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Characterization of the genes responsible for rubber degradation in Actinoplanes sp. strain OR16.

ABSTRACT: A Gram-positive rubber-degrading bacterium, Actinoplanes sp. strain OR16 (strain NBRC 114529), is able to grow on agar plates containing natural and synthetic rubber as the sole sources of carbon and energy. When this strain was grown on natural rubber latex overlay agar plates, translucent halos around the cells were observed. To identify the natural rubber degradation genes and other features of its metabolism, its complete genome sequence was determined. The genome of OR16 consists of 9,293,892 bp and comprises one circular chromosome (GenBank accession number AP019371.1) with a G + C content of 70.3%. The genome contains 8238 protein-coding and 18 rRNA genes. A homology search of the genome sequence revealed that three genes (lcp1, lcp2, and lcp3) are homologous to an extracellular latex-clearing protein (Lcp) of Streptomyces sp. K30. RT-PCR analysis revealed that lcp1 and lcp2 seem to constitute an operon. Purified lcp gene products have oxygen consumption activity toward natural rubber latex, suggesting that all these genes encode rubber-degrading enzymes in OR16. Quantitative reverse transcription-PCR analysis indicated that the transcription of these genes is induced during the growth of OR16 on natural rubber. The genes located adjacent to lcp1 and lcp3, which code for a TetR/AcrR-type transcriptional regulator, can bind to the promoter regions of these lcp genes. It is suggested that the putative regulators play a role in regulating the transcription of the lcp genes. These results strongly suggested that three lcp genes are required for the utilization of natural rubber in strain OR16. Key Points • The complete genome sequence of Actinoplanes sp. strain OR16 was determined. • Three lcp genes which are involved in the natural rubber degradation in OR16 were identified. • Transcription of these lcp genes is induced during utilization of rubber in OR16. • Two regulators, which bind to the promoter regions of lcp, were determined.

SUBMITTER: Gibu N

PROVIDER: S-EPMC7413915 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Characterization of the genes responsible for rubber degradation in Actinoplanes sp. strain OR16.

Gibu Namiko N Arata Tomoka T Kuboki Saya S Linh Dao Viet DV Fukuda Masao M Steinbüchel Alexander A Kasai Daisuke D

Applied microbiology and biotechnology 20200718 17

A Gram-positive rubber-degrading bacterium, Actinoplanes sp. strain OR16 (strain NBRC 114529), is able to grow on agar plates containing natural and synthetic rubber as the sole sources of carbon and energy. When this strain was grown on natural rubber latex overlay agar plates, translucent halos around the cells were observed. To identify the natural rubber degradation genes and other features of its metabolism, its complete genome sequence was determined. The genome of OR16 consists of 9,293,8 ...[more]

PMID: 32681242

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Project description:Carbofuran, a broad-spectrum systemic insecticide, has been extensively used for approximately 50 years. Diverse carbofuran-degrading bacteria have been described, among which sphingomonads have exhibited an extraordinary ability to catabolize carbofuran; other bacteria can only convert carbofuran to carbofuran phenol, while all carbofuran-degrading sphingomonads can degrade both carbofuran and carbofuran phenol. However, the genetic basis of carbofuran catabolism in sphingomonads has not been well elucidated. In this work, we sequenced the draft genome of Sphingomonas sp. strain CDS-1 that can transform both carbofuran and carbofuran phenol but fails to grow on them. On the basis of the hypothesis that the genes involved in carbofuran catabolism are highly conserved among carbofuran-degrading sphingomonads, two such genes, cehACDS-1 and cfdCCDS-1, were predicted from the 84 open reading frames (ORFs) that share ?95% nucleic acid similarities between strain CDS-1 and another sphingomonad Novosphingobium sp. strain KN65.2 that is able to mineralize the benzene ring of carbofuran. The results of the gene knockout, genetic complementation, heterologous expression, and enzymatic experiments reveal that cehACDS-1 and cfdCCDS-1 are responsible for the conversion of carbofuran and carbofuran phenol, respectively, in strain CDS-1. CehACDS-1 hydrolyzes carbofuran to carbofuran phenol. CfdCCDS-1, a reduced flavin mononucleotide (FMNH2)- or reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase, hydroxylates carbofuran phenol at the benzene ring in the presence of NADH, FMN/FAD, and the reductase CfdX. It is worth noting that we found that carbaryl hydrolase CehAAC100, which was previously demonstrated to have no activity toward carbofuran, can actually convert carbofuran to carbofuran phenol, albeit with very low activity.IMPORTANCE Due to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. This study revealed the genetic determinants of carbofuran degradation in Sphingomonas sp. strain CDS-1. We speculate that the close homologues cehA and cfdC are highly conserved among other carbofuran-degrading sphingomonads and play the same roles as those described here. These findings deepen our understanding of the microbial degradation mechanism of carbofuran and lay a foundation for the better use of microbes to remediate carbofuran contamination.

Dataset Information

Characterization of the genes responsible for rubber degradation in Actinoplanes sp. strain OR16.

Publications

Characterization of the genes responsible for rubber degradation in Actinoplanes sp. strain OR16.

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