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Draft genome sequence of humic substances-degrading Pseudomonas kribbensis CHA-19 from temperate forest soil
Korean J. Microbiol 2019;55(2):177-179
Published online June 30, 2019
© 2019 The Microbiological Society of Korea.

Dockyu Kim1,*, and Hyoungseok Lee2

1Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea,
2Unit of Polar Genomics, Korea Polar Research Institute, Incheon 21990, Republic of Korea
Correspondence to: E-mail: envimic@kopri.re.kr; Tel.: +82-32-760-5525; Fax: +82-32-760-5509
Received April 11, 2019; Revised May 8, 2019; Accepted May 8, 2019.
Abstract

Pseudomonas kribbensis CHA-19 was isolated from a temperate forest soil (mid latitude) in New Jersey, USA, for its ability to degrade humic acids, a main component of humic substances (HS), and subsequently confirmed to be able to decolorize lignin (a surrogate for HS) and catabolize lignin-derived ferulic and vanillic acids. The draft genome sequence of CHA-19 was analyzed to discover the putative genes for depolymerization of polymeric HS (e.g., dye-decolorizing peroxidases and laccase-like multicopper oxidases) and catabolic degradation of HS-derived small aromatics (e.g., vanillate O-demethylase and biphenyl 2,3-dioxygenase). The genes for degradative activity were used to propose a HS degradation pathway of soil bacteria.

Keywords : catabolic pathway, degradative enzyme, humic acids, soil bacteria
Body

Humic substances (HS) are a natural complex heteropolymer, which are widely distributed in various cold, temperate, and tropical soils. HS and HS-derived compounds regulate the growth of plants and microorganisms through various and continuous interactions within soils (Grinhut et al., 2011; Lipczynska-kochany, 2018). Owing to a structural similarity between lignin and HS, bacterial HS-degradative pathways were proposed based on previous studies for lignin degradation (Bugg et al., 2011; Kamimura et al., 2017; Kim et al., 2018). It is assumed that HS are depolymerized by bacterial extracellular enzymes, such as dye-decolorizing peroxidases and laccase-like multicopper oxidases, and the resulting HS-derived small aromatic compounds are uptaken into the cells and further catabolized.

A forest soil containing decaying plant material was sampled to study on the HS microbial degradation from New Jersey, USA, in September 2016. A bacterial strain (CHA-19) was isolated from the soil using an MSB minimal-agar plate owing to its ability to degrade humic acids (HA, Sigma-Aldrich; Cat. no. 53680). CHA-19 was able to decolorize lignin (Sigma-Aldrich; Cat. no. 370959) and catabolize lignin-derived mono-aromatics (ferulic and vanillic acids).

The analysis of 16S rRNA gene of CHA-19 (GenBank no. MK660005) showed that it was phylogenetically closest to Pseudomonaskribbensis 46-2T (99.93% similarity), P. koreensis Ps 9-14T (99.59% similarity) and P. moraviensis CCM 7280T (99.45% similarity). Genome sequencing of CHA-19 was performed at ChunLab, Inc. using the Illumina Miseq sequencing method and the sequence was assembled de novo into 34 contigs with SPAdes 3.10.1 (Bankevich et al., 2012). The average nucleotide identity (ANI) values between the type strains of P. kribbensis, P. koreensis, and P. moraviensis and CHA-19 were 95.88%, 88.82%, and 87.81%, respectively, by ChunLab TrueBac ID algorithm, and thus this strain was finally named Pseudomonaskribbensis CHA-19 (= KCTC 72262).

The draft genome sequence was approximately 6.4 Mb long with a G+C content of 60.6%. The resulting N50 size of contigs was 413,591 bp and the total coverage over the genome was 297–fold. Following NCBI GenBank submission, the genes in draft genome sequence were annotated with NCBI Prokaryotic Genome Annotation Pipeline (PGAP) using best-placed reference protein set; GeneMarkS-2 method (Lomsadze et al., 2018). The genome annotation revealed 5,737 coding sequences (CDSs), 64 tRNA genes, and 4 rRNA genes (two for 5S, one for 16S, and one for 23S). Several putative HS-degradative genes were detected on the CHA-19 draft genome, which were used to propose a HS-degradation pathway by CHA-19 (Fig. 1): laccase-like multicopper oxidases [GenBank accession no. TFH77958 (moxA) and TFH78995], dye-decolorizing peroxidases [TFH80052 (efeB), TFH80975 (yfeX), and TFH81056 (yfeX)], biphenyl 2,3-dioxygenase [TFH78976 (cntA), TFH79968 (hsaC), and TFH80324 (hcaE)], 2,3-dihydroxybiphenyl-1,2-dioxygenase [TFH79858 (hsaC)], vanillate O-demethylase [TFH78866 (vanB) and TFH79337 (vanA)], protocatechuate 3,4-dioxygenase for ortho-ring cleavage [TFH82232 (pcaH) and TFH82233 (pcaG)], and catechol 1,2-dioxygenase for ortho-ring cleavage [TFH78177 (catA)].

Fig. 1.

Proposed HS-degradative pathway by Pseudomonas kribbensis CHA-19. Dotted and solid lines represent multi-step reactions by different enzymes and one-step reactions by one enzyme, respectively. GenBank accession numbers for putative enzymes catalyzing the corresponding reactions are shown next to the lines.


Nucleotide sequence accession number

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession SPDQ00000000. The version described in this paper is version SPDQ01000000 and consists of sequences SPDQ01000001-SPDQ01000034.

적 요

미국 뉴저지주 중위도 산림토양에서 부식산(천연 복합유기화합물인 부식질의 주요 구성성분) 분해능이 있는 세균 균주 Pseudomonas kribbensis CHA-19를 분리하였으며, 이후 또 다른 토양 유기물인 리그닌과 리그닌 유래의 페룰산(ferulic acid)과 바릴린산(vanillic acid)의 분해능을 확인하였다. 부식질 초기 저분자화 효소(예, dye-decolorizing peroxidase와 laccase-like multicopper oxidase)와 부식질 유래의 다양한 저분자 분해산물들을 분해하는 효소(예, vanillate O-demethylase와 biphenyl 2,3-dioxygenase)를 탐색하기 위해 CHA-19 게놈 염기서열을 분석하였다. 최종 확보한 효소유전자 정보는 토양 세균의 부식질 분해경로 제안에 사용되었다.

Acknowledgements

This work was supported by a grant, Modeling responses of terrestrial organisms to environmental changes on King George Island (PE19090), funded by the Korea Polar Research Institute.

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June 2019, 55 (2)