- Open Access
Genome sequence of the Listia angolensis microsymbiont Microvirga lotononidis strain WSM3557T
Standards in Genomic Sciences volume 9, pages540–550 (2014)
Microvirga lotononidis is a recently described species of root-nodule bacteria that is an effective nitrogen- (N2) fixing microsymbiont of the symbiotically specific African legume Listia angolensis (Welw. ex Bak.) B.-E. van Wyk & Boatwr. M. lotononidis possesses several properties that are unusual in root-nodule bacteria, including pigmentation and the ability to grow at temperatures of up to 45°C. Strain WSM3557T is an aerobic, motile, Gram-negative, non-spore-forming rod isolated from a L. angolensis root nodule collected in Chipata, Zambia in 1963. This is the first report of a complete genome sequence for the genus Microvirga. Here we describe the features of Microvirga lotononidis strain WSM3557T, together with genome sequence information and annotation. The 7,082,538 high-quality-draft genome is arranged in 18 scaffolds of 104 contigs, contains 6,956 protein-coding genes and 84 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.
Legume-rhizobia symbioses are important components of southern Australian agricultural systems, in which symbiotic N2-fixation provides a significant amount of the nitrogen input that is required to boost food and animal production [1,2]. Traditionally, pasture legumes have been Mediterranean annuals such as medics and subterranean clover . However, recent changes to the rainfall patterns in south-western Western Australia, resulting in a 10–20% decrease in annual rainfall , have adversely affected production from these annual legumes. Researchers are therefore seeking to introduce alternative perennial legume species and associated rhizobia that are better adapted to the arid climate and acid, infertile soils found in these systems . Among the perennial, herbaceous forage legumes selected for further study are several species within the papilionoid legume clade Lotononis sensu lato.
Lotononis s. l. is grouped within tribe Crotalarieae, has a centre of origin in South Africa and consists of some 150 species, divided into 15 sections . The taxonomy has recently been revised and the three distinct clades within Lotononis s. l. are now recognized at the generic level as Listia, Leobordea and Lotononis s. s. . Species within the genus Listia are of agronomic interest, as they have potential as perennial pasture legumes that are able to reduce groundwater recharge and assist in preventing dry land salinity in southern Australian agricultural systems . Listia spp. produce stoloniferous roots on their lower branches (a characteristic thought to be associated with the seasonally wet habitats where these species are found)  and form lupinoid, rather than indeterminate nodules, in response to infection by rhizobia [7,8]. The symbioses between Listia species and their associated root-nodule bacteria are highly specific. All studied host species are nodulated by strains of pigmented methylobacteria [7,9,10], except for Listia angolensis, which is effectively nodulated only by newly described species of Microvirga . Microvirga lotononidis strain WSM3557T is the type strain for this species. Here we present a set of preliminary classification and general features for M. lotononidis strain WSM3557T together with the description of the genome sequence and annotation.
Classification and general features
M. lotononidis strain WSM3557T is a motile, Gram-negative, non-spore-forming rod with one to several flagella (Figure 1, left and center panel). It is a member of the family Methylobacteriaceae in the class Alphaproteobacteria (Figure 2). WSM3557T is fast growing, forming 0.5–1.5 mm diameter colonies within 2–3 days. It is moderately thermophilic and has a mean generation time of 1.6 h when grown in broth at the optimum growth temperature of 41°C . WSM3557T is pigmented, an unusual property for rhizobia. Colonies on half Lupin Agar (½LA)  are pale pink, opaque, slightly domed, moderately mucoid with smooth margins (Figure 1, right panel). The color develops after several days. WSM3557T is able to tolerate a pH range between 6.0 and 9.5 . Carbon source utilization, cellular fatty acid profiles, polar lipid analysis and respiratory lipoquinone analysis have been described previously . Minimum Information about the Genome Sequence (MIGS) is provided in Table 1.
M. lotononidis strain WSM3557T nodulates (Nod+) and fixes N2 effectively (Fix+) with Listia angolensis; nodulates and is partially effective on Leobordea platycarpa, Leobordea bolusii and Lotononis crumanina and nodulates but is unable to fix N2 (Nod+, Fix-) with Leobordea longiflora, Leobordea stipulosa and Lotononis falcata . It forms occasional ineffective nodules with Phaseolus vulgaris, but is unable to nodulate Crotalaria juncea, Indigofera patens, Lotus corniculatus, Lupinus angustifolius, or Macroptilium atropurpureum .
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the basis of its environmental and agricultural relevance to issues in global carbon cycling, alternative energy production, and biogeochemical importance, and is part of the Community Sequencing Program at the U.S. Department of Energy, Joint Genome Institute (JGI) for projects of relevance to agency missions. The genome project is deposited in the Genomes OnLine Database  and an improved-high-quality-draft genome sequence in IMG. Sequencing, finishing and annotation were performed by the JGI. A summary of the project information is shown in Table 2.
Growth conditions and DNA isolation
Microvirga lotononidis WSM3557T was grown to mid-logarithmic phase in TY rich medium  on a gyratory shaker at 28°C. DNA was isolated from 60 mL of cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method .
Genome sequencing and assembly
The improved high quality draft genome of Microvirga lotononidis WSM3557T was generated at the DOE Joint Genome Institute (JGI) using a combination of Illumina  and 454 technologies . An Illumina GAii shotgun library comprising 71,475,016 reads totaling 5,432.1 Mb reads and 1 paired end 454 library with an average insert size of 10 Kb which produced 582,107 reads totaling 113.9 Mb of 454 data were generated for this genome. All general aspects of library construction and sequencing performed at the JGI can be found at . The initial draft assembly contained 444 contigs in 1 scaffold. The 454 paired end data was assembled together with Newbler, version 2.3 PreRelease-6/30/2009. The Newbler consensus sequences were computationally shredded into 2 Kb overlapping fake reads (shreds). Illumina sequencing data was assembled with Velvet, version 1.0.13 , and the consensus sequences were computationally shredded into 1.5 Kb overlapping fake reads (shreds). The 454 Newbler consensus shreds, the Illumina Velvet consensus shreds and the read pairs in the 454 paired end library using parallel phrap, version SPS - 4.24 (High Performance Software, LLC) were integrated. The software Consed [28–30] was used in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality, using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher , or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. A total of 303 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The estimated genome size is 7.2 Mb and the final assembly is based on 59.7 Mb of 454 draft data which provides an average 8.3× coverage of the genome and 2,160 Mb of Illumina draft data which provides an average 300× coverage of the genome.
Genes were identified using Prodigal  as part of the DOE-JGI Annotation pipeline , followed by a round of manual curation using the JGI GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE , RNAMMer , Rfam , TMHMM , and SignalP . Additional gene prediction analyses and functional annotation were performed within the Integrated Microbial Genomes (IMG-ER) platform .
The genome is 7,082,538 nucleotides with 63.00% GC content (Table 3) and comprised of 18 scaffolds (Figures 3a,3b and Figure 3c) of 104 contigs. From a total of 7,040 genes, 6,956 were protein encoding and 84 RNA only encoding genes. The majority of genes (67.64%) were assigned a putative function while the remaining genes were annotated as hypothetical. The distribution of genes into COGs functional categories is presented in Table 4.
Herridge DF, Peoples MB, Boddey RM. Global inputs of biological nitrogen fixation in agricultural systems. Plant Soil 2008; 311:1–18. http://dx.doi.org/10.1007/s11104-008-9668-3
Howieson JG, Yates RJ, Foster K, Real D, Besier B. Prospects for the future use of legumes. In: Dilworth MJ, James EK, Sprent JI, Newton WE, editors. Leguminous Nitrogen-Fixing Symbioses. London, UK: Elsevier; 2008. p 363–394.
Loi A, Howieson JG, Nutt BJ, Carr SJ. A second generation of annual pasture legumes and their potential for inclusion in Mediterranean-type farming systems. Aust J Exp Agric 2005; 45:289–299. http://dx.doi.org/10.1071/EA03134
George RJ, Speed RJ, Simons JA, Smith RH, Ferdowsian R, Raper GP, Bennett DL. Long-term groundwater trends and their impact on the future extent of dryland salinity in Western Australia in a variable climate. Salinity Forum 2008. 2008.
van Wyk BE. A Synopsis of the Genus Lotononis (Fabaceae: Crotalarieae). Cape Town, South Africa: Rustica Press; 1991.
Boatwright JS, Wink M, van Wyk BE. The generic concept of Lotononis (Crotalarieae, Fabaceae): Reinstatement of the genera Euchlora, Leobordea and Listia and the new genus Ezoloba. Taxon 2011; 60:161–177.
Yates RJ, Howieson JG, Reeve WG, Nandasena KG, Law IJ, Bräu L, Ardley JK, Nistelberger HM, Real D, O’Hara GW. Lotononis angolensis forms nitrogen fixing, lupinoid nodules with phylogenetically unique, fast-growing, pinkpigmented bacteria, which do not nodulate L. bainesii or L. listii. Soil Biol Biochem 2007; 39:1680–1688. http://dx.doi.org/10.1016/j.soilbio.2007.01.025
Ardley JK. Symbiotic specificity and nodulation in the southern African legume clade Lotononis s. l. and description of novel rhizobial species within the Alphaproteobacterial genus Microvirga: Murdoch University, Murdoch, WA, Australia; 2012.
Norris DO. A red strain of Rhizobium from Lotononis bainesii Baker. Aust J Agric Res 1958; 9:629–632. http://dx.doi.org/10.1071/AR9580629
Jaftha JB, Strijdom BW, Steyn PL. Characterization of pigmented methylotrophic bacteria which nodulate Lotononis bainesii. Syst Appl Microbiol 2002; 25:440–449. PubMed http://dx.doi.org/10.1078/0723-2020-00124
Ardley JK, Parker MA, De Meyer SE, Trengove RD, O’Hara GW, Reeve WG, Yates RJ, Dilworth MJ, Willems A, Howieson J.G. Microvirga lupini sp. nov., Microvirga lotononidis sp. nov. and Microvirga zambiensis sp. nov. are alphaproteobacterial root-nodule bacteria that specifically nodulate and fix nitrogen with geographically and taxonomically separate legume hosts. Int J Syst Evol Microbiol 2011; 62:2579–2588. PubMed http://dx.doi.org/10.1099/ijs.0.035097-0
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using Maximum Likelihood, evolutionary distance, and Maximum Parsimony methods. Mol Biol Evol 2011; 28:2731–2739. PubMed http://dx.doi.org/10.1093/molbev/msr121
Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985; 39:783–791. http://dx.doi.org/10.2307/2408678
Liolios K, Mavromatis K, Tavernarakis N, Kyrpides NC. The Genomes On Line Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2008; 36(Database issue):D475–D479. PubMed http://dx.doi.org/10.1093/nar/gkm884
Ardley JK, Parker MA, De Meyer SE, Trengove RD, O’Hara GW, Reeve WG, Yates RJ, Dilworth MJ, Willems A, Howieson JG. Microvirga lupini sp. nov., Microvirga lotononidis sp. nov., and Microvirga zambiensis sp. nov. are Alphaproteobacterial root nodule bacteria that specifically nodulate and fix nitrogen with geographically and taxonomically separate legume hosts. Int J Syst Evol Microbiol 2011; 62:2579–2588. PubMed http://dx.doi.org/10.1099/ijs.0.035097-0
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen M, Angiuoli SV, et al. Towards a richer description of our complete collection of genomes and metagenomes “Minimum Information about a Genome Sequence” (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed http://dx.doi.org/10.1038/nbt1360
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed http://dx.doi.org/10.1073/pnas.87.12.4576
Garrity GM, Bell JA, Lilburn T. Class I. Alphaproteobacteria class. In: Garrity GM, Brenner DJ, Kreig NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. Second ed: New York: Springer-Verlag; 2005, p 1.
Kuykendall LD. Order VI. Rhizobiales ord. nov. In: Garrity GM, Brenner DJ, Kreig NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. Second ed: New York: Springer-Verlag; 2005. p 324.
Garrity GM, Bell JA, Lilburn TG. Family IX. Methylobacteriaceae. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. Second ed. Volume 2. New York: Springer-Verlag; 2005, p 567.
Kanso S, Patel BKC. Microvirga subterranea gen. nov., sp. nov., a moderate thermophile from a deep subsurface Australian thermal aquifer. Int J Syst Evol Microbiol 2003; 53:401–406. PubMed http://dx.doi.org/10.1099/ijs.0.02348-0
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25–29. PubMed http://dx.doi.org/10.1038/75556
Reeve WG, Tiwari RP, Worsley PS, Dilworth MJ, Glenn AR, Howieson JG. Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria. Microbiology 1999; 145:1307–1316. PubMed http://dx.doi.org/10.1099/13500872-145-6-1307
Bennett S. Solexa Ltd. Pharmacogenomics 2004; 5:433–438. PubMed http://dx.doi.org/10.1517/146224126.96.36.1993
Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 2005; 437:376–380. PubMed
Zerbino DR. Using the Velvet de novo assembler for short-read sequencing technologies. Current Protocols in Bioinformatics 2010;Chapter 11:Unit 11 5.
Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 1998; 8:175–185. PubMed http://dx.doi.org/10.1101/gr.8.3.175
Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 1998; 8:175–185. PubMed http://dx.doi.org/10.1101/gr.8.3.175
Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res 1998; 8:195–202. PubMed http://dx.doi.org/10.1101/gr.8.3.195
Han C, Chain P. Finishing repeat regions automatically with Dupfinisher. In: Valafar HRAH, editor. Proceeding of the 2006 international conference on bioinformatics & computational biology: CSREA Press; 2006. p 141–146.
Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010; 11:119. PubMed http://dx.doi.org/10.1186/1471-2105-11-119
Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM, Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci 2009; 1:63–67. PubMed http://dx.doi.org/10.4056/sigs.632
Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods 2010; 7:455–457. PubMed http://dx.doi.org/10.1038/nmeth.1457
Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997; 25:955–964. PubMed
Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 2007; 35:3100–3108. PubMed http://dx.doi.org/10.1093/nar/gkm160
Griffiths-Jones S, Bateman A, Marshall M, Khanna A, Eddy SR. Rfam: an RNA family database. Nucleic Acids Res 2003; 31:439–441. PubMed http://dx.doi.org/10.1093/nar/gkg006
Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J Mol Biol 2001; 305:567–580. PubMed http://dx.doi.org/10.1006/jmbi.2000.4315
Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004; 340:783–795. PubMed http://dx.doi.org/10.1016/j.jmb.2004.05.028
Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed http://dx.doi.org/10.1093/bioinformatics/btp393
This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396. We gratefully acknowledge the funding received from the Murdoch University Strategic Research Fund through the Crop and Plant Research Institute (CaPRI) and the Centre for Rhizobium Studies (CRS) at Murdoch University. The authors would like to thank the Australia-China Joint Research Centre for Wheat Improvement (ACCWI) and SuperSeed Technologies (SST) for financially supporting Mohamed Ninawi’s PhD project.