Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported.

Gene PA1990 of Pseudomonas aeruginosa , located downstream of pqqE and encoding a putative peptidase, was shown to be involved in excretion of PQQ into the culture supernatant. This gene is cotranscribed with the pqqABCDE cluster and was named pqqH . A PA1990::Km r mutant (VK3) did not show any effect in growth behaviour; however, in contrast to the wild-type, no excretion of PQQ into the culture supernatant was observed. The putative pqqF gene of P. aeruginosa was shown to be essential for PQQ biosynthesis. A pqqF ::Km r mutant did not grow aerobically on ethanol, because of its inability to produce PQQ. Transcription of the pqqABCDEH operon was induced upon aerobic growth on ethanol, 1-propanol, 1,2-propanediol and 1-butanol, while on glycerol, succinate and acetate, transcription was low. Transcription of the pqqABCDEH operon was also found upon anoxic growth on ethanol with nitrate as electron acceptor, but no PQQ was produced. Expression of the pqqABCDEH operon is regulated at the transcriptional level. In contrast, the pqqF operon appeared to be transcribed constitutively at a very low level under all growth conditions studied.

In addition to the known response regulator ErbR (former AgmR) and the two-component regulatory system EraSR (former ExaDE), three additional regulatory proteins have been identified as being involved in controlling transcription of the aerobic ethanol oxidation system in Pseudomonas aeruginosa. Two putative sensor kinases, ErcS and ErcS', and a response regulator, ErdR, were found, all of which show significant similarity to the two-component flhSR system that controls methanol and formaldehyde metabolism in Paracoccus denitrificans. All three identified response regulators, EraR (formerly ExaE), ErbR (formerly AgmR) and ErdR, are members of the luxR family. The three sensor kinases EraS (formerly ExaD), ErcS and ErcS' do not contain a membrane domain. Apparently, they are localized in the cytoplasm and recognize cytoplasmic signals. Inactivation of gene ercS caused an extended lag phase on ethanol. Inactivation of both genes, ercS and ercS', resulted in no growth at all on ethanol, as did inactivation of erdR. Of the three sensor kinases and three response regulators identified thus far, only the EraSR (formerly ExaDE) system forms a corresponding kinase/regulator pair. Using reporter gene constructs of all identified regulatory genes in different mutants allowed the hierarchy of a hypothetical complex regulatory network to be established. Probably, two additional sensor kinases and two additional response regulators, which are hidden among the numerous regulatory genes annotated in the genome of P. aeruginosa, remain to be identified.

All pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases contain an unusual disulfide ring formed between adjacent cysteine residues. A mutant enzyme that is lacking this structure was generated by replacing Cys105 and Cys106 with Ala in quinoprotein ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa ATCC17933. Heterologously expressed quinoprotein ethanol dehydrogenase in which Cys-105 and Cys-106 have been replaced by Ala (Cys105Ala/Cys106Ala apo-QEDH) was successfully converted to enzymatic active holo-enzyme by incorporation of its cofactor PQQ in the presence of Ca(2+). The enzymatic activity of the mutant enzyme in the artificial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 did not depend on an activating amine which is essential for wild type activity under these conditions. The mutant enzyme showed increased Michaelis constants for primary alcohols, while the affinity for the secondary alcohol 2-propanol was unaltered. Surprisingly, for all substrates tested the specific activity of the mutant enzyme in the artificial dye test was higher than that found for wild type QEDH. On the contrary, in the ferricyanide test with the natural electron acceptor cytochrome c(550) the activity of mutant Cys105Ala/Cys106Ala was 15-fold lower than that of wild type QEDH. We demonstrate for the first time unambiguously that the unusual disulfide ring is essential for efficient electron transfer at pH 7 from QEDH to its natural electron acceptor cytochrome c(550).

Gluconobacter oxydans is famous for its rapid and incomplete oxidation of a wide range of sugars and sugar alcohols. The organism is known for its efficient oxidation of D-glucose to D-gluconate, which can be further oxidized to two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, as well as 2,5-di-keto-D-gluconate. For this oxidation chain and for further oxidation reactions, G. oxydans possesses a high number of membrane-bound dehydrogenases. In this review, we focus on the dehydrogenases involved in D-glucose oxidation and the products formed during this process. As some of the involved dehydrogenases contain pyrroloquinoline quinone (PQQ) as a cofactor, also PQQ synthesis is reviewed. Finally, we will give an overview of further PQQ-dependent dehydrogenases and discuss their functions in G. oxydans ATCC 621H (DSM 2343).

Pseudomonas aeruginosa ATCC 17933 is capable of growing aerobically on ethanol as sole source of carbon and energy. This requires the glyoxylate cycle for replenishing C4-compounds to the TCA cycle. The enzyme isocitrate lyase (ICL) catalyzes the first step of this glyoxylate shunt. Its activity was induced more than 10-fold in response to the carbon sources ethanol or acetate instead of glucose or succinate. We could prove that in P. aeruginosa ICL is essential for aerobic as well as anaerobic utilization of C2-sources. Transcriptional regulation of icl gene (aceA) expression was monitored on different carbon sources by using an aceA–lacZ gene fusion. A strong correlation between promoter and ICL activity indicated regulation at the transcriptional level. But ICL was not simply induced by the mere presence of ethanol in the growth medium as was demonstrated by cultivation on mixed substrates. P. aeruginosa showed diauxic growth on media containing ethanol–succinate or ethanol–glucose mixtures and did not transcribe the aceA gene to metabolize ethanol until succinate or glucose, respectively, were exhausted. Inactivation of the chromosomal aceA gene in P. aeruginosa led to an inability to grow on ethanol and acetate. Promoter activity studies showed that all genes necessary to oxidize ethanol were downregulated in the ICL-negative mutant. But on mixed substrates like ethanol–succinate or ethanol–glucose the mutant exhibited growth and utilized ethanol as well, probably as energy source only.

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c1) and 3-methyl-dihydro-2(3H)-furanone (4c2) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.

Dehalococcoides strains reductively dechlorinate a wide variety of halogenated compounds including chlorinated benzenes, biphenyls, naphthalenes, dioxins, and ethenes. Recent genome sequencing of the two Dehalococcoides strains CBDB1 and 195 revealed the presence of 32 and 18 reductive dehalogenase homologous genes, respectively, and therefore suggested an even higher dechlorinating potential than previously anticipated. Here, we demonstrate reductive dehalogenation of chlorophenol congeners by Dehalococcoides strains CBDB1 and 195. Strain CBDB1 completely converted 2,3-dichlorophenol, all six trichlorophenols, all three tetrachlorophenols, and pentachlorophenol to lower chlorinated phenols. Observed dechlorination rates in batch cultures with cell numbers of 10(7) mL(-1) amounted up to 35 microM day(-1). Chlorophenols were preferentially dechlorinated in the ortho position, but also doubly flanked and singly flanked meta- or para-chlorine substituents were removed. We used a newly designed computer-assisted direct cell counting protocol and quantitative PCR to demonstrate that strain CBDB1 uses chlorophenols as electron acceptors for respiratory growth. The growth yield of strain CBDB1 with 2,3-dichlorophenol was 7.6 x 10(13) cells per mol of Cl- released, and the growth rate was 0.41 day(-1). For strain 195, fast ortho dechlorination of 2,3-dichlorophenol, 2,3,4-trichlorophenol, and 2,3,6-trichlorophenol was detected, with only the ortho chlorine removed. Because chlorinated phenolic compounds are widely distributed as natural components in anaerobic environments, our results reveal one mode in which the Dehalococcoides species could have survived through earth history.

The GOX1857 gene, which encodes a putative membrane-bound pyrroloquinoline quinone (PQQ)-dependent dehydrogenase in Gluconobacter oxydans ATCC 621 H, was characterized. GOX1857 was disrupted and the oxidizing potential of the resulting mutant strain was compared to that of the wild-type. In contrast to the wild-type, the mutant was unable to grow with myo-inositol as the sole energy source and did not show any myo-inositol dehydrogenase activity in vitro, indicating that GOX1857 encodes an inositol dehydrogenase. The association of inositol dehydrogenase with the membrane and the requirement for the cofactor PQQ were confirmed. Inositol dehydrogenase exhibited optimal activity at pH 8.75. As indicated by cultivation on different substrates, inositol dehydrogenase was repressed by D-glucose.

ABSTRACT In Gluconobacter oxydans, pyrroloquinoline quinone (PQQ) serves as the cofactor for various membrane-bound dehydrogenases that oxidize sugars and alcohols in the periplasm. Proteins for the biosynthesis of PQQ are encoded by the pqqABCDE gene cluster. Our reverse transcription-PCR and promoter analysis data indicated that the pqqA promoter represents the only promoter within the pqqABCDE cluster of G. oxydans 621H. PQQ overproduction in G. oxydans was achieved by transformation with the plasmid-carried pqqA gene or the complete pqqABCDE cluster. A G. oxydans mutant unable to produce PQQ was obtained by site-directed disruption of the pqqA gene. In contrast to the wild-type strain, the pqqA mutant did not grow with d-mannitol, d-glucose, or glycerol as the sole energy source, showing that in G. oxydans 621H, PQQ is essential for growth with these substrates. Growth of the pqqA mutant, however, was found with d-gluconate as the energy source. The growth behavior of the pqqA mutant correlated with the presence or absence of the respective PQQ-dependent membrane-bound dehydrogenase activities, demonstrating the vital role of these enzymes in G. oxydans metabolism. A different PQQ-deficient mutant was generated by Tn5 transposon mutagenesis. This mutant showed a defect in a gene with high homology to the Escherichia coli tldD gene, which encodes a peptidase. Our results indicate that the tldD gene in G. oxydans 621H is involved in PQQ biosynthesis, possibly with a similar function to that of the pqqF genes found in other PQQ-synthesizing bacteria.

Gluconobacter oxydans DSM 2343 (ATCC 621H)catalyzes the oxidation of glucose to gluconic acid and subsequently to 5-keto-D-gluconic acid (5-KGA), a precursor of the industrially important L-(+)-tartaric acid. To further increase 5-KGA production in G. oxydans, the mutant strain MF1 was used. In this strain the membrane-bound gluconate-2-dehydrogenase activity, responsible for formation of the undesired by-product 2-keto-D-gluconic acid, is disrupted. Therefore, high amounts of 5-KGA accumulate in the culture medium. G. oxydans MF1 was equipped with plasmids allowing the overexpression of the membrane-bound enzymes involved in 5-KGA formation. Overexpression was confirmed on the transcript and enzymatic level. Furthermore, the resulting strains overproducing the membrane-bound glucose dehydrogenase showed an increased gluconic acid formation, whereas the overproduction of gluconate-5-dehydrogenase resulted in an increase in 5-KGA of up to 230 mM. Therefore, these newly developed recombinant strains provide a basis for further improving the biotransformation process for 5-KGA production.

Binding of methanol to the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa has been studied by pulsed electron-nuclear double resonance at 9 GHz. Shifts in the hyperfine couplings of the pyrroloquinoline quinone radical provide direct evidence for a change in the environment of the cofactor when substrate is present. By performing experiments with deuteriated methanol, we confirmed that methanol was the cause of the effect. Density functional theory calculations show that these shifts can be understood if a water molecule, which is often found in x-ray structures of the active site of quinoprotein alcohol dehydrogenases, is displaced by the substrate. The difference between the binding of water and methanol is that the water molecule forms a hydrogen bond to O5 of pyrroloquinoline quinone, which the methanol, by virtue of its methyl group, does not. The results support the proposal that aspartate rather than glutamate is the catalytically active base for a hydride transfer mechanism in quinoprotein alcohol dehydrogenases.

Rhodococcus sp. strain DTB (DSM 44534) grows aerobically on diethylether as sole source of carbon and energy. Dense cell suspension experiments showed that the induced ether-cleaving enzyme system attacks a broad range of ethers like tetrahydrofuran, phenetole and chlorinated alkylethers including Cα-substituted alkylethers. Identification of metabolites revealed that degradation of the ethers started by an initial attack of the ether bond. Diethylether-grown cells degraded bis(4-chloro-n-butyl) ether via an initial ether scission followed by the transient accumulation of γ-butyrolactone as intermediate at nearly stoichiometric concentrations.

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-D-gluconic acid (2-KGA) and 5-keto-D-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important L-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO2, 0.05% pCO2) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.

Pyrroloquinoline quinone (2,7,9-tricarboxypyrroloquinoline quinone, PQQ) is one of several quinone cofactors that is utilized in a class of dehydrogenases known as quinoproteins. In this contribution, we have used continuous-wave high-field/high-frequency electron paramagnetic resonance (EPR) at 94 GHz (W-band) to study substrate binding in ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa, taking advantage of the fact that the enzyme is isolated with a substantial proportion of the PQQ cofactor in the paramagnetic semiquinone form. In the substrate-free enzyme, the principal values of the g-tensor, obtained by spectral simulation are: gx = 2.00585(2), gy = 2.00518(2), and gz = 2.00212(2), giving giso = 2.00438(2). All three principal values of the g-tensor decrease when ethanol is bound to the protein: gx = 2.00574(2), gy = 2.00511(2), and gz = 2.00207(2), giving giso = 2.00431(2). The results represent the first direct evidence for the tight binding of an alcohol to a PQQ-dependent alcohol dehydrogenase and show that ethanol also binds to the enzyme even when the PQQ cofactor is in the semiquinone form. The decrease in g is consistent with an increase in polarity in the immediate vicinity of the PQQ cofactor and probably reflects a changed geometry of the PQQ-Ca2+ complex when ethanol binds.

Quinoprotein alcohol dehydrogenases use the pyrroloquinoline quinone (PQQ) cofactor to catalyze the oxidation of alcohols. The catalytic cycle is thought to involve a hydride transfer from the alcohol to the oxidized PQQ, resulting in the generation of aldehyde and reduced PQQ. Reoxidation of the cofactor by cytochrome proceeds in two sequential steps via the PQQ radical. We have used a combination of electron nuclear double resonance and density functional theory to show that the PQQ radical is not protonated at either O-4 or O-5, a result that is at variance with the general presumption of a singly protonated radical. The quantum mechanical calculations also show that reduced PQQ is unlikely to be protonated at O-5; rather, it is either singly protonated at O-4 or not protonated at either O-4 or O-5, a result that also challenges the common assumption of a reduced PQQ protonated at both O-4 and O-5. The reaction cycle of PQQ-dependent alcohol dehydrogenases is revised in light of these findings.

The binding pocket of the pyrroloquinoline quinone (PQQ) cofactor in quinoprotein alcohol dehydrogenases contains a characteristic disulphide ring formed by two adjacent cysteine residues. To analyse the function of this unusual structural motif we have investigated the wild-type and a double cysteine:alanine mutant of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa by electron paramagnetic resonance (EPR) spectroscopy. Thus, we have obtained the principal values for the full rhombic g-tensor of the PQQ semiquinone radical by high-field (94 GHz) EPR necessary for a discrimination of radical species in dehydrogenases containing PQQ together with other redox-active cofactors. Our results show that the characteristic disulphide ring is no prerequisite for the formation of the functionally important semiquinone form of PQQ.

Hydrogen oxidation and electron transport were studied in the chlorobenzene-utilizing anaerobe Dehalococcoides sp. strain CBDB1. While Cu2+ and Hg2+ ions irreversibly inhibited hydrogenase activity in intact cells, Ni2+ ions inhibited reversibly. About 80% of the initial hydrogenase activity was inactivated within 30 s when the cells were exposed to air. In contrast, hydrogenase was active at a redox potential of + 10 mV when this redox potential was established anoxically with a redox indicator. Viologen dyes served both as electron acceptor for hydrogenase and electron donor for the dehalogenase. A menaquinone analogue, 2,3-dimethyl 1,4-naphthoquinone, served neither as electron acceptor for the hydrogenase nor as electron donor for the dehalogenase. In addition, the menaquinone antagonist 2-n-heptyl-4-hydroxyquinoline-N- oxide had no effect on dechlorination catalyzed by cell suspensions or isolated membranes with hydrogen as electron donor, lending further support to the notion that menaquinone is not involved in electron transport. The ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenylhydrazone did not inhibit dechlorination by cell suspensions, indicating that strain CBDB1 does not require reverse electron transport. The ATP-synthase inhibitor N,N'-dicyclohexylcarbodiimide inhibited the dechlorination reaction with cell suspensions; however, the latter effect was partially relieved by the addition of tetrachlorosalicylanilide. 1,2,3,4-Tetrachlorobenzene strongly inhibited dechlorination of other chlorobenzenes by cell suspensions with hydrogen as electron donor, but it did not interfere with either hydrogenase or dehalogenase activity.