Functional and transcriptional analyses of the initial oxygenase genes for acenaphthene degradation from Sphingomonas sp. strain A4

Abstract

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene as its sole carbon and energy source. To isolate the genes responsible for acenaphthene degradation, transposon mutagenesis was performed on strain A4 and four mini-Tn5-inserted mutants lacking the ability to utilize acenaphthene were isolated. In three of the four mini-Tn5 inserted mutants, the mini-Tn5s were inserted into the same locus (within about 16 kb) as the arhA1A2 genes, which had previously been identified as the genes encoding the terminal oxygenase components for the initial oxygenation of acenaphthene. The nucleotide sequence analysis of the corresponding 16?4 kb DNA fragment revealed the existence of 16 ORFs and a partial ORF. From these ORFs, the genes encoding the ferredoxin (ArhA3) and ferredoxin reductase (ArhA4) complementary to ArhA1A2 were identified. RT-PCR analysis suggested that a 13?5 kb gene cluster, consisting of 13 ORFs and including all the arhA genes, forms an operon, although it includes several ORFs that are apparently unnecessary for acenaphthene degradation. Furthermore, using gene disruption and quantitative RT-PCR analyses, the LysR-type activator, ArhR, required for expression of the 13?5 kb gene cluster was also identified. Transcription of the gene cluster by ArhR was induced in the presence of acenaphthene (or its metabolite), and a putative binding site (T-N 11 -A motif) for ArhR was found upstream from the transcription start point of arhA3.

The Sphingomonas Plasmid pCAR3 Is Involved in Complete Mineralization of Carbazole

“…Among the Pseudomonas degradative plasmids, whole nucleotide sequences of pADP-1 (37), pEST4011 (65), pJP4 (62), pUO1 (IncP-1 [56]), pCAR1 (36,54), pND6-1 (IncP-7 [35]), NAH7 (57), pDTG1 (14), and pWW0 (IncP-9 [18]) have been determined and analyzed in detail. Various Sphingomonas strains have also been identified as xenobiotic degraders and have been shown to have diverse catabolic capabilities toward acenaphthene, biphenyl, naphthalene, carbazole (CAR), dibenzop-dioxin, and many other compounds (2,12,21,34). The sequences and organization of degradative genes are remarkably similar in several sphingomonads but differ from those in pseudomonads (46).…”

Sphingobium sp. HV3 degrades both herbicides and polyaromatic hydrocarbons using ortho- and meta-pathways with differential expression shown by RT-PCR

“…4) were assayed using real-time PCR with the aim of studying the activity of the three pathways in presence of different growth substrates. The 16S rRNA gene was used as a reference gene as in similar previous studies (Kouzuma et al 2006;Parnell et al 2010). Similar results were obtained from both absolute quantification and relative quantification methods (Data not shown).…”

The development of a liquid formulation of Pseudoxanthomonas sp. RN402 and its application in the treatment of pyrene-contaminated soil

“…RN402, Sphingomonas sp. A4 (Kouzuma et al 2006), Liquid formulation of Pseudoxanthomonas for bioremediation Novosphingobium aromaticivorans F199 (Romine et al 1999), Sphingobium sp. P2 (Pinyakong et al 2000) and Acinetobacter sp.…”

“…RN402 containing the nidA gene, Sphingomonas sp. strain A4 containing arh genes (Kouzuma et al 2006), N. aromaticivorans strain F199 containing bph genes (Romine et al 1999), Sphingobium sp. strain P2 containing bph genes (Pinyakong et al 2000) and Acinetobacter sp.…”