Complete genome sequence of Xylanimonas cellulosilytica type strain (XIL07^T)

Abstract

Xylanimonas cellulosilytica Rivas et al. 2003 is the type species of the genus Xylanimonas of the actinobacterial family Promicromonosporaceae. The species X. cellulosilytica is of interest because of its ability to hydrolyze cellulose and xylan. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the large family Promicromonosporaceae, and the 3,831,380 bp long genome (one chromosome plus an 88,604 bp long plasmid) with its 3485 protein-coding and 61 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Introduction

Strain XIL07^T (= DSM 15894 = CECT 5975 = JCM 12276) is the type strain of the species Xylanimonas cellulosilytica and was first described in 2003 by Rivas et al. [1]. It was isolated from a decayed tree, Ulmus nigra, in Salamanca, Spain. X. cellulosilytica is of high interest because it produces a set of hydrolytic enzymes, cellulases and xylanases that enable the organism to hydrolyze cellulose and xylan. Cellulolytic enzymes from microorganisms have many biotechnological and industrial applications, for example in the food, detergent, paper and textile industries or in the production of biofuels. Here we present a summary classification and a set of features for X. cellulosilytica XIL07^T, together with the description of the complete genomic sequencing and annotation.

Classification and features

The most closely related 16S rRNA gene sequences from cultivated strains that are stored in Genbank originate from isolates classified into neighboring genera within the Promicromonosporaceae. Among them are some Isoptericola species isolated from different habitats, including soil, tufa, decayed wood and the hindgut of the humus-feeding larva of the beetle Pachnoda ephippiata. Sequences of 16S rRNAs from several uncultivated bacteria detected in midgut and hindgut of P. ephippiata (AJ576375, AJ576390, AJ576391 AJ576404, AJ576378, AJ576417) [2] are apparently the most closely related phylotypes, with 96–97% sequence similarity. Environmental samples from metagenomic surveys do not surpass 92% sequence similarity, indicating that members of the species are not heavily represented in the so far genomically screened habitats (status July 2009).

Figure 1 shows the phylogenetic neighborhood of X. cellulosilytica XIL07^T in a 16S rRNA based tree. The sequences of the three copies of the 16S rRNA gene in the genome differ by up to four nucleotides, and differ by up to five nucleotides from the previously published sequence generated from DSM 15894 (AF403541).

Figure 1.

Phylogenetic tree highlighting the position of X. cellulosilytica XIL07^T relative to the other type strains within the family Promicromonosporaceae. The tree was inferred from 1,393 aligned characters [3,4] of the 16S rRNA gene sequence under the maximum likelihood criterion [5] and rooted in accordance with the current taxonomy. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [6] are shown in blue, published genomes in bold.

Based on thin section EMs, cells of strain XIL07^T were described as coccoid (approximately 1.1 × 0.8 µm) that occur singly [1]. However, SEM images (Figure 2) show coccobacillary forms and short rods, as known from other members of the Promicromonosporaceae: Cellulosimicrobium, Isoptericola and Promicromonospora (Table 1). Strain XIL07^T is Gram-positive, aerobic or facultatively anaerobic, nonmotile and non-spore-forming. Colonies grown on YED are white-to-cream colored and their morphology is circular, smooth and mostly flat. Strain XIL07^T utilizes L-arabinose, carboxymethylcellulose, mannose, maltose, rhamnose, starch and xylan as sole carbon source, and produces acid from amygdalin, L-arabinose, arbutin, cellobiose, fructose, galactose, getobiose, glucose, glycerol, glycogen, lactose, lyxose, maltose, mannose, melozitose, rhamnose, salicin, sucrose, trehalose, turanose and D-xylose. No growth was observed with acetate, citrate, gluconate, inositol, malate or mannitol as carbon sources. Strain XIL07^T actively produces amylases, cellulases, gelatinase, xylanases and β-galactosidase and shows weak catalase activity. Esculin was hydrolyzed and nitrate was not reduced [1].

Figure 2.

Scanning electron micrograph of X. cellulosilytica XIL07^T

Table 1. Classification and general features of X. cellulosilytica XIL07^T according to the MIGS recommendations [7]

Chemotaxonomy

The cell wall of X. cellulosilytica XIL07^T contains A4α-type peptidoglycan (L-Lys-D-Asp). Cell wall sugars are galactose and rhamnose. Mycolic acids are absent. Strain XIL07^T contains menaquinone MK-9(H4) as the major respiratory lipoquinone and a lower amount of MK-8(H4). The cellular fatty acid pattern is composed of iso- and anteiso-branched fatty acids with anteiso-C15:0 (12-methyl tetradecanoic acid) being the predominant and iso-C15:0 the minor fatty acid. The major polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and other unidentified phosphoglycolipids [1].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position, and is part of the Genomic Encyclopedia of Bacteria and Archaea project [13]. The genome project is deposited in the Genome OnLine Database [6] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Table 2. Genome sequencing project information

Growth conditions and DNA isolation

X. cellulosilytica XIL07^T, DSM 15894, was grown in DSMZ medium 92 (Trypticase Soy Yeast Extract Medium) at 28°C [14]. DNA was isolated from 0.5–1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol without modifications.

Genome sequencing and assembly

The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov/. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 4,321 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using Arachne assembler. Possible mis-assemblies were corrected and gaps between contigs were closed by custom primer walks from sub-clones or PCR products. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 437 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 36.1× coverage of the genome. The final assembly contains 52,128 Sanger and 514,866 Pyrosequencing reads.

Genome annotation

Genes were identified using Prodigal [15] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [16]. 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. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [17].

Genome properties

The genome is 3,831,380 bp long and comprises one main circular chromosome and one plasmid with a 72.5% GC content (Table 3 and Figure 3). Of the 3,546 genes predicted, 3,485 were protein coding genes, and 61 RNAs. In addition, 42 pseudogenes were identified. The majority of the genes (68.4%) were assigned with a putative function while those remaining were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Figure 3.

Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 3. Genome Statistics

Table 4. Number of genes associated with the general COG functional categories