Draft genome sequence of Rhodomicrobium udaipurense JA643T with special reference to hopanoid biosynthesis.

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 JA643(T) isolated from a fresh water stream of Udaipur in Himachal Pradesh, India, by using the Illumina HiSeq pair end chemistry of 2 × 100 bp platform. Determined genome showed a high degree of similarity to the genome of R. vannielii ATCC17100(T) 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 containing 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 JA643(T) will be useful in understanding the functional features of hopanoids in this bacterium.

Introduction

Hopanoids are a group of natural pentacyclic triterpenoid lipids widely distributed in plant and bacterial systems, act as cell membrane rigidifiers, analogues to sterols present in eukaryotes which have tetracyclic rings. Apart from their biological occurrence, hopanoids were also observed in geo-sediments (geohopanoids) formed due to diagenetic processes and are considered as ‘molecular fossils’ for ancient bacteria.[1] Till date, ∼200 structures of hopanoids are identified from bio-/geo-sources.[2] Hopanoids play a key role in conferring membrane integrity under extreme conditions of stress and any loss in hopanoids makes the bacterium sensitive due to weakening of outer membrane integrity.[3,4] Hopanoids are synthesized from six isopentenyl units forming squalene, an immediate precursor in hopanoid synthesis.[5] In a highly complex cyclization reaction which is similar to oxido squalene to sterols conversion, the hopane skeleton is formed from squalene by the squalene hopene cyclase coded by the gene shc.[5] Though the occurrence of shc gene is widespread among bacteria, there is limited information on the hopanoids identified from different members.[1]

Among the hopanoids of phototrophic bacteria, much of the work was focused on Rhodopseudomonas palustris TIE-1, which accumulates substantial amounts of diploptene, diplopterol, aminobacteriohopanetriol and 2β-methyl-bacteriohopanepoyol.[6] A large diversity of hopanoids occur in a recently described photosynthetic proteobacterium, Rhodomicrobium udaipurense JA643T belonging to the family Hyphomicrobiaceae.[7] There are nine genomes sequenced from the family Hyphomicrobiaceae in the class Alphaproteobacteria (www.ncbi.nlm.nih.gov/genomes/MICROBES/microbial_taxtree.html), and the genus Rhodomicrobium has two recognized species names; R. vannielii and R. udaipurense, which are phototrophic.[7] While R. vannielii ATCC17100T could tolerate solvent stress and degrade aromatic hydrocarbons, R. udaipurense JA643T was psychrotolerant with diverse metabolic capabilities.[7,8] To assess the metabolic potentials with particular reference to the hopanoid biosynthetic genes of R. udaipurense JA643T, we have sequenced the whole genome of this bacterium. The present genome sequence will expand our understanding of hopanoid biosynthesis and provide more insights into the role of hopanoids in R. udaipurense JA643T.

Materials and methods

Organism and DNA preparation

Rhodomicrobium udaipurense JA643T was grown photoheterotrophically in light with 2,400 lux at 30°C  for 48 h maintaining micro-anaerobic conditions in the screw cap test tube. Culture was grown in liquid mineral medium with pyruvate as carbon source.[9] DNA was isolated with the QIAamp minikit (Qiagen) according to the manufacturer's protocol. The authenticity of the genome was confirmed by 16S rRNA gene sequencing, which was done as previously described.[10]

Genome sequencing and annotations

Genome sequencing was outsourced to the NxGenBio Life Sciences (India). In brief, genomic DNA was fragmented using Covaris system for a mean size range of 300–350 bp. Genomic DNA shotgun library was prepared using standard Illumina TruSeq protocol. Sequencing was carried out using Illumina Hiseq 2X 100 bp paired end chemistry. Two sequencing runs in total were carried out. The quality of raw reads was checked by fastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). The raw reads were trimmed by fastx toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). The sequence data were de novo assembled using Newbler assembly software. Annotations were performed with de novo assembled sequence using the RAST (Rapid Annotation using Subsystem Technology) servers.[11] tRNA and rRNA were predicted using RNAmmer.[12] In silico DNA–DNA hybridization was carried out using formula 2 of GGDC website server (http://ggdc.dsmz.de/).

Average nucleotide identity (ANI) was calculated using ANI calculator (http://enve-omics.ce.gatech.edu/ani/). SpecI server was used to confirm the species identification.[13] Phylogenetic tree was constructed by MEGA 5 software.[14] The sequence data were also mapped to the reference sequence of R. vannielii ATCC 17100T using Burrows Wheel Aligner (BWA version 0.7.5). The reference genome sequence of R. vannielli ATCC17100T (GCA_000166055.1) was taken from NCBI. The reference genome map was generated using CGView server.[15]

Extraction and characterization of hopanoid by GC–MS

Cells were grown photoheterotrophically as previously described by Venkata et al.[7] with pyruvate (34 mM) as carbon source. Cells were pelleted at 8,000g for 10 min at 4°C. Hopanoids were extracted according to Welender et al.[4] In brief, cells were sonicated in 10 ml methanol:dichloromethane (DCM):water (10:5:4) and 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 millilitres of water and DCM were added to the supernatant and centrifuged at 3,000g for 10 min. The organic phase was combined and evaporated to dryness. Hopanoids were derivatized by acetic anhydride:pyridine (1:1) and analysed by GC–MS (Agilent 7890). Separation was achieved using DB-1HT column (30 m × 0.25 mm i.d.; 0.1 µm film thickness) with helium as carrier gas. Ramping program was started with 100°C (held for 2 min), then ramped from 100 to 200°C at 10°C min−1 and from 200 to 360°C at 6°C min−1 (held for 10 min). The system was operated with the following parameters: electron voltage 70 eV, source temperature 200°C, interface temperature 350°C and acquisition delay for 120 s. Mass spectrometer was operated in full-scan mode (m/z 30–1,000).

Results and discussion

13.7 million reads of 101 bp long were obtained after sequencing the genome of R. udaipurense JA643T. Raw reads were trimmed and assembled into 256 contigs having 3,649,277 bp (3.64 Mb) in total. The largest contig size was 103,250 bp, and N50 of contig was 26,179 bp. However, the Newbler software predicted that the genome size of R. udaipurense JA643T would be ∼3.8 Mb. Thus, genome sequence has revealed 96% of the whole genome, with an average G + C content of 62.4 mol% and 3,611 open-reading frames (ORFs). The protein coding bases were 3,000,076 bp in total, covering 82.21% of the total bases determined. It contains 68 tRNAs and 3 rRNA (Table 1).

Table 1.

General features of Rhodomicrobium udaipurense JA643T draft genome sequence

Total bases	3,649,277
Average gene length	830 bp
Protein coding features	3,611
Protein coding bases	3,000,076 (82.21%)
tRNAs	68
rRNAs	3
G + C percentage	62.4 mol%
DNA–DNA hybridization (in vitro) with Rhodomicrobium vannielii DSM162T (=ATCC17100T)[1]	46.1%
In silico DNA–DNA hybridization with the genome of R. vannielii ATCC17100T [Genome accession number; GCA_000166055.1]	58.60%

Though R. udaipurense JA643T and R. vannielii ATCC17100T have 100% 16S rRNA gene sequence similarity, based on genome relatedness (46.1% after DNA–DNA hydridization) and phenotypic traits, these were described as two different species.[7] Delineation of the two species was further confirmed by in silico regression DDH analysis of the whole genomes, and the determined value of 58.6% is much lower than the gold standard value of 70% for delineation of bacterial species.[16] Further, SpecI analysis using 40 phylogenetic marker genes (pMGs) also indicated an average identity (Supplementary Table S1) of 94.69% which concludes a clear delineation between the two species.[13] Calculated ANI value of 95.26% between the genomes of R. udaipurense JA643T and R. vannielii ATCC17100T strengthens the separation into two species.

Protein-coding genes of R. udaipurense JA643T have an average length of 830 bases, ranging from 70 to 4,796 bases. Out of 3,611 ORFs identified, 2,295 (63.55%) were functionally annotated, 1,238 (34.28%) were hypothetical, 64 were putative and 14 were conserved hypothetical genes. Most of the annotated protein coding sequences [∼1,367 genes (62.74%)] were involved in metabolic and biosynthetic processes. In addition, 39 genes were involved in cell signaling and regulations, 26 genes in motility and chemotaxis, 119 genes in RNA metabolism, 74 genes in membrane transport, 168 genes in protein metabolism, 109 genes in DNA metabolism, 46 genes into defence, 88 genes in cell wall and capsule formation and 259 genes in other cellular functions, which were not distributed in subsystem category (Supplementary Figs S1 and S2).

When contig sequences of R. udaipurense JA643T were mapped on R. vannielii ATCC17100T genome sequence, only 2,989 genes got mapped, indicating that the remaining 622 genes were diverged from R. vannielii ATCC17100T counterparts or absent in R. vannielii ATCC17100T genome. Rhodomicrobium udaipurense JA643T showed phototrophic growth, and it contained sets of genes for bacteriochlorophyll biosynthesis and a set of light harvesting complexes as well as puf L,M,C,H genes. A set of light harvesting complex gene sequences were observed with one α and a β gene. For N2 fixation, two types of nitrogenase dimmers, molybdenum-dependent and vanadium-dependent nitrogenase, were present with all related cofactors and accessory genes. Secondary transport system genes included facilitator super family (MFS) transporter, resistance nodulation cell division (RND) pumps, tripartite ATP-dependent periplasmic (TRAP) transporter and organic solvent resistance transporter. The heavy metal efflux transporters and organic solvent resistance transporters present may allow the organism to survive in high concentrations of metals and other solvents.[16] Rhodomicrobium udaipurense JA643T has the genes involved in the degradation of nitrogen-containing compounds like amino acids and heterocyclic aromatic compounds and may dehalogenate with the help of different monooxygenase, dioxygenase, peroxidases and dehalogenase.

Draft genome sequence of R. udaipurense JA643T showed the presence of genes involved in benzoate degradation. Anaerobic benzoate degradation takes place by two known pathways; one occurs through –CoA ligation, while the other by hydroxylation, and R. udaipurense JA643T has genes involved in both the pathways.[17] There are 12 genes related to aromatic hydrocarbon metabolism; of these, 6 genes are involved in benzoate degradation through hydroxylation. However, the RAST subsystem classifies genes involved in benzoate degradation through hydroxylation as nonfunctional in this organism. The presence of aromatic hydrocarbon metabolizing genes and utilization of benzoate by R. udaipurense JA643T and R. vannielii ATCC17100T indicate their probable role in the aromatic hydrocarbon recycling in oxic/anoxic environments.[1,17]

Some of homologous sequences unique in R. udaipurense JA643T may compensate the function; sterol-binding domain-containing protein, hemolysins and related proteins containing CBS domain, putative homolysin, myosin cross-reactive streptococcal antigen, cobalt–zinc–cadmium/protein resistance, sporulation domain protein, sperimidine synthase, antibiotic biosynthesis monooxygenase, stage 0 sporulation protein J and sporulation initiation inhibitor protein. The summary of all these genes is given as Supplementary Table S2.

Hopanoid biosynthesis

Information on the genes involved in the biosynthesis of hopanoids is limited, and our study mainly focused on the genomic insight of hopanoid biosynthesis in R. udaipurense JA643T.[3,4,6] Hopanoids are biosynthesized from isopentenyl pyrophosphate (IPP) which is synthesized either through mevalonate pathway or methyl erythritol phosphate pathway (MEP).[18] Genes involved in the MEP pathway are present in R. udaipurense JA643T, and the genes are scattered in the genome (Fig. 1). Only 4-hydroxy-3-methylbut-2-enyl diphosphate reductase coding gene (ispH) was present in the cluster of seven hopanoid biosynthesis genes. List of seven genes involved in the MEP pathway is given in the Supplementary Table S3. IPP isomerase catalyzes the conversion of relatively unreactive IPP to electrophile dimethylallyl pyrophosphate (DMAPP).[19] Farnesyldiphosphate synthase (ispA/ispB) catalyzes the formation of (2Z, 6E)-farnesyl diphosphate from three IPP.[20] Squalene synthase (hpnC/hpnD) catalyzes stepwise tail-to-tail addition of farnesyl diphosphate (C15) which condenses to form presqualene pyrophosphate, an intermediate product.[5] Presqualene pyrophosphate is converted to squalene (I) by squalene synthase (hpnC/hpnD). Squalene synthase (hpnC/hpnD) is homologous to phytoene synthase (involved in carotenoid biosynthesis) and the function of hpnC/hpnD in the hopanoid biosynthesis is poorly understood.[21,22]

Figure 1.

Location of the hopanoid biosynthetic genes in the genome of Rhodomicrobium udaipurense JA643T. The representation is not up to the scale. erfK: ErfK/YbiS/YcfS/YnhG family gene; ispH: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; hpnH: radical SAM protein required for addition of adenosine to hopane skeleton; hpnG: adenosylhopane nucleoside/hopanoid-associated phosphorylase; shc/hpnF: squalene hopene cyclase; hpnE/crtI: squalene-associated, FAD-dependent desaturase/phytoene desaturase; hpnD: squalene synthase; hpnC: squalene synthase; hpnI/hpnB: glycosyl transferase, family 2; lepA: translation elongation factor LepA; mepM: murein endopeptidase; hpnO: acetylornithine aminotransferase; H597: hypothetical gene-597; hpnN: hopanoid-associated RND transporter; H598: hypothetical gene-598; MP: membrane protein; H230: hypothetical gene-230; hpnX/SD: sterol desaturase family protein; thiG: thiazole biosynthesis protein; H: hypothetical protein; dxs: 1-deoxy-D-xylulose 5-phosphate synthase; xseB: exodeoxyribonuclease VII small subunit; cdsA: phosphatidate cytidylyltransferase; dxr: 1-deoxy-: membrane-associated zinc metalloprotease; LIAS: lipoate synthase; ispF/ispD: 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; NR9II: nitrogen regulation protein NR(II); TRP: TPR domain protein; ispE: 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase; H138: hypothetical gene-138; znuB: zinc ABC transporter; ispG: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; H708: hypothetical gene-708; TSRP: two-component system regulatory protein; ispB: eranyltranstransferase (farnesyldiphosphate synthase); mtgA: peptidoglycan transglycosylase.

Eight genes involved in the hopanoid biosynthesis were located on contig no. 3 between 36,000 and 45,850 bp including ispH which encodes for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, an enzyme involved in the MEP pathway. These genes encode radical SAM protein (hpnH), adenosine nucleosidase (hpnG), squalene hopene cyclase (shc/hpnF), squalene-associated, FAD-dependent desaturase (hpnE/crtI), squalene synthase (hpnD and hpnC) and glycosyltransferase family protein (hpnB). Ten additional genes involved in hopanoid biosynthesis were scattered in the genome. The region having hopanoid biosynthetic genes and their upstream and downstream regions are shown in Fig. 1. The fragmented organization of the genes indicates the complex regulatory network of hopanoid biosynthesis. Squalene (I), hop-22(29)-ene (II), diplopterol (III), tetrahymanol (IV), 2-methyldiploptetrol (VI), adenosylhopane (VII), bacteriohopanetetrol (XI), aminobacteriohopanetetrol (XII), N-tryptophanyl aminobacteriohopanetetrol (XV) were identified through GC–MS analysis (Fig. 2), and their structures are given in Supplementary Fig. S3.

Figure 2.

Mass spectra of hopanoids detected after acetylation of total lipid extract from the Rhodomicrobium udaipurense JA643T. Identification of hopanoids was done by comparing the previously published data. Squalene (I), hop-22(29)-ene (II), hopan-22-ol (III), tetrahymanol (IV), 2-methyldiplopterol (VI), adenosylhopane (VII), bacteriohopanetetrol (XI), aminobacteriohopanetriol (XII) and N-tryptophanyl-35-aminobacteriohopanetriol (XV).

In the hopanoid biosynthetic pathway, squalene (I) is converted to hop-22(29)-ene/diploptene (II) or 22-hopanol/diplopterol (III) by squalene hopene cyclase (shc) or squalene hopanol cyclase. Cyclization of squalene (I) into the pentacyclic tripterpenoid is one of the complex processes catalyzed by squalene hopene cyclase (shc/hpnF). A single enzyme catalyzes the stepwise formation of 13 covalent bonds and 9 stereo centres.[5] Accumulation of squalene (I) by Δshc mutant could not produce hopanoids including tetrahymanol (IV),[4] suggesting the role of shc gene in the biosynthesis of tetrahymanol (IV) as well. Phylogenetic tree using 339 shc genes from different microorganisms showed (Supplementary Fig. S4) a distinct clade for Rhodomicrobium spp. and has a sequence similarity of 97% between shc genes of R. udaipurense JA643T and R. vannielii ATCC17100T (Table 2).

Table 2.

Genes involved in the hopanoid biosynthesis pathway

Gene name	Code	EC number	Gene length (bp)	Similaritya (%)
Squalene hopene cyclase	Shc/hpnF	5.4.99.17	2,039	97
Glycosyl transferase, family 2, hpnB	hpnI/hpnB		1,166	99
5′-Methylthioadenosine nucleosidase/S-adenosylhomocysteine nucleosidase	hpnG	3.2.2.16/3.2.2.9	701	98
Radical SAM protein required for addition of adenosine to hopane skeleton	hpnH	–	1,142	99
Acetylornithine aminotransferase	hpnO/argD	2.6.1.11	1,199	98
Hopanoid-associated RND transporter	hpnN		2,666	98
Phytoene desaturase/squalene-associated FAD-dependent desaturase	hpnE/crtI	1.14.99	1,325	99
Phytoene synthase/squalene synthase	hpnC	2.5.1.32	857	97
Phytoene synthase/squalene synthase	hpnD	2.5.1.32	881	98
Sterol desaturase family protein	erg3/hpnX		776	97
Geranyltranstransferase (farnesyldiphosphate synthase)	ispB/ispA	2.5.1.10	890	95

aSimilarity with respective genes of Rhodomicrobium vannielii ATCC17100T.

hpnP encoding 2-methyltransferase was not identified in the draft genome sequence of R. udaipurense JA643T. However, identification of a methylated hopanoid (2-methyldiplopterol; Fig 2) indicates the presence of hpnP gene in R. udaipurense JA643T which might have not been covered in the draft genome sequence. The hop-22(29)-ene (II) is further converted to adenosylhopane (VII) by the radical SAM protein (hpnH) required for the addition of adenosine to hopane skeleton. hpnH gene product is a B12-binding radical SAM protein, and mutation in hpnH resulted in the production of C30 hopanoids (diploptene and diplopterol).[6] Removal of adenine moiety from adenosylhopane (VII) carried out by adenosylhopane nucleoside/hopanoid-associated phosphorylase, encoded by hpnG gene (a putative nucleoside hydrolase) lead to the formation of ribosylhopane (VIII). Mutation in hpnG gene resulted in loss in the synthesis of bacteriohopanetetrol (XI) and aminobacteriohopanetriol (XII), and consequently an intermediate adenosylhopane (VII) was accumulated.[6]

A lactone hopanoid, ribosylhopane (VIII), was identified as an intermediate in the biosynthesis of C35 hopanoids.[23] The lactone ring of this hopanoid could be opened via a lactone hydrolysis, a reaction which is similar to the hydrolysis of the quorum-sensing molecule (acylhomeserine lactone [AHL]) by acylhomoserine lactonase.[24] However, gene O-acylhomoserine lactonase was not observed in the draft genome of R. udaipurense JA643T. The lactone ring of hopanoid might be opened via oxidation to form putative ribonylhopane (IX) precursor.[25] The presence of ribonylhopane (IX) in some R. palustris mutants suggested that the biosynthesis of the C35 hopanoids would require an oxidation of the ribosylhopane (VIII) produced by the hpnG to form ribonylhopane (IX).[6] The pathway may proceed with the interconversion of ribonylhopane (IX) to formylhopane (X), because ribonylhopane (IX) is less reactive, whereas formylhopane (X) is more reactive. In the GC–MS analysis, ribosylhopane (VIII), ribonylhopane (IX) and formylhopane were not detected. The gene involved in the interconversion of ribosylhopane to formylhopane is also not yet known.

Formylhopane (X) may undergo reductive amination leading to the formation of an amino group containing hopanoid, aminobacteriohopanetriol (XII).[26] Knockout study of ornithine: oxo-acid aminotransferase (hpnO) showed that it was involved in the formation of aminobacteriohopanetriol (XII).[6] hpnO bears homology with N-acetylornithine aminotransferase (ArgD). ArgD acts on an aldehyde rather than a hydroxyl group supporting the idea that the formylhopane (X) is more likely to be the correct substrate. In GC–MS analysis, N-tryptophanyl-35-aminobacteriohopane-32,33,34-triol was observed, in which amino acid residue is linked to the amino group of aminobacteriohopanetriol, indicating that there is a modification of aminobacteriohopanetriol in R. udaipurense JA643T. The genes involved in the synthesis of bacteriohopanetetrol and N-tryptophanyl-35-aminobacteriohopane-32,33,34-triol are not yet known, but products were identified in GC–MS analysis.

Sterol desaturase (erg32/hpnX) gene was present on contig no. 30 in the genome of R. udaipurense JA643T from 959,672 to 958,896 bp. Upstream and downstream regions are shown in Fig. 1. Sterol desaturase family protein known to catalyze formation of a C-5 double bond in the B ring of ergosterol. erg32 is involved in the biosynthesis of ergosterol which is important for the plasma membrane structure, function and localization of the plasma membrane proteins. However, in R. udaipurense JA643T sterols are not present. As hopanoids are the sterol surrogates and some hopanoids with double bond at C6 and C11 positions were reported previously (XIV),[27] it is hypothesized that sterol desaturase (hpnX) might be responsible for the incorporation of double bond in the hopanoids.

hpnI/hpnB genes are associated with hopanoid glycosyltransferase, and glycosylated hopanoids were identified with Geobacter sulfurreducens and Geobacter metallireducens.[28,29] Hopanoids with glycosyl moiety is expected in R. udaipurense JA643T since hpnI/hpnB genes were observed in the genome of this organism; however, no glycosyl group containing hopanoids were detected in the GC–MS analysis. hpnC, hpnD and hpnE genes are involved in the modification of squalene. hpnE is a squalene-associated, FAD-dependent desaturase which has oxidoreductase activity. Perzl et al. suggested that it might be involved as an oxidoreductase in hopanoid or terpenoid biosynthesis pathways.[21] However, its role in hopanoid biosynthesis is not understood. Gene hpnA encode for NAD-dependent nucleoside diphosphate–sugar epimerase/dehydrates protein, which was not observed in the draft genome of R. udaipurense JA643T. Hopanoid biosynthetic genes between R. udaipurense JA643T and R. vannielii ATCC17100T were compared. Sequence similarity ranges from 95 to 99% between them (Table 2, Supplementary Table S3). The hopanoid biosynthetic pathway was then inferred based on the genes identified, experimental validation of hopanoid molecules and available literature (Fig. 3).[3,4,15-30]

Figure 3.

Schematic representation of hopanoid biosynthetic pathway in Rhodomicrobium udaipurense JA643T based on genes identified, GC–MS analysis and previous reports.[3,4,15–26,30] This figure appears in colour in the online version of DNA Research.

Apart from hopanoid biosynthetic genes, few genes playing a key role in localization of hopanoids, hopanoid regulation and transportation were also observed in R. udaipurense JA643T. One such gene is hopanoid-associated RND transporter (hpnN) which encodes an RND-like transporter that helps to localize hopanoids to the outer membrane. hpnN and hpnO were located close together with a gene coding for hypothetical protein in between. ΔhpnN mutant no longer contain any hopanoids in the outer membrane, suggesting its importance in hopanoid transportation.[31] hpnN mutant produces more 2-methylhopanoids, possibly indicating some role for hpnN in hopanoid homeostasis.[31] Similarly, extracellular sigma factor (ECF) regulates the methylation of hopanoids at C-2 position.[32] Genome sequence of R. udaipurense JA643T showed that it has two RNA polymerase ECF-type sigma factors. Probably these sigma factors may be the regulators for the expression of hpnP gene and production of methylated hopanoids.

In summary, genome sequence of R. udaipurense JA643T has a set of genes required for degrading aromatic compounds and resistance to solvents. It has all the machinery required for photosynthesis. There were 18 genes involved in the hopanoid biosynthesis and transport of hopanoids in this organism. The MEP pathway exists in R. udaipurense JA643T which leads to the formation of IPP. Major hopanoids were diploptene, diplopterol, bacteriohopanetetrol, aminobacteriohopanetriol and N-tryptophanyl-35-aminobacteriohopanetriol. Though hpnP gene was not covered in the genome of R. udaipurense JA643T, the presence of methylated hopanoids (Fig. 2) indicates that this gene might be present in the complete genome of this organism. On the other hand, we could not find hpnP gene from the complete genome of R. vannielii ATCC17100T, and this makes the difference in the hopanoids of R. udaipurense JA643T. The genes involved in the formation of amino acid group containing hopanoids need an in-depth investigation.

Hopanoid-deficit mutants can grow at optimized laboratory conditions; however, the mutants act differently under stress conditions. Hopanoids confer membrane integrity and help the bacterium to tolerate bile salts, pH, antibiotics and temperature.[4] Hopanoids are also important for a bacterium to undergo normal cell cycle.[31] However, the role of hopanoids in R. udaipurense JA643T is yet to be revealed, and this part of the work is in progress at our laboratory, while comparisons are also being made with R. vannielii ATCC17100T, whose complete genome sequence is available.

Data access

This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JFZJ00000000 and SRR1016881. The version described in this paper is version JFZJ01000000.

Supplementary Data

Supplementary data are available at www.dnaresearch.oxfordjournals.org

Funding

This work was funded by Council of Scientific and Industrial Research (CSIR) project. T.L. is supported by research fellowship from the University Grants Commission (UGC). Funding to pay the Open Access publication charges for this article was provided by Council of Scientific and Industrial Research (CSIR) project [Grant ID 38(1351)/13/EMR-II dated on 14-2-2013].