Open Access

Complete genome sequence of Xylanimonas cellulosilytica type strain (XIL07T)

  • Brian Foster
  • , Rüdiger Pukall
  • , Birte Abt
  • , Matt Nolan
  • , Tijana Glavina Del Rio
  • , Feng Chen
  • , Susan Lucas
  • , Hope Tice
  • , Sam Pitluck
  • , Jan-Fang Cheng
  • , Olga Chertkov,
  • , Thomas Brettin,
  • , Cliff Han,
  • , John C. Detter,
  • , David Bruce,
  • , Lynne Goodwin,
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Amrita Pati
  • , Natalia Mikhailova
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Loren Hauser,
  • , Yun-Juan Chang,
  • , Cynthia D. Jeffries,
  • , Patrick Chain,
  • , Manfred Rohde
  • , Markus Göker
  • , Jim Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz
  • , Nikos C. Kyrpides
  • , Hans-Peter Klenk
  • and Alla Lapidus
Corresponding author

DOI: 10.4056/sigs.571102

Received: 28 January 2010

Published: 28 February 2010

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.

Keywords:

AerobicGram-positivenon-motilecellulasesxylanasesPromicromonosporaceaeGEBA

Introduction

Strain XIL07T (= 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 XIL07T, 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 XIL07T 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 XIL07T 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 XIL07T were described as coccoid (approximately 1.1 x 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 XIL07T 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 XIL07T 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 XIL07T 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 XIL07T

Table 1

Classification and general features of X. cellulosilytica XIL07T according to the MIGS recommendations [7]

MIGS ID

Property

Term

Evidence code

Classification

Domain Bacteria

TAS [8]

Phylum Actinobacteria

TAS [9]

Class Actinobacteria

TAS [10]

Subclass Actinobacteridae

TAS [10]

Order Actinomycetales

TAS [10]

Suborder Micrococcineae

TAS [10]

Family Promicromonosporaceae

TAS [10]

Genus Xylanimonas

TAS [1]

Species Xylanimonas cellulosilytica

TAS [1]

Type strain XIL07

Gram stain

positive

TAS [1]

Cell shape

coccoid, short rods

TAS [1] & IDA

Motility

nonmotile

TAS [1]

Sporulation

nonsporulating

TAS [1]

Temperature range

mesophile

TAS [1]

Optimum temperature

30 °C

TAS [1]

Salinity

not reported

MIGS-22

Oxygen requirement

aerobic or facultatively anaerobic

TAS [1]

Carbon source

L-arabinose, carboxymethylcellulose, mannose,maltose, rhamnose, starch and xylan

TAS [1]

Energy source

chemoorganotroph

TAS [1]

MIGS-6

Habitat

soil

TAS [1]

MIGS-15

Biotic relationship

free living

NAS

MIGS-14

Pathogenicity

non pathogenic

NAS

Biosafety level

1

TAS [11]

Isolation

decaying tree

TAS [1]

MIGS-4

Geographic location

Salamanca, Spain

TAS [1]

MIGS-5

Sample collection time

not reported

MIGS-4.1 MIGS-4.2

Latitude, Longitude

40.965, -5.663

NAS

MIGS-4.3

Depth

not reported

MIGS-4.4

Altitude

not reported

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); 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 [12]. If the evidence code is IDA, then the property was directly observed for a living isolate by one of the authors or an expert mentioned in the acknowledgements.

Chemotaxonomy

The cell wall of X. cellulosilytica XIL07T contains A4α-type peptidoglycan (L-Lys-D-Asp). Cell wall sugars are galactose and rhamnose. Mycolic acids are absent. Strain XIL07T 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

MIGS ID

     Property

     Term

MIGS-31

     Finishing quality

     Finished

MIGS-28

     Libraries used

     One Sanger libraries 8 kb pMCL200     and one 454 Pyrosequencing     standard library

MIGS-29

     Sequencing platforms

     ABI3730, 454 GS FLX

MIGS-31.2

     Sequencing coverage

     9.2× Sanger, 26.9× Pyrosequencing

MIGS-30

     Assemblers

     Newbler, Arachne

MIGS-32

     Gene calling method

     Prodigal, GenePRIMP

     GenBank ID

     CP001821 (chromosome), CP001822 (plasmid)

     GenBank Date of Release

     November 20, 2009

     GOLD ID

     Gc01153

     NCBI project ID

     19715

     Database: IMG-GEBA

     2501651194

MIGS-13

     Source material identifier

     DSM 15894

     Project relevance

     Tree of Life, GEBA

Growth conditions and DNA isolation

X. cellulosilytica XIL07T, 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 Web Site. 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.

Table 3

Genome Statistics

Attribute

Value

% of Total

Genome size (bp)

3,831,380

100%

DNA coding region (bp)

3,531,102

92.16%

DNA G+C content (bp)

2,775,913

72.45%

Number of replicons

2

Extrachromosomal elements

1

Total genes

3,546

100.00%

RNA genes

61

1.72%

rRNA operons

3

Protein-coding genes

3,485

98.28%

Pseudo genes

42

1.18%

Genes with function prediction

2,426

68.42%

Genes in paralog clusters

411

11.59%

Genes assigned to COGs

2,403

67.77%

Genes assigned Pfam domains

2,490

70.22%

Genes with signal peptides

864

24.37%

Genes with transmembrane helices

925

26.09%

CRISPR repeats

1

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 4

Number of genes associated with the general COG functional categories

Code

Value

%age

  Description

J

155

4.4

  Translation, ribosomal structure and biogenesis

A

1

0.0

  RNA processing and modification

K

243

7.0

  Transcription

L

137

3.9

  Replication, recombination and repair

B

1

0.0

  Chromatin structure and dynamics

D

25

0.7

  Cell cycle control, mitosis and meiosis

Y

0

0.0

  Nuclear structure

V

85

2.4

  Defense mechanisms

T

118

3.4

  Signal transduction mechanisms

M

131

3.8

  Cell wall/membrane biogenesis

N

6

0.2

  Cell motility

Z

0

0.0

  Cytoskeleton

W

0

0.0

  Extracellular structures

U

39

1.1

  Intracellular trafficking and secretion

O

77

2.2

  Posttranslational modification, protein turnover, chaperones

C

153

4.4

  Energy production and conversion

G

294

8.4

  Carbohydrate transport and metabolism

E

214

6.1

  Amino acid transport and metabolism

F

79

2.3

  Nucleotide transport and metabolism

H

110

3.2

  Coenzyme transport and metabolism

I

73

2.1

  Lipid transport and metabolism

P

152

4.4

  Inorganic ion transport and metabolism

Q

30

0.9

  Secondary metabolites biosynthesis, transport and catabolism

R

318

9.1

  General function prediction only

S

200

5.7

  Function unknown

-

1082

31.0

  Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Gabriele Gehrich-Schröter for growing X. cellulosilytica cultures, and Susanne Schneider for DNA extraction and quality analysis (both at DSMZ). 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, as well as German Research Foundation (DFG) INST 599/1-1.


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