Non-contiguous finished genome sequence and contextual data of the filamentous soil bacterium Ktedonobacter racemifer type strain (SOSP1-21).

Abstract

Ktedonobacter racemifer corrig. Cavaletti et al. 2007 is the type species of the genus Ktedonobacter, which in turn is the type genus of the family Ktedonobacteraceae, the type family of the order Ktedonobacterales within the class Ktedonobacteria in the phylum 'Chloroflexi'. Although K. racemifer shares some morphological features with the actinobacteria, it is of special interest because it was the first cultivated representative of a deep branching unclassified lineage of otherwise uncultivated environmental phylotypes tentatively located within the phylum 'Chloroflexi'. The aerobic, filamentous, non-motile, spore-forming Gram-positive heterotroph was isolated from soil in Italy. The 13,661,586 bp long non-contiguous finished genome consists of ten contigs and is the first reported genome sequence from a member of the class Ktedonobacteria. With its 11,453 protein-coding and 87 RNA genes, it is the largest prokaryotic genome reported so far. It comprises a large number of over-represented COGs, particularly genes associated with transposons, causing the genetic redundancy within the genome being considerably larger than expected by chance. This work is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Full text

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Standards in Genomic Sciences (2011) 5:97-111
DOI:10.4056/sigs.2114901

The Genomic Standards Consortium
Non-contiguous finished genome sequence and contextual
data of the filamentous soil bacterium Ktedonobacter
racemifer type strain (SOSP1-21T)
Yun-juan Chang1,2, Miriam Land1,2, Loren Hauser1,2, Olga Chertkov2,3, Tijana Glavina Del
Rio2, Matt Nolan2, Alex Copeland2, Hope Tice2, Jan-Fang Cheng2, Susan Lucas2, Cliff Han2,3,
Lynne Goodwin2,3, Sam Pitluck2, Natalia Ivanova2, Galina Ovchinikova2, Amrita Pati2, Amy
Chen4, Krishna Palaniappan4, Konstantinos Mavromatis2, Konstantinos Liolios2, Thomas
Brettin2,3, Anne Fiebig5, Manfred Rohde6, Birte Abt5, Markus Göker5, John C. Detter2,3, Tanja
Woyke2, James Bristow2, Jonathan A. Eisen2,7, Victor Markowitz4, Philip Hugenholtz2,8,
Nikos C. Kyrpides2, Hans-Peter Klenk5*, and Alla Lapidus2
1 Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
2 DOE Joint Genome Institute, Walnut Creek, California, USA
3 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA
4 Biological Data Management and Technology Center, Lawrence Berkeley National
Laboratory, Berkeley, California, USA
5 DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig,
Germany
6 HZI – Helmholtz Centre for Infection Research, Braunschweig, Germany
7 University of California Davis Genome Center, Davis, California, USA
8 Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The
University of Queensland, Brisbane, Australia
*Corresponding: author: Hans-Peter Klenk
Keywords: aerobic, heterotrophic, filamentous, non-motile, Gram-positive, moderately aci-
dophilic, sporulating, transposon, broken-stick distribution, entropy, Ktedonobacteraceae,
Chloroflexi, GEBA
Ktedonobacter racemifer corrig. Cavaletti et al. 2007 is the type species of the genus Ktedo-
nobacter, which in turn is the type genus of the family Ktedonobacteraceae, the type family
of the order Ktedonobacterales within the class Ktedonobacteria in the phylum ‘Chloroflexi’.
Although K. racemifer shares some morphological features with the actinobacteria, it is of
special interest because it was the first cultivated representative of a deep branching unclassi-
fied lineage of otherwise uncultivated environmental phylotypes tentatively located within
the phylum ‘Chloroflexi’. The aerobic, filamentous, non-motile, spore-forming Gram-positive
heterotroph was isolated from soil in Italy. The 13,661,586 bp long non-contiguous finished
genome consists of ten contigs and is the first reported genome sequence from a member of
the class Ktedonobacteria. With its 11,453 protein-coding and 87 RNA genes, it is the largest
prokaryotic genome reported so far. It comprises a large number of over-represented COGs,
particularly genes associated with transposons, causing the genetic redundancy within the
genome being considerably larger than expected by chance. This work is a part of the Ge-
nomic Encyclopedia of Bacteria and Archaea project.
Introduction
Strain SOSP1-21T (= DSM 44963 = NRRL B-41538)
is the type strain of the species Ktedonobacter ra-
cemifer, which is the type species of the monotypic
genus Ktedonobacter, the type genus of the family
Ktedonobacteraceae [1]. K. racemifer was first de-
scribed in 2006 [1,2] as an aerobic, non-motile,
filamentous, mesophilic, Gram-positive hetero-
troph also capable of growing under microaero-
philic conditions [1]. The genus name was derived
from the Greek word ktedon -onos, fiber, and the
Neo-Latin bacter, a rod, meaning a filamentous
rod [1]. The species epithet is derived from the
Latin adjective racemifer, carrying clusters of
grapes [1].

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Ktedonobacter racemifer type strain (SOSP1-21T)
The original spelling, Ktedobacter racemifer was
corrected in 2007 on validation according to Rule
61 and Recommendation 6(7) [2]. Strain SOSP1-
21T was originally isolated from a soil sample of a
black locust wood in Gerenzano, Northern Italy.
Ten phylogenetically (class level) related strains
were also isolated from soil samples collected at
different locations in Northern Italy [1]. Only re-
cently, a nearest cultivated neighbor, Thermospo-
rothrix hazakensis, was isolated from hot compost
in Japan [3]. Here we present a summary classifi-
cation and a set of features for K. racemifer strain
SOSP1-21T, together with the description of the
complete genomic sequencing and annotation.
Classification and features
Using NCBI BLAST [4], a representative genomic
16S rRNA sequence of K. racemifer SOSP1-21T was
compared under default settings (e.g., considering
only the high-scoring segment pairs (HSPs) from
the best 250 hits) with the most recent release of
the Greengenes database [5] and the relative fre-
quencies of taxa and keywords (reduced to their
stem [6]) were determined, weighted by BLAST
scores. The most frequently occurring genus was
'Ktedobacter' (100.0%) (1 hit in total; this
represents the original, incorrect spelling of Ktedo-
nobacter). No hits to sequences with (other) spe-
cies names were found. (Note that the Greengenes
database uses the INSDC (= EMBL/NCBI/DDBJ)
annotation, which is not an authoritative source for
nomenclature or classification.) The highest-
scoring environmental sequence was AM180157
('New lineage filamentous spore-forming soil iso-
late SOSP1-30SOSP1-30 str. SOSP1-30'), which
showed an identity of 99.0% and an HSP coverage
of 95.2%. The most frequently occurring keywords
within the labels of environmental samples which
yielded hits were 'soil' (11.2%), 'prari, tallgrass'
(4.9%), 'miner, weather' (1.9%), 'new' (1.8%) and
'filament, lineag, spore-form' (1.6%) (249 hits in
total). These keywords reflect some of the ecologi-
cal properties reported for strain SOSP1-21T in the
original description [1]. Environmental samples
which yielded hits of a higher score than the high-
est scoring species were not found.
Figure 1 shows the phylogenetic neighborhood of K.
racemifer in a 16S rRNA based tree. The sequences
of the eight 16S rRNA genes copies in the genome
differ by up to nine nucleotides from each other and
by up to five nucleotides from the previously pub-
lished 16S rRNA sequence (AM180156), which con-
tains two ambiguous base calls.
98 Standards in Genomic Sciences
K. racemifer strain SOSP1-21T cells are rod-
shaped, filamentous and grow both vegetative and
aerial mycelia on solid medium (Figure 2a). The
large aerial hyphae produce spherical spores that
cluster together with a grape-like appearance
(Figure 2b). All other K. racemifer strains pro-
duced rounded spores, although they were ar-
ranged differently on the aerial hyphae [1]. Fila-
mentous growth of strain SOSP1-21T also oc-
curred in submerged cultures, which contained
the branched mycelia known from actinomycetes
[1]. SOSP1-21T stains Gram-positive and is not ac-
id fast [1]. It produces pigments ranging from
cream to pinkish orange on all media [1]. Although
essentially aerobic, SOSP1-21T is capable of grow-
ing under microaerophilic conditions [1]. The op-
timal growth temperature is 28-33°C [1]. It grows
well at pH values between 4.8 and 6.8 with an op-
timum at pH 6 [1]. Salinity up to 10 g per liter does
not inhibit the growth of the strain [1].
Strain SOSP1-21T was capable of hydrolyzing
starch, casein, gelatin, and (to a lesser extent) kera-
tin but not cellulose, xylan, or chitin [1]. Strain
SOSP1-21T was catalase positive and produced H2S
but could not reduce nitrates [1]. It is sensitive to 5
ug/ml novobiocin or ramoplanin and to 20 mg/ml
apramycin and the glycopeptide A40926.
Chemotaxonomy
The peptidoglycan of strain SOSP1-21T contains
ornithine, alanine, glutamic acid, serine, and glycine
at a molar ratio of approximately 0.7:1.8:1.0:0.8:1.9
[1]. Serine was identified at the N-terminus of the
interpeptide bridge [1]. When originally described,
a detailed peptidoglycan structure had not been
determined but A-type cross-linkage was suggested
[1]. The cellular fatty acid pattern of strain SOSP1-
21T was reported to be characterized by an unusual
high abundance of C16:1 2-OH (30%) with other domi-
nant lipids being branched-chain saturated fatty
acids iso-C17:0 (25%), iso-C16:0 (11.5%) and anteiso-
C17:0 (9.6%), as well as C16:0 10-Me (7.8%) and C16:0
(6.7%) [1]. Our own data (DSMZ) did not confirm
this fatty acid spectrum, but revealed iso-C16:0
(20.1%) as the most frequent fatty acid, followed
by anteiso-C17:0 (18.5%), iso-C17:0 (15.0%), only
13.1% C16:1 2-OH and 11.6% C16:0 10-Me. Polar lipids
consisted of phosphatidylinisitol, phosphatitylgly-
cerol, diphosphatidylglycerol and an unknown gly-
colipid [1]. MK-9(H2) was the only menaquinone
reported for strain SOSP1-21T [1].

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Chang et al.
http://standardsingenomics.org 99

Figure 1. Phylogenetic tree highlighting the position of K. racemifer relative to the other type strains within
the phylum ‘Chloroflexi’. The tree was inferred from 1,359 aligned characters [7,8] of the 16S rRNA gene se-
quence under the maximum likelihood (ML) criterion [9]. Rooting was done initially using the midpoint me-
thod [10] and then checked for its agreement with the current classification (Table 1). The branches are
scaled in terms of the expected number of substitutions per site. Numbers above branches are support values
from 750 ML bootstrap replicates [11] (left) and from 1,000 maximum parsimony bootstrap replicates [12]
(right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [13] are
labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks [14-17] as well as
CP001337, CP000804, CP000909, CP002084, and AP012029.










Figures 2a and 2b. Scanning electron micrographs of K. racemifer SOSP1-21T mycelium and spores.

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Ktedonobacter racemifer type strain (SOSP1-21T)
Table 1. Classification and general features of K. racemifer SOSP1-21T according to the MIGS recommendations [18]
and the NamesforLife database [19].
100 Standards in Genomic Sciences
MIGS ID Property Term Evidence code








MIGS-22


MIGS-6
MIGS-15
MIGS-14


MIGS-4
MIGS-5
MIGS-4.1
MIGS-4.2
MIGS-4.3
MIGS-4.4
Current classification
Domain Bacteria
Phylum Chloroflexi
Class Ktedonobacteria
Order Ktedonobacterales
Family Ktedonobacteraceae
Genus Ktedonobacter
Species Ktedonobacter racemifer
Type strain SOSP1-21
positive
filamentous
non-motile
spherical spore-forming
mesophile
28-33°C
NaCl up to 10 g/l growth w/o problem, inhibited at 30 g/l
aerobic and microaerophilic
sugars and peptides
heterotrophic
soil
free-living
none
1
soil from a black locust wood
Gerenzano, Northern Italy
November 2001
45.64
9.00
not reported
about 210 m
TAS [20]
TAS [21,22]
TAS [1-3]
TAS [1,2]
TAS [1,2]
TAS [1,2]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
TAS [1]
NAS
NAS
TAS [23]
TAS [1]
TAS [1]
NAS
NAS
NAS

NAS
Gram stain
Cell shape
Motility
Sporulation
Temperature range
Optimum temperature
Salinity
Oxygen requirement
Carbon source
Energy metabolism
Habitat
Biotic relationship
Pathogenicity
Biosafety level
Isolation
Geographic location
Sample collection time
Latitude
Longitude
Depth
Altitude
Evidence codes - 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 of the Gene Ontology project [24].
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the
basis of its phylogenetic position [25], and is part
of the Genomic Encyclopedia of Bacteria and Arc-
haea project [26]. The genome project is depo-
sited in the Genomes OnLine Database [13] and
the complete genome sequence is deposited in
GenBank. Sequencing, finishing and annotation
were performed by the DOE Joint Genome Insti-
tute (JGI). A summary of the project information is
shown in Table 2.

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Chang et al.
http://standardsingenomics.org 101
Table 2. Genome sequencing project information
MIGS ID Property
MIGS-31 Finishing quality
Term
Non-contiguous finished
MIGS-28 Libraries used
Two Sanger 8 kb pMCL200 and fosmid libraries;
one 454 pyrosequence standard library
MIGS-29 Sequencing platforms ABI3730, 454 GS FLX
MIGS-31.2 Sequencing coverage 10.1 × Sanger; 24.6 × pyrosequence
MIGS-30 Assemblers Newbler version 1.1.02.15, phrap
Prodigal 1.4, Genemark 4.6b, tRNAScan-SE-1.23,
infernal 0.81
MIGS-32 Gene calling method





INSDC ID ADVG00000000
Genbank Date of Release June 14, 2010
GOLD ID Gi02261
NCBI project ID 27943
Database: IMG-GEBA 648276680
MIGS-13

Source material identifier DSM 44963
Project relevance Tree of Life, GEBA
Growth conditions and DNA isolation
K. racemifer SOSP1-21T, DSM 44963, was grown in
DSMZ medium 65 (GYM Streptomyces medium)
[27] adjusted to pH 6.0, at 28°C. DNA was isolated
from 0.5-1 g of cell paste using Qiagen Genomic
500 DNA Kit (Qiagen 10262) following the manu-
facturer’s protocol, with cell lysis protocol
st/LALMP as described in Wu et al. [26]. DNA is
available through the DNA Bank Network [28].
Genome sequencing and assembly
The genome was sequenced using a combination
of Sanger and 454 sequencing platforms. All gen-
eral aspects of library construction and sequenc-
ing can be found at the JGI website [29]. Pyrose-
quencing reads were assembled using the Newb-
ler assembler (Roche). The initial Newbler contigs
were broken into 14,080 overlapping fragments of
1,000 bp and entered as pseudo-reads into the
subsequence assembly. The sequences were as-
signed quality scores based on Newbler consensus
q-scores with modifications to account for overlap
redundancy and to adjust inflated q-scores. A hy-
brid 454/Sanger assembly was produced using
parallel phrap (High Performance Software, LLC).
Possible mis-assemblies were corrected with Dup-
finisher [30], or transposon bombing of bridging
clones (Epicentre Biotechnologies, Madison, WI)
[31]. Some gaps between contigs were closed by
editing in Consed [32], custom primer walking or
PCR amplification. A total of 3,354 Sanger finish-
ing reads and five shatter libraries were produced
to close gaps, to resolve some repetitive regions,
and to raise the quality of the finished sequence.
Illumina reads were also used to correct potential
base errors and increase consensus quality using a
software Polisher developed at JGI [33]. The error
rate of the completed genome sequence is less
than 1 in 100,000. Together, the combination of
the Sanger and 454 sequencing platforms pro-
vided 34.7 × coverage of the genome. The final
assembly contained 165,050 pyrosequence and
2,305,667 Illumina reads.
Genome annotation
Genes were identified using Prodigal [34] as part
of the Oak Ridge National Laboratory genome an-
notation pipeline, followed by a round of manual
curation using the JGI GenePRIMP pipeline [35].
The predicted CDSs were translated and used to
search the National Center for Biotechnology In-
formation (NCBI) non-redundant database, Uni-
Prot, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and In-
terPro databases. Additional gene prediction anal-
ysis and functional annotation were performed
within the Integrated Microbial Genomes - Expert
Review (IMG-ER) platform [36].