RESUMO
The aim of this work was to characterize bacterial ring-hydroxylating dioxygenase (RHD) diversity in a pristine microbial mat and follow their diversity changes in response to heavy fuel oil contamination. In order to describe the RHDs diversity, new degenerate primers were designed and a nested-PCR approach was developed to gain sensitivity and wider diversity. RHD diversity in artificially contaminated mats maintained in microcosms and in chronically contaminated mats was analysed by clone libraries and terminal restriction fragment length polymorphism (T-RFLP) at genomic and transcriptomic levels. The RHD diversity in the pristine microbial mat was represented by Pseudomonas putida nahAc-like genes and no increase of diversity was detected after 1 year of oil contamination. The diversity observed in a 30 year chronically polluted microbial mat was represented by four main RHD clusters and two new genes revealing higher polyaromatic hydrocarbon (PAH) degradation capacity. This study illustrates that a single petroleum contamination (such as oil spill) is not enough to involve a detectable modification of RHD diversity. The new degenerate primers described here allowed RHD gene amplification from pristine and contaminated samples thereby showing their diversity. The proposed approach solves one of the main problems of functional gene analysis providing effective amplification of the environmental diversity of the targeted genes.
Assuntos
Bactérias/enzimologia , Bactérias/genética , Dioxigenases/biossíntese , Dioxigenases/genética , Sedimentos Geológicos/microbiologia , Hidrocarbonetos Aromáticos/metabolismo , Clonagem Molecular , Análise por Conglomerados , Impressões Digitais de DNA , Primers do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Dados de Sequência Molecular , Petróleo/metabolismo , Filogenia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Poluição Química da ÁguaRESUMO
AT2433, an indolocarbazole antitumor antibiotic, is structurally distinguished by its aminodideoxypentose-containing disaccharide and asymmetrically halogenated N-methylated aglycon. Cloning and sequence analysis of AT2433 gene cluster and comparison of this locus with that encoding for rebeccamycin and the gene cluster encoding calicheamicin present an opportunity to study the aminodideoxypentose biosynthesis via comparative genomics. The locus was confirmed via in vitro biochemical characterization of two methyltransferases--one common to AT2433 and rebeccamycin, the other unique to AT2433--as well as via heterologous expression and in vivo bioconversion experiments using the AT2433 N-glycosyltransferase. Preliminary studies of substrate tolerance for these three enzymes reveal the potential to expand upon the enzymatic diversification of indolocarbazoles. Moreover, this work sets the stage for future studies regarding the origins of the indolocarbazole maleimide nitrogen and indolocarbazole asymmetry.