Genome sequence of Microvirga lupini strain LUT6(T), a novel Lupinus alphaproteobacterial microsymbiont from Texas.

Microvirga lupini LUT6(T) is an aerobic, non-motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Lupinus texensis. LUT6(T) was isolated in 2006 from a nodule recovered from the roots of the annual L. texensis growing in Travis Co., Texas. LUT6(T) forms a highly specific nitrogen-fixing symbiosis with endemic L. texensis and no other Lupinus species can form an effective nitrogen-fixing symbiosis with this isolate. Here we describe the features of M. lupini LUT6(T), together with genome sequence information and its annotation. The 9,633,614 bp improved high quality draft genome is arranged into 160 scaffolds of 1,366 contigs containing 10,864 protein-coding genes and 87 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of a DOE Joint Genome Institute 2010 Community Sequencing Project.

Introduction

is one of the most recently discovered genera of known to engage in symbiotic nitrogen fixation with legume plants, and joins a diverse set of at least twelve other lineages of that share this ecological niche [1-4]. Several genera of legume root-nodule symbionts have a world-wide distribution and interact with many legume taxa. By contrast, symbiotic strains of are currently known from two distant locations and only two legume host genera [5,6]. The limited geographic and host distribution of symbionts, along with the fact that root-nodule symbiosis is not characteristic of the genus as a whole [7], suggest a relatively recent evolutionary transition to legume symbiosis in this group.

is a specialized nodule symbiont associated with the legume Lupinus texensis, an annual plant endemic to a relatively small geographic area in central Texas and northeastern Mexico [5]. The genus Lupinus has about 270 annual and perennial species concentrated in western North America and in Andean regions of South America, and a much smaller number of species in the Mediterranean region of Europe and northern Africa [8]. Basal lineages of Lupinus all occur in the Mediterranean and are associated with bacterial symbionts in the genus [9,10]. is also the main symbiont lineage for most Lupinus species in North and South America, although a few Lupinus species utilize nodule bacteria in the genus [10-13]. Thus, the acquisition of symbionts in the genus by plants of L. texensis appears to be an unusual, derived condition for this legume genus.

L. texensis occurs in grassland and open shrub communities with an annual precipitation of 50 - 100 cm, on diverse soil types [14]. L. texensis appears to have a specialized symbiotic relationship with in that existing surveys have failed to detect nodule symbionts of any other bacterial genus associated with this plant [5]. Moreover, inoculation experiments with other North American species of Lupinus, as well as other legume genera, have so far failed to identify any plant besides L. texensis that is capable of forming an effective, nitrogen-fixing symbiosis with [5]. strain Lut6T was isolated from a nodule collected from a L. texensis plant in Travis Co., Texas in 2006. Here we provide an analysis of the complete genome sequence of strain Lut6T; one of the three described symbiotic species of [15].

Classification and general features

LUT6T is a non-motile, Gram-negative rod in the order of the class . The rod-shaped form varies in size with dimensions of 1.0 μm for width and 1.5-2.0 μm for length (Figure 1 Left and Center). It is fast growing, forming colonies within 3-4 days when grown on half strength Lupin Agar (½LA) [16], tryptone-yeast extract agar (TY) [17] or a modified yeast-mannitol agar (YMA) [18] at 28°C. Colonies on ½LA are white-opaque, slightly domed and moderately mucoid with smooth margins (Figure 1 Right).

Figure 1

Images of LUT6T using scanning (Left) and transmission (Center) electron microscopy and the appearance of colony morphology on solid medium (Right).

Minimum Information about the Genome Sequence (MIGS) is provided in Table 1. Figure 2 shows the phylogenetic neighbor-hood of LUT6T in a 16S rRNA sequence based tree. This strain shares 100% (1,358/1,358 bases) and 98% (1,344/1,367 bases) sequence identity to the 16S rRNA of Lut5 and WSM3557T, respectively.

Table 1

Classification and general features of LUT6T according to the MIGS recommendations [19,20]

MIGS ID	Property	Term	Evidence code
	Current classification	Domain Bacteria	TAS [20]
		Phylum Proteobacteria	TAS [21]
		Class Alphaproteobacteria	TAS [22,23]
		Order Rhizobiales	TAS [23,24]
		Family Methylobacteriaceae	TAS [23,25]
		Genus Microvirga	TAS [15,26-28]
		Species Microvirga lupini	TAS [15]
		Strain LUT6T
	Gram stain	Negative	TAS [15]
	Cell shape	Rod	TAS [15]
	Motility	Non-Motile	IDA
	Sporulation	Non-sporulating	TAS [15]
	Temperature range	Mesophile	TAS [15]
	Optimum temperature	39°C	TAS [15]
	Salinity	Non-halophile	TAS [15]
MIGS-22	Oxygen requirement	Aerobic	TAS [15]
	Carbon source	Varied	TAS [15]
	Energy source	Chemoorganotroph	TAS [15]
MIGS-6	Habitat	Soil, root nodule, on host	TAS [15]
MIGS-15	Biotic relationship	Free living, symbiotic	TAS [15]
MIGS-14	Pathogenicity	Non-pathogenic	NAS
	Biosafety level	1	TAS [29]
	Isolation	Root nodule of Lupinus texensis	TAS [5]
MIGS-4	Geographic location	Travis Co., Texas	TAS [5]
MIGS-5	Soil collection date	03 Jan 2006	IDA
MIGS-4.1MIGS-4.2	LatitudeLongitude	-97.838   30.459	IDA    IDA
MIGS-4.3	Depth	0-10 cm	IDA
MIGS-4.4	Altitude	270 m	IDA

Evidence codes – IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [30].

Figure 2

Phylogenetic tree showing the relationship of LUT6T (shown in bold print) to other root nodule bacteria in the order based on aligned sequences of the 16S rRNA gene (1,320 bp internal region). All sites were informative and there were no gap-containing sites. Phylogenetic analyses were performed using MEGA, version 5 [31]. The tree was built using the Maximum-Likelihood method with the General Time Reversible model [32]. Bootstrap analysis [33] with 500 replicates was performed to assess the support of the clusters. Type strains are indicated with a superscript T. Brackets after the strain name contain a DNA database accession number and/or a GOLD ID (beginning with the prefix G) for a sequencing project registered in GOLD [34]. Published genomes are indicated with an asterisk.

Symbiotaxonomy

strain Lut6T was isolated in from a nodule collected from Lupinus texensis growing near Travis Co., Texas. The symbiotic characteristics of this isolate on a range of selected hosts are provided in Table 2.

Table 2

Nodulation and N2 fixation properties of Lut6T on selected legumes†.

Legume Species	Nodulation	N2 fixation	Comment
Lupinus texensis	Nod+	Fix+	Highly effective
Lupinus perennis	Nod-	Fix-	No nodulation
Lupinus succulentus	Nod-	Fix-	No nodulation
Lupinus microcarpus	Nod-	Fix-	No nodulation
Phaseolus vulgaris	Nod-	Fix-	No nodulation
Macroptilium atropurpureum	Nod+	Fix-	No fixation
Desmodium canadense	Nod-	Fix-	No nodulation
Cytisus scoparius	Nod+	Fix-	No fixation
Mimosa pudica	Nod-	Fix-	No nodulation

†Data compiled [5]. Note that ‘+’ and ‘-’ denote presence or absence, respectively, of nodulation (Nod) or N2 fixation (Fix).

Genome sequencing and annotation information

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 [34] 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 3.

Table 3

Genome sequencing project information for LUT6T.

MIGS ID	Property	Term
MIGS-31	Finishing quality	Improved high-quality draft
MIGS-28	Libraries used	Illumina GAii shotgun and a paired end 454 libraries
MIGS-29	Sequencing platforms	Illumina GAii and 454 GS FLX Titanium technologies
MIGS-31.2	Sequencing coverage	3.5× 454 paired end, 300× Illumina
MIGS-30	Assemblers	Velvet version 1.0.13; Newbler 2.3, phrap SPS - 4.24
MIGS-32	Gene calling methods	Prodigal 1.4
	GOLD ID	Gi06478
	NCBI project ID	66529
	Database: IMG	2508501050
	Project relevance	Symbiotic N2 fixation, agriculture

Growth conditions and DNA isolation

LUT6T was cultured to mid logarithmic phase in 60 ml of TY rich media [35] on a gyratory shaker at 28°C. DNA was isolated from the cells using a CTAB (Cetyl trimethyl ammonium bromide) bacterial genomic DNA isolation method [36].

Genome sequencing and assembly

The genome of LUT6T was sequenced at the Joint Genome Institute (JGI) using a combination of Illumina [37] and 454 technologies [38]. An Illumina GAii shotgun library which generated 77,090,752 reads totaling 5,858.9 Mbp, and a paired end 454 library with an average insert size of 8 Kbp which generated 238,026 reads totaling 81.4 Mb of 454 data were generated for this genome [36].

All general aspects of library construction and sequencing performed at the JGI can be found at [36]. The initial draft assembly contained 1,719 contigs in 6 scaffolds. The 454 paired end data were assembled together with Newbler, version 2.3-PreRelease-6/30/2009. The Newbler consensus sequences were computationally shredded into 2 Kbp overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 1.0.13 [39], and the consensus sequence computationally shredded into 1.5 Kbp overlapping fake reads (shreds). The 454 Newbler consensus shreds, the Illumina VELVET consensus shreds and the read pairs in the 454 paired end library were integrated using parallel phrap, version SPS - 4.24 (High Performance Software, LLC). The software Consed [40-42] 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 [43]. Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished) or Dupfinisher [44]. Some gaps between contigs were closed by editing in Consed. The estimated genome size is 10.3 Mb and the final assembly is based on 36.2 Mb of 454 draft data which provides an average 3.5x coverage of the genome and 3,090 Mbp of Illumina draft data which provides an average 300x coverage of the genome.

Genome annotation

Genes were identified using Prodigal [45] as part of the DOE-JGI annotation pipeline [46]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. The tRNAScanSE tool [47] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [48]. Other non–coding RNAs such as the RNA components of the protein secretion complex and the RNase P were identified by searching the genome for the corresponding Rfam profiles using INFERNAL [49]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) platform [50].

Genome properties

The genome is 9,633,614 nucleotides long with 60.26% GC content (Table 4) and comprised of 160 scaffolds (Figure 3) of 1,366 contigs. From a total of 10,951 genes, 10,864 were protein encoding and 87 RNA only encoding genes. The majority of genes (63.25%) were assigned a putative function whilst the remaining genes were annotated as hypothetical. The distribution of genes into COGs functional categories is presented in Table 5.

Table 4

Genome statistics for LUT6T

Attribute	Value	% of Total
Genome size (bp)	9,633,614	100.00
DNA coding region (bp)	7,880,506	81.80
DNA G+C content (bp)	5,805,078	60.26
Number of scaffolds	160
Number of contigs	1,366
Total genes	10,951	100.00
RNA genes	87	0.79
rRNA operons	1	0.01
Protein-coding genes	10,864	99.21
Genes with function prediction	6,927	63.25
Genes assigned to COGs	6,990	63.83
Genes assigned Pfam domains	7,343	67.05
Genes with signal peptides	768	7.01
Genes with transmembrane helices	2,006	18.32
CRISPR repeats	0

Figure 3

Graphical map of the genome of LUT6T showing the four largest scaffolds. From bottom to the top of each scaffold: Genes on forward strand (color by COG categories as denoted by the IMG platform), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, sRNAs red, other RNAs black), GC content, GC skew.

Table 5

Number of protein coding genes of LUT6T associated with the general COG functional categories.

Code	Value	%age	COG Category
J	209	2.72	Translation, ribosomal structure and biogenesis
A	1	0.01	RNA processing and modification
K	571	7.43	Transcription
L	667	8.68	Replication, recombination and repair
B	10	0.13	Chromatin structure and dynamics
D	53	0.69	Cell cycle control, mitosis and meiosis
Y			Nuclear structure
V	104	1.35	Defense mechanisms
T	463	6.02	Signal transduction mechanisms
M	316	4.11	Cell wall/membrane biogenesis
N	69	0.9	Cell motility
Z	0	0	Cytoskeleton
W	1	0.01	Extracellular structures
U	95	1.24	Intracellular trafficking and secretion
O	249	3.24	Posttranslational modification, protein turnover, chaperones
C	401	5.22	Energy production conversion
G	602	7.83	Carbohydrate transport and metabolism
E	828	10.77	Amino acid transport metabolism
F	100	1.3	Nucleotide transport and metabolism
H	263	3.42	Coenzyme transport and metabolism
I	266	3.46	Lipid transport and metabolism
P	388	5.05	Inorganic ion transport and metabolism
Q	263	3.42	Secondary metabolite biosynthesis, transport and catabolism
R	976	12.70	General function prediction only
S	790	10.28	Function unknown
-	3,961	36.17	Not in COGS