A comparative study of biodegradation of vinyl acetate by environmental strains

Four Gram-negative strains, E3_2001, EC1_2004, EC3_3502 and EC2_3502, previously isolated from soil samples, were subjected to comparative studies in order to select the best vinyl acetate degrader for waste gas treatment. Comparison of biochemical and physiological tests as well as the results of fatty acids analyses were comparable with the results of 16S rRNA gene sequence analyses. The isolated strains were identified as Pseudomonas putida EC3_2001, Pseudomonas putida EC1_2004, Achromobacter xylosoxidans EC3_3502 and Agrobacterium sp. EC2_3502 strains. Two additional strains, Pseudomonas fluorescens PCM 2123 and Stenotrophomonas malthophilia KB2, were used as controls. All described strains were able to use vinyl acetate as the only source of carbon and energy under aerobic as well as oxygen deficiency conditions. Esterase, alcohol dehydrogenase and aldehyde dehydrogenase were involved in vinyl acetate decomposition under aerobic conditions. Shorter degradation times of vinyl acetate were associated with accumulation of acetic acid, acetaldehyde and ethanol as intermediates in the culture fluids of EC3_2001 and KB2 strains. Complete aerobic degradation of vinyl acetate combined with a low increase in biomass was observed for EC3_2001 and EC1_2004 strains. In conclusion, P. putida EC1_2004 is proposed as the best vinyl acetate degrader for future waste gas treatment in trickle-bed bioreactors

An Anaerobic-Type α-Ketoglutarate Ferredoxin Oxidoreductase Completes the Oxidative Tricarboxylic Acid Cycle of Mycobacterium tuberculosis

Aerobic organisms have a tricarboxylic acid (TCA) cycle that is functionally distinct from those found in anaerobic organisms. Previous reports indicate that the aerobic pathogen Mycobacterium tuberculosis lacks detectable α-ketoglutarate (KG) dehydrogenase activity and drives a variant TCA cycle in which succinyl-CoA is replaced by succinic semialdehyde. Here, we show that M. tuberculosis expresses a CoA-dependent KG dehydrogenase activity, albeit one that is typically found in anaerobic bacteria. Unlike most enzymes of this family, the M. tuberculosis KG: ferredoxin oxidoreductase (KOR) is extremely stable under aerobic conditions. This activity is absent in a mutant strain deleted for genes encoding a previously uncharacterized oxidoreductase, and this strain is impaired for aerobic growth in the absence of sufficient amounts of CO2. Interestingly, inhibition of the glyoxylate shunt or exclusion of exogenous fatty acids alleviates this growth defect, indicating the presence of an alternate pathway that operates in the absence of β-oxidation. Simultaneous disruption of KOR and the first enzyme of the succinic semialdehyde pathway (KG decarboxylase; KGD) results in strict dependence upon the glyoxylate shunt for growth, demonstrating that KG decarboxylase is also functional in M. tuberculosis intermediary metabolism. These observations demonstrate that unlike most organisms M. tuberculosis utilizes two distinct TCA pathways from KG, one that functions concurrently with β-oxidation (KOR-dependent), and one that functions in the absence of β-oxidation (KGD-dependent). As these pathways are regulated by metabolic cues, we predict that their differential utilization provides an advantage for growth in different environments within the host

High-Definition DNA Methylation Profiles from Breast and Ovarian Carcinoma Cell Lines with Differing Doxorubicin Resistance

Acquired drug resistance represents a frequent obstacle which hampers efficient chemotherapy of cancers. The contribution of aberrant DNA methylation to the development of drug resistant tumor cells has gained increasing attention over the past decades. Hence, the objective of the presented study was to characterize DNA methylation changes which arise from treatment of tumor cells with the chemotherapeutic drug doxorubicin. DNA methylation levels from CpG islands (CGIs) linked to twenty-eight genes, whose expression levels had previously been shown to contribute to resistance against DNA double strand break inducing drugs or tumor progression in different cancer types were analyzed. High-definition DNA methylation profiles which consisted of methylation levels from 800 CpG sites mapping to CGIs around the transcription start sites of the selected genes were determined. In order to investigate the influence of CGI methylation on the expression of associated genes, their mRNA levels were investigated via qRT-PCR. It was shown that the employed method is suitable for providing highly accurate methylation profiles, comparable to those obtained via clone sequencing, the gold standard for high-definition DNA methylation studies. In breast carcinoma cells with acquired resistance against the double strand break inducing drug doxorubicin, changes in methylation of specific cytosines from CGIs linked to thirteen genes were detected. Moreover, similarities between methylation profiles obtained from breast and ovarian carcinoma cell lines with acquired doxorubicin resistance were found. The expression levels of a subset of analyzed genes were shown to be linked to the methylation levels of the analyzed CGIs. Our results provide detailed DNA methylation information from two separate model systems for acquired doxorubicin resistance and suggest the occurrence of similar methylation changes in both systems upon exposure to the drug