Juanito V. Parales

Diversity of microbial toluene degradation pathways.

Publisher Summary Toluene is found naturally in petroleum and coal, and is a major component of gasoline. It is also produced by the burning of organic materials and has been detected in cigarette smoke. Toluene is also produced industrially for use as a solvent and in the production of various chemicals. In addition, toluene is produced and emitted by plants. Toluene is also produced industrially for use as a solvent and in the production of various chemicals. The chapter highlights the fact that toluene is produced and emitted by plants. Some bacteria use a dioxygenase to catalyze the formation of a cis-dihydrodiol product, which is then subjected to dehydrogenation to form 3-methylcatechol. Other bacteria have monooxygenases that catalyze the initial hydroxylation at the ortho, meta, or para position of toluene, which is then followed by a second hydroxylation by the same or a different hydroxylase to form catecholic products.

Pseudomonas putida F1 has multiple chemoreceptors with overlapping specificity for organic acids

Previous studies have demonstrated that Pseudomonas putida strains are not only capable of growth on a wide range of organic substrates, but also chemotactic towards many of these compounds. However, in most cases the specific chemoreceptors that are involved have not been identified. The complete genome sequences of P. putida strains F1 and KT2440 revealed that each strain is predicted to encode 27 methyl-accepting chemotaxis proteins (MCPs) or MCP-like proteins, 25 of which are shared by both strains. It was expected that orthologous MCPs in closely related strains of the same species would be functionally equivalent. However, deletion of the gene encoding the P. putida F1 orthologue (locus tag Pput_4520, designated mcfS) of McpS, a known receptor for organic acids in P. putida KT2440, did not result in an obvious chemotaxis phenotype. Therefore, we constructed individual markerless MCP gene deletion mutants in P. putida F1 and screened for defective sensory responses to succinate, malate, fumarate and citrate. This screen resulted in the identification of a receptor, McfQ (locus tag Pput_4894), which responds to citrate and fumarate. An additional receptor, McfR (locus tag Pput_0339), which detects succinate, malate and fumarate, was found by individually expressing each of the 18 genes encoding canonical MCPs from strain F1 in a KT2440 mcpS-deletion mutant. Expression of mcfS in the same mcpS deletion mutant demonstrated that, like McfR, McfS responds to succinate, malate, citrate and fumarate. Therefore, at least three receptors, McfR, McfS, and McfQ, work in concert to detect organic acids in P. putida F1.

Xyn10A, a thermostable endoxylanase from Acidothermus cellulolyticus 11B.

ABSTRACT We cloned and purified the major family 10 xylanase (Xyn10A) from Acidothermus cellulolyticus 11B. Xyn10A was active on oat spelt and birchwood xylans between 60°C and 100°C and between pH 4 and pH 8. The optimal activity was at 90°C and pH 6; specific activity and Km for oat spelt xylan were 350 μmol xylose produced min−1 mg of protein−1 and 0.53 mg ml−1, respectively. Based on xylan cleavage patterns, Xyn10A is an endoxylanase, and its half-life at 90°C was approximately 1.5 h in the presence of xylan.

Chemotaxis to Pyrimidines and Identification of a Cytosine Chemoreceptor in Pseudomonas putida

We developed a high-throughput quantitative capillary assay and demonstrated that Pseudomonas putida strains F1 and PRS2000 were attracted to cytosine, but not thymine or uracil. In contrast, Pseudomonas aeruginosa PAO1 was not chemotactic to any pyrimidines. Chemotaxis assays with a mutant strain of F1 in which the putative methyl-accepting chemotaxis protein-encoding gene Pput_0623 was deleted revealed that this gene (designated mcpC) encodes a chemoreceptor for positive chemotaxis to cytosine. P. putida F1 also responded weakly to cytidine, uridine, and thymidine, but these responses were not mediated by mcpC. Complementation of the F1 DeltamcpC mutant XLF004 with the wild-type gene restored chemotaxis to cytosine. In addition, introduction of this gene into P. aeruginosa PAO1 conferred the ability to respond to cytosine. To our knowledge, this is the first report of a chemoreceptor for cytosine.

Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species

ABSTRACT The bacterium Pseudonocardia dioxanivorans CB1190 grows on the cyclic ethers 1,4-dioxane (dioxane) and tetrahydrofuran (THF) as sole carbon and energy sources. Prior transcriptional studies indicated that an annotated THF monooxygenase (THF MO) gene cluster, thmADBC, located on a plasmid in CB1190 is upregulated during growth on dioxane. In this work, transcriptional analysis demonstrates that upregulation of thmADBC occurs during growth on the dioxane metabolite β-hydroxyethoxyacetic acid (HEAA) and on THF. Comparison of the transcriptomes of CB1190 grown on THF and succinate (an intermediate of THF degradation) permitted the identification of other genes involved in THF metabolism. Dioxane and THF oxidation activity of the THF MO was verified in Rhodococcus jostii RHA1 cells heterologously expressing the CB1190 thmADBC gene cluster. Interestingly, these thmADBC expression clones accumulated HEAA as a dead-end product of dioxane transformation, indicating that despite its genes being transcriptionally upregulated during growth on HEAA, the THF MO enzyme is not responsible for degradation of HEAA in CB1190. Similar activities were also observed in RHA1 cells heterologously expressing the thmADBC gene cluster from Pseudonocardia tetrahydrofuranoxydans K1.

Reconstructing the evolutionary history of nitrotoluene detection in the transcriptional regulator NtdR

Many toxic man‐made compounds have been introduced into the environment, and bacterial strains that are able to grow on them are ideal model systems for studying the evolution of metabolic pathways and regulatory systems. Acidovorax sp. strain JS42 is unique in its ability to use 2‐nitrotoluene as a sole carbon, nitrogen, and energy source for growth. The LysR‐type transcriptional regulator NtdR activates expression of the 2‐nitrotoluene degradation genes not only when nitroaromatic compounds are present, but also in the presence of a wide range of aromatic acids and analogues. The molecular determinants of inducer specificity were identified through comparative analysis with NagR, the activator of the naphthalene degradation pathway genes in Ralstonia sp. strain U2. Although NagR is 98% identical to NtdR, it does not respond to nitrotoluenes. Exchange of residues that differ between NagR and NtdR revealed that residues at positions 227 and 232 were key for the recognition of nitroaromatic compounds, while the amino acid at position 169 determined the range of aromatic acids recognized. Structural modelling of NtdR suggests that these residues are near the predicted inducer binding pocket. Based on these results, an evolutionary model is presented that depicts the stepwise evolution of NtdR.

Integration of chemotaxis, transport and catabolism in Pseudomonas putida and identification of the aromatic acid chemoreceptor PcaY.

Aromatic and hydroaromatic compounds that are metabolized through the β‐ketoadipate catabolic pathway serve as chemoattractants for Pseudomonas putida F1. A screen of P. putida F1 mutants, each lacking one of the genes encoding the 18 putative methyl‐accepting chemotaxis proteins (MCPs), revealed that pcaY encodes the MCP required for metabolism–independent chemotaxis to vanillate, vanillin, 4‐hydroxybenzoate, benzoate, protocatechuate, quinate, shikimate, as well as 10 substituted benzoates that do not serve as growth substrates for P. putida F1. Chemotaxis was induced during growth on aromatic compounds, and an analysis of a pcaY‐lacZ fusion revealed that pcaY is expressed in the presence of β‐ketoadipate, a common intermediate in the pathway. pcaY expression also required the transcriptional activator PcaR, indicating that pcaY is a member of the pca regulon, which includes three unlinked gene clusters that encode five enzymes required for the conversion of 4‐hydroxybenzoate to tricarboxylic acid cycle intermediates as well as the major facilitator superfamily transport protein PcaK. The 4‐hydroxybenzoate permease PcaK was shown to modulate the chemotactic response by facilitating the uptake of 4‐hydroxybenzoate, which leads to the accumulation of β‐ketoadipate, thereby increasing pcaY expression. The results show that chemotaxis, transport and metabolism of aromatic compounds are intimately linked in P. putida.

Taxis of Pseudomonas putida F1 toward Phenylacetic Acid Is Mediated by the Energy Taxis Receptor Aer2

ABSTRACT The phenylacetic acid (PAA) degradation pathway is a widely distributed funneling pathway for the catabolism of aromatic compounds, including the environmental pollutants styrene and ethylbenzene. However, bacterial chemotaxis to PAA has not been studied. The chemotactic strain Pseudomonas putida F1 has the ability to utilize PAA as a sole carbon and energy source. We identified a putative PAA degradation gene cluster (paa) in P. putida F1 and demonstrated that PAA serves as a chemoattractant. The chemotactic response was induced during growth with PAA and was dependent on PAA metabolism. A functional cheA gene was required for the response, indicating that PAA is sensed through the conserved chemotaxis signal transduction system. A P. putida F1 mutant lacking the energy taxis receptor Aer2 was deficient in PAA taxis, indicating that Aer2 is responsible for mediating the response to PAA. The requirement for metabolism and the role of Aer2 in the response indicate that P. putida F1 uses energy taxis to detect PAA. We also revealed that PAA is an attractant for Escherichia coli; however, a mutant lacking a functional Aer energy receptor had a wild-type response to PAA in swim plate assays, suggesting that PAA is detected through a different mechanism in E. coli. The role of Aer2 as an energy taxis receptor provides the potential to sense a broad range of aromatic growth substrates as chemoattractants. Since chemotaxis has been shown to enhance the biodegradation of toxic pollutants, the ability to sense PAA gradients may have implications for the bioremediation of aromatic hydrocarbons that are degraded via the PAA pathway.

Effects of Phenolic Monomers on Growth of Acidothermus cellulolyticus

Previous studies on biological pretreatment of switchgrass by solid‐state fermentation with Acidothermus cellulolyticus 11B have shown that inhibitory compounds prevent growth on untreated switchgrass. A. cellulolyticus was grown in liquid medium containing cellobiose with phenolic monomers added to determine if the phenolic compounds are one possible source of inhibition. Cinnamic acid derivatives (trans‐p‐coumaric, trans‐ferulic, and hydrocinnamic acids), hydroxybenzoic acids (p‐hydroxybenzoic, syringic, and vanillic acids), benzaldehydes (vanillin and p‐hydroxybenzaldehyde), and condensed tannin monomers (catechin and epicatechin) were tested at levels up to 20 mM. All compounds exhibited a dose‐response relationship and strongly inhibited growth at 20 mM. trans‐p‐Coumaric acid was found to be the strongest inhibitor of A. cellulolyticus growth, with a specific growth rate of 0.004 h−1 at 1 mM (0.18 h−1 without phenolic monomer). GC‐MS and HPLC methods were used to confirm the presence of these phenolic compounds in switchgrass and measure the amounts extracted using different conditions. The amounts of phenolic compounds measured were found to be higher than the threshold for growth inhibition. Leaching with water at 55°C was inefficient at removing bound phenolics, whereas NaOH treatment improved efficiency. Phenolic compounds spiked into alkaline pretreated switchgrass were also found to inhibit growth of A. cellulolyticus in solid‐state fermentation. However, addition of insoluble polyvinylpolypyrrolidone (PVPP) to switchgrass improved growth of A. cellulolyticus in liquid cultures, providing a possible approach for alleviating microbial inhibition due to phenolic compounds in lignocellulose.

Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

ABSTRACT Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.