Degradation of 2-bromo-, 2-chloro- and 2-fluorobenzoate by Pseudomonas putida CLB 250

Abstract Pseudomonas putida strain CLB 250 (DSM 5232) utilized 2-bromo-, 2-chloro- and 2-fluorobenzoate as sole source of carbon and energy. Degradation is suggested to be initiated by a dioxygenase liberating halide in the first catabolic step. After decarboxylation and rearomatization catechol is produced as a central metabolite which is degraded via the ortho-pathway. After inhibition of ring cleavage activities with 3-chlorocatechol, 2-chlorobenzoate was transformed to catechol in nearly stoichiometric amounts. Other ortho -substituted benzoates like anthranilate and 2-methoxybenzoate seem to be metabolized via the same route.     

Combination of azathioprine and UVA irradiation is a major source of cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine

Thiopurine antimetabolites, such as azathioprine (Aza) and 6-thioguanine (6-TG), are widely used in the treatment of cancer, inflammatory conditions and organ transplantation patients. Recent work has shown that cells treated with 6-TG and UVA generate ROS, with implied oxidatively generated modification of DNA. In a study of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in renal transplant patients, we provided the first in vivo evidence linking Aza and oxidatively damaged DNA. Using the hOGG1 comet assay, we herein demonstrate high levels of 8-oxodG and alkali-labile sites (ALS) in cells treated with biologically relevant doses of 6-TG, or Aza, plus UVA. This damage was induced dose-dependently. Surprisingly, given the involvement of 6-TG incorporation into DNA in its therapeutic effect, significant amounts of 8-oxodG and ALS were induced in quiescent cells, although less than in proliferating cells. We speculate that some activity of hOGG1 towards unirradiated, 6-TG treated cells, implies possible recognition of 6-TG or derivatives thereof. This is the first report to conclusively demonstrate oxidatively damaged DNA in cells treated with thiopurines and UVA. These data indicate that Aza-derived oxidative stress will occur in the skin of patients on Aza, following even low level UVA exposure. This is a probable contributor to the increased risk of non-melanoma skin cancer in these patients. However, as oxidative stress is unlikely to be involved in the therapeutic effects of Aza, intercepting ROS production in the skin could be a viable route by which this side effect may be minimised.     

The Degradation of Dimethoate and the Mineral Immobilizing Function for Cd2+ by Pseudomonas putida

Organophosphorus pollution and heavy metal pollution are prominent in China and have caused increasingly severe environmental pollution. This research used Pseudomonas putida to degrade dimethoate so as to induce the formation of calcium carbonate (CaCO₃) and calcium phosphate (Ca₃(PO₄)₂) in beef extract peptone medium. In addition, the mineral immobilizing function of the generated Ca₃(PO₄)₂ and CaCO₃ for Cd²⁺ was studied by adding different concentrations of Cd²⁺ to the culture solution. Meanwhile, transmission electron microscopy (TEM), scanning electronic microscopy (SEM), X-ray diffraction, gas chromatography and atomic absorption spectrophotometry were used to investigate the biodegradation of dimethoate, the concentration variation of Ca²⁺ and Cd²⁺, the mineral and chemical compositions of the precipitates. The results showed that the growth of P. Putida could increase the pH value of the culture solution and effectively degrade the organophosphorus pesticide dimethoate. Besides, the concentration of Ca²⁺ in the culture solution decreased significantly in the first four days and then tended to be stable. Moreover, the TEM and SEM results presented that there were large amounts of biogenic sedimentary CaCO₃ and a little Ca₃(PO₄)₂ in the precipitates. Furthermore, in the employed culture system, the removal rates of Cd²⁺, when added at two different concentrations (6 ppm and 15 ppm), reached 100%. Therefore, this study provided a new idea for treating wastewater polluted with organophosphorus pesticide and heavy metals by using microorganisms.     

Isotope dilution high-performance liquid chromatography–electrospray tandem mass spectrometry assay for the measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in biological samples

A sensitive and specific assay aimed at measuring 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) has been developed by associating a reversed-phase liquid chromatographic separation with an electrospray tandem mass spectrometric detection. The HPLC–MS approach in the single ion monitoring (SIM) mode and the HPLC–MS/MS assay in the multiple reaction monitoring (MRM) mode have been compared, using isotopically labeled [M+4] 8-oxodGuo as the internal standard. The limit of detection of 8-oxodGuo was found to be around 5 pmol and 20 fmol for the HPLC–MS and HPLC–MS/MS methods, respectively. The HPLC–MS/MS assay is sensitive enough to allow the determination of the level of 8-oxodGuo in cellular liver DNA and in urine samples.     

Conversion of indene to cis-(1S),(2R)-indandiol by mutants of Pseudomonas putida F1

Two mutation and selection methods were used to isolate mutants of Pseudomonas putida F1 which convert indene to cis-(1S),(2R)-indandiol in a toluene-independent fashion. Using soybean or silicone oil as a second phase to deliver indene to the culture, cis-(1S),(2R)-indandiol, cis-(1R),(2S)-indandiol, 1,2-indenediol (or the keto-hydroxy indan tautomer), and the monooxygenation products 1-indenol and 1-indanone were produced from indene as a function of time. Similarly the enantiomeric excess of the cis-(1S),(2R)-indandiol produced also increased with increasing time. In addition, mutants were isolated which produced cis-(1S),(2R)-indandiol of lower optical purity which corresponded to reduced levels of 1,2-indenediol. These data suggest this toluene dioxygenase produces cis-(1S),(2R)-indandiol of low optical purity and that cis-glycol dehydrogenase plays a role in resolving the two cis-1,2-indandiol enantiomers. Received 15 November 1996/ Accepted in revised form 09 March 1997     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

Biodegradation of cyanides, cyanates and thiocyanates to ammonia and carbon dioxide by immobilized cells of Pseudomonas putida

Pseudomonas putida utilizes cyanide as the sole source of carbon and nitrogen. Agar, alginate, and carrageenan were screened as the encapsulating matrices for P. putida. Alginate-immobilized cells of P. putida degraded sodium cyanide (NaCN) more efficiently than non-immobilized cells or cells immobilized in agar or carrageenan. The end products of biodegradation of cyanide were identified as ammonia (NH₃) and carbon dioxide (CO₂). These products changed the medium pH. In bioreactors, the rate of cyanide degradation increased with an increase in the rate of aeration. Maximum utilization of cyanide was observed at 200 ml min⁻¹ of aeration. Immobilized cells of P. putida degraded cyanides, cyanates and thiocyanates to NH₃ and CO₂. Use of Na[¹⁴C]-CN showed that 70% of carbon of Na[¹⁴C]-CN was converted into ¹⁴CO₂ and only 10% was associated with the cell biomass. The substrate-dependent kinetics indicated that the K _m and V _max values of P. putida for the substrate, NaCN were 14 mM and 29 nmol of oxygen consumed mg protein⁻¹ min⁻¹ respectively. Received 29 January 1996/ Accepted in revised form 19 September 1997     

Highly efficient production of 2-ketobutyrate from 2-hydroxybutyrate by resting cells of Pseudomonas stutzeri DEH130

Resting cells of Pseudomonas stutzeri DEH130 were found to act as biocatalysts to convert 2-hydroxybutyrate (2-HB) into 2-ketobutyrate (2-KA). Two different catalysis mechanisms of 2-KA formation were present in the cells, which were induced by glycolate (GA), and DL-lactate (DL-LA), respectively. The productivity number of 6.52?mmol g^?1 cells h^?1 was obtained when DL-LA was the sole carbon source and was the highest ever reported.     

Renal excretion of 8-oxo-7,8-dihydro-2(')-deoxyguanosine: degradation rates of RNA and metabolic rate in humans

Renally excreted 8-oxo-7,8-dihydro-2(')-deoxyguanosine (oxo(8)dG) is a potential marker of oxidative DNA damage by reactive oxygen species. Whole-body degradation rates of t- and rRNA are potential indicators of the resting metabolic rate (RMR). Excretion rates of oxo(8)dG and degradation rates of t- and rRNA were determined in healthy non-smoking adults and children. RMR (indirect calorimetry; 14 children, 16 adults), total energy expenditure (TEE; doubly labelled water technique; 4 children, 6 adults), and lean body mass (LBM; dual-energy X-ray absorptiometry; 14 children, 16 adults) were also measured. Degradation of t- and rRNA (micromol/d/kg LBM; 4 children, 6 adults) was highly correlated with RMR (kJ/d/kg LBM), r=0.867 (p<0.005) and 0.959 (p<0.001), respectively. Excretion of oxo(8)dG (pmol/d/kg LBM; 14 children, 16 adults) was not significantly correlated with RMR (p>0.05). Neither excretion of oxo(8)dG nor degradation of RNA was significantly correlated with TEE (kJ/d/ kg LBM) (p>0.05). In healthy subjects further factors, other than the metabolic rate, seem to influence the excretion rate of oxo(8)dG. The degradation rates of t- and rRNA seem to be appropriate indicators of the RMR.     

Putting cells under pressure: a simple and efficient way to enhance the productivity of medium-chain-length polyhydroxyalkanoate in processes with Pseudomonas putida KT2440

The success of bioprocess implementation relies on the ability to achieve high volumetric productivities and requires working with high‐cell‐density cultivations. Elevated atmospheric pressure might constitute a promising tool for enhancing the oxygen transfer rate (OTR), the major growth‐limiting factor for such cultivations. However, elevated pressure and its effects on the cellular environment also represent a potential source of stress for bacteria and may have negative effects on product formation. In order to determine whether elevated pressure can be applied for enhancing productivity in the case of medium‐chain‐length polyhydroxyalkanoate (mcl‐PHA) production by Pseudomonas putida KT2440, the impact of a pressure of 7 bar on the cell physiology was assessed. It was established that cell growth was not inhibited by this pressure if dissolved oxygen tension (DOT) and dissolved carbon dioxide tension (DCT) were kept below ～30 and ～90 mg L^?1, respectively. Remarkably, a little increase of mcl‐PHA volumetric productivity was observed under elevated pressure. Furthermore, the effect of DCT, which can reach substantial levels during high‐cell‐density processes run under elevated pressure, was investigated on cell physiology. A negative effect on product formation could be dismissed since no significant reduction of mcl‐PHA content occurred up to a DCT of ～540 mg L^?1. However, specific growth rate exhibited a significant decrease, indicating that successful high‐cell‐density processes under elevated pressure would be restricted to chemostats with low dilution rates and fed‐batches with a small growth rate imposed during the final part. This study revealed that elevated pressure is an adequate and efficient way to enhance OTR and mcl‐PHA productivity. We estimate that the oxygen provided to the culture broth under elevated pressure would be sufficient to triple mcl‐PHA productivity in our chemostat system from 3.4 (at 1 bar) to 11 g L^?1 h^?1 (at 3.2 bar). Biotechnol. Bioeng. 2012; 109:451–461. © 2011 Wiley Periodicals, Inc.     

Purification and characterization of a salicylate hydroxylase involved in 1-hydroxy-2-naphthoic acid hydroxylation from the naphthalene and phenanthrene-degrading bacterial strain Pseudomonas putida BS202-P1

1-Hydroxy-2-naphthoate is formed as an intermediate in the bacterial degradation of phenanthrene. A monooxygenase which catalyzed the oxidation of 1-hydroxy-2-naphthoateto 1,2-dihydroxynaphthalene was purified from the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1. The purified protein had a molecular weight of45 kDa and required NAD(P)H and FAD as cofactors. The purified enzyme also catalysed the oxidation of salicylate and various substituted salicylates. The comparison of the K_mand V_max values for 1-hydroxy-2-naphthoate and salicylate demonstrated a higher catalytic efficiency of the enzyme for salicylate as a substrate. A significant substrate-inhibition was detected with higher concentrations of 1-hydroxy-2-naphthoate.The aminoterminal amino acid sequence of the purified enzyme showed significant homologies to salicylate 1-monooxygenases from other Gram negative bacteria. It was therefore concluded that during the degradation of phenanthrene the conversion of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene is catalysed by a salicylate1-monooxygenase. Together with previous studies, this suggested that the enzymes of the naphthalene pathway are sufficient to catalyse also the mineralization of phenanthrene.     

Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta

In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) damage, many DNA polymerases exhibit a dual coding potential which facilitates efficient incorporation of matched dCTP or mismatched dATP. This also holds true for the insertion of 8-oxodGTP opposite template bases dC and dA. Employing single-turnover kinetic methods, we examined human DNA polymerase beta and its novel X-family homolog, human DNA polymerase lambda, to determine which nucleotide and template base was preferred when encountering 8-oxodG and 8-oxodGTP, respectively. While DNA polymerase beta preferentially incorporated dCTP over dATP, DNA polymerase lambda did not modulate a preference for either dCTP or dATP when opposite 8-oxodG in single-nucleotide gapped DNA, as incorporation proceeded with essentially equal efficiency and probability. Moreover, DNA polymerase lambda is more efficient than DNA polymerase beta to fill this oxidized single-nucleotide gap. Insertion of 8-oxodGTP by both DNA polymerases lambda and beta occurred predominantly against template dA, thereby reiterating how the asymmetrical design of the polymerase active site differentially accommodated the anti and syn conformations of 8-oxodG and 8-oxodGTP. Although the electronegative oxygen at the C8 position of 8-oxodG may induce DNA structural perturbations, human DNA ligase I was found to effectively ligate the incorporated 8-oxodGMP to a downstream strand, which sealed the nicked DNA. Consequently, the erroneous nucleotide incorporations catalyzed by DNA polymerases lambda and beta as well as the subsequent ligation catalyzed by a DNA ligase during base excision repair are a threat to genomic integrity.     

Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products

Epidemiological studies testing the effect of β-carotene in humans have found a relative risk for lung cancer in smokers supplemented with β-carotene. We investigated the reactions of retinal and β-apo-8′-carotenal, two β-carotene oxidation products, with 2′-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N²-etheno-2′-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with β-carotene or β-carotene oxidation products, significantly increased levels of 1,N²-etheno-2′-deoxyguanosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of β-carotene oxidation products.     

Inhibition by singlet oxygen quenchers of oxidative damage to DNA produced in cultured cells by exposure to a quinolone antibiotic and ultraviolet A irradiation

The photogenotoxicity mechanism of quinolone antibiotics was investigated by measuring oxidative DNA damage in lomefloxacin- and UVA-exposed cultured liver-derived cells. The combination of lomefloxacin and UVA irradiation produced a dose-dependent increase in 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo-dG) in cell DNA. This DNA damage was substantially inhibited by co-incubation with sodium azide (NaN₃) or 2,2,6,6-tetramethyl-4-piperadone (TMP), chemicals that specifically quench singlet oxygen. No significant reduction of 8-oxo-dG formation was produced by N-t-butyl--phenylnitrone (TBP) or -tocopherol, which primarily scavenge hydroxyl radicals. We conclude that the photodynamic generation of 8-oxo-dG by quinolones is mediated, at least in part, by singlet oxgen.     

Degradation of 2-methylnaphthalene by free and immobilized cells of Pseudomonas sp. strain NGK1

A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of utilizing 2-methylnaphthalene (2-MN) was immobilized in various matrices namely, polyurethane foam (PUF), alginate, agar and polyvinyl alcohol (PVA) (1.5 × 10¹² c.f.u. g^–1 beads). The degradation rates of 25 and 50 mM 2-MN by freely suspended cells (2 × 10¹¹ c.f.u. ml^–1) and immobilized cells in batches, semi-continuous with shaken culture and continuous degradation in a packed-bed reactor were compared. The PUF-immobilized cells achieved higher degradation of 25 and 50 mM of 2-MN than freely suspended cells and the cells immobilized in alginate, agar or PVA. The PVA- and PUF-immobilized cells could be reused for more than 30 and 20 cycles respectively, without losing any degradation capacity. The effect of dilution rates on the rate of degradation of 25 and 50 mM 2-MN with freely suspended and immobilized cells were compared in the continuous system. Increase in dilution rate increased the degradation rate only up to 1 h^–1 in free cells with 25 mM 2-MN and no significant increase was observed with 50 mM 2-MN. With immobilized cells, the degradation rate increased with increase in dilution rate up to 1.5 h^–1 for 25 mM and 1 h^–1 for 50 mM 2-MN. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for biodegradation of 2-MN.     

Combined analysis of polymorphism variants in hMTH1, hOGG1 and MUTYH genes on the risk of type 2 diabetes in the Chinese population

Reactive oxygen species are considered to play a role in the development of type 2 diabetes mellitus (T2DM) and its complications. 8-Oxoguanine, which is one of the major oxidation base lesions produced by reactive oxygen species, may cause G:C to T:A transversion mutations because it can mispair with adenine. hMTH1 (human mutT homolog 1), hOGG1 (human 8-oxoguanine glycosylase 1) and MUTYH (human mutY homolog) genes constitute the 8-oxoG repair pathway. In this study, we screened for the polymorphism variants Val83Met (c.247G>A, rs4866) in hMTH1; c.-53G>C (rs56387615), c.-23A>G (rs1801129) and c.-18G>T (rs1801126) in the 5′-UTR of hOGG1; and AluYb8 insertion in MUTYH (AluYb8MUTYH, rs10527342) and investigated their synergistic effect on the risk of T2DM in the Chinese population. The genotypes were determined by electrophoresis, a high-resolution melting technique and sequencing of PCR products. Our results showed that the c.247G>A variant in the hMTH1 gene increased the risk of T2DM in > 55 years of age groups (OR = 1.579; 95%CI: 1.029–2.421). The set of c.-53G>C, c.-23A>G and c.-18G>T variants detected in the 5′-UTR of the hOGG1 gene and the AluYb8 insertion in the MUTYH gene were each associated with an increased risk of T2DM (OR = 1.507, 95%CI: 1.122–2.024; OR = 1.229, 95%CI: 1.030–1.466, respectively). Combined analysis of the variations among the three genes suggested that the c.247G>A variant in hMTH1 combined with AluYb8MUTYH variant had a synergistic effect on increasing the risk of T2DM (OR = 1.635; 95%CI: 1.147–2.330). This synergy was also observed between the variants in the 5′-UTR of the hOGG1 and the AluYb8MUTYH variant (OR = 1.804; 95%CI: 1.254–2.595). Our results suggest, for the first time, the combined effects of AluYb8MUTYH with either hMTH1 c.247G>A or variants in the 5′-UTR of the hOGG1 on the risk of T2DM.     

Transformation of 2-aminoacetophenone to (S)-2-amino-1-phenylethanol by Arthrobacter sulfureus

A new isolate of Arthrobacter sulfureus , when incubated at 50 g resting cells (dry cell wt) l(-1) with 50 g glucose l(-1) and 1 g 2-aminoacetophenone l(-1) in 50 mm potassium buffer (pH 7, 4 ml) at 30 degrees C, produced ( S )-2-amino-1-phenylethanol (e.e. >99%) with 75% yield in 6 h.