Journal of Biotechnology 136 (2008) 22–30
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Journal of Biotechnology
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Ultrafast pyrosequencing of Corynebacterium kroppenstedtii DSM44385
revealed insights into the physiology of a lipophilic corynebacterium
that lacks mycolic acids
Andreas Taucha,∗, Jessica Schneidera,b, Rafael Szczepanowskia, Alexandra Tilkerc,
Prisca Viehoeverd, Karl-Heinz Gartemanne, Walter Arnoldc, Jochen Blomb,
Karina Brinkrolfa,f, Iris Brunea, Susanne G¨ otkera, Bernd Weisshaard,
Alexander Goesmannb, Marcus Dr¨ ogeg, Alfred P¨ uhlerh
aInstitut f¨ ur Genomforschung und Systembiologie, Centrum f¨ ur Biotechnologie, Universit¨ at Bielefeld, Universit¨ atsstraße 25, D-33615 Bielefeld, Germany
bBioinformatics Resource Facility, Centrum f¨ ur Biotechnologie, Universit¨ at Bielefeld, Universit¨ atsstraße 25, D-33615 Bielefeld, Germany
cInstitut f¨ ur Innovationstransfer an der Universit¨ at Bielefeld GmbH, Universit¨ atsstraße 25, D-33615 Bielefeld, Germany
dLehrstuhl f¨ ur Genomforschung, Fakult¨ at f¨ ur Biologie, Universit¨ at Bielefeld, Universit¨ atsstraße 27, D-33615 Bielefeld, Germany
eLehrstuhl f¨ ur Gentechnologie und Mikrobiologie, Fakult¨ at f¨ ur Biologie, Universit¨ at Bielefeld, Universit¨ atsstraße 25, D-33615 Bielefeld, Germany
fInternational NRW Graduate School in Bioinformatics and Genome Research, Centrum f¨ ur Biotechnologie, Universit¨ at Bielefeld,
Universit¨ atsstraße 25, D-33615 Bielefeld, Germany
gRoche Applied Science, Nonnenwald 2, D-82372 Penzberg, Germany
hLehrstuhl f¨ ur Genetik, Fakult¨ at f¨ ur Biologie, Universit¨ at Bielefeld, Universit¨ atsstraße 25, D-33615 Bielefeld, Germany
a r t i c l ei n f o
Received 12 December 2007
Received in revised form 20 February 2008
Accepted 11 March 2008
Mycolic acid biosynthesis
a b s t r a c t
Corynebacterium kroppenstedtii is a lipophilic corynebacterial species that lacks in the cell envelope the
characteristic ?-alkyl-?-hydroxy long-chain fatty acids, designated mycolic acids. We report here the
bioinformatic analysis of genome data obtained by pyrosequencing of the type strain C. kroppenstedtii
DSM44385 that was initially isolated from human sputum. A single run with the Genome Sequencer
FLX system revealed 560,248 shotgun reads with 110,018,974 detected bases that were assembled into
a contiguous genomic sequence with a total size of 2,446,804bp. Automatic annotation of the complete
genome sequence resulted in the prediction of 2122 coding sequences, of which 29% were considered as
specific for C. kroppenstedtii when compared with predicted proteins from hitherto sequenced pathogenic
corynebacteria. This comparative content analysis of the genome data revealed a large repertoire of genes
involved in sugar uptake and central carbohydrate metabolism and the presence of the mevalonate route
for isoprenoid biosynthesis. The lack of mycolic acids and the lipophilic lifestyle of C. kroppenstedtii are
apparently caused by gene loss, including a condensase gene cluster, a mycolate reductase gene, and a
microbial type I fatty acid synthase gene. A complete ?-oxidation pathway involved in the degradation
of fatty acids is present in the genome. Evaluation of the genomic data indicated that lipophilism is the
dominant feature involved in pathogenicity of C. kroppenstedtii.
© 2008 Elsevier B.V. All rights reserved.
The species Corynebacterium kroppenstedtii is part of a long
isolated subline in the genus Corynebacterium (Collins et al.,
1998). Members of this species are phenotypically readily distin-
guished from most other corynebacteria in lacking the so-called
(coryno)mycolic acids, ?-alkyl-?-hydroxy long-chain fatty acids
with approximately 22–35 carbon atoms that are important struc-
tural components of the corynebacterial cell envelope (Daff´ e,
∗Corresponding author. Tel.: +49 521 106 5605; fax: +49 521 106 5626.
E-mail address: Andreas.Tauch@Genetik.Uni-Bielefeld.DE (A. Tauch).
2005). Moreover, C. kroppenstedtii is characterized by its lipophilic
(lipid-requiring) phenotype and is thus unable to grow on syn-
thetic media lacking lipid supplementation (Riegel et al., 2004).
The taxonomic description of C. kroppenstedtii was validly pub-
from a single clinical isolate (Collins et al., 1998). C. kroppenstedtii
is rarely recognized in human clinical samples, but it was recov-
ered from respiratory specimens as well as from breast tissue, pus
or deep wound swaps of patients with mastitis (Bernard et al.,
2002; Paviour et al., 2002). Pathological data revealed an associa-
disease (Taylor et al., 2003; Riegel et al., 2004; Kieffer et al., 2006).
The pathogenicity of C. kroppenstedtii might be associated with its
0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
A. Tauch et al. / Journal of Biotechnology 136 (2008) 22–30
ability to colonize fat globules, in which the organism can appar-
ently multiply irrespective of the neutrophil response (Paviour et
al., 2002; Taylor et al., 2003).
In this report, we present the bioinformatic evaluation of the
complete genome sequence from the type strain C. kroppenst-
edtii DSM44385 that was originally isolated from sputum of an
82-year-old female with pulmonary disease (Collins et al., 1998).
The genomic sequence data were obtained by pyrosequencing and
subsequently analyzed with the microbial genome annotation sys-
tem GenDB (Meyer et al., 2003). The functional annotation of
the genome sequence from C. kroppenstedtii DSM44385 provided
insights into the physiology of this unusual corynebacterium.
2. Materials and methods
2.1. Pyrosequencing of C. kroppenstedtii DSM44385
C. kroppenstedtii DSM44385 (Collins et al., 1998) was obtained
as a lyophilized culture from DSMZ (Braunschweig) and was rou-
tinely cultured at 37◦C in BYT complex medium (Tauch et al.,
2004) for 36–48h. Genomic DNA was purified from a 20ml cul-
ture, using a published protocol (Hopwood, 1985) with two minor
lysozyme at 37◦C for 60min. (ii) Cell lysis was achieved by adding
0.7ml of a 10% (w/v) sodium dodecyl sulfate solution and incubat-
DNA was used for the construction of the single-stranded template
DNA (sstDNA) library to be sequenced with the Genome Sequencer
FLX (Roche Applied Science). Preparation of the single-stranded
template DNA library and shotgun sequencing were carried out
as described by the manufacturer, using the following modifica-
tions of the protocol: (i) The DNA library selected for shotgun
sequencing was established without extrusion of small DNA frag-
ments by using the AMPure 60ml kit (Agentcourt Bioscience).
(ii) The DNA concentration was determined with the Agilent RNA
6000 Nano kit (Agilent Technologies). Assembly of the shotgun
reads was performed with the GS Assembler software (version
The assembled genomic contigs were linked by long-range PCR
assays, using the Phusion hot start high-fidelity DNA polymerase
(Finnzymes) and a TProfessional PCR thermocycler (Biometra). PCR
conditions were as follows: initial denaturation was carried out at
98◦C for 2min followed by 30s annealing at 64◦C, 210s elongation
at 72◦C, and 10s denaturation at 98◦C. This cycle was repeated 32
assays were sequenced by IIT Biotech (Bielefeld, Germany).
2.2. Bioinformatic analysis of the genome sequence
The complete genome sequencefrom
DSM44385 was uploaded into the GenDB database system
(Meyer et al., 2003) that guided an automated annotation process.
Gene prediction and bioinformatic characterization of the pre-
dicted proteins were performed as described previously (Tauch et
al., 2005). Calculation of orthologs among the proteins encoded
by pathogenic corynebacteria was based on bidirectional best
BLASTP hits (Altschul et al., 1997). Two proteins were considered
as orthologs if BLASTP matches with at least 30% sequence identity
and a minimum coverage of 50% of the query sequence length were
detected in both directions, and the orthologous pair of proteins
represented the best hits for the respective query sequence in the
target genomes. A gene was assigned to the core genome upon
detection of orthologous genes in the complete set of selected
corynebacterial genomes. Accordingly, genes were classified as
Overview of the Corynebacterium kroppenstedtii DSM44385 pyrosequencing project
Feature Project data obtained after
Number of GS FLX runs
Number of shotgun reads
Number of detected bases
Mean read length (bp)
Number of large contigs (>500bp)
Assembled bases of large contigs
Mean contig size (bp)
Number of small contigs (<500bp)
Assembled bases of small contig
Total assembled bases in project
singletons if none of the BLAST hits met the criteria defined above
in any of the other genomes.
The annotatedgenome sequence
DSM44385 was submitted to the EMBL database.
from C. kroppenstedtii
3. Results and discussion
3.1. Pyrosequencing of the C. kroppenstedtii genome resulted in
seven genomic contigs
The genome of the type strain C. kroppenstedtii DSM44385
(Collins et al., 1998) was investigated by an ultrafast pyrosequenc-
ing approach in combination with an almost fully automated
annotation process. The resulting data of the genome project are
summarized in Tables 1 and 2. Shotgun sequencing and assembly
of the reads revealed one small contig (349bp) and six large con-
sequence of the ribosomal RNA (rrn) operons of C. kroppenstedtii
(Fig. 1). The genomic sequence contigs were ordered into a circular
chromosome after manual inspection of the assembled data and by
Corynebacterium glutamicum ATCC 13032 (Kalinowski et al., 2003).
range PCR assays, thereby indicating a tandem duplication of the
small contig and the presence of three rrn copies in the genome of
C. kroppenstedtii DSM44385 (Fig. 1). The low number of genomic
contigs is remarkable when considering that repetitive sequences
such as IS elements and rrn operons are the main cause of gaps in
pyrosequencing projects due to the short length of the sequenc-
ing reads. This unexpected result suggested that the genome of
C. kroppenstedtii DSM44385 lacks any repetitive sequences with
the exception of the duplicated small contig and the rrn operons
(Fig. 1). This observation is consistent with the annotation of the
genome sequence, indicating the presence of only two transposase
DSM44385. The tnpF2 pseudogene is located adjacent to a short
integration of foreign DNA occurred at this site of the genome.
Features of the C. kroppenstedtii DSM44385 genome
FeatureData obtained after
sequencing and annotation
Genome size (bp)
Mean G+C content
Predicted coding sequences
Average gene length (bp)
Average length of intergenic regions (bp)
A. Tauch et al. / Journal of Biotechnology 136 (2008) 22–30
Fig. 1. Linear representation of the ordered genomic contigs obtained by pyrosequencing of Corynebacterium kroppenstedtii DSM44385. The genomic contigs are shown by
boxes; the length of large contigs (1–5) is indicated. Contigs containing rRNA operons are specifically labeled (rrn); the arrow indicates the direction of transcription of the
rRNA operon genes (16S–23S–5S). The tandem duplication of the small contig (349bp) is shown by green boxes. The size of sequence gaps is indicated between the contigs
(red boxes). The gap size varied between 0 and 58bp. The position of the dnaA gene, the origin of replication (oriC), and the replication termination region (dif) are included
as landmarks. Genomic regions amplified by long-range PCR are marked below the contigs. Note that the graph is not drawn to scale. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of the article.)
The high G+C region includes genes for a multicopper oxidase
(mco), a two-component system (tcsR4 and tcsS4), and a copper-
transporting P-type ATPase (copB) and revealed similarity to a DNA
segment of the corynebacterial plasmid pLEW279b (Williams et
al., 2006). The apparent absence of mobile DNA elements in the
genome of C. kroppenstedtii DSM44385 is in contrast to the gene
content of C. glutamicum ATCC 13032, C. glutamicum R, Corynebac-
terium jeikeium K411, and Corynebacterium urealyticum DSM7109
that are characterized by the presence of a diverse set of inser-
tion sequences and potential prophages (Kalinowski et al., 2003;
Yukawa et al., 2007; Tauch et al., 2005, 2006). However, a remain-
der of a putative prophage with a size of approx. 52kb is present
in C. kroppenstedtii DSM44385 (Fig. 2) and is flanked at the 3?junc-
tion by the integrase gene int9. In principle, the lack of a mycolic
acid bilayer at the cell surface (and thus of a physical barrier) and
the apparent lack of genes for typical DNA restriction-modification
systems in C. kroppenstedtii DSM44385 should favor the exchange
of genetic material by horizontal gene transfer, although C. krop-
penstedtii DSM44385 might otherwise have evolved an efficient
mechanism to prevent the incoming or integration of foreign DNA.
It is however conceivable that the transfer of foreign genes to
C. kroppenstedtii occurs only with low frequency in the hitherto
unknown habitat of this species. Since most antibiotic resistance
determinants known from pathogenic corynebacteria are located
stedtii to major clinically relevant antibiotics (Collins et al., 1998;
Riegel et al., 2004).
3.2. General architecture of the C. kroppenstedtii DSM44385
The genome of C. kroppenstedtii DSM44385 has a total size of
than the value determined during the taxonomic examination of
this strain (Collins et al., 1998). The size of the C. kroppenstedtii
DSM44385 chromosome is thus in the range of genomes known
from pathogenic corynebacteria (Cerde˜ no-T´ arraga et al., 2003;
Tauch et al., 2005, 2006). Plotting of the G/C skew [(G−C)/(G+C)]
with a sliding window revealed characteristic features of a bidirec-
tional replication mechanism, with an origin of replication (oriC)
sequence with similarity to actinobacterial replication termination
regions (dif) (Hendrickson and Lawrence, 2007) was detected at
about 1057kb of the chromosomal map, indicating different sizes
of the C. kroppenstedtii DSM44385 replichores (Fig. 2).
Synteny analysis by reciprocal best BLASTP matches revealed
a conserved order of orthologous genes between C. kroppenstedtii
DSM44385 and other pathogenic corynebacteria (Fig. 3). Nev-
ertheless, several breakpoints were detectable in the X-shaped
scatterplot, indicating the occurrence of genomic inversions dur-
ing the evolution of the C. kroppenstedtii DSM44385 chromosome
(Eisen et al., 2000). The presence of synteny breakpoints in the
chromosomal organization of C. kroppenstedtii is remarkable, since
moderate rearrangements in the corynebacterial genome architec-
ture were hitherto only detected in C. jeikeium and C. urealyticum
(Tauch et al., 2005, in press), whereas the genomes of C. glutam-
icum, Corynebacterium efficiens, and Corynebacterium diphtheriae
lack any detectable inversion within their chromosomal sequences
C. kroppenstedtii chromosome demonstrated that a reorganization
of the corynebacterial genome is not restricted to species of the
C. jeikeium/C. urealyticum subline. The initially detected stability of
corynebacterial genomes with respect to the order of orthologous
genes (Nakamura et al., 2003) may be limited to species belonging
to the main lineages within the genus Corynebacterium.
3.3. The protein content and regulatory repertoire of C.
Automatic annotation of the genome sequence with the GenDB
system identified 46 tRNA genes as well as 2122 protein-coding
regions that were classified into COG categories (Tatusov et al.,
2000). The deduced proteins of C. kroppenstedtii DSM44385 were
compared by reciprocal best BLASTP hits with the complete set
of predicted proteins encoded by other pathogenic corynebacte-
ria, comprising C. diphtheriae NCTC 13129, C. jeikeium K411, and C.
urealyticum DSM7109 (Fig. 2). This comparative content analysis at
the protein level revealed a core genome of 1016 genes and 614
genes from C. kroppenstedtii DSM44385 (29%) that were consid-
A. Tauch et al. / Journal of Biotechnology 136 (2008) 22–30
Fig. 2. Circular representations of the C. kroppenstedtii DSM44385 genome. The circles represent from the outside in: circle 1, DNA base position; circles 2 and 3, predicted
coding sequences transcribed clockwise and anticlockwise; circles 4–6, genes encoding orthologous proteins in C. jeikeium K411 (red), C. urealyticum DSM7109 (blue), and C.
diphtheriae NCTC 13129 (green); circle 7, G/C skew [(G−C)/(G+C)] plotted using a 10-kb window (blue, positive skew; green, negative skew); circle 8, G+C content plotted
are color coded according to the functional classification by COG categories (Tatusov et al., 2000). A putative prophage region is located at around 1900kb of the chromosomal
map. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
ered as specific (singletons) for this species (data not shown). The
size of the core genome and the number of singletons is similar to
values deduced from a recent comparative study of corynebacte-
rial genome sequences, indicating a core set of 835 genes among
pathogenic and non-pathogenic corynebacteria and 160–502 spe-
cific genes for each individual species (Yukawa et al., 2007). The
characteristic features of C. kroppenstedtii DSM44385 are not only
based on the presence of singletons, since gene loss played also
an important role in defining the metabolism of this species as
Moreover, the transcriptional regulatory repertoire of C. krop-
penstedtii DSM44385 was deduced from the annotated genome
sequence and functionally compared with the predicted regula-
tory proteins from sequenced corynebacteria (Brune et al., 2005;
Brinkrolf et al., 2007). This data mining approach revealed 43
DNA-binding transcriptional regulators that were grouped into
20 distinct regulatory protein families (Fig. 4). The C. kroppenst-
edtii DSM44385 genome codes for nine additional transcriptional
regulators, seven response regulators, and seven sigma factors,
resulting in a total number of 66 proteins potentially involved
in transcriptional regulation (Fig. 4). This number of transcrip-
tional regulators represents 3.1% of the predicted proteins from C.
kroppenstedtii DSM44385 and is in the range known from other
pathogenic corynebacteria (Brinkrolf et al., 2007). It is notewor-
thy that the set of detected DNA-binding transcriptional regulators
includes only 24 out of the 25 proteins that so far constituted
the core of transcriptional regulators in corynebacteria, since C.
kroppenstedtii DSM44385 lacks a gene for an ortholog of the con-
served GntR regulator Cg3261 (Brinkrolf et al., 2007; Gao et al.,
2007). Another prominent regulatory gene missing in C. kroppenst-
A. Tauch et al. / Journal of Biotechnology 136 (2008) 22–30
Fig. 3. Synteny between the genomic organization of C. kroppenstedtii DSM44385 and the complete genomes of Corynebacterium diphtheriae NCTC 13129, Corynebacterium
jeikeium K411, and Corynebacterium urealyticum DSM7109. The X–Y plots show syntenic regions between the corynebacterial genomes. Each dot represents a predicted C.
kroppenstedtii DSM44385 protein having an ortholog in another corynebacterial genome. The co-ordinates correspond to the position of the respective coding region in each
genome. Orthologous proteins were detected by reciprocal best matches with BLASTP (Altschul et al., 1997). Color code: C. diphtheriae NCTC 13129, green dots; C. jeikeium
K411, red; C. urealyticum DSM7109, blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
edtii DSM44385 is amtR encoding the master regulator of nitrogen
the genomes of C. jeikeium and C. urealyticum, indicating different
strategies among corynebacteria to control nitrogen metabolism
(Brune et al., 2005; Brinkrolf et al., 2007; Walter et al., 2007).
3.4. Metabolic features of the C. kroppenstedtii DSM44385
The taxonomic description of C. kroppenstedtii DSM44385
already indicated that this species is able to utilize some sugars as
carbon and energy sources, such as glucose, sucrose, and maltose
(Collins et al., 1998). Annotation of the genome sequence revealed
a much larger potential for the utilization of carbohydrates (Fig. 5).
Nine genes encoding components of the bacterial phosphotrans-
ferase system (PTS) (Barabote and Saier, 2005) were identified in
the genome of C. kroppenstedtii DSM44385, including genes for
the general energy-coupling proteins (ptsH and ptsI) and sugar-
specific permeases (EII proteins) for glucose (ptsG), fructose (ptsF),
sucrose (ptsS), and ?-glucosides (bglF). Three further enzyme II
genes (manP, nagE, and bglP) encoding proteins of the fructose,
glucose and glucoside (sub)families of PTS permeases (Barabote
Fig. 4. The transcriptional regulatory repertoire of C. kroppenstedtii DSM44385. The classification of predicted transcriptional regulators into families of regulatory proteins
A. Tauch et al. / Journal of Biotechnology 136 (2008) 22–30
Fig. 5. Reconstruction of pathways involved in carbohydrate uptake and metabolism of C. kroppenstedtii DSM44385. The metabolic reconstruction was supported by the
CellDesigner software (Funahashi et al., 2007). Genes encoding transporters or enzymes involved in carbohydrate uptake and metabolism are indicated. Key metabolites are
shown by circles. Abbreviations: AAcetyl-CoA, acetoacetyl-CoA; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; DMAPP, dimethylallyl diphosphate; Ery-4P,
erythrose-4-phosphate; FattyA-CoA, fatty acyl-CoA; Fru-1P, fructose-1-phosphate; Fru-1,6PP, fructose-1,6-bisphosphate; Fru-6P, fructose-6-phosphate; Gal-1P, galactose-1-
6-phosphate; Glu-6P, glucosamine-6-phosphate; GPDE, glycerophosphodiester; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; IPP, isopentenyl diphosphate; ManNAc, N-
NeuNAc(t), terminal N-acetyl-neuraminic acid; P-Mev, phosphomevalonate; PPE, phosphoenolpyruvate; PP-Mev, diphosphomevalonate; P-?-Glcs, phospho-?-glycoside;
6P-Gct, 6-phosphogluconate; 6P-Gll, 6-phosphogluconolactone; 6P-?-Glcs, 6-phospho-?-glycoside; ?-Keto-G, ?-ketoglutarate.
A. Tauch et al. / Journal of Biotechnology 136 (2008) 22–30
and Saier, 2005) were also detected (Fig. 5). The genome of C.
kroppenstedtii DSM44385 encodes moreover transporters for lac-
tate (lldT), gluconate (gntP), glycerol (glpF), glycerol-3-phosphate
(glpT and ugpAEBC), and N-acetyl-neuraminic acid (nanT). The bio-
conversion of the respective carbohydrates into intermediates of
the glycolysis is ensured by appropriate gene content (Fig. 5),
including for instance 6-phospho-?-glucosidase (bglA), sucrose-
6-phosphate hydrolase (scrB), mannose-6-phosphate isomerase
(manA), l-lactate dehydrogenase (lldD), gluconate kinase (gntK),
glycerol kinase (glpK), and glycerol-3-phosphate dehydrogenase
(glpD). N-Acetyl-neuraminic acid is converted into fructose-6-
phosphate through the N-acetyl-d-mannosamine route (nanLKE
and nagAB). Likewise, galactose (galTKEU), glycerate (glrK), and
dihydroxyacetone (dhaKLM) can be converted into glycolysis path-
way components (Fig. 5).
Further analysis of the central metabolism in C. kroppenstedtii
DSM44385 revealed a complete set of genes involved the pentose
phosphate pathway (Fig. 5). Gluconeogenesis occurs via phospho-
(pfkB), whereas anaplerosis is accomplished by pyruvate carboxy-
lase (pyc). The set of enzymes involved in additional reactions
around pyruvate includes pyruvate:quinone oxidoreductase (pqo),
d-lactate dehydrogenase (ldh), malic enzyme (mez), and a puta-
cycle of C. kroppenstedtii DSM44385 is incomplete and lacks the
supporting the view that a variant of the TCA cycle exists in
pathogenic corynebacteria (Cerde˜ no-T´ arraga et al., 2003; Tauch et
al., 2005). The gap in the TCA cycle might be closed by using a
putative succinyl-CoA:CoA transferase (cat1) that is homologous
to the experimentally characterized Cat1 protein from Clostridium
kluyveri (Cordwell, 1999).
A remarkable feature of the C. kroppenstedtii DSM44385
metabolism is the presence of the mevalonate pathway for
the biosynthesis of isoprene units instead of the 2-C-methyl-d-
erythritol 4-phosphate pathway (Kuzuyama, 2002). The respective
CoA to isopentenyl diphosphate and dimethylallyl diphosphate
(Fig. 5). Moreover, the genome of C. kroppenstedtii DSM44385 is
equipped with genes for biotin biosynthesis (Streit and Entcheva,
2003), including the bioABD genes and a bioF gene that may code
for a bifunctional protein involved in the generation of pimeloyl-
CoA and 8-amino-7-oxopelargonic acid. Genes for a biotin uptake
system (bioYMN) (Hebbeln et al., 2007) were also detected in the
3.5. The genome of C. kroppenstedtii lacks key genes involved in
mycolic acid biosynthesis
Currently, C. kroppenstedtii, Corynebacterium amycolatum, and
Corynebacterium atypicum, all recovered from human clinical
sources, are the only recognized species of the genus Corynebac-
terium that lack mycolic acids (Collins et al., 1988, 1998; Hall et al.,
2003). The key step in mycolic acid biosynthesis is catalyzed by a
condensase that is encoded by the Cg-pks gene in C. glutamicum.
This gene is flanked by two coding regions predicted to encode an
acyl-CoA synthetase (Cg-fadD) and an acyl-CoA carboxylase sub-
unit (Cg-accD3) that are involved in activation reactions (Gande et
al., 2004; Portevin et al., 2004, 2005). The respective gene region is
ing at the respective position in the C. kroppenstedtii DSM44385
genome. In addition, no cmrA gene for a Corynebacterineae myco-
sequence from C. kroppenstedtii DSM44385, providing evidence
that key parts for the biosynthesis of mycolic acids are absent in C.
kroppenstedtii thereby causing the unusual feature of mycolic acid
deficiency. On the other hand, genes encoding subunits of a puta-
tive carboxylase involved in mycolic acid synthesis (accBC, accD,
and genes for mycolyltransferases (cmtABC). The latter enzymes
can transfer a mycoloyl residue on the cell arabinogalactan and
from trehalose monomycolate to another molecule of trehalose
monomycolate to yield trehalose dimycolate (Daff´ e, 2005). This
gene composition makes C. kroppenstedtii an ideal candidate for
the detailed analysis of distinct steps in mycolic acid metabolism
and the development of the corynebacterial cell envelope.
3.6. The repertoire of genes involved in fatty acid utilization by C.
C. kroppenstedtii is a lipophilic species whose growth depends
on the supplementation of synthetic media with appropriate lipids
(Riegel et al., 2004). The lipophilic phenotype of C. kroppenstedtii is
apparently caused by the absence of a gene for a microbial type I
fatty acid synthase, the key enzyme for de novo fatty acid synthesis
(Schweizer and Hofmann, 2004). The genome sequence of C. krop-
penstedtii DSM44385 codes for two secreted lipases (lipA and lipE)
for growth. The predicted LipE protein is a member of the SGNH-
hydrolase superfamily of lipolytic enzymes (Akoh et al., 2004),
whereas the LipA gene product belongs to the class 2 of lipases
(Finn et al., 2008). The latter protein family consists of enzymes
that hydrolyze ester bonds in triacylglycerol giving di- or monoa-
cylglycerol, glycerol, and free fatty acids. Moreover, the deacylation
of phospholipids by C. kroppenstedtii DSM44385 may result in the
formation of free fatty acids and glycerophosphodiesters that can
be hydrolyzed by glycerophosphodiester phosphodiesterase (glpQ)
into glycerol-3-phosphate and the corresponding alcohol (Patton-
Vogt, 2007). The physiological role of the detected uptake systems
for glycerol (glpF) and glycerol-3-phosphate (glpT and ugpAEBC) is
thus linked to the lipophilic phenotype of C. kroppenstedtii and the
acquisition of free fatty acids for growth. The released molecules of
glycerol and glycerol-3-phosphate can be utilized either as precur-
sors for the synthesis of new phospholipids or as carbon sources
for glycolysis (Fig. 5).
Annotation of the genome sequence revealed several genes
that are apparently involved in the activation of exogenous fatty
acids and their degradation by the ?-oxidation pathway (Black
and DiRusso, 2003; Hiltunen and Qin, 2000). This set of coding
sequences includes: (i) five fadD-like genes encoding fatty acyl-
CoA synthetases involved in the activation of fatty acids by forming
acyl-CoA esters; (ii) four fadE-like genes encoding long-chain and
short-chain acyl-CoA dehydrogenases; (iii) two fadB-like genes
encoding enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase
activities; and (iv) three fadA-like genes encoding 3-ketoacyl-CoA
genome lacks genes for auxillary enzymes that generally link
the degradation of structurally modified acyl-CoA esters with the
?-oxidation pathway (Hiltunen and Qin, 2000) with the excep-
tion of four echA-like genes coding for proteins of the enoyl-CoA
hydratase/isomerase family. The small repertoire of genes involved
ently reflects the different role of fatty acid substrates for the
bacterial metabolism when compared with the large repertoires
detected in the genomes of the lipophilic species C. jeikeium and
C. urealyticum. In these corynebacteria, exogenous fatty acids are
not only necessary to supplement the fatty acid auxotrophy, but
they are also required as carbon and energy sources and as build-
A. Tauch et al. / Journal of Biotechnology 136 (2008) 22–30
ing blocks for the biosynthesis of corynomycolic acids (Tauch et
al., 2005, in press). Since C. kroppenstedtii is able to utilize a large
variety of sugars as carbon and energy sources and additionally
lacks corynomycolic acids in the cell envelope, lower amounts of
exogenous fatty acids might be required for growth of this species.
This different type of lipophilism may represent a key feature for C.
kroppenstedtii to colonize a wider range of habitats on the human
body than C. jeikeium and C. urealyticum, which are predominantly
A pyrosequencing strategy in conjunction with an established
genome annotation pipeline provided rapid access to the genetic
information encoded by the C. kroppenstedtii DSM44385 genome.
The most prominent feature of this species from the taxonomical
viewpoint is the lack of corynomycolic acids (Collins et al., 1998)
that is apparently caused by the loss of a condensase gene cluster
and a mycolate reductase gene, whereas the lipophilic phenotype
is due to the absence of a microbial type I fatty acid synthase
gene. The loss of the latter gene resulted in the observed require-
ment for exogenous fatty acids for cellular growth (Riegel et al.,
2004). Although the habitat of C. kroppenstedtii on the human body
is currently unknown, colonization may be limited to body sites
that provide sufficient amounts of exogenous fatty acids. Since C.
kroppenstedtii is able to utilize a diverse range of sugars as carbon
and energy sources, its habitat might not be restricted to moist
and oily regions of the human skin that are frequently colonized
by lipophilic corynebacteria (McGinley et al., 1985). According to
the functional annotation of the genome sequence, C. kroppenst-
edtii DSM44385 lacks typical virulence factors with the exception
of a neuraminidase gene (nanI), which is consistent with the low
pathogenic potential of this species (Riegel et al., 2004). The neu-
raminidase might be involved in pathogenicity of C. kroppenstedtii
DSM44385 by cleaving terminal sialic acid residues from surface
host (Jedrzejas, 2001; Vimr and Lichtensteiger, 2002). This obser-
vation raises the question which metabolic or structural features of
the C. kroppenstedtii cell are moreover involved in virulence, since
infection by C. kroppenstedtii is apparently associated with inflam-
et al., 2006). The ability of C. kroppenstedtii to multiply in fat glob-
providing access to exogenous fatty acids for growth and thereby
escaping the neutrophil response (Paviour et al., 2002; Taylor et al.,
2003). The persistence of the bacterium in fat globules also lim-
its the use of antimicrobials for the treatment of C. kroppenstedtii
infections, since only the lipophilic antibiotic doxycycline turned
out to be effective in vivo (Skinner et al., 2007).
The authors thank Inga B¨ oll (Roche Applied Science) for expert
training of the sequencing team and Burkhard Linke for preparing
the EMBL file of the genome sequence.
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