Structural basis of the stereospecificity of bacterial B12-dependent 2-hydroxyisobutyryl-CoA mutase.

Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile(A90) and Asp(A117). Asp(A117) determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.

Thermophilic Coenzyme B12-Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of (R)-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA.

The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502-15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of thioesterase II, are discussed.

Exploiting mixtures of H2, CO2, and O2 for improved production of methacrylate precursor 2-hydroxyisobutyric acid by engineered Cupriavidus necator strains.

Current manufacturing of most bulk chemicals through petrochemical routes considerably contributes to common concerns over the depletion of fossil carbon sources and greenhouse gas emissions. Sustainable future production of commodities thus requires the shift to renewable feedstocks in combination with established or newly developed synthesis routes. In this study, the potential of Cupriavidus necator H16 for autotrophic synthesis of the building block chemical 2-hydroxyisobutyric acid (2-HIBA) is evaluated. A novel biosynthetic pathway was implemented by heterologous expression of the 2-hydroxyisobutyryl-coenzyme A (2-HIB-CoA) mutase from Aquincola tertiaricarbonis L108, relying on a main intermediate of strain H16's C4 overflow metabolism, 3-hydroxybutyryl-CoA. The intention was to direct the latter to 2-HIBA instead or in addition to poly-3-hydroxybutyrate (PHB). Autotrophic growth and 2-HIBA (respectively, PHB) synthesis of wild-type and PHB-negative mutant strains were investigated producing maximum 2-HIBA titers of 3.2 g L(-1) and maximum specific 2-HIBA synthesis rates (q 2-HIBA) of about 16 and 175 µmol g(-1) h(-1), respectively. The obtained specific productivity was the highest reported to date for mutase-dependent 2-HIBA synthesis from heterotrophic and autotrophic substrates. Furthermore, expression of a G protein chaperone (MeaH) in addition to the 2-HIB-CoA mutase subunits yielded improved productivity. Analyzing the inhibition of growth and product synthesis due to substrate availability and product accumulation revealed a strong influence of 2-HIBA, when cells were cultivated at high titers. Nevertheless, the presented results imply that at the time the autotrophic synthesis route is superior to thus far established heterotrophic routes for production of 2-HIBA with C. necator.

Adaptation of Delftia acidovorans for degradation of 2,4-dichlorophenoxyacetate in a microfluidic porous medium.

Delftia acidovorans MC1071 can productively degrade R-2-(2,4-dichlorophenoxy)propionate (R-2,4-DP) but not 2,4-dichlorophenoxyacetate (2,4-D) herbicides. This work demonstrates adaptation of MC1071 to degrade 2,4-D in a model two-dimensional porous medium (referred to here as a micromodel). Adaptation for 2,4-D degradation in the 2 cm-long micromodel occurred within 35 days of exposure to 2,4-D, as documented by substrate removal. The amount of 2,4-D degradation in the adapted cultures in two replicate micromodels (~10 and 20 % over 142 days) was higher than a theoretical maximum (4 %) predicted using published numerical simulation methods, assuming instantaneous biodegradation and a transverse dispersion coefficient obtained for the same pore structure without biomass present. This suggests that the presence of biomass enhances substrate mixing. Additional evidence for adaptation was provided by operation without R-2,4-DP, where degradation of 2,4-D slowly decreased over 20 days, but was restored almost immediately when R-2,4-DP was again provided. Compared to suspended growth systems, the micromodel system retained the ability to degrade 2,4-D longer in the absence of R-2,4-DP, suggesting slower responses and greater resilience to fluctuations in substrates might be expected in the soil environment than in a chemostat.

Bacterial acyl-CoA mutase specifically catalyzes coenzyme B12-dependent isomerization of 2-hydroxyisobutyryl-CoA and (S)-3-hydroxybutyryl-CoA.

Coenzyme B(12)-dependent acyl-CoA mutases are radical enzymes catalyzing reversible carbon skeleton rearrangements in carboxylic acids. Here, we describe 2-hydroxyisobutyryl-CoA mutase (HCM) found in the bacterium Aquincola tertiaricarbonis as a novel member of the mutase family. HCM specifically catalyzes the interconversion of 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA. Like isobutyryl-CoA mutase, HCM consists of a large substrate- and a small B(12)-binding subunit, HcmA and HcmB, respectively. However, it is thus far the only acyl-CoA mutase showing substrate specificity for hydroxylated carboxylic acids. Complete loss of 2-hydroxyisobutyric acid degradation capacity in hcmA and hcmB knock-out mutants established the central role of HCM in A. tertiaricarbonis for degrading substrates bearing a tert-butyl moiety, such as the fuel oxygenate methyl tert-butyl ether (MTBE) and its metabolites. Sequence analysis revealed several HCM-like enzymes in other bacterial strains not related to MTBE degradation, indicating that HCM may also be involved in other pathways. In all strains, hcmA and hcmB are associated with genes encoding for a putative acyl-CoA synthetase and a MeaB-like chaperone. Activity and substrate specificity of wild-type enzyme and active site mutants HcmA I90V, I90F, and I90Y clearly demonstrated that HCM belongs to a new subfamily of B(12)-dependent acyl-CoA mutases.

Cultivation of Aquincola tertiaricarbonis L108 on the fuel oxygenate intermediate tert-butyl alcohol induces aerobic anoxygenic photosynthesis at extremely low feeding rates.

Aerobic anoxygenic photosynthesis (AAP) is found in an increasing number of proteobacterial strains thriving in ecosystems ranging from extremely oligotrophic to eutrophic. Here, we have investigated whether the fuel oxygenate-degrading betaproteobacterium Aquincola tertiaricarbonis L108 can use AAP to compensate kinetic limitations at low heterotrophic substrate fluxes. In a fermenter experiment with complete biomass retention and also during chemostat cultivation, strain L108 was challenged with extremely low substrate feeding rates of tert-butyl alcohol (TBA), an intermediate of methyl tert-butyl ether (MTBE). Interestingly, formation of photosynthetic pigments, identified as bacteriochlorophyll a and spirilloxanthin, was only induced in growing cells at TBA feeding rates less than or equal to maintenance requirements observed under energy excess conditions. Growth continued at rates between 0.001 and 0.002 h(-1) even when the TBA feed was decreased to values close to 30 % of this maintenance rate. Partial sequencing of genomic DNA of strain L108 revealed a bacteriochlorophyll synthesis gene cluster (bchFNBHL) and photosynthesis regulator genes (ppsR and ppaA) typically found in AAP and other photosynthetic proteobacteria. The usage of light as auxiliary energy source enabling evolution of efficient degradation pathways for kinetically limited heterotrophic substrates and for lowering the threshold substrate concentration Smin at which growth becomes zero is discussed.

Constitutive expression of the cytochrome P450 EthABCD monooxygenase system enables degradation of synthetic dialkyl ethers in Aquincola tertiaricarbonis L108.

In Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005, and Gordonia sp. strain IFP 2009, the cytochrome P450 monooxygenase EthABCD catalyzes hydroxylation of methoxy and ethoxy residues in the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). The expression of the IS3-type transposase-flanked eth genes is ETBE dependent and controlled by the regulator EthR (C. Malandain et al., FEMS Microbiol. Ecol. 72:289-296, 2010). In contrast, we demonstrated by reverse transcription-quantitative PCR (RT-qPCR) that the betaproteobacterium Aquincola tertiaricarbonis L108, which possesses the ethABCD genes but lacks ethR, constitutively expresses the P450 system at high levels even when growing on nonether substrates, such as glucose. The mutant strain A. tertiaricarbonis L10, which is unable to degrade dialkyl ethers, resulted from a transposition event mediated by a rolling-circle IS91-type element flanking the eth gene cluster in the wild-type strain L108. The constitutive expression of Eth monooxygenase is likely initiated by the housekeeping sigma factor σ(70), as indicated by the presence in strain L108 of characteristic -10 and -35 binding sites upstream of ethA which are lacking in strain IFP 2001. This enables efficient degradation of diethyl ether, diisopropyl ether, MTBE, ETBE, TAME, and tert-amyl ethyl ether (TAEE) without any lag phase in strain L108. However, ethers with larger residues, n-hexyl methyl ether, tetrahydrofuran, and alkyl aryl ethers, were not attacked by the Eth system at significant rates in resting-cell experiments, indicating that the residue in the ether molecule which is not hydroxylated also contributes to the determination of substrate specificity.

Synthesis of the building block 2-hydroxyisobutyrate from fructose and butyrate by Cupriavidus necator H16.

2-Hydroxyisobutyryl-coenzyme A mutase, originally discovered in the context of methyl tert-butyl ether degradation in Aquincola tertiaricarbonis L108, catalyzes the isomerization of 3-hydroxybutyryl-coenzyme A (3-HB-CoA) to 2-hydroxyisobutyryl-CoA. It thus constitutes the basis for a biotechnological route from practically any renewable carbon to 2-hydroxyisobutyrate (2-HIB) via the common metabolite 3-hydroxybutyrate. At first sight, recombinant Cupriavidus necator H16 expressing the mutase seems to be well suited for such a synthesis process, as a strong overflow metabolism via (R)-3-HB-CoA is easily induced in this bacterium possessing the poly-3-hydroxybutyrate metabolism. However, the recently established stereospecificity of the mutase, dominantly preferring the (S)-enantiomer of 3-HB-CoA, calls for a closer investigation of C. necator as potential 2-HIB production strain and raised the question about the strain's potential to yield 2-HIB from substrates directly providing (S)-3-HB-CoA. We compared two mutase-expressing C. necator H16 strains for their capability to synthesize 2-HIB from fructose and butyrate, delivering either (R)- or (S)-3-HB-CoA. Our results indicate that due to the enantiospecificity of the mutase, fructose is a weaker substrate for 2-HIB synthesis than butyrate. Production rates achieved with the PHB-negative strain H16 PHB(-)4 on butyrate were higher than on fructose. Using the wild-type did not significantly improve the production rates as the latter showed a 34-fold and a 5-fold lower 2-HIB synthesis rate compared to H16 PHB(-)4 on fructose and butyrate, respectively. Moreover, both strains showed concomitant excretion of undesired side products, such as pyruvate and 3-hydroxybutyrate, significantly decreasing the 2-HIB yield.

Bacterial degradation of tert-amyl alcohol proceeds via hemiterpene 2-methyl-3-buten-2-ol by employing the tertiary alcohol desaturase function of the Rieske nonheme mononuclear iron oxygenase MdpJ.

Tertiary alcohols, such as tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA) and higher homologues, are only slowly degraded microbially. The conversion of TBA seems to proceed via hydroxylation to 2-methylpropan-1,2-diol, which is further oxidized to 2-hydroxyisobutyric acid. By analogy, a branched pathway is expected for the degradation of TAA, as this molecule possesses several potential hydroxylation sites. In Aquincola tertiaricarbonis L108 and Methylibium petroleiphilum PM1, a likely candidate catalyst for hydroxylations is the putative tertiary alcohol monooxygenase MdpJ. However, by comparing metabolite accumulations in wild-type strains of L108 and PM1 and in two mdpJ knockout mutants of strain L108, we could clearly show that MdpJ is not hydroxylating TAA to diols but functions as a desaturase, resulting in the formation of the hemiterpene 2-methyl-3-buten-2-ol. The latter is further processed via the hemiterpenes prenol, prenal, and 3-methylcrotonic acid. Likewise, 3-methyl-3-pentanol is degraded via 3-methyl-1-penten-3-ol. Wild-type strain L108 and mdpJ knockout mutants formed isoamylene and isoprene from TAA and 2-methyl-3-buten-2-ol, respectively. It is likely that this dehydratase activity is catalyzed by a not-yet-characterized enzyme postulated for the isomerization of 2-methyl-3-buten-2-ol and prenol. The vitamin requirements of strain L108 growing on TAA and the occurrence of 3-methylcrotonic acid as a metabolite indicate that TAA and hemiterpene degradation are linked with the catabolic route of the amino acid leucine, including an involvement of the biotin-dependent 3-methylcrotonyl coenzyme A (3-methylcrotonyl-CoA) carboxylase LiuBD. Evolutionary aspects of favored desaturase versus hydroxylation pathways for TAA conversion and the possible role of MdpJ in the degradation of higher tertiary alcohols are discussed.

Synthesis of short-chain diols and unsaturated alcohols from secondary alcohol substrates by the Rieske nonheme mononuclear iron oxygenase MdpJ.

The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain Aquincola tertiaricarbonis L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an mdpJ knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed.

Formation of alkenes via degradation of tert-alkyl ethers and alcohols by Aquincola tertiaricarbonis L108 and Methylibium spp.

Bacterial degradation pathways of fuel oxygenates such as methyl tert-butyl and tert-amyl methyl ether (MTBE and TAME, respectively) have already been studied in some detail. However, many of the involved enzymes are still unknown, and possible side reactions have not yet been considered. In Aquincola tertiaricarbonis L108, Methylibium petroleiphilum PM1, and Methylibium sp. strain R8, we have now detected volatile hydrocarbons as by-products of the degradation of the tert-alkyl ether metabolites tert-butyl and tert-amyl alcohol (TBA and TAA, respectively). The alkene isobutene was formed only during TBA catabolism, while the beta and gamma isomers of isoamylene were produced only during TAA conversion. Both tert-alkyl alcohol degradation and alkene production were strictly oxygen dependent. However, the relative contribution of the dehydration reaction to total alcohol conversion increased with decreasing oxygen concentrations. In resting-cell experiments where the headspace oxygen content was adjusted to less than 2%, more than 50% of the TAA was converted to isoamylene. Isobutene formation from TBA was about 20-fold lower, reaching up to 4% alcohol turnover at low oxygen concentrations. It is likely that the putative tert-alkyl alcohol monooxygenase MdpJ, belonging to the Rieske nonheme mononuclear iron enzymes and found in all three strains tested, or an associated enzymatic step catalyzed the unusual elimination reaction. This was also supported by the detection of mdpJK genes in MTBE-degrading and isobutene-emitting enrichment cultures obtained from two treatment ponds operating at Leuna, Germany. The possible use of alkene formation as an easy-to-measure indicator of aerobic fuel oxygenate biodegradation in contaminated aquifers is discussed.

Biosynthesis of 2-hydroxyisobutyric acid (2-HIBA) from renewable carbon.

Nowadays a growing demand for green chemicals and cleantech solutions is motivating the industry to strive for biobased building blocks. We have identified the tertiary carbon atom-containing 2-hydroxyisobutyric acid (2-HIBA) as an interesting building block for polymer synthesis. Starting from this carboxylic acid, practically all compounds possessing the isobutane structure are accessible by simple chemical conversions, e. g. the commodity methacrylic acid as well as isobutylene glycol and oxide. During recent years, biotechnological routes to 2-HIBA acid have been proposed and significant progress in elucidating the underlying biochemistry has been made. Besides biohydrolysis and biooxidation, now a bioisomerization reaction can be employed, converting the common metabolite 3-hydroxybutyric acid to 2-HIBA by a novel cobalamin-dependent CoA-carbonyl mutase. The latter reaction has recently been discovered in the course of elucidating the degradation pathway of the groundwater pollutant methyl tert-butyl ether (MTBE) in the new bacterial species Aquincola tertiaricarbonis. This discovery opens the ground for developing a completely biotechnological process for producing 2-HIBA. The mutase enzyme has to be active in a suitable biological system producing 3-hydroxybutyryl-CoA, which is the precursor of the well-known bacterial bioplastic polyhydroxybutyrate (PHB). This connection to the PHB metabolism is a great advantage as its underlying biochemistry and physiology is well understood and can easily be adopted towards producing 2-HIBA. This review highlights the potential of these discoveries for a large-scale 2-HIBA biosynthesis from renewable carbon, replacing conventional chemistry as synthesis route and petrochemicals as carbon source.

Structures of 2-Hydroxyisobutyric Acid-CoA Ligase Reveal Determinants of Substrate Specificity and Describe a Multi-Conformational Catalytic Cycle.

2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B12-dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154° rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50° and 130° from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases.

The two enantiospecific dichlorprop/alpha-ketoglutarate-dioxygenases from Delftia acidovorans MC1--protein and sequence data of RdpA and SdpA.

Two alpha-ketoglutarate-dependent dioxygenases carrying enantiospecific activity for the etherolytic cleavage of racemic phenoxypropionate herbicides [(RS)-2-(2,4-dichlorophenoxy)propionate and (RS)-2-(4-chloro-2-methylphenoxy)propionate] from Delftia acidovorans MC1 were characterized with respect to protein and sequence data. The (S)-phenoxypropionate/alpha-ketoglutarate-dioxygenase (SdpA) appeared as a monomeric enzyme with a molecular weight of 32 kDa in the presence of SDS. N-terminal sequences revealed relationship to alpha-ketoglutarate-dependent taurine dioxygenase (TauD) and to 2,4-dichlorophenoxyacetate/alpha-ketoglutarate-dioxygenase (TfdA). The (R)-phenoxypropionate/alpha-ketoglutarate-dioxygenase (RdpA) referred to 36 kDa in the presence of SDS and to 108 kDa under native conditions. Internal sequences of fragments obtained after digestion made evident relationship to TfdA and TauD. Two-dimensional electrophoretic separation resulted in the resolution of up to 3 individual spots with almost identical molecular weights but different isoelectric points with both RdpA and SdpA. The structural differences of these isoenzyme forms are not yet clear.

Declining capacity of starving Delftia acidovorans MC1 to degrade phenoxypropionate herbicides correlates with oxidative modification of the initial enzyme.

Bioremediation relies on the stability of enzymatic activities, particularly when bioavailable contaminant concentrations do not permit much renewal of microbial biomass. Starving Delftia acidovorans MC1 were found to lose specific degradation activity, while accumulating variants of the alpha-ketoglutarate-dependent dioxygenase RdpA, the enzyme initiating the degradation of (RS)-2-(2,4-dichlorophenoxy)propionate. These variants differed in their pI and originated from post-translational modification, since there is only one rdpA gene in the genome. It was tested if RdpA modification resulted from carbonylation by reactive oxygen species, known side products of dioxygenase reactions. Carbonylated amino acids in proteins of starved cells were specifically derivatized with 2,4-dinitrophenylhydrazine. Subsequent immunolabeling of the resulting hydrazones and mass spectrometry of tryptic digests confirmed different levels of carbonylation of RdpA.

Effects of pore-scale heterogeneity and transverse mixing on bacterial growth in porous media.

Microbial degradation of contaminants in the subsurface requires the availability of nutrients; this is impacted by porous media heterogeneity and the degree of transverse mixing. Two types of microfluidic pore structures etched into silicon wafers (i.e., micromodels), (i) a homogeneous distribution of cylindrical posts and (ii) aggregates of large and small cylindrical posts, were used to evaluate the impact of heterogeneity on growth of a pure culture (Delftia acidovorans) that degrades (R)-2-(2,4-dichlorophenoxy)propionate (R-2,4-DP). Following inoculation, dissolved O2 and R-2,4-DP were introduced as two parallel streams that mixed transverse to the direction of flow. In the homogeneous micromodel, biomass growth was uniform in pore bodies along the center mixing line, while in the aggregate micromodel, preferential growth occurred between aggregates and slower less dense growth occurred throughout aggregates along the center mixing line. The homogeneous micromodel had more rapid growth overall (2 times) and more R-2,4-DP degradation (9.5%) than the aggregate pore structure (5.7%). Simulation results from a pore-scale reactive transport model indicate mass transfer limitations within aggregates along the center mixing line decreased overall reaction; hence, slower biomass growth rates relative to the homogeneous micromodel are expected. Results from this study contribute to a better understanding of the coupling between mass transfer, reaction rates, and biomass growth in complex porous media and suggest successful implementation and analysis of bioremediation systems requires knowledge of subsurface heterogeneity.

Degradation of fuel oxygenates and their main intermediates by Aquincola tertiaricarbonis L108.

Growth of Aquincola tertiaricarbonis L108 on the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), as well as on their main metabolites tert-butyl alcohol (TBA), tert-amyl alcohol (TAA) and 2-hydroxyisobutyrate (2-HIBA) was systematically investigated to characterize the range and rates of oxygenate degradation by this strain. The effective maximum growth rates for MTBE, ETBE and TAME at pH 7 and 30 degrees C were 0.045 h(-1), 0.06 h(-1) and 0.055 h(-1), respectively, whereas TAA, TBA and 2-HIBA permitted growth at rates up to 0.08 h(-1), 0.1 h(-1) and 0.17 h(-1), respectively. The experimental growth yields with all these substrates were high. Yields of 0.55 g dry mass (dm) (g MTBE)(-1), 0.53 g dm (g ETBE)(-1), 0.81 g dm (g TAME)(-1), 0.48 g dm (g TBA)(-1), 0.76 g dm (g TAA)(-1) and 0.54 g dm (g 2-HIBA)(-1) were obtained. Maximum specific degradation rates were 0.92 mmol MTBE h(-1) (g dm)(-1), 1.11 mmol ETBE h(-1) g(-1), 0.66 mmol TAME h(-1) g(-1), 1.19 mmol TAA h(-1) g(-1), 2.82 mmol TBA h(-1) g(-1), and 3.27 mmol 2-HIBA h(-1) g(-1). The relatively high rates with TBA, TAA and 2-HIBA indicate that the transformations of these metabolites did not limit the metabolism of MTBE and the related ether compounds. Despite the fact that these metabolites still carry a tertiary carbon atom that is commonly suspected to confer recalcitrance to the ether oxygenates, the transformation rates were in the same range as those with succinate and fructose. With MTBE, strain L108 grew at pHs between 5.5 and 8.0 at near-maximal rate, whereas no growth was found below pH 5.0 and above pH 9.0. The optimum growth temperature was 30 degrees C, but at 5 degrees C still about 15 % of the maximum rate remained, whereas no growth occurred at 42 degrees C. This indicates that MTBE metabolites are valuable substrates and that A. tertiaricarbonis L108 is a good candidate for bioremediation purposes. The possible origin of its exceptional metabolic capability is discussed in terms of the evolution of enzymic activities involved in the conversion of compounds carrying tertiary butyl groups.

Carbon conversion efficiency and limits of productive bacterial degradation of methyl tert-butyl ether and related compounds.

The utilization of the fuel oxygenate methyl tert-butyl ether (MTBE) and related compounds by microorganisms was investigated in a mainly theoretical study based on the Y(ATP) concept. Experiments were conducted to derive realistic maintenance coefficients and K(s) values needed to calculate substrate fluxes available for biomass production. Aerobic substrate conversion and biomass synthesis were calculated for different putative pathways. The results suggest that MTBE is an effective heterotrophic substrate that can sustain growth yields of up to 0.87 g g(-1), which contradicts previous calculation results (N. Fortin et al., Environ. Microbiol. 3:407-416, 2001). Sufficient energy equivalents were generated in several of the potential assimilatory routes to incorporate carbon into biomass without the necessity to dissimilate additional substrate, efficient energy transduction provided. However, when a growth-related kinetic model was included, the limits of productive degradation became obvious. Depending on the maintenance coefficient m(s) and its associated biomass decay term b, growth-associated carbon conversion became strongly dependent on substrate fluxes. Due to slow degradation kinetics, the calculations predicted relatively high threshold concentrations, S(min), below which growth would not further be supported. S(min) strongly depended on the maximum growth rate mu(ma)(x), and b and was directly correlated with the half maximum rate-associated substrate concentration K(s), meaning that any effect impacting this parameter would also change S(min). The primary metabolic step, catalyzing the cleavage of the ether bond in MTBE, is likely to control the substrate flux in various strains. In addition, deficits in oxygen as an external factor and in reduction equivalents as a cellular variable in this reaction should further increase K(s) and S(min) for MTBE.

Aquincola tertiaricarbonis gen. nov., sp. nov., a tertiary butyl moiety-degrading bacterium.

Strains L10(T), L108 and CIP I-2052 were originally obtained from methyl tert-butyl ether (MTBE)-contaminated groundwater and from a wastewater treatment plant, respectively. All share the ability to grow on tert-butanol, an intermediate of MTBE degradation. Cells are strictly aerobic, motile by a polar flagellum and exhibit strong pili formation. Poly beta-hydroxybutyrate (PHB) granules are formed. The DNA G+C content is 69-70.5 mol% and the main ubiquinone is Q-8. The major cellular fatty acids are 16 : 1 cis-9 and 16 : 0 and the only hydroxy fatty acid is 10 : 0 3-OH. The major phospholipids are phosphatidylethanolamine (PE) 16 : 1/16 : 1 and phosphatidylglycerol 16 : 0/16 : 1. A significant amount of PE 17 : 0/16 : 1 is present. The 16S rRNA gene sequences of these strains are almost identical and form a separate line of descent in the Rubrivivax-Roseateles-Leptothrix-Ideonella-Aquabacterium branch of the Betaproteobacteria with 97 % similarity to 16S rRNA genes of the type strains of Rubrivivax gelatinosus, Leptothrix mobilis and Ideonella dechloratans. However, physiological properties, DNA-DNA relatedness values and the phospholipid and cellular fatty acid profiles distinguish the novel isolates from the three closely related genera. Therefore, it is concluded that strains L10(T), L108 and CIP I-2052 represent a new genus and novel species for which the name Aquincola tertiaricarbonis gen. nov., sp. nov., is proposed. The type strain is strain L10(T) (=DSM 18512(T)=CIP 109243(T)).

Uptake kinetics of 2,4-dichlorophenoxyacetate by Delftia acidovorans MC1 and derivative strains: complex characteristics in response to pH and growth substrate.

The present investigation showed that active processes were involved in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Delftia acidovorans MC1. With 2,4-D-grown cells, uptake at pH 6.8 was highly affine and showed a complex pattern-forming intermediary plateau at 20-100 microM 2,4-D. The kinetics became increasingly sigmoidal with raising of the pH to 7.5 and 8.5, and complexity disappeared. The apparent maximum was obtained at around 400 microM 2,4-D at either pH, and amounted to 15-20 nmol/min x mg protein. Higher substrate concentrations resulted in significant inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate, 2,4-D uptake increased significantly and reached 45 nmol/min x mg, hinting at induction of a specific carrier(s). The kinetic characteristics made it apparent that several proteins contribute to 2,4-D uptake in MC1. An open reading frame was detected which has similarity to genes encoding major facilitator superfamily (MFS) transporters. Mutant strains that lacked this gene showed altered kinetics with decreased affinity to 2,4-D at pH 6.8. A mutant with complete deficiency in phenoxyalkanoate utilization showed an almost linear uptake pattern hinting at sole diffusion. Cloning of tfdK encoding a specific transporter for 2,4-D resulted in an increased uptake rate and, above all, higher affinity at slightly alkaline conditions due to hyperbolic kinetics. The presence of carbonylcyanide m-chlorophenylhydrazone led to the subsequent strong inhibition of 2,4-D uptake, suggesting proton symport as the likely active mechanism.