DNA RESEARCH 21, 639–647, (2014) doi:10.1093/dnares/dsu026 Advance Access publication on 12 August 2014 Draft Genome Sequence of Rhodomicrobium udaipurense JA643T with Special Reference to Hopanoid Biosynthesis L. Tushar1,CH. Sasikala2, and CH. V. Ramana1,* Department of Plant Sciences, School of Life Sciences, University of Hyderabad, P.O.Central University, Hyderabad 500 046, India1 and Bacterial Discovery Laboratory, Centre for Environment, IST, JNT University, Hyderabad, Kukatpally, Hyderabad 500 085, India2 *To whom correspondence should be addressed. Tel. þ91 40-23-134-502. Fax. þ91 40-23-010-120. Email: [email protected] Edited by Dr Naotake Ogasawara (Received 29 January 2014; accepted 4 July 2014) Abstract Hopanoids are present in vast amounts as integral components of bacteria and plants with their primary function to strengthen rigidity of the plasma membrane. To establish their roles more precisely, we conducted sequencing of the whole genome of Rhodomicrobium udaipurense JA643T isolated from a fresh water stream of Udaipur in Himachal Pradesh, India, by using the Illumina HiSeq pair end chemistry of 2 3 100 bp platform. Determined genome showed a high degree of similarity to the genome of R. vannielii ATCC17100T and the 13.7 million reads generated a sequence of 3,649,277 bp possessing 3,611 putative genes. The genomic data were subsequently investigated with respect to genes involved in various features. The machinery required for the degradation of aromatic compounds and resistance to solvents as well as all that required for photosynthesis are present in this organism. Also, through extensive functional annotation, 18 genes involved in the biosynthesis of hopanoids are predicted, namely those responsible for the synthesis of diploptene, diplopterol, adenosylhopane, ribosylhopane, aminobacteriohopanetriol, glycosyl group contain- ing hopanoids and unsaturated hopanoids. The hopanoid biosynthetic pathway was then inferred based on the genes identified and through experimental validation of individual hopanoid molecules. The genome data of R. udaipurense JA643T will be useful in understanding the functional features of hopanoids in this bacterium. Key words: Rhodomicrobium udaipurense JA643T; genome sequence; Illumina Hiseq; hopanoid biosynthesis pathway 1. Introduction integrity.3,4 Hopanoids are synthesized from six isopentenyl units forming squalene, an immediate Hopanoids are a group of natural pentacyclic triter- precursor in hopanoid synthesis.5 In a highly complex penoid lipids widely distributed in plant and bacterial cyclization reaction which is similar to oxido squalene systems, act as cell membrane rigidifiers, analogues to to sterols conversion, the hopane skeleton is formed sterols present in eukaryotes which have tetracyclic from squalene by the squalene hopene cyclase coded rings. Apart from their biological occurrence, hopa- by the gene shc.5 Though the occurrence of shc gene noids were also observed in geo-sediments (geohopa- is widespread among bacteria, there is limited in- noids) formed due to diagenetic processes and are formation on the hopanoids identified from different considered as ‘molecular fossils’ for ancient bacteria.1 members.1 Till date, 200 structures of hopanoids are identified Among the hopanoids of phototrophic bacteria, from bio-/geo-sources.2 Hopanoids play a key role in much of the work was focused on Rhodopseudomonas conferring membrane integrity under extreme condi- palustris TIE-1, which accumulates substantial amounts tions of stress and any loss in hopanoids makes the bac- of diploptene, diplopterol, aminobacteriohopanetriol terium sensitive due to weakening of outer membrane and 2b-methyl-bacteriohopanepoyol.6 A large diversity # The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the CreativeCommons AttributionNon-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 640 Draft Genome of Rhodomicrobium udaipurense [Vol. 21, of hopanoids occur in a recently described photosyn- MEGA 5 software.14 The sequence data were also thetic proteobacterium, Rhodomicrobium udaipurense mapped to the reference sequence of R. vannielii ATCC JA643T belonging to the family Hyphomicrobiaceae.7 17100T using Burrows Wheel Aligner (BWA version There are nine genomes sequenced from the family 0.7.5). The reference genome sequence of R. vannielli Hyphomicrobiaceae in the class Alphaproteobacteria ATCC17100T (GCA_000166055.1) was taken from (www.ncbi.nlm.nih.gov/genomes/MICROBES/microbial_ NCBI. The reference genome map was generated using taxtree.html), and the genus Rhodomicrobium has two CGView server.15 recognized species names; R. vannielii and R. udaipurense, which are phototrophic.7 While R. vannielii ATCC17100T could tolerate solvent stress and degrade aromatic hydro- 2.3. Extraction and characterization of hopanoid carbons, R. udaipurense JA643T was psychrotolerant with by GC–MS diverse metabolic capabilities.7,8 To assess the metabolic Cells were grown photoheterotrophicallyas previous- 7 potentials with particular reference to the hopanoid ly described by Venkata et al. with pyruvate (34 mM) biosynthetic genes of R. udaipurense JA643T,wehave as carbon source. Cells were pelleted at 8,000g for sequenced the whole genome of this bacterium. The 10 min at 48C. Hopanoids were extracted according to 4 present genome sequence will expand our understand- Welender et al. In brief, cells were sonicated in 10 ml ing of hopanoid biosynthesis and provide more insights methanol:dichloromethane (DCM):water (10:5:4) and into the role of hopanoids in R. udaipurense JA643T. centrifuged at 3,000g for 10 min. Supernatant was transferred to a new tube; sonication was repeated one more time and the supernatants were pooled. Ten milli- 2. Materials and methods litres of waterand DCM were added to the supernatant and centrifuged at 3,000g for 10 min. The organic phase was 2.1. Organism and DNA preparation combined and evaporated to dryness. Hopanoids were Rhodomicrobium udaipurense JA643T was grown derivatized by acetic anhydride:pyridine (1:1) and photoheterotrophically in light with 2,400 lux at analysed by GC–MS (Agilent 7890). Separation was 308C for 48 h maintaining micro-anaerobic conditions achieved using DB-1HT column (30 m  0.25 mm i.d.; in the screw cap test tube. Culture was grown in liquid 0.1 mm film thickness) with helium as carrier gas. mineral medium with pyruvate as carbon source.9 Ramping program was started with 1008C (held for DNA was isolated with the QIAamp minikit (Qiagen) 2 min), then ramped from 100 to 2008Cat108C according to the manufacturer’s protocol. The authen- min21 and from 200 to 3608Cat68Cmin21 (held for ticity of the genome was confirmed by 16S rRNA gene 10 min). The system was operated with the following sequencing, which was done as previously described.10 parameters: electron voltage 70 eV, source tempera- ture 2008C, interface temperature 3508C and acquisi- 2.2. Genome sequencing and annotations tion delay for 120 s. Mass spectrometer was operated Genome sequencing was outsourced to the NxGenBio in full-scan mode (m/z 30–1,000). Life Sciences (India). Inbrief,genomic DNAwasfragmen- ted using Covaris system for a mean size range of 300– 350 bp. Genomic DNA shotgun library was prepared 3. Results and discussion using standard Illumina TruSeq protocol. Sequencing was carried out using Illumina Hiseq 2X 100 bp paired 13.7 million reads of 101 bp long were obtained after end chemistry. Two sequencing runs in total were sequencing the genome of R. udaipurense JA643T.Raw carried out. The quality of raw reads was checked by reads were trimmed and assembled into 256 contigs fastQC (www.bioinformatics.babraham.ac.uk/projects/ having 3,649,277 bp (3.64 Mb) in total. The largest fastqc/). The raw reads were trimmed by fastx toolkit contig size was 103,250 bp, and N50 of contig was (http://hannonlab.cshl.edu/fastx_toolkit/). The sequence 26,179 bp. However, the Newbler software predicted data were de novo assembled using Newbler assembly that the genome size of R. udaipurense JA643T would software. Annotations were performed with de novo be 3.8 Mb. Thus, genome sequence has revealed assembledsequenceusingtheRAST(RapidAnnotation 96% of the whole genome, with an average G þ C using Subsystem Technology) servers.11 tRNA and rRNA content of 62.4 mol% and 3,611 open-reading frames werepredictedusingRNAmmer.12 In silico DNA–DNA hy- (ORFs). The protein coding bases were 3,000,076 bp bridization was carried out using formula 2 of GGDC in total, covering 82.21% of the total bases determined. websiteserver(http://ggdc.dsmz.de/). It contains 68 tRNAs and 3 rRNA (Table 1). Average nucleotide identity (ANI) was calculated Though R. udaipurense JA643T and R. vannielii using ANI calculator (http://enve-omics.ce.gatech. ATCC17100T have 100% 16S rRNA gene sequence edu/ani/). SpecI server was used to confirm the species similarity, based on genome relatedness (46.1% after identification.13 Phylogenetic tree was constructed by DNA–DNA hydridization) and phenotypic traits, these No. 6] L. Tushar et al. 641 T Table 1. General features of Rhodomicrobium udaipurense JA643 were observed with one a and a b gene. For N2 fixation, draft genome sequence two types of nitrogenase dimmers, molybdenum-de-