RESEARCH LETTER
Complete mitochondrial genome of compactin-producing
fungus Penicillium solitum and comparative analysis of
Trichocomaceae mitochondrial genomes
Michael A. Eldarov, Andrey V. Mardanov, Alexey V. Beletsky, Vakhtang V. Dzhavakhiya,
Nikolai V. Ravin & Konstantin G. Skryabin
Centre ‘Bioengineering,’ Russian Academy of Sciences, Moscow, Russia
Correspondence: Michael A. Eldarov,
Laboratory of Genetic Engineering, Centre
‘Bioengineering,’ Russian Academy of
Sciences, Prosp. 60-let Oktyabrya, bld. 7-1,
Moscow, 117312, Russia.
Tel.: +74991356219; fax: +74991350571;
e-mail: [email protected]
Received 23 October 2011; revised 8
December 2011; accepted 1 January 2012.
Final version published online 8 February
2012.
DOI: 10.1111/j.1574-6968.2012.02497.x
Abstract
We determined the complete mitochondrial genome sequence of the compactin-producing fungus Penicillium solitum strain 20-01. The 28 601-base pair
circular-mapping DNA molecule encodes a characteristic set of mitochondrial
proteins and RNA genes and is intron-free. All 46 protein- and RNA-encoding
genes are located on one strand and apparently transcribed in one direction.
Comparative analysis of this mtDNA and previously sequenced but unannotated mitochondrial genomes of several medically and industrially important species of the Aspergillus/Penicillium group revealed their extensive similarity in
terms of size, gene content and sequence, which is also reflected in the almost
perfect conservation of mitochondrial gene order in Penicillium and Aspergillus.
Phylogenetic analysis based on concatenated mitochondrial protein sequences
confirmed the monophyletic origin of Eurotiomycetes.
MICROBIOLOGY LETTERS
Editor: Michael Galperin
Keywords
mitochondrial DNA; filamentous fungi; statin
biosynthesis; gene order; sequence analysis;
genome evolution.
Introduction
Fungal mitochondrial genomics is a rapidly evolving field
initiated to a large extent by the efforts of organelle genome sequencing programs (Korab-Laskowska et al., 1998;
O’Brien et al., 2009) and fungal mitochondrial genome
project (Paquin et al., 1997). More than 80 fungal mitogenomes have been sequenced and analysed up to now,
providing invaluable information on mitochondrial genome organization, evolution, replication and expression,
while phylogenetic and taxonomic studies have also been
conducted in all major fungal lineages (Paquin & Lang,
1996; Kouvelis et al., 2004; Bullerwell & Lang, 2005;
Nosek et al., 2006; Juhasz et al., 2008; Lee & Young,
2009; Wu et al., 2009).
The standard approach for mitochondrial genome
sequencing involves isolation of mitochondria, library
construction and sequencing of individual clones, and
gap closure using PCR. This labour-intensive approach is
FEMS Microbiol Lett 329 (2012) 9–17
surpassed by next-generation sequencing technologies,
such as pyrosequencing (Margulies et al., 2005). The vast
amount of sequencing data generated by these platforms is
usually sufficient to provide several-fold coverage of
10–30-MB size fungal nuclear genomes and simultaneously
sequence mitochondrial genomes as ‘by-products’ of whole
genome shotgun (WGS) sequencing approach. Because of
their high copy number and topological independence,
mitochondrial (mt) genomes are readily assembled as
separate contigs, covered by multiple sequence reads.
This strategy has been successfully applied to sequence
Glomus mitochondrial genomes (Lee & Young, 2009),
Pichia farinosa mt genome (Jung et al., 2010) and, by us,
mitochondrial genomes of the diatom algae Synedra
acus (Ravin et al., 2010) and the methylotrophic yeast
Hansenula polymorpha (Eldarov et al., 2011). WGS does
not provide information on mtDNA topology in vivo
(circular versus linear) or the presence of alternative
mtDNA isoforms (Williamson, 2002; Valach et al., 2011),
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
10
but has a clear advantage of ease and speed, and also
the ability to identify important patterns of nuclearmitochondrial interactions, for example, the identification
of nuclear-mitochondrial DNA sequences resulting from
random insertions of mtDNA fragments into chromosomal regions (Hazkani-Covo et al., 2010) and the
requirements for the import of specific RNA and protein
molecules from the cytosol to the mitochondria, which is
important for RNA splicing and translation in mitochondria, involving mechanisms for speciation in fungi (Merz
& Westermann, 2009; Chou & Leu, 2010).
We used WGS to determine the complete mitochondrial genome of the compactin-producing fungus Penicillium solitum strain 20-01. Compactin is a well-known
statin that is converted by biotransformation into pravastain, the pharmaceutically active HMG-CoA reductase
inhibitor widely used to treat hyperlipidemia and other
cardiovascular disorders (Barrios-González & Miranda,
2010). Based on nuclear rRNA operon and mitochondrial
sequences, we previously confirmed the identification of
our strain 20-01 as a representative of P. solitum (Frisvad
& Samson, 2004), rather than another compactin-producing species, Penicillium citrinum (Endo et al., 1976).
Penicillium citrinum and P. solitum belong to the Penicillium genus of the Trichocomaceae family of Eurtotiales, an
order within the Pezizomycotina (filamentous fungi) subphylum of ascomycete fungi, which include many common
and well-known species of major ecological, medical and
commercial importance. The extreme metabolic and fermentative versatility of eurotialean fungi explains their role
in food spoilage, as well as in the food and pharmaceutical
industries as producers of various biopolymer-degrading
enzymes and medically active compounds.
Here, we describe the general organization of P. solitum
20-01 mtDNA, gene order and content and analyse its
phylogenetic relationships with other members of Pezizomyctotina.
To extend the comparative study of Trichocomaceae
mitochondrial genomes, we included the mitochondrial
genomes of several medically and industrially important
species in our analysis, namely the penicillin-producing
strain Penicillium chrysogenum (van den Berg et al.,
2008), the plant pathogenic fungus Penicillium digitatum
(Eckert & Eaks, 1989), the lovastatin-producing strain
Aspergillus terreus (Hajjaj et al., 2001), and Aspergillus
oryzae, used in the production of fermented foods in Chinese and Japanese cuisine (Machida et al., 2005). These
mitochondrial genomes are available as completely assembled and partially annotated or unannotated contigs generated from corresponding genome sequencing projects
and have not been analysed since then.
Our phylogenetic and gene order analysis confirmed
the current view of taxonomic positions of these strains
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M.A. Eldarov et al.
based on the analysis of nuclear-encoded genes, which
revealed conserved genus-specific patterns of mitochondrial genome architecture within Eurotiales and indicated
possible mechanisms of gene rearrangements involved in
the evolution of these genomes.
Materials and methods
Strains and DNA preparation
The compactin-producing strain P. solitum 20-01 was
obtained from the laboratory collection of Centre ‘Bioengineering’ RAS. Culture conditions and harvesting of
mycelia were as described before (Dzhavakhiya & Voinova,
2006). Mycelia were freeze-dried and ground to fine
powder. Total DNA was isolated using DNeasy Plant Mini
Kit (Qaigen) according to the manufacturer’s instructions.
Sequencing, assembly and annotation
Mitochondrial genome sequencing was performed using a
total genomic DNA sample without prior isolation of the
mitochondrial DNA. The genome was sequenced on a
Roche Genome Sequencer FLX using Titanium protocol
for a shotgun genome library. The GS FLX run resulted
in the generation of about 470 MB of sequences of an
average read length of 379 bp. The GS FLX reads were
assembled into contigs using the ‘GS de novo assembler’.
Sequence coverage was 229. A single 28 601-bp contig
was identified as representing the mtDNA on the basis of
extensive sequence similarity to known yeast mitochondrial genomes.
MFANNOT tool (http://megasun.bch.umontreal.ca/cgi-bin/
mfannot/mfannotInterface.pl) with default settings was
used for mitochondrial genome annotation, which was
manually adjusted by sequence alignment of deduced genes
with their intronless orthologs from related species. Putative proteins encoded by gene models with no similarity to
characterized genes were analysed by BLAST homology
search against NCBI protein database The codon frequency
was determined with CODONW (Peden, 2005) for concatenated ORFs for all protein-coding genes in the P. solitum
mitochondrial genome.
Genome contigs, corresponding to P. chrysogenum,
A. oryzae and A. terreus mitochondrial genomes, all contained ‘extra’ sequences that were actually duplications of
a region of rnL gene and adjacent tRNA gene cluster.
These ‘extra’ sequences were considered as assembly
artefacts and were manually deleted in the course of
annotation.
The complete mtDNA sequence of P. solitum 20-01
mtDNA is available in GenBank (JN696111, BioProject
ID: PRJNA72889).
FEMS Microbiol Lett 329 (2012) 9–17
11
Penicillium solitum mtDNA
Genome comparison and phylogenetic analysis
Whole genome DNA comparison was performed using
MEGABLAST (Altschul et al., 1997) against NCBI database
and mVISTA genome visualization and comparison tool
(Frazer et al., 2004). Genome visualization, search for
conserved sequence motifs and DNA repeats were performed with Vector NTI (Lu & Moriyama, 2004) and
Ugene (http://ugene.unipro.ru/).
For phylogenetic analysis, 14 mitochondrial proteins,
including subunits of the respiratory chain complexes
(cox1-cox3, cob), ATPase subunits (atp6, atp8 and atp9),
and seven NADH:quinone reductase subunits (nad1, nad2,
nad3, nad4, nad4L, nad5 and nad6), were concatenated
and aligned using the MUSCLE algorithm included in the
MEGA5 package (Tamura et al., 2011). The sequence data
for 25 filamentous fungi and yeast species with complete
mitochondrial genomes were used as follows: Arthroderma
obtusum (FJ385029), A. oryzae (AP007176), Aspergillus
tubingensis (DQ217399), A. terreus (AAJN01000268.1),
Candida orthopsilosis (NC_006972), Epidermophyton
floccosum (AY916130), Harpochytrium sp. (NC_004760),
Hypocrea jecorina (AF447590), Lecanicillium muscarium
(AF487277), Metarhizium anisopliae (AY884128), Arthroderma otae (FJ385030), Millerozyma farinosa (NC_013255),
P. solitum (JN696111), P. chrysogenum (AM920464),
P. digitatum (HQ622809), Penicillium marneffei (AY347307),
Phakopsora meibomiae (GQ338834), Pichia angusta
(NC_014805), Pneumocystis carinii (GU133622), Rhizopus
oryzae (NC_006836), Trichophyton mentagrophytes
(FJ385027), Trichophyton rubrum (FJ385026), Verticillium
dahliae (DQ351941), Yarrowia lipolytica (NC_002659).
Phylogenetic analysis was performed with maximum
likelihood (ML) and Bayesian methods. The Whelan and
Goldman + Freq. model was used to infer evolutionary
history using the ML algorithms provided in the MEGA5
package. The bootstrap consensus trees inferred from 100
replicates were taken to represent the evolutionary history
of the taxa analysed. Branches corresponding to partitions
reproduced in < 50% of bootstrap replicates were
collapsed. Initial trees for the heuristic search were
automatically obtained as follows. A discrete gamma distribution was used to model evolutionary rate differences
between sites (five categories (+G, parameter = 1.0399).
All positions that contained gaps or missing data were
eliminated. There were a total of 3414 sites in the final
data set.
Bayesian phylogenetic analysis was performed using
PhyloBayes with a CAT substitution model (Lartillot &
Philippe, 2004), discrete gamma distribution rate variation; trees were sampled every two of 2958 generations
and the first 500 trees were discarded as burn-in.
FEMS Microbiol Lett 329 (2012) 9–17
Results
Strain taxonomy, general features and gene
content
Statin-producing species are found in many fungal genera
(Chakravarti & Sahai, 2004). It is generally considered
that the industrial compactin-producing strain is P. citrinum. However, original papers describing the discovery
of this strain lack molecular taxonomic data (Endo et al.,
1976; Hosobuchi et al., 1993). Initial taxonomic evaluation of our strain was made based on nuclear rRNA gene
sequence, obtained as a separate contig in the course of
WGS sequencing (Genbank Acc# JN642222). A BLAST
search clearly demonstrated that the ITS-5, 8s-ITS2 region
of this sequence was identical to the corresponding
sequences of various P. solitum isolates and differed from
P. citrinum rDNA sequences. This observation was confirmed by multiple sequence alignment of the 1080-bp
region of the P. solitum 20-01 rDNA gene with selected
P. solitum and P. citrinum rDNA sequences (Supporting
Information, Fig. S1).
This taxonomic evaluation was also supported by comparison of mitochondrial cox1 and small subunit ribosomal RNA gene sequences (not shown). It is noteworthy
that the sequence of the compactin-producing gene cluster in our strain (not shown) was almost identical to the
published one (Abe et al., 2002).
The mitochondrial genome of the P. solitum 20-01
strain has a highly compact and rather simple genome
organization. It is a circular-mapping DNA molecule of
28 601 bp with a low GC content of 25%. It contains the
usual set of mitochondrial protein and RNA genes characteristic of the majority of sequenced filamentous fungi
mitochondrial genomes (Table S1). RNA-encoding genes
include 27 tRNA genes and genes for large and small
ribosomal RNA (rnS, rnL), as well as a predicted rnpB
gene encoding the subunit of mitochondrial RNase P
(mtP-RNA), known to be responsible for tRNA processing (Seif et al., 2003).
Protein-encoding genes include those for ATP-synthase
subunits 6, 8 and 9 (atp6, atp8 and atp9), subunits of
cytochrome oxidase (cox1, cox2 and cox3), apocytochrome
b (cob), one ribosomal protein (rps5) and NADH dehydrogenase subunits (nad1, nad2, nad3, nad4, nad4L, nad5
and nad6). Group I or group II introns, frequently interrupting yeast and filamentous fungi mitochondrial genes
(Lang et al., 2007), are not found. Two open reading
frames (ORFs) located between cox2 and tRNA-R, and
between tRNA-H and atp9 could encode for hypothetical
proteins without apparent homology to any known proteins in the GenBank database. All genes are located on
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
12
M.A. Eldarov et al.
Fig. 1. Penicillium solitum mtDNA gene map.
Genes and ORFs are shown as solid blocks.
The colour of the box designates the type of
gene: genes of the ATPase subunits (atp6,
atp8 and atp9) are green, genes of respiratory
chain complexes (cox1-cox3, cob) are red,
genes encoding subunits of NADH
dehydrogenase (nad1-6 and nad4L) are blue,
ORFs are yellow, the tRNA genes are black,
and the rRNA genes and rps3 are grey. Direct
repeats flanking the cox1-trnH gene pair
shown as arrows.
one strand and apparently transcribed in one direction
(Fig. 1).
To extend our analysis of mitochondrial genome
organization to other members of the Penicillium/Aspergillus
clade, we included mitochondrial genomes that have already
been sequenced in whole genome sequencing programs,
such as the mitochondrial genomes of P. chrysogenum,
A. terreus and A. oryzae. These genomes are available
from GenBank as partially annotated or unannotated
contigs. The general features of all compared genomes are
summarized in Table 1. It is evident that all compared
Penicillium and Aspergillus species possess conserved
features of mitochondrial genome organization, including
gene content. Genome size variation is low and is explained
by the length of intergenic regions and the presence of one
intron in the A. oryzae and P. digitatum mitochondrial
genomes.
Transfer RNAs, the genetic code and codon
usage
The majority of P. solitum mitochondrial tRNA genes are
organized into two dense gene clusters, a feature common
to many sequenced mitochondrial genomes of filamenª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
tous fungi. This set of 27 tRNA genes is sufficient to
decode all codons present in the predicted ORFs, alleviating the need for tRNA import into the mitochondria
from the cytoplasm (Kolesnikova et al., 2000), as is the
case for some yeast, plant and protist mitochondrial
genomes. The presence of tRNA-W (anticodon UCA)
recognizing the TGA codon, as well as the TGG codon, and
the absence of abnormal tRNA-T (anticodon CUN)
indicate that P. solitum mitochondrial protein-encoding
genes are translated according to genetic code 4 (Fox,
1987), as shown for other Pezizomycotina mitochondrial
genomes. All protein-encoding sequences start with the
ATG codon, except cox1, which starts with the codon TTG.
The frequency of codon usage, summarized in Table
S2, shows that all possible codons are used at least once,
except CTC and CGC for Leu and Arg, respectively. ATrich codons are much more abundant, reflecting the high
AT content of the P. solitum mitochondrial genome. Codons for amino acids with nonpolar side chains (Phe, Leu
and Ile) are very frequent, which is not surprising given
the hydrophobic nature of encoded proteins of respiratory membrane complexes. Among the 27 tRNA genes,
there are several isoacceptor tRNAs for glycine, arginine,
leucine, serine and isoleucine. The abundant ATA codons
FEMS Microbiol Lett 329 (2012) 9–17
13
Penicillium solitum mtDNA
Table 1. General features of Aspergillus and Penicillium mitochondrial genomes
Fungal species
Aspergillus oryzae
Aspergillus terreus
Penicillium solitum
Penicillium chrysogenum
Penicillium digitatum
Accession
Genome size, bp
GC (%)
Protein-coding regions (%)
rRNA-coding regions (%)
tRNA gene number
Intron number
‘ORFs’
Genome project
AP007176
29 202
26
56
20
26
1
2
PRJNA28175
AAJN01000268
24 729
27
52
23
26
0
0
PRJNA15631
JN696111
28 601
25
53
22
27
0
2
This study PRJNA72889
AM920464
28 874
25
53
26
27
0
1
PRJNA39879
HQ622809
28 978
25
50
17
27
1
3
PRJNA62571
for isoleucine are probably read by one of the three predicted tRNA-M following the cytosine to lysidine modification of the CAU anticodon, like in fungal, protist and
fission yeast mitochondrial genomes (Bullerwell et al.,
2003; Grayburn et al., 2004).
Phylogenetic analyses
Phylogenetic relationships among Eurotiales based on multigene comparison of nuclear-encoded genes are well established (Spatafora et al., 2006). Our phylogenetic analysis
based on concatenated mitochondrial protein sequences
confirmed the monophletic origin of Eurotiomycetidae and
the current view of the taxonomic position of Aspergilli
and Penicilli within Onygenales and related taxa (Geyser,
2006). Phylogenetic trees constructed using both ML and
Bayesian approaches were essentially congruent (Fig. 2 and
Fig. S4). Aspergillus and Penicillium species were divided
into two well-resolved clades with high support. Interestingly, the determined phylogenetic position of the pathogenic dimorphic fungus P. marneffei suggests that this
species is more distantly related to the studied members of
Trichocomaceae. The higher degree of divergence of mitochondrial protein sequences between P. marneffei and
other members of Trichocomaceae correlates with the difference of gene order in P. marneffei mitochondrial genome relative to the mitochondrial genomes of A. nidulans
and other Aspergillus and Penicillium mtDNAs described
here.
Altogether, these observations question the current taxonomic position of P. marneffei and suggest that this fungus
may represent a separate genus within Trichocomaceae, as
suggested earlier during nuclear genome comparisons (van
den Berg et al., 2008).
Fig. 2. Phylogenetic analysis of Penicilli and
Aspergilli based on mitochondrial protein
sequences. Phylogenetic tree was constructed
using Bayesian approach from multiple
sequence alignment of 14 concatenated
mtDNA encoded proteins. Numbers above the
nodes indicate posterior probabilities (%). The
tree is drawn to scale, with branch lengths
measured by the number of substitutions per
site.
FEMS Microbiol Lett 329 (2012) 9–17
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Published by Blackwell Publishing Ltd. All rights reserved
14
Genome comparison and gene order synteny
The extensive similarity of Aspergillus and Penicillium
mitochondrial genomes in terms of gene size, content
and sequence homology (Table 1) was also reflected in
the almost perfect conservation of mitochondrial gene
order in compared species. The genus-specific syntenic
regions cover whole genomes, include all main proteinand RNA-encoding genes and are only interrupted by
insertions of several ORFs with unknown functionality.
The very high degree of colinearity of Aspergillus and Penicillium genomes is also evident from the intergenera gene
order comparison (Fig. S2). The main architectural features, such as the presence of two clusters of tRNA genes
flanking the rnL gene and clusters of atp and nad genes
characteristic of syntenic patterns and specific to Pezizomycotina mitochondrial genomes, are present (Ghikas
et al., 2006). More detailed analysis of syntenic blocks
M.A. Eldarov et al.
showed that only three simple rearrangement steps were
required to interconvert gene order between the prototype Aspergillus and Penicillium mitochondrial genomes.
On the other hand, more extensive rearrangements are
required to build P. marneffei mitochondrial gene order
(Woo et al., 2003) from the most recent common ancestor
of the compared species. These data, together with phylogenetic analysis, justify the early separation of P. marneffei
from the most recent common ancestor of Penicillium and
Aspergillus species. Interestingly, the divergent cox1-trnH
gene pair, which is shuffled in Aspergillus and Penicillium
mitochondrial genomes, is flanked by two 100-bp direct
repeats in Penicillium mtDNA – a sign of a recent recombination event or a substrate for pop-out excision of an
intervening fragment (Fig. S3).
Graphical representation of variation among Penicillium and Aspergillus genomes was performed using mVISTA and P. solitum as a reference sequence (Fig. 3).
Fig. 3. Comparison of Penicillium solitum, Penicillium digitatum and Aspergillus oryzae mitochondrial genomes using mVISTA. Penicillium solitum
was used a reference sequence. Conserved nucleotide sequences are shown in grey, arrows indicate genes, black rectangles – repeats. The
height of the peaks indicates the per cent identity of sequences for a 100-bp moving window.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiol Lett 329 (2012) 9–17
Penicillium solitum mtDNA
Conserved syntenic regions were unambiguously visible,
while divergent regions mainly included intergenic spacers, rearranged genes and ORFs with unknown function.
Vista comparisons including the mitochondrial genome
of P. chrysogenum or A. oryzae gave similar results (data
not shown).
Discussion
Our comparative analysis of complete mitochondrial
genome of P. solitum strain 20-01 and other Aspergillus
and Penicillium mitogenomes have revealed several shared
specific features that confirm close phylogenetic relationships and recent evolutionary divergence of the two
genera. These features include extreme conservation of
gene composition and gene order in analysed genomes,
the very high degree of their colinearity and similarity of
coding sequences, compact genome organization, presence of syntenic genus-, family, class- and order-specific
gene blocks, identified before (see, for instance, Pantou
et al., 2008) including clustered tRNA genes. The tRNA
gene set is sufficient to decode all codons present in
protein-coding genes, includes additional isoacceptor
tRNAs and does not require import of missing tRNAs
from cytosol. Introns are rare and intergenic regions
occupy less genome space as compared to large mitogenomes of Neurospora crassa (~65 kb; http://www.broad.
mit.edu/cgi-bin/annotation/fungi/neurospora_crassa_7/down
load_license.cgi) or Podospora anserina (~100 kb, Cummings et al., 1990).
This pattern of mitochondrial genome organization is
likely to be beneficial for an efficient mitochondrial function and to support metabolic versatility of Trichocomacea
that include many industrially important species.
With more and more Trichocomaceae genome projects
close to completion (Nitsche et al., 2011), new mt genomic sequences of Aspergillus and Penicillium species are
likely to be available in near future that should aid in
more detailed understanding the mechanisms of mitochondrial genetic variation in these genera and their phylogenetic studies.
Acknowledgements
This work was supported by the Ministry of Education
and Sciences of Russia (contract 16.552.11.7035).
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Taxonomic evaluation of P. solitum strain 20-01.
Fig. S2. Gene order conservation in mt genomes of Penicillium and Aspergillus species.
Fig. S3. Homologous direct repeats flanking cox1-tRNAH gene pair in Penicillium mitochondrial genomes.
Fig. S4. Evolutionary relationships of P. solitum inferred
using the ML method.
Table S1. Gene content of P. solitum mitochondrial genome.
Table S2. Codon usage in P. solitum mitochondrial genome.
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