14,FEMSmicro,Genome sequence of Kosakonia radicincitans

G E N O M E A N N O U N C E M E N T

Genome sequence of Kosakonia radicincitans UMEnt01/12,a bacterium associated with bacterial wilt diseased banana plant

Nurul Shamsinah Mohd Suhaimi,Kien-Pong Yap,Noni Ajam &Kwai-Lin Thong

Institute of Biological Sciences,Faculty of Science,University of Malaya,Kuala Lumpur,Malaysia

Correspondence:Kwai Lin Thong,Institute of Biological Sciences,Faculty of Science,University of Malaya,50603Kuala Lumpur,Malaysia.

Tel.:+60379674437;fax:+60379675908;

e-mail:thongkl@https://www.360docs.net/doc/4c3401396.html,.my

Received 3June 2014;revised 7July 2014;accepted 12July 2014.Final version published online 4August 2014.DOI:10.1111/1574-6968.12537Editor:David Studholme

Keywords

Enterobacteriaceae ;gene;Type VI secretion system;Bio?lm;DNA;annotation.

Abstract

Kosakonia radicincitans (formerly known as Enterobacter radicincitans ),an endo-phytic bacterium was isolated from the symptomatic tissues of bacterial wilt dis-eased banana (Musa spp.)plant in Malaysia.The total genome size of K.radicincitans UMEnt01/12is 5783769bp with 5463coding sequences (CDS),75tRNAs,and 9rRNAs.The annotated draft genome of the K.radicinci-tans UMEnt01/12strain might shed light on its role as a bacterial wilt-associated bacterium.

Bacterial wilt disease is an important plant disease with a worldwide distribution.In this study,Kosakonia radicinci-tans ,UMEnt01/12was previously isolated from the stem tissues of the bacterial wilt banana plant with clear symp-toms of wilting and yellowing of foliage,appearance of narrow dark stripes in vascular bundles,and rotting fruit.This UMEnt01/12strain,grew on the casamino peptone glucose (CPG)agar (casamino acid 1g L à1,peptone 10g L à1,glucose 5g L à1,agar 18g L à1),produced cream colored,mucoid colonies,with typical surface irregularity.To date,only one genome of K.radicincitans was sequenced,which associated with plant growth pro-moting in its host plant (Witzel et al.,2012).However,its roles in plant colonization and pathogenesis have not been clearly established.Kosakonia radicincitans shares a relatively recent common ancestor with Enterobacter cloa-cae that is associated with wilting symptoms in ginger plant (Nishijima et al.,2004;Brady et al.,2013).

Genomic DNA was extracted from K.radicincitans UMEnt01/12using the Wizard Genomic DNA Puri?ca-tion kit according to the manufacturer’s instructions (Promega).The whole genome sequencing was performed using the Illumina Hiseq2000.About 1.6million reads and 580Mb of data were obtained with 37x genome average coverage.The resulting sequence data were qual-ity assessed,trimmed,and assembled de novo in the same way as described previously (Yap et al.,2012a)into 71contigs using CLCBio Genomic Workbench 5.1(CLCBio,Denmark)with n50value of 204410bp.While tRNA and rRNA genes were identi?ed with RNAmmer 1.2(Lagesen et al.,2007)and TRNASCAN -SE 1.2(Lowe &Eddy,1997),respectively.The open reading frames (ORFs)were predicted and subsequently annotated as described previ-ously (Yap et al.,2012b).

The genome size of UMEnt01/12is 5783769bp with a G +C content of 53.9%.The genome annotation revealed a total of 5463coding sequences (CDS),75tRNAs,and 9rRNAs,accounted for 89.0%of the total genome size.A total of 4957CDS could be assigned to biological functions,while 506CDS are annotated as hypothetical proteins.

The genome of K.radicincitans UMEnt01/12contains genes coding for pectin acetylesterase,pectate disaccharide-lyase (PATE),and pectate lyase,PelB which are related to

FEMS Microbiol Lett 358(2014)11–13

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M I C R O B I O L O G Y L E T T E R S

pectate degradation,an important process in the cell inter-spatial colonization (Taghavi et al.,2010).UMEnt01/12genome also well-equipped with ?agellar biosynthesis (?gABCDEFGHIJKLNM ),?agellar transcriptional regulator (?hCD ),?agellar motor,(motAB ),and ?agellin genes (?iACDEFGHJKLMOPQR ).Some putative genes encoding proteins related to bio?lm formation such as the bio?lm formation regulatory protein,BssR,bio?lm stress,and bio?lm motility protein A were also detected in K.radi-cincitans UMEnt01/12.The toxin –antitoxin (TA)-related genes that encode for TabA and HipA which are involved in bio?lm formation are present in the genome (Kim et al.,2009;Zhao et al.,2013).Large number of heavy metal resistance genes was also identi?ed in the genome which include genes encoding heavy metal-translocating P-type ATPase involved in Cd/Co/Hg/Pb/Zn resistance,heavy metal RND transporter,and heavy metal sensor kinase family protein.The genome also contains genes for cobalt-precorrin,cobGJLM and cbiCETDJ,and cobalt transporters.Few pathogenic-associated genes also found in UMEnt01/12genome which encode lipoate-protein ligase,LplA,RNA polymerase sigma factor,RpoS,pyrroline-5-carboxylate reductase,and GntR family regulators.

The presence of nif operon,comprising the nifY-UBAMSNEHXWZQ genes,in our UMEnt01/12strain also suggests that this strain has the potential to biologi-cally ?x atmospheric nitrogen.Similarly,such ability has been reported in plant growth-promoting endophyte,K.radicincitans DSM 16656T (previously known as E.radicincitans DSM 16656T )that is associated with phyllosphere of winter wheat (Witzel et al.,2012).Approximately,5%of the genes in the K.radicincitans UMEnt01/12genome was not identi?ed in K.radicinci-tans DSM 16656T (Fig.1).These genes shared highest degree of sequence similarity with those of the opportu-nistic plant pathogen E.cloacae .Genes with homologues in E.cloacae are mostly encode for membrane-related proteins,transporters,regulatory proteins,phages,enzymes,stress-related proteins,and large numbers of uncharacterized proteins.

We also identi?ed a Type VI secretion system (T6SS)gene clusters in UMEnt01/12genome.The T6SS-related genes,such as vasA encoding structural protein present in Virulence Associated (VAS)cluster of Vibrio cholerae (Pukatzki et al.,2006),together with effector proteins,VgrG,and Hcp were found in K.radicincitans UMEnt01/12.Previous genome study of various bacteria including the plant pathogenic,Xanthomonas spp.suggested that the T6SS is strongly associated with pathogenicity of the bac-teria (Shrivastava &Mande,2008).Although it has been shown that T6SS can be deployed as a versatile weapon to compete with other bacteria or capable of attacking simple or higher eukaryotes,this intriguing system is far from being well-understood (Coulthurst,2013).To

the

Fig.1.A phylogenomic tree and its corresponding heat-plot showing the genome comparison of the Kosakonia radicincitans UMEnt01/12with closely and distantly related phytopathogenic and nonphytopathogenic bacteria.The UMEnt01/12was aligned using BLASTN all-to-all to 20phytopathogenic and nonphytopathogenic bacterial genomes using GEGENEES 2.0.3(Agren et al.,2012)(GenBank Accession No:AAHI01000000,NC014307,FN666575,BAGV00000000,AJVN00000000,NZ_ADCU00000000,CP004142,CP003683,ADBP01000000,CP002910,NZ_AMFN01000000,CP006731,JDYJ00000000,AKYD00000000,CP001918,AFHR00000000,ALNS00000000,AEXB00000000,BADM00000000,NZ_AKXM01000000,NZ_BAFF01000000).The size of the alignments core is set at 40%.The phylogenomic tree was constructed with SPLITSTREE 4V4.13.1(Bryant &Moulton,2004)using the neighbor joining method.The tree shows that the UMEnt01/12strain is clustered together with K.radicincitans DSM 16656with 98%similarity.The UMEnt01/12strain is also closely related to potential phytopathogens,Enterobacter cloacae,and E.mori .

FEMS Microbiol Lett 358(2014)11–13

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12N.S.Mohd Suhaimi et al.

best of our knowledge,no study to date has shown the presence of a well-characterized T6SS in Kosakonia spp. Future studies are needed to elucidate the functions of the T6SS system as well as other uncharacterized putative genes found in UMEnt01/12genome.

The genome sequence of this strain will provide new insight into the possible mechanisms of this poorly stud-ied bacterium,such as successful colonization,establish-ment,and interaction process in the vasculature system as well as potential pathogenic properties leading to symptomatic bacterial wilt diseased banana plant. Nucleotide sequence accession numbers:This Whole Genome Shotgun project has been deposited at DDBJ/ EMBL/GenBank under the Accession Number JDYJ00000000.The version described in this paper is the version,JDYJ00000000.The BioProject designation for this project is PRJNA236629.

Acknowledgements

We thank University of Malaya for facilities and support. This project was supported by University of Malaya High Impact Research,Grant No.UM.C/625/1/MOHE/02,and PPP Grant,PV123-2012A.The?rst author is supported by Ministry of Higher Education,Malaysia and SLAI Scheme,University of Malaya.We also wish to con?rm that there is no known con?ict of interest associated with this publication.

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Published by John Wiley&Sons Ltd.All rights reserved Genome sequence of Kosakonia radicincitans13