DE NOVO ASSEMBLY OF COMPLETE GENOME SEQUENCE OF PLANOCOCCUS

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De novo assembly of complete genome sequence of Planococcus kocurii ATCC 43650T, a potential plant growth promoting bacterium


Wah-Seng See-Tooa,b, Peter Conveyb,c, David A. Pearceb,c,d, Tan Jia Yia, Yan-Lue Lima, Robson Eea, Wai-Fong Yina , Kok-Gan Chana,e*


a Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia


b National Antarctic Research Centre (NARC), Institute of Postgraduate Studies, University of Malaya, 50603 Kuala Lumpur, Malaysia


c British Antarctic Survey, NERC, High Cross, Madingley Road, Cambridge CB3 OET, UK


d Faculty of Health and Life Sciences, University of Northumbria, Newcastle Upon Tyne NE1 8ST, UK


e UM Omics Centre, University of Malaya, Kuala Lumpur


*Corresponding author Tel.: +603–79675162; E-mail address: [email protected]



Abstract

Planococcus kocurii ATCC 43650T is a halotolerant and psychrotolerant bacterium isolated from the skin of a North sea cod. Here, we present the first complete genome and annotation of P. kocurii ATCC 43650T, identifying its potential as a plant growth promoting bacterium and its capability in the biosynthesis of butanol.


Keyword

Halotolerant, psychrotolerant, butanol biosynthesis, glutamate dehydrogenase, SMRT sequencing


  1. Introduction

Planococcus spp. are motile, cocci-shaped, Gram-positive bacteria in the family Planococcaceae. To date, the Planococcus genus includes 17 recognised species (http://www.bacterio.net/planococcus.html). Predominantly isolated from cold and/or marine environments, Planococcus spp. have demonstrated various degradation capabilities including starch degradation in P. donghaensis JH1T (Choi et al., 2007), aromatic compounddegradation in Planococcus sp. ZD22 and S5 (Dalal et al., 2012; Hupert-Kocurek et al., 2012; Li et al., 2006) and alkane degradation in P. alkanoclasticus MAE2T (Engelhardt et al., 2001). Some Planococcus species also display remarkable heavy metal resistance and a capability to produce biosurfactants while utilizing olive oil as the sole carbon source (Chowdhury et al., 2003; Hupert-Kocurek et al., 2012; Nithya et al., 2011). As most Planococcus species are also halotolerant and psychrotolerant, members of the genus have been proposed to be useful as potential bioremediation candidates of significant biotechnological value (Hupert-Kocurek et al., 2012).


P. kocurii ATCC 43650T (=JCM 2569T =HK 701T) is an understudied member of the Planococcus genus originally isolated from the skin of a North Sea cod by Hao and Komagata (1985). To date, no biotechnological evaluation has been conducted for this taxon. Here, we explore the biotechnological potential of P. kocurii ATCC 43650T by sequencing the complete genome (3,487,770bp, with a G+C content of 40.9%) using Single Molecule Real-Time (SMRT) sequencing technology. The functional annotation performed unravelled its potential as a plant growth promoting bacterium and in butanol biosynthesis. The availability of this complete genome will also help to facilitate robust comparative genome analyses in this genus.


  1. Data Description

P. kocurii ATCC 43650T was cultured aerobically for 48h at 20°C on seawater yeast peptone agar. Genomic DNA was extracted using the MasterPureTM Gram Positive DNA purification kit (Epicenter, WI, USA) following the manufacturer's protocols. A template library was constructed according to the “Guidelines for Preparing 20kb SMRTbellTM Templates” and sequenced in 1 SMRT cells using the PacBio RS II single-molecule real-time (SMRT) sequencing technology (Pacific Biosciences, CA, USA). An output data of 1,180,039,791 bases and 88,826 reads with 279.99 fold coverage was generated from the SMRT sequencing in which de novo assembly was performed using the Hierarchical Genome-Assembly Process (HGAP) version 3 in SMRT Analysis v2.3.0. The open reading frame (ORF) was subsequently predicted using Prodigal v2.6.1 (Hyatt et al., 2010) and coding DNA sequences (CDS) were translated and searched against the UniProt, Pfam and COG (Clusters of Orthologous Group) databases. Non-coding genes and miscellaneous features were predicted using RNAmmer v1.2 (Lagesen et al., 2007), tRNAscan-SE v1.21 (Lowe & Eddy, 1997), SignalP v4.1 (Petersen et al., 2011) and TMHMM v2.0 (Krogh et al., 2001). The graphical circular map of the genome was constructed and visualized using Circos v0.67 (Krzywinski et al., 2009).


The complete genome of P. kocurii ATCC 43650T is made up of a single circular chromosome (3,487,770bp, 40.9% G+C content) and a circular plasmid of (21,505bp, 40.0% G+C content) (Table 1). Briefly, a total of 3,328 genes, 3237 proteins, 45 pseudogenes and 28 rRNAs were identified in the chromosome, with a further 22 genes, 16 proteins and 6 pseudogenes identified in the plasmid. A graphical map along with the COG distribution of both the chromosome and plasmid genomes of P. kocurii ATCC 43650T are given in Figure 1. A MIxS mandatory information of the MIxS checklist and environmental packages containing the phenotypic, physiological and biochemical key features of P. kocurii ATCC 43650T is presented in Table 2.


Table 1. General features of P. kocurii ATCC 43650T

Attribute

Chromosome (CP013661.1)

Plasmid (CP013660.1)

Genome size (bp)

3,487,770

21,505

Contigs

1

1

G+C content (%)

40.9

40.0

Genes

3,328

22

Protein

3,237

16

Pseudogenes

45

6

rRNA (5S, 16S, 23S)

28 (9, 10, 9)

-

tRNA

72

-

ncRNA

4

-


Figure 1. Graphical map of the chromosome (left) and plasmid (right) genomes of P. kocurii ATCC 43650T

DE NOVO ASSEMBLY OF COMPLETE GENOME SEQUENCE OF PLANOCOCCUS


Table 2. MIGS mandatory information for P. kocurii ATCC 43650T.

Item

Description

General feature of classification

Classification

Domain: Bacteria


Phylum: Firmicutes


Class: Bacilli


Order: Bacillales


Family: Planococcaceae


Genus: Planococcus


Species: kocurii

Gram stain

Positive

Cell shape

Cocci

Motility

Motile

Temperature range (°C)

5-30°C

NaCl concentration (optimum) (%)

0-10%



Investigation

Submitted to INSDC

Chromosome accession number: CP013661.1

Plasmid accession number: CP013660.1

Investigation type

MIGS BA

Project name

Complete genome sequencing of Planococcus kocurii ATCC 43650T



Environment

Geographic location (latitude and longitude)

Not collected

Geographic location (depth)

Not collected

Geographic location (country)

Not collected

Collection date

1st January 1957

Environment (biome)

Skin

Environment (feature)

North Sea cod



MIGS/MIMS/MIMARKS extension

Environmental package

Skin of the North Sea cod (host-associated)



Nucleic acid sequence source

Isolation and growth conditions

Hao & Komagata (1985)



Sequencing

Target gene or locus

Complete genome sequence of Planococcus kocurii ATCC 43650T

Sequencing method

Single Molecules Real-Time (SMRT) sequencing

Sequencing platform

PacBio RS II sequencer



Assembly

Assemble method

De novo assembly

Assembly name

Hierarchical Genome Assembly Process (HGAP) version 3

Finishing strategy

Complete genome, finished

Coverage

230.5×

Assembly size (bp)

Chromosome: 3,487,770bp

Plasmid: 21,505bp

Contig

2 (1 chromosome, 1 plasmid)


Two pathways involved in assimilation of ammonia were identified in the genome. The first pathway was a glutamate dehydrogenase (GDH) pathway using NAD-specific glutamate dehydrogenase (NCBI locus tag: AUO94_11260, AUO94_13090, AUO94_15260). The second was a glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway which involved glutamine synthetase (NCBI locus tag: AUO94_06480; EC 6.3.1.2), glutamate synthase (NCBI locus tag: AUO94_02135, AUO94_02130; EC 1.4.1.13), serine hydroxymethyltransferase (NCBI locus tag: AUO94_03600; EC 2.1.2.1), glycine dehydrogenase (NCBI locus tag: AUO94_09395, AUO94_09400; EC 1.4.4.2) and glutamine amidotransferase (NCBI locus tag: AUO94_00170).


Functional annotation also revealed 18 genes for the biosynthesis of butanol using the acetone/isopropanol butanol–ethanol (A/IBE) pathway, which included acetyl-coA acetyl transferase (NCBI locus tag: AUO94_03485, AUO94_06550, AUO94_11520, AUO94_13195, AUO94_14735), 3-hydroxybutyryl-coA-dehydrogenase (NCBI locus tag: AUO94_02245, AUO94_03490, AUO94_04680), enoyl-coA-hydrotase (NCBI locus tag: AUO94_02695, AUO94_05775, AUO94_10305, AUO94_10505, AUO94_11770, AUO94_11910), butryl-coA dehydrogenase (NCBI locus tag: AUO94_13135), acetaldehyde dehydrogenase (NCBI locus tag: AUO94_02290), alcohol dehydrogenase (NCBI locus tag: AUO94_12930), and butanol dehydrogenase (NCBI locus tag: AUO94_11255). The availability of this genome may serve as a fundamental resource to further study the full biotechnological potential of P. kocurii ATCC 43650T.


Nucleotide sequence accession number

The complete genome sequence of P. kocurii ATCC 43650T (=JCM 2569T =HK 701T) has been deposited at DDBJ/EMBL/GenBank under the accession number of CP013661.1 (chromosome) and CP013660.1 (plasmid).


Acknowledgements

This work was supported by the University of Malaya High Impact Research Grants (UMMOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, Grant No. H-50001-A000027; UMMOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/01, Grant No. A-000001-50001) awarded to Kok-Gan Chan. Peter Convey is supported by NERC core funding to the British Antarctic Survey’s ‘Biodiversity, Evolution and Adaptation’ Team.


References

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Chowdhury R, Sen AK, Karak P, Chatterjee R. Giri AK & K., C. (2003). Isolation and characterization of an arsenic-resistant bacterium from a bore-well in West Bengal, India. Annual Microbiology 59, 253–258.

Dalal, S., Panigrahi, D. P., Randhawa, G. S. & Dubey, R. C. (2012). Molecular characterisation of high-strength polycyclic aromatic hydrocarbon (PAH)-degrading and phenol-tolerant bacteria obtained from thermal power plant wastewater. Chemistry and Ecology 28, 187-192.

Engelhardt, M. A., Daly, K., Swannell, R. P. & Head, I. M. (2001). Isolation and characterization of a novel hydrocarbon-degrading, Gram-positive bacterium, isolated from intertidal beach sediment, and description of Planococcus alkanoclasticus sp. nov. Journal of Applied Microbiology 90, 237-247.

Hao, M. V. & Komagata, K. (1985). A new species of Planococcus, P. kocurii isolated from fish, frozen foods, and fish curing brine. Journal of General and Applied Microbiology 31, 41-455.

Hupert-Kocurek, K., Guzik, U. & Wojcieszynska, D. (2012). Characterization of catechol 2,3-dioxygenase from Planococcus sp. strain S5 induced by high phenol concentration. Acta Biochimica Polonica 59, 345-351.

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Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of Molecular Biology 305, 567-580.

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Lowe, T. M. & Eddy, S. R. (1997). tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Research 25, 955-964.

Nithya, C., Gnanalakshmi, B. & Pandian, S. K. (2011). Assessment and characterization of heavy metal resistance in Palk Bay sediment bacteria. Marine Environmental Research 71, 283-294.

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Tags: assembly of, 230.5× assembly, assembly, planococcus, complete, genome, sequence