Proteomics analysis of aromatic catabolic pathways in thermophilic Geobacillus thermodenitrificans NG80-2

Geobacillus thermodenitrificans NG80-2 is a crude oil-degrading thermophilic bacterium isolated from an oil reservoir in China. In this study, the gene clusters and pathways for the degradation of benzoate (via benzoyl-CoA), phenylacetate (via phenylacetyl-CoA), 4-hydroxyphenylacetate (via 3,4-dihydroxyphenylacetate) and anthranilate (via 3-hydroxyanthranilate) were confirmed using combined in silico analysis and proteomics approaches. It was observed that synthesis of the enzymes catalyzing initial activation, ring oxidation and ring cleavage reactions were generally induced specifically by their respective substrates, while many of the enzymes catalyzing downstream reactions exhibited broader substrate specificities. Novel genes encoding benzoyl-CoA epoxidase and 3,4-dihydroxyphenylacetate 2,3-dioxygenase, and a paaX homologue that serves as a positive regulator of benzoate degradation were proposed. Downregulation of the glycolysis pathway, along with upregulation of the gluconeogenesis pathway and the glyoxylate bypass (phenylacetate) were detected in association with the utilization of the aromatics. This novel proteomics approach confirmed the presence of multiple metabolic pathways for aromatic compounds in NG80-2, which is highly advantageous to the survival of this thermophilic bacterium under reservoir conditions.     

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24

Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.     

Characterization of a Spontaneously Occurring Mutant of the TOL20 Plasmid in Pseudomonas putida MT20: Possible Regulatory Implications

Pseudomonas putida MT20 carries a plasmid (TOL20) that codes for the enzymes responsible for the catabolism of toluene, m- and p-xylene to benzoate, and m- and p-toluate, respectively, followed by meta cleavage of the aromatic ring. Growth on 5 mM benzoate selects very strongly for (i) strains that have been cured of the plasmid and (ii) strains with an intermediate growth pattern (the B3 phenotype) that retain the ability to grow on toluene, m-xylene, and benzoate but are unable to grow on m-toluate. Both types of strains were selected because they are no longer able to oxidize benzoate by the plasmid pathway but instead use an alternative route, the ortho or β-ketoadipate pathway, which is chromosomally coded and supports faster growth. Evidence that one strain with the B3 phenotype, MT20-B3, has a regulatory mutation that prevents induction of the meta-pathway enzymes by benzoate and m-toluate, but which enables them to be induced by toluene and m-xylene, is presented. The plasmid in this strain, as in most of the others with the same phenotype, is nonconjugative. Analysis of MT20-B3, together with revertants of it and other noninducible mutants, has led to a model for the regulation of the plasmid-coded enzymes in MT20, in which it is proposed that the early enzymes for degradation of m-toluate and benzoate are positively controlled by two regulator molecules, one of which interacts with toluene and m-xylene as inducers and the other of which interacts with benzoate and m-toluate. It is argued that MT20-B3 and strains with a similar phenotype arose as a result of a deletion of the gene coding for the second regulator molecule.     

Construction of a partial diploid for the degradative pathway encoded by the TOL plasmid (pWW0) from Pseudomonas putida mt-2: Evidence for the positive nature of the regulation by the xylR gene

Summary The hypothesis that the early enzymes of the degradative pathway determined by the TOL plasmid pWW0 are positively regulated by the product of the xylR gene has been tested by constructing a strain which is a partial diploid for the TOL genes. Two parental plasmids were first constructed by in vivo methods, neither of which could determine the ability to grow on m-xylene, one of the primary substrates of the plasmid degradative pathway, because of mutations. One of these, pWW0-216, was a derivative of pWW0 but carried a xylR ^- allele and a copy of the Tn401 transposon, encoding carbenicillin resistance. The other plasmid, pWW0-152, was a derivative of the promiscuous R plasmid RP4 into which had been translocated part of a pWW0 plasmid carrying a wild type xylR ⁺ allele but with a defective xylA, the structural gene for xylene oxidase. When these two plasmids were mated into the same strain, all the transconjugants examined grew on m-xylene and one representative of these, PaW 219, was shown to contain induced levels of xylene oxidase when grown under inducing conditions. The possibility that ability to utilise m-xylene was due to recombination between or reversion of the coexisting plasmids was eliminated by demonstrating that the two parental plasmids segregated on mating out from PaW 219. It is concluded therefore that xylR ⁺ is transdominant to xylR ^-, and that its gene product is a positive regulator.     

Initiation of anaerobic degradation of p-cresol by formation of 4-hydroxybenzylsuccinate in desulfobacterium cetonicum

The anaerobic bacterium Desulfobacterium cetonicum oxidized p-cresol completely to CO₂ with sulfate as the electron acceptor. During growth, 4-hydroxybenzylsuccinate accumulated in the medium. This finding indicated that the methyl group of p-cresol is activated by addition to fumarate, analogous to anaerobic toluene, m-xylene, and m-cresol degradation. In cell extracts, the formation of 4-hydroxybenzylsuccinate from p-cresol and fumarate was detected at an initial rate of 0.57 nmol min⁻¹ (mg of protein)⁻¹. This activity was specific for extracts of p-cresol-grown cells. 4-Hydroxybenzylsuccinate was degraded further to 4-hydroxybenzoyl-coenzyme A (CoA), most likely via β-oxidation. 4-Hydroxybenzoyl-CoA was reductively dehydroxylated to benzoyl-CoA. There was no evidence of degradation of p-cresol via methyl group oxidation by p-cresol-methylhydroxylase in this bacterium.     

The iscS gene deficiency affects the expression of pyrimidine metabolism genes

Inactivation of iscS encoding cysteine desulfurase results in a slow growth phenotype associated with the deficiency of iron-sulfur clusters, thiamine, NAD, and tRNA thionucleosides in Escherichia coli. However, the other roles of iscSin vivo are unknown. By using differential screening strategies, we identified 2 pyrimidine salvage enzymes, namely, uridine phosphorylase and cytidine deaminase, which were down-regulated in the iscS mutant. Both enzymes are positively regulated by the cAMP receptor protein (CRP). We also identified a novel protein complex, namely, YeiT-YeiA, whose expression level was decreased in the iscS mutant. The recombinant YeiT-YeiA complex exhibited NADH-dependent dihydropyrimidine dehydrogenase activity, indicating its role in pyrimidine metabolism. The presence of a CRP-binding consensus sequence on the 5′-upstream of the yeiT-YeiA gene suggests its regulation by CRP. These results provide a clue to the possible role of iscS in pyrimidine metabolism by gene regulation.     

A butenolide signaling system synergized with biosynthetic gene modules led to effective activation and enhancement of silent oviedomycin production in Streptomyces

Secondary metabolic gene clusters widely exist in the genomes of Streptomyces but mostly remain silent. To awaken this hidden reservoir of natural products, various strategies concerning secondary metabolic pathways are applied. Here, we describe that butenolide signaling molecule deficiency and glucose addition can interdependently activate the expression of silent oviedomycin biosynthetic gene clusters in Streptomyces ansochromogenes and Streptomyces antibioticus. Since oviedomycin is a promising anti-tumor lead compound, in order to improve its yield, we use the cluster-situated genes (ovmF, ovmG, ovmI and ovmH) encoding the enzymes for acyl carrier protein modification and precursor biosynthesis, and the discrete precursor biosynthetic genes (pyk2, gap1 and accA2) involved in glycolysis to assemble two gene modules (pFGIH and pPGA). Their co-overexpression in ΔsabA (a disruption mutant of sabA encoding SAB synthase) has superimposed effect on the yield of oviedomycin, which can be further increased to 59-fold in the presence of galactose as optimal carbon source. This is the most unambiguous evidence that butenolide signaling system can synergize with the optimization of primary metabolism to regulate the expression of secondary metabolic gene clusters, providing efficient strategies for mining natural products of Streptomyces.     

Deepening TOL and TOU catabolic pathways of Pseudomonas sp. OX1: Cloning,sequencing and characterization of the lower pathways

Pseudomonas sp. OX1 is able to metabolize toluene and o-xylene through the TOU catabolic pathway, whereas its mutant M1 strain was found to be able to use m- and p-xylene as carbon and energy source, using the TOL catabolic pathway. Here we report the complete nucleotide sequence of the phe lower operon of the TOU catabolic pathway, and the sequence of the last four genes of the xyl-like lower operon of the TOL catabolic pathway. DNA sequence analysis shows the gene order within the operons to be pheCDEFGHI (phe operon) and xyl-likeQKIH (xyl-like operon), identical to the order found for the isofunctional genes of meta operons in the toluene/xylene pathway of TOL plasmid pWW0 from Pseudomonas putida mt-2 and the phenol/methylphenol pathway of pVIl50 from Pseudomonas sp. CF600. The nucleotide and the deduced amino acid sequences are homologous to the equivalent gene and enzyme sequences from other Pseudomonas meta pathways. Recombinant 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), coded by pheCD genes, respectively, and ADA and HOA enzymes from both phe and xyl operons were expressed in E. coli and steady-state kinetic analysis was carried out. The analysis of the kinetic parameters of HMSD and HMSH showed that the enzymes from Pseudomonas sp. OX1 are more specialized to channel metabolites into the two branches of the lower pathway than homologous enzymes from other pseudomonads. The kinetics parameters of recombinant ADA from phe and xyl-like operon were found to be similar to those of homologous enzymes from other Pseudomonas strains. In addition, the enzyme from xyl-like operon showed a substrate affinity three times higher than the enzyme from phe operon.     

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the ¹⁴C-labeled crystalline polymeric substrates ¹⁴C chitin nanowhiskers and ¹⁴C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed K_i(obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the K_i(obs) of Cel7A was an order of magnitude higher than the true K_i value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation.     

Rapid Microbial Mineralization of Toluene and 1,3-Dimethylbenzene in the Absence of Molecular Oxygen

Up to 0.4 mM 1,3-dimethylbenzene (m-xylene) was rapidly mineralized in a laboratory aquifer column operated in the absence of molecular oxygen with nitrate as an electron acceptor. Under continuous flow conditions, the degradation rate constant (pseudo-first order) was >0.45 h⁻¹. Based on a carbon mass balance with [ring-¹⁴C]m-xylene and a calculation of the electron balance, m-xylene was shown to be quantitatively (80%) oxidized to CO₂ with a concomitant reduction of nitrate. The mineralization of m-xylene in the column also took place after reducing the redox potential, E′, of the inflowing medium with sulfide to <−0.11 V. Microorganisms adapted to growth on m-xylene were also able to degrade toluene under denitrifying conditions. These results suggest that aromatic hydrocarbons present in anoxic environments such as lake sediments, sludge digestors, and groundwater infiltration zones from landfills and polluted rivers are not necessarily persistent but may be mineralized in the absence of molecular oxygen.     

Degradation of <Emphasis Type="Italic">o</Emphasis>-xylene and <Emphasis Type="Italic">m</Emphasis>-xylene by a novel sulfate-reducer belonging to the genus <Emphasis Type="Italic">Desulfotomaculum</Emphasis>

A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO₂. Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbon-oxidizing bacterium in this genus.     

Studies on the Utilization of Hydrocarbons by Microorganisms

In the course of study on the utilization of methyl-substituents of mono-cyclic aromatic hydrocarbons by Pseudomonas aeruginosa S668B2, some organic acids and phenolic compounds were found to be produced in culture broth.

Strain S668B2 was capable of producing ultraviolet absorbing and fluorescent substances from m-xylene. These substances were isolated in the form of crystal and identified as 3-methyl salicylic acid and m-toluic acid.

Strain S668B2 also produced ultraviolet absorbing and fluorescent substances from pseudocumene (1,2,4-trimethyl benzene). These substances were isolated in the crystalline form and identified as 3,4-dimethyl benzoic acid and 3,4-dimethyl phenol.

Strain S668B″ did not attack o-xylene. Under the similar conditions Pseudomonas desmolytica S449B3, which produced a large amount of cumic acid from p-cymene, did not oxidize o-xylene, but grew on p-xylene, m-xylene and 1,2,4-trimethyl benzene.

None out of 364 soil samples gave microorganisms which utilize o-xylene as a sole carbon source.     

Mutation of Glutamic Acid 103 of Toluene o-Xylene Monooxygenase as a Means To Control the Catabolic Efficiency of a Recombinant Upper Pathway for Degradation of Methylated Aromatic Compounds

Toluene o-xylene monooxygenase (ToMO) and phenol hydroxylase (PH) of Pseudomonas stutzeri OX1 act sequentially in a recombinant upper pathway for the degradation of aromatic hydrocarbons. The catalytic efficiency and regioselectivity of these enzymes optimize the degradation of growth substrates like toluene and o-xylene. For example, the sequential monooxygenation of o-xylene by ToMO and PH leads to almost exclusive production of 3,4-dimethylcatechol (3,4-DMC), the only isomer that can be further metabolized by the P. stutzeri meta pathway. We investigated the possibility of producing ToMO mutants with modified regioselectivity compared with the regioselectivity of the wild-type protein in order to alter the ability of the recombinant upper pathway to produce methylcatechol isomers from toluene and to produce 3,4-DMC from o-xylene. The combination of mutant (E103G)-ToMO and PH increased the production of 4-methylcatechol from toluene and increased the formation of 3,4-DMC from o-xylene. These data strongly support the idea that the products and efficiency of the metabolic pathway can be controlled not only through mutations that increase the catalytic efficiency of the enzymes involved but also through tuning the substrate specificity and regioselectivity of the enzymes. These findings are crucial for the development of future metabolic engineering strategies.     

Cap analog substrates reveal three clades of cap guanine-N2 methyltransferases with distinct methyl acceptor specificities

The Tgs proteins are structurally homologous AdoMet-dependent eukaryal enzymes that methylate the N2 atom of 7-methyl guanosine nucleotides. They have an imputed role in the synthesis of the 2,2,7-trimethylguanosine (TMG) RNA cap. Here we exploit a collection of cap-like substrates to probe the repertoire of three exemplary Tgs enzymes, from mammalian, protozoan, and viral sources, respectively. We find that human Tgs (hTgs1) is a bona fide TMG synthase adept at two separable transmethylation steps: (1) conversion of m⁷G to m^2,7G, and (2) conversion of m^2,7G to m^2,2,7G. hTgs1 is unable to methylate G or m²G, signifying that both steps require an m⁷G cap. hTgs1 utilizes a broad range of m⁷G nucleotides, including mono-, di-, tri-, and tetraphosphate derivatives as well as cap dinucleotides with triphosphate or tetraphosphate bridges. In contrast, Giardia lamblia Tgs (GlaTgs2) exemplifies a different clade of guanine-N2 methyltransferase that synthesizes only a dimethylguanosine (DMG) cap structure and cannot per se convert DMG to TMG under any conditions tested. Methylation of benzyl⁷G and ethyl⁷G nucleotides by hTgs1 and GlaTgs2 underscored the importance of guanine N7 alkylation in providing a key π-cation interaction in the methyl acceptor site. Mimivirus Tgs (MimiTgs) shares with the Giardia homolog the ability to catalyze only a single round of methyl addition at guanine-N2, but is distinguished by its capacity for guanine-N2 methylation in the absence of prior N7 methylation. The relaxed cap specificity of MimiTgs is revealed at alkaline pH. Our findings highlight both stark and subtle differences in acceptor specificity and reaction outcomes among Tgs family members.     

Expression,purification and crystallization of adenosine 1408 aminoglycoside-resistance rRNA methyltransferases for structural studies

High-level resistance to a broad spectrum of aminoglycoside antibiotics can arise through either N7-methyl guanosine 1405 (m⁷G1405) or N1-methyl adenosine 1408 (m¹A1408) modifications at the drug binding site in the bacterial 30S ribosomal subunit decoding center. Two distinct families of 16S ribosomal RNA (rRNA) methyltransferases that incorporate these modifications were first identified in aminoglycoside-producing bacteria but were more recently identified in both human and animal pathogens. These resistance determinants thus pose a new threat to the usefulness of aminoglycosides as antibiotics, demanding urgent characterization of their structures and activities. Here, we describe approaches to cloning, heterologous expression in Escherichia coli, and purification of two A1408 rRNA methyltransferases: KamB from the aminoglycoside-producer Streptoalloteichus tenebrarius and NpmA identified in a clinical isolate of pathogenic E. coli ARS3. Antibiotic minimum inhibitory concentration (MIC) assays and in vitro analysis of KamB and NpmA using circular dichroism (CD) spectroscopy, S-adenosyl-l-methionine (SAM) binding by isothermal titration calorimetry and 30S subunit methylation assays showed both enzymes were soluble, folded and active. Finally, crystals of each enzyme complexed with SAM were obtained, including selenomethionine-derived KamB, that will facilitate high-resolution X-ray crystallographic analyses of these important bacterial antibiotic-resistance determinants.