Non-contiguous finished genome sequence of the opportunistic oral pathogen Prevotella multisaccharivorax type strain (PPPA20).

Abstract

Prevotella multisaccharivorax Sakamoto et al. 2005 is a species of the large genus Prevotella, which belongs to the family Prevotellaceae. The species is of medical interest because its members are able to cause diseases in the human oral cavity such as periodontitis, root caries and others. Although 77 Prevotella genomes have already been sequenced or are targeted for sequencing, this is only the second completed genome sequence of a type strain of a species within the genus Prevotella to be published. The 3,388,644 bp long genome is assembled in three non-contiguous contigs, harbors 2,876 protein-coding and 75 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Full text

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Standards in Genomic Sciences (2011) 5:41-49
DOI:10.4056/sigs.2164949

The Genomic Standards Consortium
Non-contiguous finished genome sequence of the
opportunistic oral pathogen Prevotella multisaccharivorax
type strain (PPPA20T)
Amrita Pati1, Sabine Gronow2, Megan Lu1,3, Alla Lapidus1, Matt Nolan1, Susan Lucas1, Nancy
Hammon1, Shweta Deshpande1, Jan-Fang Cheng1, Roxanne Tapia1,3, Cliff Han1,3, Lynne
Goodwin1,3 Sam Pitluck1, Konstantinos Liolios1, Ioanna Pagani1, Konstantinos Mavromatis1,
Natalia Mikhailova1, Marcel Huntemann1, Amy Chen4, Krishna Palaniappan4, Miriam
Land1,5, Loren Hauser1,5, John C. Detter1,3, Evelyne-Marie Brambilla2, Manfred Rohde6,
Markus Göker2, Tanja Woyke1, James Bristow1, Jonathan A. Eisen1,7, Victor Markowitz4,
Philip Hugenholtz1,8, Nikos C. Kyrpides1, Hans-Peter Klenk2*, and Natalia Ivanova1
1 DOE Joint Genome Institute, Walnut Creek, California, USA
2 DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig,
Germany
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 Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
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: obligately anaerobic, non-motile, Gram-negative, mesophilic, chemoorganotroph-
ic, opportunistic pathogen, Prevotellaceae, GEBA
Prevotella multisaccharivorax Sakamoto et al. 2005 is a species of the large genus Prevotella,
which belongs to the family Prevotellaceae. The species is of medical interest because its
members are able to cause diseases in the human oral cavity such as periodontitis, root caries
and others. Although 77 Prevotella genomes have already been sequenced or are targeted for
sequencing, this is only the second completed genome sequence of a type strain of a species
within the genus Prevotella to be published. The 3,388,644 bp long genome is assembled in
three non-contiguous contigs, harbors 2,876 protein-coding and 75 RNA genes and is a part
of the Genomic Encyclopedia of Bacteria and Archaea project.
Introduction
Strain PPPA20T (= DSM 17128 = JCM 12954) is the
type strain of Prevotella multisaccharivorax [1].
Currently, there are about 50 species placed in the
genus Prevotella [1]. The species epithet is derived
from the Latin adjective
‘many/much’, the Latin noun saccharum meaning
‘sugar’ and the Latin adjective vorax meaning ‘liking
to eat’ referring to the metabolic properties of the
species to digest a variety of carbohydrates [2]. P.
multisaccharivorax strain PPPA20T is considered to
be an opportunistic pathogen and was isolated
from subgingival plaque from a patient with chron-
ic periodontitis. Additionally, five more strains iso-
lated from the human oral cavity were placed in the
multus meaning
species P. multisaccharivorax [2]. Using non-culture
techniques on sites affected by endodontic and pe-
riodontal diseases, a large number of sequences
have been found that belong to Prevotella and Pre-
votella-like bacteria. Many of those species have
never been isolated or described [3]. The complex
microbial community living in the rich ecological
niche of the human oral cavity and its interaction
with consumed food will be of lasting interest for
medical and ecological reasons [4,5]. Here we
present a summary classification and a set of fea-
tures for P. multisaccharivorax PPPA20T, together
with the description of the non-contiguous finished
genomic sequencing and annotation.

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Prevotella multisaccharivorax type strain (PPPA20T)
Classification and features
A representative genomic 16S rRNA sequence of P.
multisaccharivorax PPPA20T was compared using
NCBI BLAST [6] under default settings (e.g., consi-
dering only the high-scoring segment pairs (HSPs)
from the best 250 hits) with the most recent release
of the Greengenes database [7] and the relative fre-
quencies of taxa and keywords (reduced to their
stem [8]) were determined, weighted by BLAST
scores. The most frequently occurring genus was
Prevotella (100.0%) (14 hits in total). Regarding the
single hit to sequences from members of the spe-
cies, the average identity within HSPs was 100.0%,
whereas the average coverage by HSPs was 98.0%.
Regarding the nine hits to sequences from other
members of the genus, the average identity within
HSPs was 90.3%, whereas the average coverage by
HSPs was 66.5%. Among all other species, the one
yielding the highest score was Prevotella ruminicola
(AF218618), which corresponded to an identity of
91.5% and an HSP coverage of 66.3%. (Note that
the Greengenes database uses the INSDC (=
EMBL/NCBI/DDBJ) annotation, which is not an au-
thoritative source for nomenclature or classifica-
tion.) The highest-scoring environmental sequence
was AY550995 ('human carious dentine clone IDR-
CEC-0032'), which showed an identity of 99.8% and
an HSP coverage of 94.5%. The most frequently oc-
curring keywords within the labels of environmen-
tal samples which yielded hits were 'fecal' (4.4%),
'beef, cattl' (4.1%), 'anim, coli, escherichia, feedlot,
habitat, marc, pen, primari, secondari, stec, syneco-
log' (4.0%), 'neg' (2.5%) and 'fece' (2.4%) (236 hits
in total). The most frequently occurring keywords
within the labels of environmental samples which
yielded hits of a higher score than the highest scor-
ing species were 'fece' (7.9%), 'goeldi, marmoset'
(4.8%), 'microbiom' (4.3%), 'aspect, canal, oral,
root' (3.9%) and 'rumen' (3.8%) (54 hits in total).
While some of these keywords correspond to the
well known habitat of P. multisaccharivorax, others
indicate additional habitats related to animals.
Figure 1 shows the phylogenetic neighborhood of P.
multisaccharivorax in a 16S rRNA based tree. The
sequences of the four 16S rRNA gene copies in the
genome differ from each other by up to two nucleo-
tides, and differ by up to two nucleotides from the
previously published
AB200414.
The cells of P. multisaccharivorax generally have the
shape of rods (0.8 × 2.5-8.3 µm) and occur singly or
42 Standards in Genomic Sciences
16S rRNA sequence
in pairs (Figure 2). They can also form longer fila-
ments. P. multisaccharivorax is a Gram-negative,
non spore-forming bacterium (Table 1). The organ-
ism is described as non-motile and only four genes
associated with motility were identified in the ge-
nome (see below). P. multisaccharivorax grows well
at 37°C, is strictly anaerobic, chemoorganotrophic
and is able to ferment cellobiose, glucose, glycerol,
lactose, maltose, mannose, melezitose, raffinose,
rhamnose, sorbitol, sucrose, trehalose and xylose
[2]. Acid production from arabinose and salicin is
variable. The organism does not reduce nitrate or
produce indole from tryptophan but it hydrolyzes
esculin and digests gelatin [2]. Growth of P. multi-
saccharivorax is inhibited by the addition of 20%
bile. Major fermentation products are succinic and
acetic acid, isovaleric acid is produced in small
amounts [2]. Activities of glucose-6-phosphate de-
hydrogenase (G6PDH) and 6-phosphogluconate
dehydrogenase (6GPDH) were not detected in iso-
lates of this species, whereas malate dehydrogenase
and glutamate dehydrogenase activities were de-
tected in all strains. P. multisaccharivorax produces
acid and alkaline phosphatase, β-galactosidase, α-
and β-glucosidase, N-acetyl-β-glucosaminidase, α-
aminofuranosidase and alanine aminopeptidase.
The organism has no demonstrable esterase (C4),
esterase lipase (C4), lipase (C4), leucine arylami-
dase, valine arylamidase, cystine arylamidase, py-
roglutamic acid arylamidase, trypsin, chymotrypsin,
β-glucuronidase, α-mannosidase, α-fucosidase, ar-
ginine aminopeptidase, leucine aminopeptidase,
proline aminopeptidase, tyrosine aminopeptidase,
phenylalanine aminopeptidase, urease or catalase
activity [2].
Chemotaxonomy
In contrast to other Prevotella species all strains of
P. multisaccharivorax harbor the menaquinones
MK-12 (40-55%) and MK-13 (40-45%) in large
amounts, whereas MK-10 (1-3%) and MK-11 (8-
10%) were found only in small amounts [2]. The
fatty acid pattern for all strains of P. multisacchari-
vorax revealed C18:1 ω9c (21.7%) and C16:0 (12.9%) as
major compounds as well as iso-C17:0 3-OH (9.2%),
anteiso-C15:0 (7.8%), C18:2 ω6,9c (7.5%) and iso-C15:0
(6.4%) in smaller amounts [2]. Additionally, the
unusual dimethyl acetals were found with C16:0 di-
methyl aldehyde in the highest amount of 8.2%.
This clearly distinguishes the species of P. multisac-
charivorax from other related Prevotella species
[2].

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Pati et al.
http://standardsingenomics.org 43

Figure 1. Phylogenetic tree highlighting the position of P. multisaccharivorax relative to the type strains of the oth-
er species within the family. The tree was inferred from 1,425 aligned characters [9,10] of the 16S rRNA gene se-
quence under the maximum likelihood (ML) criterion [11]. Rooting was done initially using the midpoint method
[12] 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 adjacent to the branches are support values from 600
ML bootstrap replicates [13] (left) and from 1,000 maximum parsimony bootstrap replicates [14] (right) if larger
than 60%. Lineages with type strain genome sequencing projects registered in GOLD [15] are labeled with one
asterisk, those also listed as 'Complete and Published' should be labeled with two asterisks: P. ruminicola [16] and
P. melaninogenica (CP002122/CP002123)

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Prevotella multisaccharivorax type strain (PPPA20T)
44 Standards in Genomic Sciences

Figure 2. Scanning electron micrograph of P. multisaccharivorax PPPA20T
Table 1. Classification and general features of P. multisaccharivorax PPPA20T according the MIGS
recommendations [17] and the NamesforLife database [1].
MIGS ID
Property
Term
Domain Bacteria
Phylum “Bacteroidetes”
Class “Bacteroidia”
Order “Bacteroidales”
Family “Prevotellaceae”
Genus Prevotella
Species Prevotella multisaccharivorax
Type strain PPPA20

Gram stain
negative

Cell shape
rod-shaped

Motility
non-motile

Sporulation
none

Temperature range
mesophilic

Optimum temperature
37°C

Salinity
physiological
MIGS-22
Oxygen requirement
obligately anaerobic

Carbon source
carbohydrates

Energy metabolism
chemoorganotrophic
MIGS-6
Habitat
host, human oral microflora
MIGS-15
Biotic relationship
free-living
MIGS-14
Pathogenicity
opportunistic pathogen

Biosafety level
2

Isolation
subgingival plaque, chronic periodontitis
MIGS-4
Geographic location
Japan
MIGS-5
Sample collection time
December 9, 2002
MIGS-4.1
Latitude
not reported
MIGS-4.2
Longitude
not reported
MIGS-4.3
Depth
not reported
MIGS-4.4
Altitude
not reported
Evidence code
TAS [18]
TAS [19]
TAS [20]
TAS [21]
TAS [21]
TAS [22,23]
TAS [2]
TAS [2]
TAS [2]
TAS [2]
TAS [2]
TAS [2]
TAS [2]
TAS [2]
TAS [2]
TAS [2]
TAS [2]
TAS [2]
TAS [2]
NAS
TAS [2]
TAS [24]
TAS [2]
TAS [2]
IDA





Current classification
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 liv-
ing, 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 [25]. If the evidence code is IDA, the property was directly
observed by one of the authors or an expert mentioned in the acknowledgements.

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Pati et al.
http://standardsingenomics.org 45
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the
basis of its phylogenetic position [26], and is part
of the Genomic Encyclopedia of Bacteria and Arc-
haea project [27]. The genome project is depo-
sited in the Genomes On Line Database [15] 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.
Table 2. Genome sequencing project information
MIGS ID Property
MIGS-31 Finishing quality
Term
Non-contiguous finished
Three genomic libraries: one 454 pyrosequence standard library,
one 454 PE library (10 kb insert size), one Illumina library
Illumina GAii, 454 GS FLX Titanium
290.0 × Illumina; 48.0 × pyrosequence
Newbler version 2.3, Velvet 0.7.63, phrap SPS 4.24
Prodigal 1.4, GenePRIMP
AFJE00000000 GL945015-GL945017
Genbank Date of Release June 20, 2011
GOLD ID Gi05358
NCBI project ID 41513
Database: IMG-GEBA 2503754046
Source material identifier DSM 17128
Project relevance Tree of Life, GEBA
MIGS-28 Libraries used
MIGS-29
MIGS-31.2
MIGS-30
MIGS-32





MIGS-13

Sequencing platforms
Sequencing coverage
Assemblers
Gene calling method
INSDC ID
Growth conditions and DNA isolation
P. multisaccharivorax PPPA20T, DSM 17128, was
grown anaerobically in DSMZ medium 104 (PYG-
medium) [28] at 37°C. DNA was isolated from 0.5-
1 g of cell paste using MasterPure Gram-positive
DNA purification kit (Epicentre MGP04100) fol-
lowing the standard protocol as recommended by
the manufacturer with modification st/DL for cell
lysis as described in Wu et al. 2009 [27]. DNA is
available through the DNA Bank Network [29].
Genome sequencing and assembly
The genome was sequenced using a combination
of Illumina and 454 sequencing platforms. All
general aspects of library construction and se-
quencing can be found at the JGI website [30]. Py-
rosequencing reads were assembled using the
Newbler assembler (Roche). The initial Newbler
assembly consisting of 154 contigs in five scaffolds
was converted into a phrap [31] assembly by mak-
ing fake reads from the consensus, to collect the
read pairs in the 454 paired end library. Illumina
GAii sequencing data (1,043.6 Mb) was assembled
with Velvet [32] and the consensus sequences
were shredded into 2.0 kb overlapped fake reads
and assembled together with the 454 data. The
454 draft assembly was based on 135.4 Mb 454
standard data and all of the 454 paired end data.
Newbler parameters are -consed -a 50 -l 350 -g -m
-ml 20. The Phred/Phrap/Consed software pack-
age [31] was used for sequence assembly and
quality assessment in the subsequent finishing
process. After the shotgun stage, reads were as-
sembled with parallel phrap (High Performance
Software, LLC). Possible mis-assemblies were cor-
rected with gapResolution [30], Dupfinisher [33],
or sequencing cloned bridging PCR fragments with
subcloning or transposon bombing (Epicentre
Biotechnologies, Madison, WI). Gaps between con-
tigs were closed by editing in Consed, by PCR and
by Bubble PCR primer walks (J.-F. Chang, unpub-
lished). A total of 218 additional reactions were
necessary to close gaps 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 de-
veloped at JGI [34].
The error rate of the completed genome sequence
is less than 1 in 100,000. Together, the combination
of the Illumina and 454 sequencing platforms pro-
vided 338 × coverage of the genome. The final as-
sembly contained 325,939 pyrosequence and
28,989,384 Illumina reads.

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Prevotella multisaccharivorax type strain (PPPA20T)
Genome annotation
Genes were identified using Prodigal [35] as part
of the Oak Ridge National Laboratory genome an-
notation pipeline, followed by a round of manual
curation using the JGI GenePRIMP pipeline [36].
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 was performed
within the Integrated Microbial Genomes - Expert
Review (IMG-ER) platform [37].

46 Standards in Genomic Sciences
Genome properties
The assembled genome sequence consists of three
non-contiguous contigs with a length of 3,334,154
bp, 47,474 bp and 7,016 bp with a G+C content of
48.3% (Figure 3 and Table 3). Of the 2,951 genes
predicted, 2,876 were protein-coding genes, and
75 RNAs; 166 pseudogenes were also identified.
The majority of the protein-coding genes (60.5%)
were assigned with a putative function while the
remaining ones were annotated as hypothetical
proteins. The distribution of genes into COGs func-
tional categories is presented in Table 4.

Figure 3. Graphical map of the largest scaffold. 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.

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Pati et al.
http://standardsingenomics.org 47
Table 3. Genome Statistics
Attribute
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Number of scaffolds
Total genes
RNA genes
rRNA operons
Protein-coding genes
Pseudo genes
Genes in paralog clusters
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
CRISPR repeats
Value % of Total
100.00%
87.66%
48.31%
3,388,644
2,970,483
1,636,375
3

2,951 100.00%
2.54% 75
4-6

2,876
166
438
1,659
1,864
782
588
97.46%
5.63%
14.84%
56.22%
63.17%
26.50%
19.93%
3


Table 4. Number of genes associated with the general COG functional categories
Code value %age Description
J 138 7.7 Translation, ribosomal structure and biogenesis
A 0 0.0 RNA processing and modification
K 102 5.7 Transcription
L 183 10.1 Replication, recombination and repair
B 0 0.0 Chromatin structure and dynamics
D 26 1.4 Cell cycle control, cell division, chromosome partitioning
Y 0 0.0 Nuclear structure
V 46 2.6 Defense mechanisms
T 63 3.5 Signal transduction mechanisms
M 155 8.6 Cell wall/membrane/envelope biogenesis
N 4 0.2 Cell motility
Z 0 0.0 Cytoskeleton
W 0 0.0 Extracellular structures
U 31 1.7 Intracellular trafficking, secretion, and vesicular transport
O 69 3.8 Posttranslational modification, protein turnover, chaperones
C 90 5.0 Energy production and conversion
G 145 8.0 Carbohydrate transport and metabolism
E 132 7.3 Amino acid transport and metabolism
F 59 3.3 Nucleotide transport and metabolism
H 74 4.1 Coenzyme transport and metabolism
I 56 3.1 Lipid transport and metabolism
P 120 6.7 Inorganic ion transport and metabolism
Q 27 1.5 Secondary metabolites biosynthesis, transport and catabolism
R 202 11.2 General function prediction only
S 82 4.6 Function unknown
- 1,292 43.8
Not in COGs

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Prevotella multisaccharivorax type strain (PPPA20T)
Acknowledgements
We would like to gratefully acknowledge the help of
Sabine Welnitz (DSMZ) for growing P. multisaccharivo-
rax cultures. This work was performed under the aus-
pices of the US Department of Energy Office of Science,
Biological and Environmental Research Program, and
by the University of California, Lawrence Berkeley Na-
tional Laboratory under contract No. DE-AC02-
48 Standards in Genomic Sciences
05CH11231, Lawrence Livermore National Laboratory
under Contract No. DE-AC52-07NA27344, and Los
Alamos National Laboratory under contract No. DE-
AC02-06NA25396, UT-Battelle and Oak Ridge National
Laboratory under contract DE-AC05-00OR22725, as
well as German Research Foundation (DFG) INST
599/1-2.
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