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Evidence for multiple adaptive peaks from populations of bacteria evolving in a structured habitat.

Natural selection tends to promote the divergence of populations living in different environments. Even in identical environments, however, replicate populations may diverge if they find alternative adaptive solutions. We describe the evolution of 18 bacterial populations (Comamonas sp.) founded from a single progenitor genotype and propagated separately for 1000 generations in two distinct environments, one physically unstructured (mass-action liquid) and the other structured (agar surfaces). Phenotypic diversity, as reflected in colony morphology, was greater in the structured habitat than in the unstructured habitat. More importantly, the trajectories for mean fitness, as measured by competition against the common ancestor, were more divergent for populations in the structured habitat than those in the unstructured habitat. Structured environments may accelerate evolutionary diversification by promoting genetic polymorphisms within populations, thereby increasing the complexity of genetic constraints that allow divergence among replicate populations.

Adaptation, Biological↗

Steady-state kinetic characterization and crystallization of a polychlorinated biphenyl-transforming dioxygenase.

The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe(2)S(2) center and catalytic iron. Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant. X-ray diffraction was measured to 1.6 A. Steady-state kinetics assays demonstrated that BPDO had an apparent k(cat)/K(m) for biphenyl of (1.2 +/- 0.1) x 10(6) M(-1) s(-1) in air-saturated buffer. Moreover, BPDO transformed dichlorobiphenyls (diClBs) in the following order of apparent specificities: 3,3'- > 2,2'- > 4, 4'-diClB. Strikingly, the ability of BPDO to utilize O(2) depended strongly on the biphenyl substrate: k(cat)/K(m(O(2))) = (3.6 +/- 0. 3), (0.06 +/- 0.02), and (0.4 +/- 0.07) x 10(5) M(-1) s(-1) in the presence of biphenyl and 2,2'- and 3,3'-diClBs, respectively. Moreover, biphenyl/O(2) consumed was 0.97, 0.44, 0.63, and 0.48 in the presence of biphenyl and 2,2'-, 3,3'-, and 4,4'-diClBs, respectively. Within experimental error, the balance of consumed O(2) was detected as H(2)O(2). Thus, PCB congeners such as 2, 2'-diClB exact a high energetic cost, produce a cytotoxic compound (H(2)O(2)), and can inhibit degradation of other congeners. Each of these effects would be predicted to inhibit the aerobic microbial catabolism of PCBs.

Dioxygenases↗

Bacterial diversity in spent mushroom compost assessed by amplified rDNA restriction analysis and sequencing of cultivated isolates.

Spent mushroom compost (SMC) is the residual by-product of commercial Agaricus spp. cultivation, and it is mainly composed of a thermally treated cereal straw/animal manure mixture colonized by the fungal biomass. Research on the valorization of this material is mainly focusing on its use as soil conditioner and plant fertilizer. An investigation of the bacterial diversity in SMC was performed using molecular techniques in order to reveal the origin of SMC microflora and its potential effect on soil microbial communities after incorporation into agricultural soils. The bacterial population was estimated by the plate count method to a mean of 2.7 10(9) colony forming units (cfu) per g of dry weight, while the numbers of Gram-positive and Gram-negative bacteria were 1.9 10(9) and 4.9 10(8) cfu per g dw respectively as estimated by enumeration on semi-selective media. Fifty bacterial isolates were classified into 14 operational taxonomic units (OTUs) following ARDRA-PCR of the 16S rDNA gene. Sequencing of the 16S rDNA amplicon assigned 12 of the 14 OTUs to Gram-positive bacteria, associated with the genera Bacillus, Paenibacillus, Exiguobacterium, Staphylococcus, Desemzia, Carnobacterium, Brevibacterium, Arthrobacter and Microbacterium of the bacterial divisions Firmicutes and Actinobacteria. Two bacterial groups have phylogenetic links with the genera Comamonas and Sphingobacterium, which belong to beta-Proteobacteria and Bacteroidetes respectively. Two potentially novel bacteria are reported, which are associated with the genera Bacillus and Microbacterium. Most of the bacteria identified are of environmental origin, while strains related to species usually isolated from insects, animal and clinical sources were also detected. It appears that bacterial diversity in SMC is greatly affected by the origin of the initial material, its thermal pasteurization treatment and the potential unintended colonization of the mushroom substrate during the cultivation process.

Agaricus↗

Physiological and genetic characteristics of two bacterial strains utilizing phenoxypropionate and phenoxyacetate herbicides.

Two strains, Rhodoferax sp. P230 and Delftia (Comamonas) acidovorans MCI, have previously been shown to carry activities for the degradation of the two enantiomers of (RS)-2-(2,4-dichlorophenoxy-)propionate (dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy-)propionate (mecoprop) and, in addition, are capable of degrading phenoxyacetate derivatives 2.4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA). Metabolism of the herbicides is initiated by alpha-ketoglutarate-dependent dioxygenases for both enantiomers of the phenoxypropionate herbicides and for 2,4-D. These activities were constitutively expressed for both enantiomers of dichlorprop in strain MC1 and for the Renantiomer in strain P230. Enzyme activities for the complete degradation of phenoxyacetate and phenoxypropionate herbicides were induced during incubation on either of these herbicides. Strain MC1 has about threefold higher activities for the degradation of dichlorprop and for growth on this substrate (mumax = 0.15 h(-1)) than strain P230; the maximum growth rate on 2,4-D amounts to 0.045 h(-1) with strain MC1. Dichlorprop is utilized faster than mecoprop and the R-enantiomers are cleaved with higher rates than the S-enantiomers. The degradation of the chlorophenolic intermediates seems to proceed via the modified ortho cleavage pathway as indicated by activities of the respective enzymes. The enzymatic results were supported by genetic investigations by which the presence of the genes tfdB (encoding a dichlorophenol hydroxylase), tfdC (encoding a chlorocatechol 1,2-dioxygenase) and tfdD (encoding a chloromuconate cycloisomerase) could be demonstrated in both strains by PCR after application of respective primers. The presence of the tfdA gene (encoding a 2,4-D/alpha-ketoglutarate dioxygenase) was only shown for strain P230 but was lacking in strain MC1. Sequence analysis of the tfd gene fragments revealed high homology to the degradative genes of other proteobacterial strains degrading chloroaromatic compounds. Strain MC1 carries a plasmid of about 120 kb which apparently harbors herbicide degradative genes as concluded from deletion mutants which have lost 2,4-D[phenoxalkanoate]/alpha-ketoglutarate dioxygenase activities for cleavage of the R- and S-enantiomer, and of 2,4-D. For strain P230, no plasmid could be demonstrated; the activity was stably conserved in this strain during growth under nonselective conditions.

2,4-Dichlorophenoxyacetic Acid↗

Investigation of product quality between extemporaneously compounded progesterone vaginal suppositories and an approved progesterone vaginal gel.

The purpose of this investigation was to compare quality parameters, including product appearance, content uniformity, pH, weight uniformity, microbial limit testing and preservative effectiveness testing on extemporaneously compounded progesterone vaginal suppositories obtained from 10 randomly chosen compounding pharmacies (90 suppositories each) across the United States, to the Food and Drug Administration (FDA) approved prescription progesterone gel product (Prochieve/Crinone) which is manufactured in a cGMP regulated facility. The content uniformity and pH were determined using qualified methods. The microbial limits testing and preservative effectiveness testing were conducted according to compendial methods. Only one pharmacy provided suppositories that were all within the potency limits required for the prescription progesterone gel product. The other pharmacies provided at least some suppositories where progesterone content was either subpotent or superpotent for progesterone. The pH of most of the compounded suppository products was in the range of 4.22 to 7.68 with a median of 6.30 (normal vaginal pH is <5), whereas the gel product was 2.80. For compounded product from one of the pharmacies, microbial limits testing indicated CDC group IVC-2 and Comamonas acidovorans were detected. This data indicates that pharmacy compounded delivery systems for progesterone should be used with caution.

Chemistry, Pharmaceutical↗

Molecular breeding of 2,3-dihydroxybiphenyl 1,2-dioxygenase for enhanced resistance to 3-chlorocatechol.

3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 1.13.11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.

Catechols↗

New inhibitors of iron-containing nitrile hydratases.

There is growing evidence in the literature emphasizing the significance of the post-translational modification of cysteine thiols to sulfenic acids (SOH), which have been found in a number of proteins. Crystallographic and mass spectrometric evidence has shown the presence of this group in an inactive form of the industrially important enzyme nitrile hydratase (NHase). This oxidized cysteine is unique in that it forms part of the coordination sphere of the low-spin iron III at the active site of the enzyme. The presence of this unstable sulfenic group in the active form of NHase is the subject of some controversy. To try to detect this function in NHase, we have studied the inhibitory effect on nitrile hydration of reagents known to react with sulfenic acids. Two NHases were studied, namely, Rhodococcus rhodochrous R312 NHase and Comamonas testosteroni NI1 NHase, and the reagents used were meta-chlorocarbonyldicyano-phenylhydrazone (m-ClCP), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), and 2-nitro-5-thiocyanato-benzoic acid (NTBA). Following this approach we report three novel inhibitors of NHases. In addition, we report thiocyanate reagents that can be used to monitor NHase activity spectroscopically.

4-Chloro-7-nitrobenzofurazan↗

Telluria mixta (Pseudomonas mixta Bowman, Sly, and Hayward 1988) gen. nov., comb. nov., and Telluria chitinolytica sp. nov., soil-dwelling organisms which actively degrade polysaccharides.

Pseudomonas mixta (type strain, ACM 1762 [= ATCC 49108], an actively dextranolytic species that possesses both lateral and polar flagella, was compared with the strictly aerobic, rod-shaped, chitinolytic bacterium "Pseudomonas chitinolytica" ACM 3522T (= CNCM I-804) (T = type strain), which has a similar flagellation pattern, by performing phenotypic characterization and DNA-DNA hybridization studies and by analyzing DNA base compositions and 16S rRNA sequences. Our results indicated that "P. chitinolytica" ACM 3522T was phenotypically and genotypically distinct from P. mixta and other phenotypically analogous Pseudomonas spp., Xanthomonas maltophilia, and other aerobic chitin degraders. The 16S rRNA sequences of strains ACM 1762T and ACM 3522T were found to be very similar (97%) to each other and indicated that these organisms are proteobacteria that belong to the beta subclass. The strains were deeply branched in the beta subclass and were distinct from other pseudomonads, including Pseudomonas cepacia, and from Comamonas testosteroni. On the basis of phenotypic, genotypic, and phylogenetic evidence, it is proposed that P. mixta and "P. chitinolytica" ACM 3522T represent two distinct species in a new genus called Telluria. Thus, the genus Telluria gen. nov. contains Telluria mixta comb. nov. and Telluria chitinolytica sp. nov., which are strictly aerobic, rod-shaped, soil-dwelling bacteria that are active polysaccharide degraders.

Base Composition↗

Sutterella wadsworthensis gen. nov., sp. nov., bile-resistant microaerophilic Campylobacter gracilis-like clinical isolates.

Campylobacter gracilis (formerly Bacteroides gracilis) is an asaccharolytic, nitrate-positive, urease-negative organism that requires formate and fumarate or hydrogen as a growth additive and may pit agar media. Clinical isolates that were obtained primarily from appendiceal and peritoneal fluid specimens and initially were identified in our laboratory as B. gracilis were later found to include "unusual" strains that could be distinguished by biochemical and genetic criteria. These unusual C. gracilis strains were bile resistant, could not reduce tetrazolium chloride under aerobic conditions if formate and fumarate were added to the medium, and could grow in the presence of 2 or 6% oxygen if no blood was added to the medium. C. gracilis, other campylobacters, and the unusual strains produced distinctive dehydrogenase patterns when gels were incubated anaerobically. A cellular fatty acid analysis revealed that the cluster formed by the unusual organisms was distinct from the (separate) clusters formed by C. gracilis, Bacteroides ureolyticus, and other Campylobacter species. 16S rRNA sequence data indicated that these organisms are not related phylogenetically to either C. gracilis or other Campylobacter species; the most closely related taxa as determined by rRNA sequence analysis were unrelated aerobes (members of the genera Bordetella, Alcaligenes, Rhodocyclus, and Comamonas). DNA homology data confirmed that these taxa are separate groups. Our data indicate that the unusual organisms are members of a new genus and new species, for which we propose the name Sutterella wadsworthensis. The type strain of S. wadsworthensis is strain WAL 9799 (= ATCC 51579).

Base Sequence↗

Microvirgula aerodenitrificans gen. nov., sp. nov., a new gram-negative bacterium exhibiting co-respiration of oxygen and nitrogen oxides up to oxygen-saturated conditions.

A denitrifier micro-organism was isolated from an upflow denitrifying filter inoculated with an activated sludge. The cells were Gram-negative, catalase- and oxidase-positive curved rods and very motile. They were aerobic as well as anoxic heterotrophs that had an atypical respiratory type of metabolism in which oxygen and nitrogen oxides were used simultaneously as terminal electron acceptors. The G&C content was 65 mol%. Our isolate was phenotypically similar to Comamonas testosteroni, according to classical systematic classification systems. However, a phylogenetic analysis based on the 165 rRNA sequence showed that the aerobic denitrifier could not be assigned to any currently recognized genus. For these reasons a new genus and species, Microvirgula aerodenitrificans gen. nov., sp. nov., is proposed, for which SGLY2T is the type strain.

Base Sequence↗

In situ detection of bacteria in continuous-flow cultures of seawater sediment suspensions with fluorescently labelled rRNA-directed oligonucleotide probes.

rRNA-targeted and fluorescently labelled oligonucleotide probes were used to study the composition of natural bacterial populations in continuous-flow cultures of seawater sediment suspensions. The cultures were run as enrichment cultures with increasing dilution rates, and hexadecane as the sole carbon source. Total cell numbers were analysed by counting DAPI (4',6-diamidino-2-phenylindole)-stained cells. To differentiate the population composition, oligonucleotide probes for eubacteria, for Cytophaga/Flavobacteria, and for four subclasses of the Proteobacteria (alpha, beta, gamma and delta) were used. About 40-80% of the DAPI-stained cells could be detected with the EUB338 probe. Moreover, it was possible to detect a shift in the composition of the natural bacterial population with increasing dilution rate of the continuous culture, from large amounts of Cytophaga/Flavobacteria to large numbers of members of the gamma-Proteobacteria. The cell recovery rate for bacteria labelled with specific oligonucleotide probes was analysed with defined cell numbers of Rhodospirillum rubrum, Comamonas testosteroni and Desulfovibrio vulgaris subsp. vulgaris introduced into the seawater sediment suspension, and was determined to be 13.9-33.5%. The standard deviation determined for this method applied to sediment suspensions was +/- 8.3%. The results suggest that the application of the in situ hybridization technique allows a good insight into the structure of populations growing in sediment suspensions.

Alkanes↗

An inducible 1-butanol dehydrogenase, a quinohaemoprotein, is involved in the oxidation of butane by "Pseudomonas butanovora".

Butane-grown "Pseudomonas butanovora" expressed two soluble alcohol dehydrogenases (ADHs), an NAD(+)-dependent secondary ADH and an NAD(+)-independent primary ADH. Two additional NAD(+)-dependent secondary ADHs could be detected when cells were grown on 2-butanol and lactate. The inducible NAD(+)-independent 1-butanol dehydrogenase (BDH) of butane-grown cells was primarily responsible for 1-butanol oxidation in the butane metabolism pathway. BDH was purified to near homogeneity and identified as a quinohaemoprotein, containing, per mol enzyme, 1.0 mol pyrroloquinoline quinone (PQQ) and 0.25 mol haem c as prosthetic groups. BDH was synthesized as a monomer of approximately 66 kDa. It has a broad substrate range, including primary alcohols, secondary alcohols, aldehydes, C(4) diols and aromatic alcohols. It exhibited the lowest K:(m) (7+/-1 microM) and highest k(cat)/K:(m) (72x10(4) M(-1) s(-1)) value towards 1-butanol. BDH exhibited ferricyanide-dependent ADH activity. Calcium ions (up to 10 mM) increased BDH activity substantially. Two BDH internal amino acid sequences showed 73 and 62% identity and 83 and 66% similarity, respectively, when compared with an amino acid sequence of ethanol dehydrogenase from Comamonas testosteroni. The presence of the inducible BDH and secondary ADH may indicate that the terminal and subterminal oxidation pathways are involved in butane degradation of butane-grown "P. butanovora".

Alcohol Oxidoreductases↗

RP4::Mu3A-mediated in vivo cloning and transfer of a chlorobiphenyl catabolic pathway.

Chromosomal DNA fragments encoding the ability to utilize biphenyl as sole carbon source (Bph+) were mobilized by means of plasmid RP4::Mu3A from strain JB1 (tentatively identified as Burkholderia sp.) to Alcaligenes eutrophus CH34 at a frequency of 10(-3) per transferred plasmid. The mobilized DNA integrated into the recipient chromosome or was recovered as catabolic prime plasmids. Three Bph+ prime plasmids were transferred from A. eutrophus to Escherichia coli and back to A. eutrophus without modification of the phenotype. The transferred Bph+ DNA segments allowed metabolism of biphenyl, 2-, 3- and 4-chlorobiphenyl, and diphenylmethane. Genes involved in biphenyl degradation were identified on the prime plasmids by DNA-DNA hybridization and by gene cloning. Bph+ prime plasmids were transferred to Burkholderia cepacia, Pseudomonas aeruginosa, Comamonas testosteroni and A. eutrophus and the catabolic genes were expressed in those hosts. Transfer of the plasmid to the 3-chlorobenzoate-degrading bacterium Pseudomonas sp. B13 allowed the recipient to mineralize 3-chlorobiphenyl. Other catabolic prime plasmids were obtained from JB1 by selection on m-hydroxybenzoate and tyrosine as carbon sources. 16S rRNA sequence data demonstrated that the in vivo transfer of bph was achieved between bacteria belonging to two different branches of the beta-Proteobacteria.

Alcaligenes↗

Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes.

Bacteria with ability to degrade polyaromatic hydrocarbons (PAHs), isolated from wastewater and soil samples, were investigated for their taxonomic, physiological and genetic diversity. Eighteen isolates able to metabolize naphthalene or phenanthrene as sole carbon source were taxonomically affiliated to different subclasses of the Proteobacteria (Sphingomonas spp., Acidovorax spp., Comamonas spp. and Pseudomonas spp.) and to phyla of Gram-positive bacteria with low and high DNA G + C content (Paenibacillus sp. and Rhodococcus spp., respectively). Representatives of the genera Pseudomonas and Sphingomonas formed a remarkably high fraction of these isolates; 9 out of 18 strains belonged to these groups. Tests for enzyme activities showed that the majority of the isolates growing with PAHs as sole sources of carbon and energy had an active catechol 2,3-dioxygenase (C230). C230 specific activities were very diverse, ranging from 0.1 to 650 mU (mg protein)-1. Pseudomonas and Sphingomonas strains showed considerably higher activities than the other isolates. All PAH degraders were examined for the presence of an initial PAH dioxygenase and C230, which catalyse key steps of PAH degradation, by PCR amplification of gene fragments and subsequent hybridization. PCR primers and internal oligonucleotide probes were developed for the specific detection of the genes of Pseudomonas and Sphingomonas strains.

Catechol 2,3-Dioxygenase↗

Herbaspirillum chlorophenolicum sp. nov., a 4-chlorophenol-degrading bacterium.

A 4-chlorophenol-degrading bacterial strain, formerly designated as a strain of Comamonas testosteroni, was reclassified as a member of the genus Herbaspirillum based on its phenotypic and chemotaxonomic characteristics, as well as phylogenetic analysis using 16S rDNA sequences. Phylogenetic inference based on 16S rDNA sequences showed that strain CPW301(T) clusters in a phylogenetic branch that contains Herbaspirillum species. 16S rDNA sequence similarity of strain CPW301(T) to species of the genus Herbaspirillum with validly published names is in the range 98.7-98.9 %. Despite the considerably high 16S rDNA sequence similarity, strain CPW301(T) could be distinguished clearly from type strains of Herbaspirillum species with validly published names by DNA-DNA relatedness values, which were <15.7 %. The genomic DNA G+C content of strain CPW301(T) is 61.3 mol%. The predominant ubiquinone is Q-8 and the major cellular fatty acids are C(16 : 0) and cyclo-C(17 : 0). The strain does not fix nitrogen and is not plant-associated. It is an aerobic rod with one unipolar flagellum. On the basis of these characteristics, a novel Herbaspirillum species, Herbaspirillum chlorophenolicum sp. nov., is proposed. The type strain of the novel species is strain CPW301(T) (=KCTC 12096(T)=IAM 15024(T)).

Base Composition↗

Characterization of a soil-derived bacterial consortium degrading 4-chloroaniline.

A bacterial consortium comprising four different species was isolated from an Indonesian agricultural soil using a mixture of aniline and 4-chloroaniline (4CA) as principal carbon sources. The four species were identified as Chryseobacterium indologenes SB1, Comamonas testosteroni SB2, Pseudomonas corrugata SB4 and Stenotrophomonas maltophilia SB5. Growth studies on aniline and 4CA as single and mixed substrates demonstrated that the bacteria preferred to grow on and utilize aniline rather than 4CA, although both compounds were eventually depleted from the culture supernatant. However, despite 100 % disappearance of the parent substrates, the degradation of 4CA was always characterized by incomplete dechlorination and 4-chlorocatechol accumulation. This result suggests that further degradation of 4-chlorocatechol may be the rate-limiting step in the metabolism of 4CA by the bacterial consortium. HPLC-UV analysis showed that 4-chlorocatechol was further degraded via an ortho-cleavage pathway by the bacterial consortium. This hypothesis was supported by the results from enzyme assays of the crude cell extract of the consortium revealing catechol 1,2-dioxygenase activity which converted catechol and 4-chlorocatechol to cis,cis-muconic acid and 3-chloro-cis,cis-muconic acid respectively. However, the enzyme had a much higher conversion rate for catechol [156 U (g protein)(-1)] than for 4-chlorocatechol [17.2 U (g protein)(-1)], indicating preference for non-chlorinated substrates. Members of the bacterial consortium were also characterized individually. All isolates were able to assimilate aniline. P. corrugata SB4 was able to grow on 4CA solely, while S. maltophilia SB5 was able to grow on 4-chlorocatechol. These results suggest that the degradation of 4CA in the presence of aniline by the bacterial consortium was a result of interspecies interactions.

Aniline Compounds↗

Cloning and expression of the gene for periplasmic poly(vinyl alcohol) dehydrogenase from Sphingomonas sp. strain 113P3, a novel-type quinohaemoprotein alcohol dehydrogenase.

A gene for periplasmic poly(vinyl alcohol) (PVA) dehydrogenase (PVADH) was cloned, based on the N-terminal amino acid sequence of the purified PVADH from Sphingomonas sp. 113P3 and the sequence of the gene for PVADH (pvaA, GenBank accession no. AB190288). The recombinant PVADH tagged with hexahistidine was expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme had the same characteristics as the purified enzyme from Sphingomonas sp. strain 113P. In addition to PVA, the recombinant PVADH could oxidize glycols such as polypropylene glycols and 1,3-butane/cyclohexanediol and 2,4-pentanediol, but neither primary nor secondary alcohols. The amino acid sequence of the recombinant PVADH showed similarity with those of PVADH from Pseudomonas sp. strain VM15C, putative PVADHs from Azoarcus sp. EbN1, and Xanthomonas species (54-25 % identity), and the quinohaemoprotein alcohol dehydrogenases (QH-ADHs) from Comamonas testosteroni, Ralstonia eutropha and Pseudomonas putida (25-29 % identity). PVADHs from strains 113P3 and VM15C have a conserved superbarrel domain (SD), probable PQQ-binding amino acids in the SD and a haem-binding domain (HBD) (they should be designated QH-PVADHs), but the positions of the amino acid sequences for the HBD and SD are the reverse of those of QH-ADHs. A protein structure of QH-PVADHs is proposed. Results of dot-blot hybridization and RT-PCR indicated that the three genes encoding oxidized PVA hydrolase, PVADH and cytochrome c are expressed constitutively and form an operon.

Alcohol Oxidoreductases↗

The atomic resolution structure of methanol dehydrogenase from Methylobacterium extorquens.

The crystal structure of methanol dehydrogenase (MDH) from Methylobacterium extorquens has been refined without stereochemical restraints at a resolution of 1.2 A. The high-resolution data have defined the conformation of the tricyclic pyrroloquinoline quinone (PQQ) cofactor ring as entirely planar. The detailed definition of the active-site geometry has shown many features that are similar to the quinohaemo-protein alcohol dehydrogenases from Comamonas testosteroni and Pseudomonas putida, both of which possess MDH-like and cytochrome c-like domains. Conserved features between the two types of PQQ-containing enzyme suggest a common pathway for electron transfer between MDH and its physiological electron acceptor cytochrome cL. A pathway for proton transfer from the active site to the bulk solvent is also suggested.

Alcohol Dehydrogenase↗