Acyl-CoA carboxylases (ACC) are involved in important primary or secondary metabolic pathways such as fatty acid and/or polyketides synthesis. In the 62 kb fragment of pccB gene locus of Streptomyces toxytricini producing a pancreatic inhibitor lipstatin, 3 distinct subunit genes of presumable propionyl-CoA carboxylase (PCCase) complex, assumed to be one of ACC responsible for the secondary metabolism, were identified along with gene for a biotin protein ligase (Bpl). The subunits of PCCase complex were a subunit (AccA3), P subunit (PccB), and auxiliary ɛ subunit (PccE). In order to disclose the involvement of the PCCase complex in secondary metabolism, some biochemical characteristics of each subunit as well as their complex were examined. In the test of substrate specificity of the PCCase complex, it was confirmed that this complex showed much higher conversion of propionyl-CoA rather than acetyl-CoA. It implies the enzyme complex could play a main role in the production of methylmalonyl-CoA from propionyl-CoA, which is a precursor of secondary polyketide biosynthesis.
Streptomyces toxytricini produces lipstatin, a specific inhibitor of pancreatic lipase, which is derived from two fatty acid moieties with eight and 14 carbon atoms. The pccB gene locus in 10.6 kb fragment of S. toxytricini chromosomal DNA contains three genes for acyl-coenzyme A carboxylase (ACCase) complex accA3, pccB, and pccE that are presumed to be involved in secondary metabolism. The pccB gene encoding a β subunit of ACCase [carboxyltransferase (CT)] was identified upstream of pccE gene for a small protein of ε subunit. The accA3 encoding the α subunit of ACCase [biotin carboxylase (BC)] was also identified downstream of pccB gene. When the pccB and pccE genes were inactivated by homologous recombination, the lipstatin production was reduced as much as 80%. In contrast, the accumulation of another compound, tetradeca-5.8-dienoic acid (the major lipstatin precursor), was 4.5-fold increased in disruptant compared with wild-type. It implies that PccB of S. toxytricini is involved in the activation of octanoic acid to hexylmalonic acid for lipstatin biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-010-2587-2) contains supplementary material, which is available to authorized users.
The nitrogen cycle and the associated microbes play an important role in natural ecosystems, including terrestrial habitats; they also have a major effect on climate change. The aim of this study was to explore microbial communities in rice paddy soil by detecting and quantifying some key functional genes involved in the nitrogen cycle using molecular techniques such as conventional polymerase chain reaction (PCR), clone library construction, sequencing, phylogenetic analysis, and real-time PCR. The genes analyzed were as follows: nitrogenase reductase gene (nifH), hydrazine synthase gene (hzsA), nitrous oxide reductase gene (nosZ), copper-containing (nirK) and cytochrome cd1-containing (nirS) nitrite reductase genes, nitrite oxidoreductase gene (nxrB), and ammonium monooxygenase gene (amoA). The sequence assessment using the clone library targeting these genes revealed high diversity and dominance of bacterial communities. Furthermore, real-time PCR using SYBR green dye and some primers specific for each gene revealed the high abundance of nxrB (4.1 × 10 9 ± 0.4 × 10 9 copies g −1 soil) and low abundance of hzsA (4.0 × 10 5 ± 1.1 × 10 5 copies g −1 soil). The findings of our study will be useful to explore microbial communities in terrestrial habitats, such as agricultural paddy fields.
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Hexavalent chromium (Cr(VI)) is recognized to be carcinogenic and toxic and registered as a contaminant in many drinking water regulations. It occurs naturally and is also produced by industrial processes. The reduction of Cr(VI) to Cr(III) has been a central topic for chromium remediation since Cr(III) is less toxic and less mobile. In this study, fermentative Fe(III)-reducing bacterial strains (Cellu-2a, Cellu-5a, and Cellu-5b) were isolated from a groundwater sample and were phylogenetically related to species of
Cellulomonas
by 16S rRNA gene analysis. One selected strain, Cellu-2a showed its capacity of reduction of both soluble iron (ferric citrate) and solid iron (hydrous ferric oxide, HFO), as well as aqueous Cr(VI). The strain Cellu-2a was able to reduce 15 μM Cr(VI) directly with glucose or sucrose as a sole carbon source under the anaerobic condition and indirectly with one of the substrates and HFO in the same incubations. The heterogeneous reduction of Cr(VI) by the surface-associated reduced iron from HFO by Cellu-2a likely assisted the Cr(VI) reduction. Fermentative features such as large-scale cell growth may impose advantages on the application of bacterial Cr(VI) reduction over anaerobic respiratory reduction.
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