RESUMO
The marine gamma-Proteobacterium Alcanivorax borkumensis is highly specialized in the assimilation of aliphatic hydrocarbons, and makes up a large part of the biomass in oil-polluted marine environments. In addition to the previously identified alkane hydroxylase AlkB1, a second alkane hydroxylase (AlkB2) showing 65% identity to the Pseudomonas aeruginosa AlkB2 alkane hydroxylase was identified. Unlike alkB1, alkB2 is not flanked by genes involved in alkane metabolism. Heterologous expression of the A. borkumensis AP1 alkB1 and alkB2 genes showed that they encode functional alkane hydroxylases with substrate ranges similar to those of their P. putida and P. aeruginosa homologues. The transcription initiation sites and levels of the alkB1, alkB2 and alkS mRNA transcripts were determined. Expression of both alkB1 and alkB2 was induced by alkanes, but transcripts corresponding to alkB1 were much more abundant than those of alkB2. An inverted repeat similar to the binding site for the P. putida GPo1 transcriptional activator AlkS was present upstream of the promoters for alkB1 and alkB2, although that of alkB2 was less well conserved, and only the transcriptional fusion of promoter PalkB1 to the reporter gene lacZ efficiently responded to n-octane. Contrary to what has been found for the P. putida GPo1 alkane degradation pathway, expression of the A. borkumensis AP1 alkS gene was not induced by alkanes, and an AlkS binding site was not present upstream of the promoter for alkS. This indicates that, in spite of the clear similarities, the A. borkumensis alk-genes are regulated by a strategy different from that of the P. putida GPo1 alk genes.
Assuntos
Citocromo P-450 CYP4A/genética , Citocromo P-450 CYP4A/metabolismo , Halomonadaceae/enzimologia , Halomonadaceae/genética , Alcanos/metabolismo , Fusão Gênica Artificial , Biodegradação Ambiental , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Genes Reporter , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Ácido Nucleico , Água do Mar/microbiologia , Análise de Sequência de DNA , Homologia de Sequência , Especificidade por Substrato , Sítio de Iniciação de Transcrição , Poluição Química da Água , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Oxidation of n-alkanes in bacteria is normally initiated by an enzyme system formed by a membrane-bound alkane hydroxylase and two soluble proteins, rubredoxin and rubredoxin reductase. Pseudomonas aeruginosa strains PAO1 and RR1 contain genes encoding two alkane hydroxylases (alkB1 and alkB2), two rubredoxins (alkG1 and alkG2), and a rubredoxin reductase (alkT). We have localized the promoters for these genes and analyzed their expression under different conditions. The alkB1 and alkB2 genes were preferentially expressed at different moments of the growth phase; expression of alkB2 was highest during the early exponential phase, while alkB1 was induced at the late exponential phase, when the growth rate decreased. Both genes were induced by C(10) to C(22)/C(24) alkanes but not by their oxidation derivatives. However, the alkG1, alkG2, and alkT genes were expressed at constant levels in both the absence and presence of alkanes.
