Novel 4-Chlorophenol Degradation Gene Cluster and Degradation Route via Hydroxyquinol in Arthrobacter chlorophenolicus A6

Abstract

Arthrobacter chlorophenolicus A6, a previously described 4-chlorophenol-degrading strain, was found to degrade 4-chlorophenol via hydroxyquinol, which is a novel route for aerobic microbial degradation of this compound. In addition, 10 open reading frames exhibiting sequence similarity to genes encoding enzymes involved in chlorophenol degradation were cloned and designated part of a chlorophenol degradation gene cluster (cph genes). Several of the open reading frames appeared to encode enzymes with similar functions; these open reading frames included two genes, cphA-I and cphA-II, which were shown to encode functional hydroxyquinol 1,2-dioxygenases. Disruption of the cphA-I gene yielded a mutant that exhibited negligible growth on 4-chlorophenol, thereby linking the cph gene cluster to functional catabolism of 4-chlorophenol in A. chlorophenolicus A6. The presence of a resolvase pseudogene in the cph gene cluster together with analyses of the G؉C content and codon bias of flanking genes suggested that horizontal gene transfer was involved in assembly of the gene cluster during evolution of the ability of the strain to grow on 4-chlorophenol.Arthrobacter chlorophenolicus A6 is a gram-positive actinobacterium that was previously isolated from a soil slurry enriched with increasing concentrations of 4-chlorophenol (4-CP) (28). This bacterium can degrade unusually high concentrations of 4-CP (up to 2.7 mM) and other p-substituted phenols, such as 4-nitrophenol and 4-bromophenol (28). In addition, A. chlorophenolicus A6 can degrade 4-CP at low temperatures (5°C) and during temperature fluctuations between 28°C and 5°C (3). Strain A6 was previously tagged with marker genes encoding the green fluorescent protein (gfp) or firefly luciferase (luc) in order to specifically monitor its ability to survive in nonsterile soil as it degraded 4-CP (9). The tagged cells survived well and were metabolically active in soil contaminated with 4-CP (9, 12). A larger fraction of the cell population remained viable after incubation in soil at 5°C than after incubation in soil at 28°C (4).There have been several reports of 4-CP degradation in other bacteria, but none of these bacteria have been shown to degrade concentrations of 4-CP as high as those degraded by A. chlorophenolicus A6. Successful mineralization of 4-CP by bacteria is usually accomplished by the oxidation of 4-CP to 4-chlorocatechol, followed by ortho cleavage of the aromatic ring. After ring cleavage, the chlorine is removed and the carbon skeleton is transformed into products that are assimilated into the central metabolism of the cell. By contrast, two strains belonging to the actinobacterium group, Arthrobacter ureafaciens CPR706 (5) and Nocardioides sp. strain NSP41 (8), were reported to transform 4-CP into hydroquinone instead of 4-chlorocatechol. However, the degradation pathways in these strains have not been described further.The aim of the present study was to elucidate the biochemistry and genetics of 4-chlorophenol degradation in A. chlorophenolicus A6. Our...

Diversity of ‘benzenetriol dioxygenase’ involved in p-nitrophenol degradation in soil bacteria

“…at the 16S rRNA gene sequence level, however, both of them exhibited a high degree of BtD amino acid sequence similarity (98 % identity). Recently, Nordin et al [9]. also reported a similar observation with maleylacetate reductase (MaR); another enzyme involved in the degradation of aromatic compounds.…”

“…Several bacterial strains belonging to diverse taxonomic groups have been isolated and characterized for degradation of the above compounds [5,6]. The molecular regulation of some of these degradation pathways have also been elucidated in detail [7][8][9]. Aromatic ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes that catalyze aromatic ring cleavage for complete degradation of these compounds.…”

“…Amongst all the enzymes involved in PNP degradation in either of the above pathways, BtD is an important target for detailed characterization. Furthermore, this enzyme is also involved in microbial degradation of several other mono-and bi-phenolic compounds including chlorophenols and catechols [9,14,15]. One of the recent approaches for characterization of functional/ catabolic genes is to evaluate their sequence diversity amongst various sources [10].…”

Overexpression and Purification of Monooxygenases Cloned from Arthrobacter chlorophenolicus A6 for Enzymatic Decomposition of 4-Chlorophenol

“…Chlorophenol을 분해하는 효소들은 주로 호기성 박테리 아로부터 추출하여 연구되어 왔으며 대표적으로 Rhodococcus, Pseudomonas, Comamonas, Fusarium, Azobacter, Arthrobacter가 있다 (Hollender et al, 1997;Li et al, 2011;Nordin et al, 2005;Olaniran and Igbinosa, 2011;Sahoo et al, 2010). 이러한 호기성 박 테리아 중에서 Arthrobacter chlorophenolicus A6는 4-chlorophenol (4-CP)을 완전히 분해하는 것으로 알려졌으 며 monooxygenase인 CphC-I, CphC-II, CphB와 dioxygenase인 CphA-I의 4가지 산화분해효소들에 의해 4-CP 가 분해되는 것으로 제안된 바 있다 (Nordin et al, 2005).…”

“…이러한 호기성 박 테리아 중에서 Arthrobacter chlorophenolicus A6는 4-chlorophenol (4-CP)을 완전히 분해하는 것으로 알려졌으 며 monooxygenase인 CphC-I, CphC-II, CphB와 dioxygenase인 CphA-I의 4가지 산화분해효소들에 의해 4-CP 가 분해되는 것으로 제안된 바 있다 (Nordin et al, 2005). 이들 연구에 따르면, monooxygenase가 4-CP를 초기분해 대사과정의 중간산물로 추측한 benzoquinone 또 는 4-chlorocatechol(4-CC)로 변환시키고 다시 monooxygenase가 2~3개의 분해단계에 순차적으로 작용하여 기질 을 분해하는 것으로 보고한 바 있다 (Fig.…”