The crystal structure of D-threonine aldolase from Alcaligenes xylosoxidans provides insight into a metal ion assisted PLP-dependent mechanism.

Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various ß-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the ß-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

Microbial abundance and community composition influence production performance in a low-temperature petroleum reservoir.

Enhanced oil recovery using indigenous microorganisms has been successfully applied in the petroleum industry, but the role of microorganisms remains poorly understood. Here, we investigated the relationship between microbial population dynamics and oil production performance during a water flooding process coupled with nutrient injection in a low-temperature petroleum reservoir. Samples were collected monthly over a two-year period. The microbial composition of samples was determined using 16S rRNA gene pyrosequencing and real-time quantitative polymerase chain reaction analyses. Our results indicated that the microbial community structure in each production well microhabitat was dramatically altered during flooding with eutrophic water. As well as an increase in the density of microorganisms, biosurfactant producers, such as Pseudomonas, Alcaligenes, Rhodococcus, and Rhizobium, were detected in abundance. Furthermore, the density of these microorganisms was closely related to the incremental oil production. Oil emulsification and changes in the fluid-production profile were also observed. In addition, we found that microbial community structure was strongly correlated with environmental factors, such as water content and total nitrogen. These results suggest that injected nutrients increase the abundance of microorganisms, particularly biosurfactant producers. These bacteria and their metabolic products subsequently emulsify oil and alter fluid-production profiles to enhance oil recovery.

Metabolism of [14C]-2,4-dichlorophenol in edible plants.

Several 2,4-dichlorophenoxyacetic acid (2,4-D)-sensitive plants have been modified by genetic engineering with tfdA gene to acquire 2,4-D tolerance. The expression product of this gene degrades 2,4-D to 2,4-dichlorophenol (DCP), which is less phytotoxic but could cause a problem of food safety. After a comparison of 2,4-D and DCP metabolism in transgenic 2,4-D-tolerant and wild cotton (Gossypium hirsutum L.), a direct study of DCP metabolism in edible plants was performed. After petiolar uptake of a [U-phenyl-(14)C]-DCP solution followed by a 48 h water chase, aqueous extracts were analysed by high-performance liquid chromatography. Metabolites were thereafter isolated and their structural identities were determined by enzymatic and chemical hydrolyses and mass spectrometry analyses. The metabolic fate of DCP was equivalent to 2,4-D metabolism in transgenic 2,4-D-tolerant cotton. In addition, DCP metabolism was similar in transgenic and wild cotton. The major terminal metabolites were DCP-saccharide conjugates in all species, essentially DCP-(6-O-malonyl)-glucoside or its precursor DCP-glucose. The significance of this metabolic pathway with regard to food safety is discussed.

Transformation of thiodiglycol by resting cells of Alcaligenes xylosoxydans PGH10.

A new strain of Alcaligenes xylosoxydans able to aerobically cometabolize thiodiglycol, the primary hydrolysis product of sulfur mustard, was isolated and tested in a laboratory scale stirred tank reactor. The strain, named PGH10, cannot use TDG as sole carbon and energy source for growth, but resting cells previously grown on either rich broth or defined mineral media efficiently metabolize this compound through [(2-hydroxyethyl)thio]acetic acid and thiodiacetic acid as intermediates. Degradation of TDG by PGH10 is shown to take place at late exponential and stationary phase but is not triggered by carbon exhaustion. Cultures pregrown to saturation for 48 h in the absence of TDG can be stored and used for degradation of TDG, reducing significantly the time required to achieve the reduction of the compound concentration to undetectable levels. Degradation can take place in buffered media with no carbon source added, although best results were obtained in mineral media supplemented with citrate or fructose. Oxidation to [(2-hydroxyethyl)thio]acetic acid and thiodiacetic acid was proposed to be catalyzed by a butanol-dehydrogenase activity. Inhibition of TDG transformation in the presence of several alcohols is also shown.

Anaerobic utilization of essential oils by denitrifying bacteria.

Plant volatile organic compounds are a major carbon source in nature. We studied the degradability of these substances by anaerobic microorganisms in enrichment cultures with representative essential oils as organic substrates and nitrate as electron acceptor. Lemon and pine needle oil supported microbial growth in the presence of pure oil, whereas parsley seed, camphor, sage, fennel, and mint oil supported growth only when the essential oils were dissolved in an overlying phase of 2,2,4,4,6,8,8-heptamethylnonane. Thyme oil did not support denitrification. Analyses of the microbially degraded oils revealed the disappearance of monoterpenes, of several monoterpenoids, and of methoxy-propenyl-benzenes, including apiole and myristicin. Most-probable-number determinations for denitrifying communities in sewage sludge and forest soil yielded 10(6) to 10(7) monoterpene-utilizing cells ml(-1), representing 0.7 to 100% of the total cultivable nitrate-reducing microorganisms. The utilization of essential oils together with the common occurrence of this metabolic trait are indications for an environmentally important, but currently unexplored anaerobic turnover of plant volatile organic compounds in soil.

Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis.

We investigated the use of multiplex polymerase chain reaction (PCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWW0), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWW0, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10(0) to 10(6) cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 microL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradative cells, at a lower detection limit of approximately 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.