butanivorans to grow uninhibited in natural gas environments. Although longer-chain-length gaseous alkane-utilizing organisms have been previously identified to grow in natural gas seepages, the data presented here represent the first detailed characterization of the physiological effects associated with inadvertent methane oxidation by a non-methanotroph, and suggest the presence of a well-evolved series of biochemical processes that allow them to grow in natural gas deposits without the need for developing the unique metabolic machinery characteristic of methanotrophs.

Thauera butanivorans sp. nov., a C2-C9 alkane-oxidizing bacterium previously referred to as 'Pseudomonas butanovora'

The placement of 'Pseudomonas butanovora' in the genus Thauera was proposed previously, based on 16S rRNA gene sequence analysis, upon further studies of taxonomical characteristics. In this study, physiological characteristics and DNA-DNA reassociation data are presented and the transfer of 'P. butanovora' to the genus Thauera is proposed. The original description of the strain (strain Bu-B1211) indicated that it was capable of denitrification but not anaerobic growth. 'P. butanovora' is capable of anaerobic respiration and growth, utilizing nitrate as a terminal electron acceptor during the oxidation of organic acids and alcohols, but not aromatic hydrocarbons or open-chain terpenoids. The total fatty acid composition supported the assignment of strain Bu-B1211 to the Betaproteobacteria and resembled that of members of the genus Thauera. The combination of 16S rRNA gene phylogenetic evidence, physiological and taxonomical characteristics and DNA-DNA reassociation data supported the placement of 'Pseudomonas butanovora' Bu-B1211 in the genus Thauera as representing a novel species, for which the name Thauera butanivorans sp. nov. is proposed. The type strain is Bu-B1211(T) (=IAM 12574(T)=ATCC 43655(T)=DSM 2080(T)).

Evidence for involvement of copper ions and redox state in regulation of butane monooxygenase in Pseudomonas butanovora.

Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C(2)-C(9) alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of beta-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O(2) when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH(4)Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O(2) (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O(2), CuSO(4) (0.5 microM) repressed 1-butanol-dependent induction of beta-galactosidase activity. Under oxic conditions (20% O(2) [vol/vol]), significantly higher concentrations of CuSO(4) (2 microM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu(2+) reducing agent Na ascorbate (100 microM) and CuSO(4) (0.5 microM) repressed the induction of beta-galactosidase activity under oxic conditions to the same extent that 0.5 microM CuSO(4) alone repressed it under anoxic conditions. Under oxic conditions, 2 microM CuSO(4) repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO(4) repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

Involvement of BmoR and BmoG in n-alkane metabolism in ‘Pseudomonas butanovora’

'Pseudomonas butanovora' uses an alcohol-inducible alkane monooxygenase (BMO) to grow on C(2)-C(9) n-alkanes. Five ORFs were identified flanking the BMO structural genes. Two of the ORFs, bmoR, encoding a putative sigma(54)-transcriptional regulator BmoR, and bmoG, encoding a putative GroEL chaperonin BmoG, were analysed by gene-inactivation experiments. The BmoR-deficient mutant grew at slower growth rates than the wild-type on C(2)-C(5) n-alkanes and showed little to no growth on C(6)-C(8) n-alkanes within 7 days. A BmoR-deficient mutant was constructed in the 'P. butanovora' bmoX : : lacZ reporter strain and used to test whether bmoR was involved in bmoX induction after growth on C(2)-C(8) carbon sources. In acetate- or lactate-grown cells, C(2)-C(8) n-alcohols failed to induce beta-galactosidase activity. In contrast, in propionate-, butyrate- or pentanoate-grown cells, n-butanol induced approximately 45 % of the beta-galactosidase activity observed in the control bmoX : : lacZ strain. In propionate-grown cells, C(2)-C(5) n-alcohols induced beta-galactosidase activity, whereas C(7) and C(8) n-alcohols did not. BmoR may act as a sigma(54)-transcriptional regulator of bmo that is controlled by the n-alcohol produced in the alkane oxidation. During growth on short-chain-length fatty acids, however, another BMO regulatory system seems to be activated to promote transcription of bmo by short-chain-length alcohols (i.e. </=C(6)). The bmoG-deficient mutant did not grow on C(2)-C(8) n-alkanes; however, it was capable of transcribing bmoX and bmoC of the BMO operon. BmoG may act as a chaperonin to assemble competent BMO.

Comparative phylogenies of Burkholderia, Ralstonia, Comamonas, Brevundimonas and related organisms derived from rpoB, gyrB and rrs gene sequences

Phylogenetic analysis of strains from Burkholderia, Ralstonia, Cupriavidus, Comamonas, Delftia, Acidovorax, Brevundimonas, Herbaspirillum huttiense and “ Pseudomonas butanovora” was performed based on the protein-coding genes rpoB and gyrB and on the 16S rRNA-coding gene rrs. Overall, the phylogenies deduced from the three genes were concordant among themselves and with current taxonomy. However, a few differences among individual gene phylogenies were noted. For example, the separation of Cupriavidus from Ralstonia was not supported in the rpoB tree, as Ralstonia was nested within Cupriavidus. Similarly, the separation of Delftia from Comamonas was not supported in the gyrB tree. Based on rrs and rpoB, the genus Burkholderia contained four groups: (i) the B. cepacia complex, (ii) the B. pseudomallei- B. thailandensis group, (iii) a 6-species group including B. caledonica and B. glathei and (iv) the B. plantarii- B. glumae- B. gladioli group. However, B. caribensis and B. glathei stood as a fifth group based on gyrB. It appears that a phylogeny cannot be reliably based on a single gene. Using rpoB and gyrB, better separation of closely related species was obtained compared to rrs, indicating the potential of these two genes for identification and species definition. Nevertheless, intraspecific sequence diversity will need to be determined to fully establish the value of these genes for strain identification.

Propionate inactivation of butane monooxygenase activity in ‘Pseudomonas butanovora’: biochemical and physiological implications

Butane monooxygenase (BMO) catalyses the oxidation of alkanes to alcohols in the alkane-utilizing bacterium 'Pseudomonas butanovora'. Incubation of alkane-grown 'P. butanovora' with butyrate or propionate led to irreversible time- and O2-dependent loss of BMO activity. In contrast, BMO activity was unaffected by incubation with lactate or acetate. Chloramphenicol inhibited the synthesis of new BMO, but did not change the kinetics of propionate-dependent BMO inactivation, suggesting that the propionate effect was not simply due to it acting as a repressor of BMO transcription. BMO was protected from propionate-dependent inactivation by the presence of its natural substrate, butane. Although both the time and O2 dependency of propionate inactivation of BMO imply that propionate might be a suicide substrate, no evidence was obtained for BMO-dependent propionate consumption, or 14C labelling of BMO polypeptides by [2-(14)C]propionate during inactivation. Propionate-dependent BMO inactivation was also explored in mutant strains of 'P. butanovora' containing single amino acid substitutions in the alpha-subunit of the BMO hydroxylase. Propionate-dependent BMO inactivation in two mutant strains with amino acid substitutions close to the catalytic site differed from wild-type (one was more sensitive and the other less), providing further evidence that propionate-dependent inactivation involves interaction with the BMO catalytic site. A putative model is presented that might explain propionate-dependent inactivation of BMO when framed within the context of the catalytic cycle of the closely related enzyme, soluble methane monooxygenase.

Butane monooxygenase of ‘Pseudomonas butanovora’: purification and biochemical characterization of a terminal-alkane hydroxylating diiron monooxygenase

Butane monooxygenase (sBMO) has been purified to homogeneity from the Gram-negative beta-proteobacterium 'Pseudomonas butanovora' and confirmed to be a three-component diiron monooxygenase system. The reconstituted enzyme complex oxidized C(3)-C(6) linear and branched aliphatic alkanes, which are growth substrates for 'P. butanovora'. The sBMO complex was composed of an iron-containing hydroxylase (BMOH), a flavo-iron sulfur-containing NADH-oxidoreductase (BMOR) and a small regulatory component protein (BMOB). The physical characteristics of sBMO were remarkably similar to the sMMO family of soluble multicomponent diiron monooxgenases. However, the catalytic properties of sBMO were quantitatively different in regard to inactivation in the presence of substrate and product distribution. BMOH was capable of ethene oxidation when supplied with H(2)O(2) and ethene (known as the peroxide shunt), but this activity was at least three orders of magnitude less than that observed for the hydroxylase of sMMO of Methylosinus trichosporium OB3b. BMOH and BMOR were efficient in the oxidation of ethene in the absence of BMOB with regard to rate of reaction and product yield. Regiospecificity of sBMO was strongly biased towards primary hydroxylation, with > or = 80 % of the hydroxylations occurring at the terminal carbon atom.

Site-directed amino acid substitutions in the hydroxylase alpha subunit of butane monooxygenase from Pseudomonas butanovora: Implications for substrates knocking at the gate.

Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase alpha subunit of butane monooxygenase (BMOH-alpha) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-alpha were altered to the corresponding residues from the alpha subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains G113N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to 1-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 microM methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 microM. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-alpha.

Effects of Dichloroethene Isomers on the Induction and Activity of Butane Monooxygenase in the Alkane-Oxidizing Bacterium “ Pseudomonas butanovora ”

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium "Pseudomonas butanovora." Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of approximately 20 nmol mg protein(-1) of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein(-1). Oxidation of similar amounts of each DCE isomer ( approximately 20 nmol mg protein(-1)) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown "P. butanovora" did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.

Trichloroethylene degradation by butane-oxidizing bacteria causes a spectrum of toxic effects

The physiological consequences of trichloroethylene (TCE) transformation by three butane oxidizers were examined. Pseudomonas butanovora, Mycobacterium vaccae, and Nocardioides sp. CF8 utilize distinctly different butane monooxygenases (BMOs) to initiate degradation of the recalcitrant TCE molecule. Although the primary toxic event resulting from TCE cometabolism by these three strains was loss of BMO activity, species differences were observed. P. butanovora and Nocardioides sp. CF8 maintained only 4% residual BMO activity following exposure to 165 microM TCE for 90 min and 180 min, respectively. In contrast, M. vaccae maintained 34% residual activity even after exposure to 165 microM TCE for 300 min. Culture viability was reduced 83% in P. butanovora, but was unaffected in the other two species. Transformation of 530 nmol of TCE by P. butanovora (1.0 mg total protein) did not affect the viability of BMO-deficient P. butanovora cells, whereas transformation of 482 nmol of TCE by toluene-grown Burkholderia cepacia G4 caused 87% of BMO-deficient P. butanovora cells to lose viability. Together, these results contrast with those previously reported for other bacteria carrying out TCE cometabolism and demonstrate the range of cellular toxicities associated with TCE cometabolism.

Nitrate-dependent salicylate degradation by Pseudomonas butanovora under anaerobic conditions

Nitrate-dependent salicylate degradation by the denitrifying Pseudomonas butanovora was investigated and the molar ratio of the cometabolism under anaerobic circumstances was determined. The bacterium was able to utilize salicylate as an electron donor for the reduction of nitrate. Salicylate was eliminated via catechol, which is degraded by means of catechol 2,3-oxygenases (meta-cleavage), forming 2-hydroxymuconic semialdehyde. The molar ratios of NO 3 − -N:salicylate existing during the experiment accorded well with the assumed 1:1 molar ratio. The tolerances of the growth, the salicylate degradation and the denitrification of P. butanovora to various heavy metal ions were also studied. Although the strain was tolerant to Pb 2+ and Cu 2+ up to 1 mM in complete medium, salicylate utilization took place only up to a concentration of 0.1 mM for both heavy metal ions. Of the heavy metal ions investigated, Cd 2+ (at a concentration of 0.05 mM) displayed the highest inhibitory effect on salicylate degradation by P. butanovora.

Proposed involvement of a soluble methane monooxygenase homologue in the cyclohexane‐dependent growth of a new Brachymonas species

High-throughput mRNA differential display (DD) was used to identify genes induced by cyclohexane in Brachymonas petroleovorans CHX, a recently isolated beta-proteobacterium that grows on cyclohexane. Two metabolic gene clusters were identified multiple times in independent reverse transcription polymerase chain reactions (RT-PCR) in the course of this DD experiment. These clusters encode genes believed to be required for cyclohexane metabolism. One gene cluster (8 kb) encodes the subunits of a multicomponent hydroxylase related to the soluble butane of Pseudomonas butanovora and methane monooxygenases (sMMO) of methanotrophs. We propose that this butane monooxygenase homologue carries out the oxidation of cyclohexane into cyclohexanol during growth. A second gene cluster (11 kb) contains almost all the genes required for the oxidation of cyclohexanol to adipic acid. Real-time PCR experiments confirmed that genes from both clusters are induced by cyclohexane. The role of the Baeyer-Villiger cyclohexanone monooxygenase of the second cluster was confirmed by heterologous expression in Escherichia coli.

Biological denitrification in a continuous-flow pilot bioreactor containing immobilized Pseudomonas butanovora cells.

Pseudomonas butanovora, a novel denitrifying bacterium, was immobilized in composite beads and filled into a reactor system. The pilot bioreactor average denitrification activity was at ethanol-C:nitrate-N ratios of 3:1 and 1.5:1 0.88 and 0.54 kg NO3(-)-Nm(-3) d(-1), respectively. The denitrification was stable in spite of the relatively low hydraulic retention times of 2.47 and 3 h. The nitrate content of the influent was almost completely reduced at the first level of the bioreactor and the nitrite formed underwent reduction in the upper part of the reactor. The experimentally determined optimum ethanol-C:nitrate-N ratio was 1.41 +/- 0.41. In consequence of the aerobic conditions, the acetic acid produced by the oxygenation of ethanol was also detectable in the reactor effluent. The pH of the effluent (7.58) never exceeded the acceptable maximum (8.5). The nitrate removal efficiency of the cells was nearly 1000% at both C:N ratios, and the nitrite content of the effluent was around the prescribed limit throughout the continuous operation. This continuous-flow pilot bioreactor containing immobilized P. butanovora cells proved an efficient denitrification system with a relatively low retention time.

Molecular analysis of the soluble butane monooxygenase from 'Pseudomonas butanovora'.

'Pseudomonas butanovora' is capable of growth with butane via the oxidation of butane to 1-butanol, which is catalysed by a soluble butane monooxygenase (sBMO). In vitro oxidation of ethylene (an alternative substrate for sBMO) was reconstituted in the soluble portion of cell extracts and was NADH-dependent. Butane monooxygenase was separated into three components which were obligately required for substrate oxidation. The N-terminal sequences of the peptides associated with butane monooxygenase led to the cloning and sequencing of the 5797 nucleotide bmo gene cluster. Comparisons of the deduced amino acid sequences with other multicomponent monooxygenases suggest that sBMO is a multimeric hydroxylase with 61, 45 and 19 kDa subunits encoded by bmoXYZ, a 40 kDa oxidoreductase encoded by bmoC, and a 15 kDa regulatory protein encoded by bmoB. A sixth structural gene (bmoD) encodes a 9.6 kDa protein with similarity exclusively to mmoD (orfY), a putative metal centre assembly protein of the soluble methane monooxygenases. Insertional inactivation of bmoX resulted in a mutant 'P. butanovora' strain incapable of growth with butane. A putative promoter element characteristic of promoters associated with sigma(54)-dependent transcription initiation was located upstream of the bmo genes. Expression of all six genes was detected in butane-induced cells. Butane monooxygenase from 'P. butanovora' aligns most closely with non-haem carboxylate-bridged diiron monooxygenases and, moreover, contains the characteristic iron-binding motif. The structural and mechanistic implications of the high sequence identity (up to 64%) between the peptides of butane monooxygenase and methane monooxygenases are discussed.