JOURNAL OF BACTERIOLOGY, Feb. 2009, p. 1180–1190 Vol. 191, No. 40021-9193/09/$08.00�0 doi:10.1128/JB.01058-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Complete Genome Sequence of Macrococcus caseolyticus StrainJSCS5402, Reflecting the Ancestral Genome of the

Human-Pathogenic Staphylococci�

Tadashi Baba,1* Kyoko Kuwahara-Arai,1 Ikuo Uchiyama,2 Fumihiko Takeuchi,3Teruyo Ito,1 and Keiichi Hiramatsu1

Department of Microbiology and Infection Control Science, Juntendo University, 2-1-1 Hongo, Bunkyo, Tokyo 113-8421, Japan1;National Institute for Basic Biology, National Institutes of Natural Sciences, Nishigonaka 38, Myodaiji, Okazaki 444-8585,

Japan2; and Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom3

Received 30 July 2008/Accepted 3 December 2008

We isolated the methicillin-resistant Macrococcus caseolyticus strain JCSC5402 from animal meat in asupermarket and determined its whole-genome nucleotide sequence. This is the first report on the genomeanalysis of a macrococcal species that is evolutionarily closely related to the human pathogens Staphylo-coccus aureus and Bacillus anthracis. The essential biological pathways of M. caseolyticus are similar tothose of staphylococci. However, the species has a small chromosome (2.1 MB) and lacks many sugar andamino acid metabolism pathways and a plethora of virulence genes that are present in S. aureus. On theother hand, M. caseolyticus possesses a series of oxidative phosphorylation machineries that are closelyrelated to those in the family Bacillaceae. We also discovered a probable primordial form of a Macrococcusmethicillin resistance gene complex, mecIRAm, on one of the eight plasmids harbored by the M. caseolyticusstrain. This is the first finding of a plasmid-encoding methicillin resistance gene. Macrococcus is consid-ered to reflect the genome of ancestral bacteria before the speciation of staphylococcal species and may beclosely associated with the origin of the methicillin resistance gene complex of the notorious humanpathogen methicillin-resistant S. aureus.

Among various bacterial genera, Macrococcus is the mostclosely related to the genus Staphylococcus. Historically, it hadbeen included in the staphylococcal family until it was reas-signed to an independent genus because of its distinctivelysmaller genome size than that of staphylococci (19). Currently,seven species are included in genus Macrococcus: Macrococcusbovicus, M. carouselicus, M. caseolyticus, M. equipercicus, M.brunensis, M. hajekii, and M. lamae (26). Unlike staphylococcalspecies, macrococci do not cause human or animal diseasesand are typically isolated from animal skin and food such asmilk and meat. The physiological features of this organism are,however, largely unknown, and only a small number of reportson macrococci have been published.

M. caseolyticus, previously classified as Staphylococcus caseo-lyticus (34), was reportedly isolated from cow’s milk, bovineorgans and food-processing factories. Phylogenetic relation-ship analysis based on 16S rRNA sequences revealed that, inaddition to Staphylococcus, Bacillus species are also closelyrelated to M. caseolyticus. The morphology of this organism isglobular; however, the cell size is larger than for staphylococci.

Since the genomes of macrococci are much smaller thanthose of staphylococci (34), we considered that knowledge ofthe macrococcal genome would be important in elucidating theevolution of staphylococci along with its acquisition of patho-genic potential to humans. We describe here a unique meta-

bolic feature of M. caseolyticus as inscribed in its genome andthe discovery of a primordial form of a mecA gene complex, thecausative genetic determinant of the notorious hospital patho-gen methicillin-resistant S. aureus (MRSA).

MATERIALS AND METHODS

Isolation and characterization of M. caseolyticus strain JCSC5402. The M.caseolyticus strain sequenced in the present study was initially isolated from askin swab of a domestic chicken during our pursuit of animal-borne antibiotic-resistant bacteria. One of the colonies grown on a mannitol-salt agar plate in thepresence of 10 mg of ceftizoxime/liter was subjected to analysis using a Staphyo-gram kit (Wako, Tokyo, Japan), which is designed to identify staphylococcalspecies by providing a colorimetric matrix of species-specific biochemical path-ways by using chromogenic indicator reagents. The strain isolated was deter-mined to be M. caseolyticus, and we confirmed this by determination of the 16Sribosomal DNA sequence amplified from the genomic DNA of the strain usingthe primers described in a previous report (36). The speciation was verified bysearching its matching sequence in the Ribosomal Database Project II (http://rdp.cme.msu.edu/).

Shotgun sequencing and contig assembly. The whole-genome sequencing wasperformed basically as described previously (1, 2, 40) with slight modifications.Briefly, total DNA was fragmented to 2 and 6 kb in length to be cloned intopUC118 plasmid vector for the construction of a genomic library, followed bysequencing. Gaps unable to be sequenced were closed with primer walking byreading 30 to 40 kb of the total DNA library cloned into a fosmid vectorpCC1FOS (Epicentre Biotechnologies, Madison, WI). The sequencing was car-ried out by Hitachi High-Tech Fielding Co. (Tokyo, Japan) and Takara Bio, Inc.(Otsu, Japan). The total length of the shotgun sequence read was �13 Mbp, orthe redundancy was 6 since the genome size was found to be 2.1 Mbp. Whole-genome sequence was assembled from each shotgun sequence read by usingArachne 2 (15). The accuracy of the assembly was confirmed by a physicalmapping using visualization of restriction digestion pattern of the chromosomalDNA (OpGen, Madison, WI). We have deposited the whole-genome sequenceof M. caseolyticus strain JCSC5402 to the DNA Database of Japan, with acces-sion numbers AP009484 (chromosome) and AP009485 through AP009492 (plas-mids pMCCL1 to 8), respectively.

* Corresponding author. Mailing address: Department of Microbi-ology and Infection Control Science, Juntendo University, 2-1-1Hongo, Bunkyo, Tokyo 113-8421, Japan. Phone: 81-3 580 21041. Fax:81-3-5684-7830. E-mail: tbaba@juntendo.ac.jp.

� Published ahead of print on 12 December 2008.

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Determination of ORFs, structural RNA, insertion sequences, phages, andplasmids. Determination of open reading frames (ORFs), structural RNAs, andannotations were performed as described previously (2). Briefly, ORF candidateswere initially extracted with the genome analysis-oriented In Silico molecularcloning software (v1.5; Insilico Biology, Yokohama, Japan), followed by individ-ual inspection of each ORF candidate by manual searching for a start codon onthe basis of ribosome-binding motifs, and then the ORFs coding for proteinswere determined. tRNA and tmRNA genes were identified by usingtRNAscan-SE (24) and by a web-based software (http://www.indiana.edu/�tmrna/), respectively. An illustration of the GC contents on a genome mapand the G-C skew [(G � C)/(G � C)] were drawn by using In Silico molecularcloning software.

Insertion sequences were also found by the web-based software IS Finder(http://www-is.biotoul.fr/is.html). Insertion of prophage sequences was identifiedby the presence of typical phage gene structures, including genes for phageintegrases, terminases, capsid, tail, and phage-related amidases (4). Plasmidswere recognized since the sequence redundancy increased more rapidly thanother regions upon shotgun sequencing, and their sequences were circularizedwith relatively small numbers of the shotgun reads upon assembly. Plasmids werealso recognizable by sequences with genes for plasmid replication proteins.

Cloning of mecAm gene from M. caseolyticus JCSC5402 plasmid pMCCL2.Whole genome was isolated from M. caseolyticus JCSC5402 as described previ-ously (2). By using this as a template, PCR was performed with the primers5�-CCCGGATCCAGTACAATAACCCAAGGAA-3� and 5�-CCCGGATCCGGGAACAATAGTTCCTCATT-3�, which allow amplification of mecAm struc-tural gene and its promoter region. After digesting the PCR product with arestriction endonuclease BamHI, the Macrococcus mecA (mecAm)-containingfragment was cloned into the corresponding site of E. coli-S. aureus shuttle vectorpYT3 (20), yielding pYMMA.

Elimination of pMCCL2 from M. caseolyticus JCSC5402. A 10�6 dilution of anovernight culture of M. caseolyticus JCSC5402 in antibiotic-free tryptic soy brothmedium was grown overnight again and repeated this for six times. A 0.1-mlportion of the final 10�6-diluted culture was then plated onto drug-free trypticsoy agar. A few hundred of the grown colonies were replica plated onto bothdrug-free and oxacillin-containing (10 �g/ml) tryptic soy agar plates, followed bythe isolation of oxacillin-susceptible colonies. A �-lactamase test using nitro-cephin was performed, and negative colonies were tested to determine whetherthey possessed pMCCL2 by treatment of the cells with S1 nuclease followed byanalysis by pulsed-field gel electrophoresis (5) compared to native JCSC5402.Loss of pMCCL2 was also confirmed by pulsed-field gel electrophoresis-basedSouthern hybridization using the PCR-amplified mecAm gene fragment as aprobe.

Genomes used for comparative analysis and chromosome structure. Genomesequences used for comparative analysis and chromosomal structure analysis arelisted in Table 1.

Identification of biochemical pathways in M. caseolyticus strain JCSC5402.Product names of the identified genes in M. caseolyticus strain JCSC5402 werereferred to biochemical pathways available in KEGG (Kyoto Encyclopedia ofGenes and Genomes) databases (16) (http://www.genome.jp/kegg/). Functionalclassification of ORFs was based on previous work on Bacillus subtilis genomeproject (21).

Chromosomal structure analysis, phylogenetic analysis, and the tree diagramdisplay. Orthologous genes among various species were aligned by using aclustering algorithm named DomClust (42, 43), which is based on hierarchicalclustering procedure for constructing orthologous groups at a domain level, andthen constructed an alignment of syntenically conserved regions designated“core structure,” using the CoreAligner program (44). This procedure allowsidentifying the order of orthologous groups that retains the conserved geneorders to the greatest possible extent in each genome. Among the core genesextracted by the CoreAligner, those that were conserved in all of the testedgenomes in one-to-one correspondence were extracted (297 genes). Multiplesequence alignments were generated by CLUSTAL W program (41). From thesealignments, the conserved aligned blocks suitable for phylogenetic analysis werethen extracted by Gblocks program (7). The resulting sequences were concate-nated and were subjected to the CLUSTAL W program to construct a phyloge-netic tree using the neighbor-joining method (33). The phylogenetic tree wasdrawn using the FigTree program (http://tree.bio.ed.ac.uk/software/figtree/).

RESULTS

M. caseolyticus genome comparison with other organisms.After closing the sequence gaps, the length of the determinedchromosome was found to be 2,102,324 bp, and 1,957 ORFswere assigned (Table 2). Four ribosomal DNA clusters werefound in the genome, which are smaller in number than staph-ylococci carrying five or six clusters, or B. subtilis with tenribosomal gene clusters. The GC contents of M. caseolyticusare 36.9%, a value that is between those of S. aureus and B.subtilis (Table 2).

Figure 1 is a circular representation of M. caseolyticus chro-mosomal DNA. The ORFs unique to the M. caseolyticus chro-

TABLE 1. Chromosomal sequences of various species used in this study

Species Strain GenBankaccession no.

Source orreference

Macrococcus caseolyticus JCSC5402Chromosome AP009484 This studyPlasmid pMCCL1-8 AP009485-92 This study

Staphylococcus aureus MW2 BA000033 2Staphylococcus epidermidis ATCC 12228 AE015929 45Staphylococcus haemolyticus JCSC1435 AP006716 40Staphylococcus saprophyticus ATCC 15305 AP008934 23Bacillus subtilis 168 AL009126 21Bacillus anthracis Ames AE016879 29Bacillus thuringiensis serovar Konkukian 97-27 AE017355 11Bacillus cereus ATCC 14579 AE016877 14Bacillus licheniformis ATCC 14580 CP000002 32Bacillus clausii KSM-K16 AP006627 Y. Takaki et al.,

unpublishedBacillus halodurans C-125 BA000004 37Geobacillus kaustophilus HTA426 BA000043 39Geobacillus thermodenitrificans NG80-2 CP000557 9Oceanobacillus iheyensis HTE831 BA000028 38Listeria innocua Clip11262 AL592022 10Clostridium perfringens 13 BA000016 35Lactobacillus plantarum WCFS1 AL935263 18Lactococcus lactis subsp. lactis Il1403 AE005176 6

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mosome (indicated in green bars) are small in number (129 of1,957 [6.6%]), and about half of them (69 of 129) are carried byprophages (Table 2). As many as 1,270 ORFs (64.9%) werefound to have genes in staphylococcal species as the closestorthologs by BLAST best-hit analysis described below. On theother hand, 333 ORFs (17.0%, orange in Fig. 1) first hit thosein Bacillus species, which include 20 ORFs (red bars in Fig. 1)whose orthologs are not found in staphylococcal species.

It is noted that the G-C skew is asymmetric on the chromo-some map (seventh circle of Fig. 1), which corresponds to anasymmetric distribution of the genes with opposite orientation(second and third circles of Fig. 1). Such an asymmetric G-Cskew is also seen in the genomes of some coagulase-negativestaphylococci, such as Staphylococcus haemolyticus strainJCSC1435 (40); the skew is presumably due to the accumula-tion of many exogenous genes in certain chromosomal regions.In case of the chromosome of M. caseolyticus JCSC5402, theasymmetricity is probably caused by the insertion of prophages(red arcs of the first circle of Fig. 1) that occurs slightly up-stream of the replication termination site (terC). Three pro-phages (�MCCL1 to -3) were identified in the chromosome ofstrain JCSC5402. Of 113 ORFs carried by the phages, 33 areunique to M. caseolyticus, whereas 45 are similar to staphylo-coccal phage proteins, 23 are similar to those of Bacillus spp.,and 12 are similar to those of other species such as Enterococ-cus, Streptococcus, Listeria, Lactococcus, and Mycoplasma spp.This may mean that the prophages of M. caseolyticus are mo-bile across great ranges of gram-positive bacterial genera.

Three insertion sequences (ISs) were found in the JCSC5402genome. The number is much smaller than that in S. haemo-lyticus strain JCSC1435, in which the insertion sequences causefrequent genome rearrangements (40). On the other hand, thestrain JCSC5402 harbors as many as eight plasmids (pMCCL1to -8) that range in size from 2 to 80 kb. Each of the plasmidscarries a unique replication unit; therefore, the replications ofthe plasmids seem to be compatible with one another. None ofthe plasmids except pMCCL2 harbor known antibiotic resis-tance genes. Some genes were found to be involved in DNAmodification and plasmid mobilization; however, most of othergenes remain functionally unknown. Therefore, how they aremaintained in the cells and what benefit each plasmid confers

to the strain are unknown. Genes for replication proteins (rep)of the eight plasmids are most homologous to those of S.aureus (pMCCL3, -4, and -5), Bacillus thuringiensis (pMCCL1and -2), Pediococcus acidilactici (pMCCL6), Tetragenococcushalophilus (pMCCL7), or Enterococcus faecalis (pMCCL8).The taxonomic distribution of the most similar genes for the 98ORFs in the eight plasmids is as follows: 31 ORFs, Staphylo-coccaceae; 12 ORFs, Enterococcaceae; 11 ORFs, Bacillaceae; 3ORFs, Lactobacillaceae; 3 ORFs, Listeriaceae; 4 ORFs, gram-positive bacterial families; 3 ORFs, mycoplasmas; and 3 ORFs,gram-negative bacterial families, in addition to 28 uniquegenes to M. caseolyticus strain JCSC5402. This suggests thatthe macrococcal plasmids are also omnipresent mobile ele-ments in large varieties of bacteria belonging to the phylumFirmicutes (gram-positive bacteria) and frequently recombinewith other plasmids.

Figure 2 illustrates similarity plot data of the chromosome ofM. caseolyticus strain JCSC5402 with that of S. aureus strainMW2 (top panel) or B. subtilis strain 168 (bottom panel). Dotsare rarely found in the MW2 chromosomal regions of ca. 0 to500 kb and ca. 2,250 to 2,830 kb (Fig. 2, top panel), indicatingthat the regions contain genes whose orthologs are absent fromthe chromosome region of JCSC5402. The region correspondsto the “oriC environ” unique in staphylococcal species. TheoriC environ contains many exogenous genes acquired acrossspecies barrier and is postulated to contribute to the evolutionof staphylococcal species (40). The similarities to the macro-coccal chromosome are distributed in the other regions ofMW2 (Fig. 2, top panel). There is an inversion in the 2,000-kbregion in the M. caseolyticus genome that corresponds to ap-proximately 500 to 600 kb in the S. aureus MW2 genome. Sincethis region includes ribosomal and tRNA genes at the bothends, an inversion may take place due to a homologous recom-bination. Figure 2 (bottom panel) shows that the similarity withB. subtilis chromosome is much smaller than with S. aureuschromosome, but the homologous sequences are diffusely dis-tributed throughout the B. subtilis genome. These data recon-firm that the genes in the M. caseolyticus chromosome aremore similar to those in S. aureus chromosomes than to thosein B. subtilis chromosomes.

Functional classification of ORFs and their phylogenic re-latedness to other organisms. We annotated the functions ofORFs of M. caseolyticus strain JCSC5402 by their BLASTbest-hit genes found in databases, and the ORFs were classi-fied according to B. subtilis genome project (21). We alsoanalyzed the taxonomic distribution of the BLAST best-hitentries to each ORF (Table 3). We regarded as “hit” whenORFs of M. caseolyticus strain JCSC 5402 were 25% or moreidentical to genes in the databases in their amino acid se-quences. Most of the genes best hit the orthologs in staphylo-coccal genomes. In particular, the components of machineriesessential for life such as those for DNA replication, RNAtranscription, protein translation, glycolysis, and TCA cyclebest hit those in the family Staphylococcaceae, followed bythose in the family Bacillaceae (including the genera Bacillus,Oceanobacillus, and Geobacillus). It is noteworthy that abouthalf of the ORFs belonging to class I-4 (membrane bioener-getics, including respiratory chain) best hit the orthologs in thefamily Bacillaceae. This indicates that M. caseolyticus and Ba-

TABLE 2. Comparison of the M. caseolyticus chromosome to thoseof S. aureus and B. subtilis

ParameterM.

caseolyticusJCSC5402

S. aureusMW2

B. subtilis168

Length of sequence (bp) 2,102,324 2,826,402 4,214,630G�C content (%) 36.9 32.8 43.5ORFs

No. of protein codingregions

1,957 2,632 4,106

% Coding 87.9 83.5 87.2No. of rRNAs

16S 4 6 1023S 4 6 105S 5 7 10

No. of transfer RNAs 48 61 86No. of insertion sequences 3 6No. of prophages 3 2 10

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cillus species share the similar machineries for electron trans-port chain as described more below.

M. caseolyticus pathways absent in the Staphylococcus spe-cies. Despite their phylogenetic proximity, M. caseolyticus andstaphylococci have different sets of electron transducer com-ponents for oxidative phosphorylation from each other. Staph-

ylococci do not possess the genes for cytochrome c oxidasecomplex, which are present in both M. caseolyticus and B.subtilis genomes. This explains the fact that M. caseolyticus ispositive for the “cytochrome oxidase test” (data not shown)that is frequently used to identify microorganisms. M. caseo-lyticus and B. subtilis also carry menaquinol-cytochrome c ox-

FIG. 1. Circular representation of the chromosome of M. caseolyticus JCSC5402. The first (outermost) circle shows the nucleotide position inbase pairs. Red arcs indicate prophages, whereas yellow shows the insertion sequences. The second circle shows ORFs oriented in the forwarddirection, whereas the third circle indicates those oriented in the reverse direction. Green bars in the second and the third circles represent uniqueORFs of M. caseolyticus, whereas blue bars show the ORFs that are the most similar to staphylococcal genes. Orange bars indicate the first-hitBacillus ORFs, and red bars indicate those whose homologs are not found in staphylococcal genomes and yet first-hit Bacillus ORFs. Black barsindicate ORFs whose most homologous genes were not found in either Bacillus or Staphylococcus. The fourth and fifth circles show genes fortransfer RNAs and rRNAs, respectively. The sixth circle represents the regions with high GC content (�50%; purple) and low GC content (blue).The window size and shift increment for GC content were 5 kbp and 0.1 kb, respectively. The seventh circle shows the G-C skew with positive(yellow) and negative (blue) values.

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idoreductase subunits that are absent in staphylococci, suggest-ing that the electron transducer components of M. caseolyticusare rather closer to those of family Bacillaceae. Instead, staph-ylococci possess quinol oxidase (encoded by qox genes, absentin M. caseolyticus) that is also found in B. subtilis. Cytochromebd ubiquinol oxidase components (encoded by cyd genes) arecommonly seen in B. subtilis, M. caseolyticus, and staphylo-

cocci, indicating that B. subtilis has flexibility in utilizing eitherone of the electron transducers.

The starch-digesting amylase gene of Bacillus species is alsopresent in M. caseolyticus but is absent from staphylococcalgenomes. The reason why M. caseolyticus possesses amylasegene is unknown. Interestingly, we also identified glycogenbiosynthesis genes in M. caseolyticus, suggesting that the or-

FIG. 2. Genome alignment between M. caseolyticus JCSC5402 and S. aureus MW2 (top) or between M. caseolyticus JCSC5402 and B. subtilis168 (bottom). Red dots indicate homologous regions that were drawn by using In Silico molecular cloning software. The top panel includesgenomic island locations and their names in either purple (unique to M. caseolyticus JCSC5402, vertical scripts) or sky blue (unique to S. aureus,horizontal scripts). SCCmec is a determinant for methicillin resistance (17). The �Sa and �Sa� are pathogenicity islands present in all of the S.aureus genomes thus far sequenced, whereas the �Sa3 element is found in some S. aureus strains, including MW2 (2, 3). �Sa elements representprophages.

TABLE 3. Functional classification of ORFs in the M. caseolyticus JCSC5402 chromosome and taxonomic distribution of BLAST best-hitentries to the ORFs

Class Functional classification

No. of most similar genes in a taxonomic family toeach M. caseolyticus ORF

Total FamilyBacillaceae

FamilyStaphylococceae Others

I-1 Cell wall 58 4 49 5I-2 Transport/binding proteins 196 44 129 23I-3 Sensors 16 1 13 2I-4 Membrane bioenergetics 63 28 30 5I-5 Protein secretion 8 1 7 0I-6 Cell division 15 0 15 0I-7 Transformation/competence 10 1 8 1II-1–1 Specific pathways 92 28 55 9II-1–2 Main glycolytic pathways 18 3 15 0II-1–3 Tricarboxylic acid cycle 11 3 8 0II-2 Metabolism of amino acids 81 10 63 8II-3 Metabolism of nucleotides and nucleic acids 63 7 54 2II-4 Metabolism of lipids 35 6 19 10II-5 Metabolism of coenzymes and prosthetic groups 80 8 64 8II-6 Metabolism of phosphate 8 2 6 0II-7 Metabolism of sulfur 2 1 1 0III-1 DNA replication 24 1 22 1III-2 DNA restriction/modification and repair 47 6 31 10III-3 DNA recombination 15 2 12 1III-4 DNA packaging and segregation 7 2 5 0III-5–1 RNA synthesis initiation 7 1 6 0III-5–2 RNA synthesis regulation 97 22 66 9III-5–3 RNA synthesis elongation 2 0 2 0III-5–4 RNA synthesis termination 5 0 5 0III-6 RNA modification 41 4 34 3III-7–1 Ribosomal proteins 48 5 43 0III-7–2 Aminoacyl-tRNA synthetases 25 1 24 0III-7–3 Protein synthesis initiation 4 0 4 0III-7–4 Protein synthesis elongation 5 0 5 0III-7–5 Protein synthesis termination 3 1 2 0III-8 Protein modification 71 12 48 11III-9 Protein folding 11 2 9 0IV-1 Adaptation to atypical conditions 29 2 25 2IV-2 Detoxification 58 14 39 5IV-3 Antibiotic production 1 1 0 0IV-4 Phage-related functions 69 16 9 44IV-5 Transposon and IS 6 0 0 6IV-6 Miscellaneous 89 20 55 14V Similar to unknown proteins 408 74 288 48VI No similarity 129

Total 1,957 333 1,270 227

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ganism could store glucose as its polymerized form. The abilityof M. caseolyticus to polymerize glucose and digest starch im-plies its efficient utilization of glucose in an environment whereglucose shortage is a great concern. Both amylase and glycogenbiosynthesis genes are the most similar to those of Bacillusspecies.

Staphylococcal pathways absent in M. caseolyticus. Reflect-ing its shorter genome size, M. caseolyticus lacks many bio-chemical pathways that are present in staphylococci. We failedto find genes similar to siderophore biosynthesis genes in M.caseolyticus JCSC5402 genome. The organism also lacks mostof the known iron uptake transporters found in staphylococci.These facts indicate that M. caseolyticus colonizes in the envi-ronments where the organism does not have to be “aggressive”upon iron import. Such environments should also help M.caseolyticus to live in the absence of many vitamins and aminoacid biosynthesis enzymes. Strain JCSC5402 lacks the full setsof genes required for panthothenate and biotin synthesis. Italso lacks glutamate, tryptophan, leucine, and histidine biosyn-thetic pathways, indicating that the strain requires these vita-mins and amino acids for its growth. Sugar utilization ability isalso limited: the strain lacks complete sets of sugar metabolismpathways for trehalose, maltose, mannitol, and lactose.

We also found that M. caseolyticus lacks all virulence deter-minants identified in S. aureus (3) except for one gene showing54% amino acid sequence identity to hemolysin A of Bacilluscereus, explaining the fact that this organism has not been thusfar recognized as a human pathogen.

Regulatory systems. Recent studies have revealed that mostof the virulence genes in S. aureus are expressed under thecontrol of regulatory systems known as global gene regulators.With regard to such systems, no homologs for agr (28) or sarA(8) regulator genes are present in M. caseolyticus, which isconsistent with the fact that the organism does not have toxingenes whose expression is known to be under regulation of agrand sarA. Therefore, it is likely that S. aureus virulence geneswere acquired together with their regulatory systems after thedivergence of the genera Staphylococcus and Macrococcus.

Two-component systems. Eleven sets of two-component sys-tems were identified in the M. caseolyticus chromosome. Ten ofthem are the most similar to those in staphylococcal speciesthat are well conserved throughout the entire sequence length.Six of the ten were found in both genera Staphylococcus andBacillus, whereas four are not present in Bacillus spp. Theother set, a sensor kinase (MCCL1698) and a response regu-lator (MCCL1701), hit those of Clostridium tetani and Clos-tridium perfringens, respectively, as the most similar orthologs.

The orthologs for the two-component system vraSR thatserves as an upregulator of cell wall synthesis in S. aureus(22) were found in M. caseolyticus JCSC5402 (vraR,MCCL1614; vraS, MCCL1615). The vraSR system is widelydistributed in gram-positive bacteria, suggesting that theregulator system is an important system originated early inthe evolutionary history of gram-positive bacteria, althoughthey are nonessential genes for cell viability (22). The or-thologs for S. aureus two-component systems, phoPR(MCCL1358-9), nreBC (MCCL0141-2), lysSR (MCCL0109-10), srrAB (MCCL1137-8), and arlSR (MCCL1065-6) thatare involved in alkaline phosphatase synthesis, nitrate re-duction system, control of the cell wall lysis rate, respiratoryresponse, and sugar transport, respectively, are also presentin M. caseolyticus. The physiological function(s) of otherfour two-component systems are unknown.

A recent study has revealed that a two-component systemgraSR of S. aureus is involved in the vancomycin resistancemechanism in vancomycin-intermediate S. aureus (27). ThegraSR system was not found in the M. caseolyticus genome,supporting the notion that the system is dispensable for theviability in S. aureus (27).

Antibiotic resistance. We initiated the present study aimingto pursue the dissemination of drug-resistant microbes throughthe stock farm products. Quite a few beta-lactam resistantbacteria were obtained (K. Kuwahara-Arai et al., unpublisheddata), in which M. caseolyticus strain JCSC5402 was unique inits multiple-drug resistance profile. Table 4 shows that strainJCSC5402 harbors various antibiotic resistance determinantsincluding beta-lactams, aminoglycosides, and macrolides.

The largest plasmid pMCCL2 carries multiple genes thathave similarity to B. anthracis plasmid pXO2 (30). Surprisingly,the plasmid harbors a mecA gene homolog, designated mecAm,encoding a penicillin-binding protein similar to PBP2� ofMRSA, with 72% identity in its amino acid sequence (Fig. 3).The mecAm gene is flanked by beta-lactam regulator genes,similar to blaI-blaR1 (designated mecIm-mecR1m), and a beta-lactamase gene similar to blaZ (blaZm) (Fig. 3, middle). Inorder to show whether the mecAm gene confers methicillinresistance to S. aureus, we introduced the mecAm gene underthe control of its own promoter from a plasmid pYMMAharboring tetracycline-resistant gene as a marker into the beta-lactam-susceptible S. aureus strain N315ex. As expected, theintroduction of pYMMA resulted in beta-lactam resistance inN315ex (Table 4, N315ex/pYMMA). On the other hand, elim-ination of pMCCL2 from M. caseolyticus JCSC5402 led to aloss of not only beta-lactams but also aminoglycosides and

TABLE 4. MICs of various antibiotics for S. aureus N315 and N315/pYMMA (mecAm) and M. caseolyticus JCSC5402 andJCSC5402/pMCCL2

StrainAntibiotic MIC (�g/ml)a

OXA CZX AMP VAN KM GM TOB EM TC IMP NFLX

S. aureus N315ex 0.25 4 32 0.5 2 0.5 0.5 128� 0.5 �0.063 2S. aureus N315ex/pYMMA 16 128� 64 1 2 0.5 0.5 128� 128� 1 2M. caseolyticus JCSC5402 64 128� 16 0.5 32 4 4 128� 0.5 2 0.5M. caseolyticus JCSC5402/pMCCL2 0.25 0.5 0.125 0.5 0.5 �0.063 0.125 0.5 0.5 �0.063 0.5

a The MIC was determined by the agar dilution method recommended by the Clinical and Laboratory Standards Institute. OXA, oxacillin; CZX, ceftizoxime; AMP,ampicillin; VAN, vancomycin; KM, kanamycin; GM, gentamicin; TOB, tobramycin; EM, erythromycin; TC, tetracycline; IMP, imipenem; NFLX, norfloxacin.

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macrolides (Table 4, M. caseolyticus JCSC5402/pMCCL2), afinding consistent with the fact that the plasmid pMCCL2 alsocarries resistance genes to aminoglycosides (aacA) and mac-rolides (ermB). The results suggest that the plasmid pMCCL2is responsible for acquisition of resistance to those antibiotics,although coelimination of other plasmids was not completelyassessed, and therefore it is still possible that the other plas-mids harbor uncharacterized antibiotic resistance genes. Nev-ertheless, it is highly likely that pMCCL2 is the only plasmidthat confers antibiotic resistance to the macrococcal cells be-cause we found no resistant genes in other plasmids.

The unique overall structure of mecAm gene complex,mecIm-mecR1m-mecAm-blaZm, seems to coincide with Matsu-hashi’s speculation on the historical make-up of the mecA genecomplex of MRSA: that is, the recombination between a blaI-blaR1-blaZ operon and a gene encoding PBP generated themecA gene complex, mecI-mecR1-mecA (12) (Fig. 3). It is

noteworthy that this is the first report of identifying a plasmid-conveying methicillin resistance.

Relatedness of chromosomal structure among species andtheir phylogenic relationship. We identified orthologous genesand their arrangements in the chromosomes of M. caseolyticusand related bacteria to infer the evolutional relationshipamong these bacterial species. We initially identified orthologsamong various species by using a clustering algorithm Dom-Clust (42, 43), which is based on hierarchical clustering proce-dure for constructing orthologous groups at a domain level,and then constructed an alignment of the syntenically con-served regions, or “core structure,” by using the CoreAlignerprogram (44), which finds the order of orthologous groups thatretains the conserved gene orders to the greatest possible ex-tent in each genome. (Fig. 4). In Fig. 4, colored lines acrossspecies indicate locations of the genes in the resulting corestructures, and the color gradation (from red through yellow to

FIG. 3. Tandem location of beta-lactam resistant genes mecA (designated mecAm) and blaZ in M. caseolyticus JCSC5402/pMCCL2 comparedto mec and bla complexes in S. aureus N315. The numbers in the purple shaded areas represent mutual amino acid sequence identities.

FIG. 4. Distribution of conserved ortholog groups among Bacillaceae and Staphylococcaceae families and Listeria innocua. After identifyingcommon orthologs among various species, we constructed the conserved chromosomal structure (“core structure”) on the basis of the consensusarrangement of the conserved orthologs. An ortholog group in the resulting core structure is indicated as a colored line across horizontal blackline representing a chromosome. To simplify the figure, only universally conserved, one-to-one correspondence ortholog groups are shown. Tovisualize chromosomal rearrangement of the core structure, color gradation is assigned according to the location on the M. caseolyticus JCSC5402chromosome from red to yellow to green. The replication origins (oriC) are located at the center.

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green) shows the location of each set of orthologs on the M.caseolyticus chromosome. This analysis can identify the con-served chromosomal structures among M. caseolyticus and thegenera Staphylococcus and Bacillus, structures that are likely tohave been vertically inherited from their common ancestor.Although several large-scale chromosome inversions are visi-ble with certain species, it is apparent that the latter generahave extra chromosomal regions studded among the corestructures where the genetic information useful for their ownhabitats and biological activity are stored.

In order to investigate the relationship among the generaBacillus, Staphylococcus, and Macrococcus further, the phylo-genetic relationship among gram-positive bacteria was studiedby using the protein sequences of 297 core genes that consti-tute the chromosomal core structures used for the Fig. 4 anal-yses, and the findings are displayed as a tree diagram (Fig. 5).The phylogenetic distance calculated by this analysis supportsthe close relatedness of the three genera. It is therefore evidentthat the three genera have been derived from a common gram-positive bacterium.

The analysis in Fig. 4 also illuminated the oriC environ,which is not found in the M. caseolyticus chromosome. Theformation of the oriC environ is considered to be due to theaccumulation of multiple exogenous genes in the downstreamof orfX by serial tandem integration of staphylococcal cassettechromosomes (SCCs) (17). Each SCC is considered to haveconferred a certain growth or survival advantage to the recip-ient bacterium. Around the oriC, M. caseolyticus does haveorfX homolog, but no SCC or its remnants were found down-stream of the orfX. It seems that the establishment of the SCCgene transfer system might have to await the divergence ofgenera Staphylococcus and Macrococcus.

DISCUSSION

The BLAST analysis of the predicted gene products of M.caseolyticus strain JCSC5402 revealed that the organism ismore closely related to the genus Staphylococcus than to anyother genera. The growth-essential genes that are involved inreplication, transcription, and translation are the most akin tothose in staphylococci, which expounds well the historic clas-sification of the species as a member of genus Staphylococcus(34). The BLAST analysis also showed that Bacillus species arethe second closest group of bacteria to M. caseolyticus. Themacrococcal genes belonging to such metabolic pathways asthe respiratory chain, which is essential for the viability underaerobic conditions, were found to be the most similar to theorthologs of Bacillus species. These facts suggest that the ge-nome of M. caseolyticus retains a part of the genome of thecommon ancestor for genera Bacillus and Macrococcus.

The shorter chromosomes of M. caseolyticus compared tothose of staphylococcal species are rather similar to those ofstreptococci, which are known to lack many genes for thebiosynthesis of small molecules, and therefore require manynutrients. It is, however, unknown which M. caseolyticus genescompensate for the shortage of such biosynthetic pathways,since the substrates of many of the genes regarded as trans-porters due to their possession of the motif sequences areunknown. For example, only one gene was identified as aferrichrome transporter in the M. caseolyticus genome in con-trast to S. aureus, in which more than ten genes are involved iniron transport. It is therefore still unknown how M. caseolyticusutilizes iron efficiently based on analysis of the whole-genomesequence. Nevertheless, it may be that the genome becameshortened after the divergence of the genera Bacillus and Mac-

FIG. 5. Phylogenetic relationships among gram-positive bacteria. The tree diagram was generated by using the concatenated protein sequenceof 297 orthologous core genes that are conserved in all of the tested genomes in one-to-one correspondence. These genes are shown as coloredlines in Fig. 4.

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rococcus, presumably because Macrococcus adapted to a nu-trient-rich habitat.

The phylogenicity of the 297 core genes and our chromo-some-clustering algorithm showed a close evolutionary relat-edness of strain JCSC5402 with Staphylococcus and Bacillusspp., despite the great differences in genome size and morphol-ogy. The small chromosome of the strain JCSC5402 may re-flect the essential ancestral chromosome before its divergenceinto the three species. This view is consistent with the result ofthe chromosome clustering analysis (Fig. 4), which clearly il-lustrates the well-conserved distribution of orthologous geneclusters across the chromosomes representing the three sepa-rate genera.

By assuming this view, it seems that the ancestral bacteriumfor family Staphylococcaceae must have downsized its chromo-some by losing the genes that were not essential in the newhabitat, or animal body, after the divergence from the familyBacillaceae. The large genomes of the Bacillus species, as beingrepresented by 4.2 Mb with B. subtilis or 5.2 Mb with B. an-thracis, harbor the genes encoding such features as rod-shapedcell morphology, spore formation, the production of insecttoxins, peptide antibiotics, and antifungals. Such genes shouldhave been unnecessary in the new environment to which theancestor for macrococcal and staphylococcal species wasadapted. Following parasitization of an animal body, the spe-ciation into macrococcal and staphylococcal species wouldhave started. Although Macrococcus remained avirulent to theanimal, the speciation into Staphylococcus, especially S. aureus,was an aggressive one accompanied by the increase of genomesize with accumulation of arrays of virulence genes against theanimal host. The oriC environ is considered to have contrib-uted much to this increase of genome size. In the case of S.aureus, the oriC environ contains genes encoding capsularpolysaccharide biosynthesis (25), the restriction-modificationsystem (13), immunoglobulin-binding protein A, tissue-bindingproteins, and some superantigens (31). A notorious methicillinresistance determinant is also one of the genes transferred bySCC into the oriC environ (17). Thus, it is tempting to specu-late that the differentiation of staphylococcal species wasgreatly promoted by the development of the SCC-mediatedgene transfer system in an early phase of the speciation ofgenus Staphylococcus.

We found that strain JCSC5402 possesses various genesassociated with drug resistance. This reconfirms the presenceof active selective pressure by chemical agents in the animalhusbandry. Figure 3 illustrates the most remarkable finding inthe JCSC5402 genome: the mecA-carrying plasmid with aunique cluster of genes, i.e., mecIm-mecR1m-mecAm-blaZm.This may be the ancestral form of the methicillin-resistancedeterminant of the intractable hospital pathogen MRSA. Anintensive study on the mobility and detailed nature of themecAm gene complex is currently under way.

ACKNOWLEDGMENTS

This study was supported by a 21st Century COE research grant-in-aid and a grant-in-aid for scientific research (grant 18590438) from theMinistry of Education, Science, Sports, Culture, and Technology ofJapan.

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JOURNAL OF BACTERIOLOGY, May 2009, p. 3429 Vol. 191, No. 100021-9193/09/$08.00�0 doi:10.1128/JB.00366-09

ERRATUM

Complete Genome Sequence of Macrococcus caseolyticus Strain JCSC5402,Reflecting the Ancestral Genome of the Human-Pathogenic Staphylococci�

Tadashi Baba,1* Kyoko Kuwahara-Arai,1 Ikuo Uchiyama,2 Fumihiko Takeuchi,3†Teruyo Ito,1 and Keiichi Hiramatsu1

Department of Microbiology and Infection Control Science, Juntendo University, 2-1-1 Hongo, Bunkyo, Tokyo 113-8421, Japan1;National Institute for Basic Biology, National Institutes of Natural Sciences, Nishigonaka 38, Myodaiji, Okazaki 444-8585,

Japan2; and Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom3

Volume 191, no. 4, 2009, p. 1180–1190. Page 1180: The title and byline should appear as shown above.Page 1190: The following should appear below the corresponding author footnote:

†Present address: Department of Gene Diagnostics and Therapeu-tics Research Institute, International Medical Center of Japan, 1-21-1Toyama, Shinjuku-ku Tokyo 162-8655, Japan.

Page 1186, column 2, paragraph 4, line 5: “identity” should read “similarity.”Page 1187: In the legend to Fig. 3, the last word should be “similarities.”

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