Metabolic commensalism and competition in a two-species microbial consortium

We analyzed metabolic interactions and the importance of specific structural relationships in a benzyl alcohol-degrading microbial consortium comprising two species, Pseudomonas putida strain R1 and Acinetobacter strain C6, both of which are able to utilize benzyl alcohol as their sole carbon and energy source. The organisms were grown either as surface-attached organisms (biofilms) in flow chambers or as suspended cultures in chemostats. The numbers of CFU of P. putida R1 and Acinetobacter strain C6 were determined in chemostats and from the effluents of the flow chambers. When the two species were grown together in chemostats with limiting concentrations of benzyl alcohol, Acinetobacter strain C6 outnumbered P. putida R1 (500:1), whereas under similar growth conditions in biofilms, P. putida R1 was present in higher numbers than Acinetobacter strain C6 (5:1). In order to explain this difference, investigations of microbial activities and structural relationships were carried out in the biofilms. Insertion into P. putida R1 of a fusion between the growth rate-regulated rRNA promoter rrnBP1 and a gfp gene encoding an unstable variant of the green fluorescent protein made it possible to monitor the physiological activity of P. putida R1 cells at different positions in the biofilms. Combining this with fluorescent in situ hybridization and scanning confocal laser microscopy showed that the two organisms compete or display commensal interactions depending on their relative physical positioning in the biofilm. In the initial phase of biofilm development, the growth activity of P. putida R1 was shown to be higher near microcolonies of Acinetobacter strain C6. High-pressure liquid chromatography analysis showed that in the effluent of the Acinetobacter strain C6 monoculture biofilm the metabolic intermediate benzoate accumulated, whereas in the biculture biofilms this was not the case, suggesting that in these biofilms the excess benzoate produced by Acinetobacter strain C6 leaks into the surrounding environment, from where it is metabolized by P. putida R1. After a few days, Acinetobacter strain C6 colonies were overgrown by P. putida R1 cells and new structures developed, in which microcolonies of Acinetobacter strain C6 cells were established in the upper layer of the biofilm. In this way the two organisms developed structural relationships allowing Acinetobacter strain C6 to be close to the bulk liquid with high concentrations of benzyl alcohol and allowing P. putida R1 to benefit from the benzoate leaking from Acinetobacter strain C6. We conclude that in chemostats, where the organisms cannot establish in fixed positions, the two strains will compete for the primary carbon source, benzyl alcohol, which apparently gives Acinetobacter strain C6 a growth advantage, probably because it converts benzyl alcohol to benzoate with a higher yield per time unit than P. putida R1. In biofilms, however, the organisms establish structured, surface-attached consortia, in which heterogeneous ecological niches develop, and under these conditions competition for the primary carbon source is not the only determinant of biomass and population structure.     

Characterization of a Pseudomonas putida rough variant evolved in a mixed-species biofilm with Acinetobacter sp. strain C6

Genetic differentiation by natural selection is readily observed among microbial populations, but a more comprehensive understanding of evolutionary forces, genetic causes, and resulting phenotypic advantages is not often sought. Recently, a surface population of Pseudomonas putida bacteria was shown to evolve rapidly by natural selection of better-adapted variants in a mixed-species biofilm consortium (S. K. Hansen, P. B. Rainey, J. A. Haagensen, and S. Molin, Nature 445:533-536, 2007). Adaptation was caused by mutations in a wapH homolog (PP4943) involved in core lipopolysaccharide biosynthesis. Here we investigate further the biofilm physiology and the phenotypic characteristics of the selected P. putida rough colony variants. The coexistence of the P. putida population in a mixed-species biofilm with Acinetobacter sp. strain C6 is dependent on the benzoate excreted from Acinetobacter during the catabolism of benzyl alcohol, the sole carbon source. Examination of biofilm development and the dynamics of the wild-type consortium revealed that the biofilm environment became oxygen limited, possibly with low oxygen concentrations around Acinetobacter microcolonies. In contrast to P. putida wild-type cells, which readily dispersed from the mixed-species biofilm in response to oxygen starvation, the rough variant cells displayed a nondispersal phenotype. However, in monospecies biofilms proliferating on benzoate, the rough variant (like the wild-type population) dispersed in response to oxygen starvation. A key factor explaining this conditional, nondispersal phenotype is likely to be the acquired ability of the rough variant to coaggregate specifically with Acinetobacter cells. We further show that the P. putida rough variant displayed enhanced production of a cellulose-like polymer as a consequence of the mutation in wapH. The resulting phenotypic characteristics of the P. putida rough variant explain its enhanced fitness and ability to form tight structural associations with Acinetobacter microcolonies.     

Metabolic Commensalism and Competition in a Two-Species Microbial Consortium

We analyzed metabolic interactions and the importance of specific structural relationships in a benzyl alcohol-degrading microbial consortium comprising two species, Pseudomonas putida strain R1 and Acinetobacter strain C6, both of which are able to utilize benzyl alcohol as their sole carbon and energy source. The organisms were grown either as surface-attached organisms (biofilms) in flow chambers or as suspended cultures in chemostats. The numbers of CFU of P. putida R1 and Acinetobacter strain C6 were determined in chemostats and from the effluents of the flow chambers. When the two species were grown together in chemostats with limiting concentrations of benzyl alcohol, Acinetobacter strain C6 outnumbered P. putida R1 (500:1), whereas under similar growth conditions in biofilms, P. putida R1 was present in higher numbers than Acinetobacter strain C6 (5:1). In order to explain this difference, investigations of microbial activities and structural relationships were carried out in the biofilms. Insertion into P. putida R1 of a fusion between the growth rate-regulated rRNA promoter rrnBP1 and a gfp gene encoding an unstable variant of the green fluorescent protein made it possible to monitor the physiological activity of P. putida R1 cells at different positions in the biofilms. Combining this with fluorescent in situ hybridization and scanning confocal laser microscopy showed that the two organisms compete or display commensal interactions depending on their relative physical positioning in the biofilm. In the initial phase of biofilm development, the growth activity of P. putida R1 was shown to be higher near microcolonies of Acinetobacter strain C6. High-pressure liquid chromatography analysis showed that in the effluent of the Acinetobacter strain C6 monoculture biofilm the metabolic intermediate benzoate accumulated, whereas in the biculture biofilms this was not the case, suggesting that in these biofilms the excess benzoate produced by Acinetobacter strain C6 leaks into the surrounding environment, from where it is metabolized by P. putida R1. After a few days, Acinetobacter strain C6 colonies were overgrown by P. putida R1 cells and new structures developed, in which microcolonies of Acinetobacter strain C6 cells were established in the upper layer of the biofilm. In this way the two organisms developed structural relationships allowing Acinetobacter strain C6 to be close to the bulk liquid with high concentrations of benzyl alcohol and allowing P. putida R1 to benefit from the benzoate leaking from Acinetobacter strain C6. We conclude that in chemostats, where the organisms cannot establish in fixed positions, the two strains will compete for the primary carbon source, benzyl alcohol, which apparently gives Acinetobacter strain C6 a growth advantage, probably because it converts benzyl alcohol to benzoate with a higher yield per time unit than P. putida R1. In biofilms, however, the organisms establish structured, surface-attached consortia, in which heterogeneous ecological niches develop, and under these conditions competition for the primary carbon source is not the only determinant of biomass and population structure.     

Allylic or benzylic stabilization is essential for catalysis by bacterial benzyl alcohol dehydrogenases.

Benzyl alcohol dehydrogenase from Acinetobacter calcoaceticus (AC-BADH) and TOL plasmid-encoded benzyl alcohol dehydrogenase from Pseudomonas putida (TOL-BADH) have previously been shown to oxidize a variety of aromatic alcohols but not aliphatic substrates. Here, we have expressed the genes for AC-BADH and TOL-BADH in Escherichia coli, purified the resulting over-expressed enzymes, and shown that each is an effective catalyst of both benzylic and allylic alcohol oxidation, but not of oxidation of nonallylic analogs. Enzyme specificity (kcat/Km) for both enzymes was higher with an aliphatic, allylic alcohol (3-methyl-2-buten-1-ol) than with benzyl alcohol. These results suggest that bacterial benzyl alcohol dehydrogenases use the resonance stabilization provided by allylic and benzylic alcohols to promote catalysis.     

Distribution of bacterial growth activity in flow-chamber biofilms.

In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated Escherichia coli rrnBP1 promoter and different variant gfp genes. It is shown that the P1 promoter is regulated in the same way in E. coli and Pseudomonas putida, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.     

Inducible uptake and metabolism of glucose by the phosphorylative pathway in Pseudomonas putida CSV86

Pseudomonas putida CSV86 utilizes glucose, naphthalene, methylnaphthalene, benzyl alcohol and benzoate as the sole source of carbon and energy. Compared with glucose, cells grew faster on aromatic compounds as well as on organic acids. The organism failed to grow on gluconate, 2-ketogluconate, fructose and mannitol. Whole-cell oxygen uptake, enzyme activity and metabolic studies suggest that in strain CSV86 glucose utilization is exclusively by the intracellular phosphorylative pathway, while in Stenotrophomonas maltophilia CSV89 and P. putida KT2442 glucose is metabolized by both direct oxidative and indirect phosphorylative pathways. Cells grown on glucose showed five- to sixfold higher activity of glucose-6-phosphate dehydrogenase compared with cells grown on aromatic compounds or organic acids as the carbon source. Study of [14C]glucose uptake by whole cells indicates that the glucose is taken up by active transport. Metabolic and transport studies clearly demonstrate that glucose metabolism is suppressed when strain CSV86 is grown on aromatic compounds or organic acids.     

Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoaceticus chromosomal benABC and Pseudomonas putida TOL pWW0 plasmid xylXYZ, genes encoding benzoate dioxygenases.

The xylXYZ DNA region is carried on the TOL pWW0 plasmid in Pseudomonas putida and encodes a benzoate dioxygenase with broad substrate specificity. The DNA sequence of the region is presented and compared with benABC, the chromosomal region encoding the benzoate dioxygenase of Acinetobacter calcoaceticus. Corresponding genes from the two biological sources share common ancestry: comparison of aligned XylX-BenA, XylY-BenB, and XylZ-BenC amino acid sequences revealed respective identities of 58.3, 61.3, and 53%. The aligned genes have diverged to assume G+C contents that differ by 14.0 to 14.9%. Usage of the unusual arginine codons AGA and AGG appears to have been selected in the P. putida xylX gene as it diverged from the ancestor it shared with A. calcoaceticus benA. Homologous A. calcoaceticus and P. putida genes exhibit different patterns of DNA sequence repetition, and analysis of one such pattern suggests that mutations creating different DNA slippage structures made a significant contribution to the evolutionary divergence of xylX.     

cis-diol dehydrogenases encoded by the TOL pWW0 plasmid xylL gene and the Acinetobacter calcoaceticus chromosomal benD gene are members of the short-chain alcohol dehydrogenase superfamily.

In the aerobic degradation of benzoate by bacteria, benzoate is first dihydroxylated by a ring-hydroxylating dioxygenase to form a cis-diol (1,2-dihydroxycyclohexa-3,4-diene carboxylate) which is subsequently transformed to a catechol by an NAD(+)-dependent cis-diol dehydrogenase. The structural gene for this dehydrogenase, encoded on TOL plasmid pWW0 of Pseudomonas putida (xylL) and that encoded on the chromosome of Acinetobacter calcoaceticus (benD), were sequenced. They encode polypeptides of about 28 kDa in size. These proteins are similar to each other, exhibiting 58% sequence identity. They are also similar to other proteins of at least 20 different functions, which are members of the short-chain alcohol dehydrogenase family. The alignment of these proteins suggest two amino acids, lysine and tyrosine, as catalytically important residues.     

Characterization of a Pseudomonas putida allylic alcohol dehydrogenase induced by growth on 2-methyl-3-buten-2-ol.

We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacterium Pseudomonas putida MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD+-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of P. putida. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase of Acinetobacter calcoaceticus were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB.     

Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil

The potential for real-time PCR (RTm-PCR) detection of the genetically engineered strain Pseudomonas putida GN2 was studied during 2-chlorobenzoate (2-CB) degradation in three different soils. The strain contained the constructed plasmid pGN2 which encoded genes for 2-CB oxidation (cbdA) and the green fluorescent protein (gfp). P. putida GN2 numbers were assessed by plating onto 2-CB minimal media and also by RTm-PCR detection of cbdA and gfp. Addition of P. putida GN2 decreased the time required to degrade 2-CB in all tested soils by more than 7 days. The RTm-PCR estimations of P. putida GN2 numbers strongly correlated with those obtained from plate count methods during active 2-CB degradation. However, after 2-CB degradation in the soils had ceased, RTm-PCR estimations of cbdA and gfp genes were generally one order of magnitude lower than those from plate counts. These results indicate the potential for RTm-PCR to rapidly determine degrader numbers in soil following bioaugmentation but also the need to exercise caution when attempting to determine cell numbers of degraders from the RTm-PCR quantification of plasmid encoded genes after substrate is depleted.     

Construction of biofilms with defined internal architecture using dielectrophoresis and flocculation

A novel approach was developed for the construction of biofilms with defined internal architecture using AC electrokinetics and flocculation. Artificial structured microbial consortia (ASMC) consisting of localized layered microcolonies of different cell types were formed by sequentially attracting different cell types to high field regions near microelectrodes using dielectrophoresis. Stabilization of the microbial consortia on the electrode surface was achieved by crosslinking the cells using the flocculant polyethyleneimine (PEI). Consortia of Escherichia coli, Micrococcus luteus, and Saccharomyces cerevisiae were made as model systems. Also, more natural consortia were made of the bacteria Pseudomonas putida, Clavibacter michiganense, and Methylobacterium mesophilum, which are found together in consortia during biodegradation of metal-cutting waste fluids.     

Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid.

Pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, TOL. These compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the TOL plasmid. The specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three respective substrates are independent of the carbon source used for growth, suggesting that a single set of nonspecific enzymes is responsible for the dissimilation of the breakdown products of toluene and m- and p-xylene. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are coincidently and possible coordinately induced by toluene and the xylenes, and by the corresponding alcohols and aldehydes. They are not induced in cells grown on m-toluate but catechol 2,3-oxygenase can be induced by m-xylene.     

Metabolism of benzyl alcohol via catechol ortho-pathway in methylnaphthalene-degrading Pseudomonas putida CSV86

Pseudomonas putida CSV86 metabolizes 1- and 2-methylnaphthalene through distinct catabolic and detoxification pathways. In spite of the similarity in the steps involved in the methylnaphthalene detoxification and the toluene side-chain hydroxylation pathways, the strain failed to utilize toluene or xylenes. However, it could grow on benzyl alcohol, 2- and 4-hydroxybenzyl alcohol. Metabolic studies suggest that the benzyl alcohol metabolism proceeds via the benzaldehyde, benzoate, and catechol ortho-cleavage pathway, in contrast to the well established catechol meta-cleavage pathway. Carbon source-dependent enzyme activity studies suggest that the degradation of aromatic alcohol involves two regulons. Aromatic alcohol induces the upper regulon, which codes for aromatic alcohol- and aromatic aldehyde-dehydrogenase and converts alcohol into acid. The aromatic acid so generated induces the specific lower regulon and is metabolized via either the ortho- or the meta-cleavage pathway. CSV86 cells transform 1- and 2-methylnaphthalene to 1- and 2-hydroxymethyl naphthalene, which are further converted to the respective naphthoic acids due to the basal level expression and broad substrate specificity of the upper regulon enzymes.     

Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium

Acinetobacter sp. strain ADP1 is a nutritionally versatile soil bacterium closely related to representatives of the well-characterized Pseudomonas aeruginosa and Pseudomonas putida. Unlike these bacteria, the Acinetobacter ADP1 is highly competent for natural transformation which affords extraordinary convenience for genetic manipulation. The circular chromosome of the Acinetobacter ADP1, presented here, encodes 3325 predicted coding sequences, of which 60% have been classified based on sequence similarity to other documented proteins. The close evolutionary proximity of Acinetobacter and Pseudomonas species, as judged by the sequences of their 16S RNA genes and by the highest level of bidirectional best hits, contrasts with the extensive divergence in the GC content of their DNA (40 versus 62%). The chromosomes also differ significantly in size, with the Acinetobacter ADP1 chromosome <60% of the length of the Pseudomonas counterparts. Genome analysis of the Acinetobacter ADP1 revealed genes for metabolic pathways involved in utilization of a large variety of compounds. Almost all of these genes, with orthologs that are scattered in other species, are located in five major 'islands of catabolic diversity', now an apparent 'archipelago of catabolic diversity', within one-quarter of the overall genome. Acinetobacter ADP1 displays many features of other aerobic soil bacteria with metabolism oriented toward the degradation of organic compounds found in their natural habitat. A distinguishing feature of this genome is the absence of a gene corresponding to pyruvate kinase, the enzyme that generally catalyzes the terminal step in conversion of carbohydrates to pyruvate for respiration by the citric acid cycle. This finding supports the view that the cycle itself is centrally geared to the catabolic capabilities of this exceptionally versatile organism.     

Stereospecificity in meta-fission catabolic pathways

The stereospecificities of 2-keto-4-pentenoate hydratase and 4-hydroxy-2-ketovalerate aldolase were studied in Escherichia coli, Pseudomonas putida, and Acinetobacter sp. Hydration was stereospecific in all three; however, only P. putida and Acinetobacter sp. showed stereospecificity in their aldolase reactions.     

Benzyl alcohol metabolism by Pseudomonas putida: a paradox resolved

Evidence is presented for the existence in Pseudomonas putida of two NAD-linked dehydrogenases that function sequentially to oxidize benzyl alcohol. Induction of muconate lactonizing enzyme, a 3-oxoadipate pathway enzyme, indicated that P. putida oxidized benzyl alcohol to benzoate. Polyacrylamide gel electrophoresis with activity staining and enzymatic assays for an NAD-dependent dehydrogenase both showed that cells contained a single, constitutive alcohol dehydrogenase capable of oxidizing benzyl alcohol. This enzyme was shown to have the same specificity in extracts of glucose-grown as in benzy alcoholgrown cells. An NAD-aldehyde dehydrogenase oxidized benzaldehyde but was most active with normal alkyl aldehydes. This aldehyde dehydrogenase was shown to be induced, by enzymatic assays and by activity staining of polyacrylamide gel electropherograms, not only in cells grown on benzyl alcohol, but also in cells grown on ethanol. These experiments suggested that the aldehyde dehydrogenase was induced by the alcohol being oxidized rather than the substrate aldehyde.In sum, the evidence from enzyme assays and polyacrylamide gel electrophoresis of extracts indicates that Pseudomonas putida catabolizes benzyl alcohol slowly when it is the sole carbon and energy source, by the action of a constitutive, nonspecific, alcohol dehydrogenase and an alcohol-induced, nonspecific aldehyde dehydrogenase to yield benzoate, which is further metabolized via the 3-oxoadipate (beta-ketoadipate) pathway.In memory of R. Y. Stanier     

Regulation of growth of Acinetobacter calcoaceticus NCIB8250 on L-mandelate in batch culture.

Batch culture of Acinetobacter calcoaceticus in L-mandelate- or phenylglyoxylate-salts medium showed an unusual non-exponential pattern unless the inoculum had been grown on benzyl alcohol. There were transient accumulations of benzaldehyde and benzyl alcohol caused by the limitation of L-mandelate oxidation by low activities of benzaldehyde dehydrogenase and the diversion of reducing power to the formation of benzyl alcohol. In vivo enzymic activities were estimated from patterns of substrate utilization in batch cultures containing pairs of substrates. When bacteria previously grown in L-mandelate-salts medium were inoculated into media containing L-mandelate and a second carbon source, metabolism of L-mandelate was arithmetical in the presence of benzoate, catechol or succinate, but accelerated on exhaustion of the second substrate. This indicated repression of the enzymes involved in L-mandelate oxidation. Inoculation of bacteria grown in benzoate-salts medium into medium containing L-mandelate and benzoate gave diauxie with initial utilization of benzoate. Similar experiments showed that benzoate oxidation was not repressed by catechol and only partially repressed by succinate. Measurement of L-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I in bacterial extracts showed no evidence for feedback inhibition by intermediates of the pathway. The rates of L-mandelate and benzoate utilization by bacterial suspensions were inhibited by succinate and catechol but not by other intermediates of the pathway.     

In vivo enzymology: a deuterium NMR study of formaldehyde dismutase in Pseudomonas putida F61a and Staphylococcus aureus

High-resolution deuterium NMR spectroscopy has been used to follow the detoxifying metabolism of [D2]formaldehyde in vivo in several bacterial species. Production of [D2]methanol in Escherichia coli confirms that the oxidation and reduction pathways of metabolism are independent in this organism. Efficient production of equimolar quantities of [D]formate and [D3]methanol in Pseudomonas putida F61a and Staphylococcus aureus implicates a formaldehyde dismutase, or "cannizzarase", activity. These observations imply that the unusual formaldehyde resistance in P. putida F61a is a direct result of efficient dismutation acting as a route for detoxification. Cross-dismutation experiments yield an enzymic kinetic isotope effect of ca. 4 for H vs D transfer and a similar spectrum of substrate specificity to the isolated enzyme. [D]benzyl alcohol produced by cross-dismutation of [D2]formaldehyde and benzaldehyde in P. putida is demonstrated to have the R configuration by a novel deuterium NMR assay. Additionally, S. aureus produces methyl formate as a product of formaldehyde detoxification, apparently by oxidizing the methanol hemiacetal of formaldehyde.     

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Substrate specificities and inhibition studies.

The apparent Km and maximum velocity values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus were determined for a range of alcohols and aldehydes and the corresponding turnover numbers and specificity constants were calculated. Benzyl alcohol was the most effective alcohol substrate for benzyl alcohol dehydrogenase. Perillyl alcohol was the second most effective substrate, and was the only non-aromatic alcohol oxidized. The other substrates of benzyl alcohol dehydrogenase were all aromatic in nature, with para-substituted derivatives of benzyl alcohol being better substrates than other derivatives. Coniferyl alcohol and cinnamyl alcohol were also substrates. Benzaldehyde was much the most effective substrate for benzaldehyde dehydrogenase II. Benzaldehydes with a single small substituent group in the meta or para position were better substrates than any other benzaldehyde derivatives. Benzaldehyde dehydrogenase II could also oxidize the aliphatic aldehydes hexan-1-al and octan-1-al, although poorly. Benzaldehyde dehydrogenase II was substrate-inhibited by benzaldehyde when the assay concentration exceeded approx. 10 microM. Benzaldehyde dehydrogenase II, but not benzyl alcohol dehydrogenase, exhibited esterase activity with 4-nitrophenyl acetate as substrate. Both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were inhibited by the thiol-blocking reagents iodoacetate, iodoacetamide, 4-chloromercuribenzoate and N-ethylmaleimide. Benzyl alcohol or benzaldehyde respectively protected against these inhibitions. NAD+ also gave some protection. Neither benzyl alcohol dehydrogenase nor benzaldehyde dehydrogenase II was inhibited by the metal-ion-chelating agents EDTA, 2,2'-bipyridyl, pyrazole or 2-phenanthroline. Neither enzyme was inhibited by a range of plausible metabolic inhibitors such as mandelate, phenylglyoxylate, benzoate, succinate, acetyl-CoA, ATP or ADP. Benzaldehyde dehydrogenase II was sensitive to inhibition by several aromatic aldehydes; in particular, ortho-substituted benzaldehydes such as 2-bromo-, 2-chloro- and 2-fluoro-benzaldehydes were potent inhibitors of the enzyme.