抗酸菌の分類同定指標として有効なコアーハウスキーピング遺伝子

誌名誌名 日本微生物資源学会誌

ISSNISSN 13424041

著者著者

水野, 卓也名取, 達矢金澤, 泉Eldesouky, I.福永, 肇江崎, 孝行

巻/号巻/号 32巻1号

掲載ページ掲載ページ p. 25-37

発行年月発行年月 2016年6月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Microb. Resour. Syst. 32(1): 25-37, 2016

Core housekeeping proteins useful for identification and classification of mycobacteria

Takuya Mizuno1・ 2l*, Tatsuya Natori1l, Izumi Kanazawa1l, Ibrahim Eldesouky3l, Hajime Fukunaga1l and Takayuki Ezaki1l

1lDepartment of Microbiology, Regeneration and Advanced Medical Science, Gifu University

Graduate School of Medicine, 1-1 Yanagido, Gifu, 501-1194, Japan

2lGifu Prefectural Institute of Health and Environmental Sciences

1-1 N aka-fudogaoka, Kakamigahara, 504-0838, Japan

3lDepartment of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine,

Kafrelsheikh University, 33516, Egypt

We generated and analyzed draft genom巳sfor 42 1lゐ1cobacteriumstrains representing 30 different species to select core housekeeping proteins (HKPs) that would be useful for differ巴ntiatingamong closely related Mycobacterium species.

HKPs of 1lゐ1cobacteriumtuberculosis H37Rv were selected as reference proteins, and values (designated diversity

values) representing the amino acid differences between these H37Rv HKPs and those of other individual 1lゐ1cobacteriumspecies were calculated for each HKP. From seven NCBI protein categories, we analyzed the 107 proteins commonly found in all 30 Mycobacterium species. We then selected the 12 most variable HKPs to construct a concatenated protein

sequence (designated Cl2HKP). The average Cl2HKP diversity value for these. 30 Mycobacterium species was 22.50%. Phylogenetic trees constructed with either Cl2HKP or C50RP (50 concatenated sequences from 50S and 30S ribosomal proteins) had r巴liablebootstrap values that were higher than those of a 16S rRNA gene tree. Of the three entities

(Cl2HKP, 16S rRNA gene, and C50RP), Cl2HKP exhibited the greatest diversity. To d1妊erentiateamong closely related species within the g巴nus1lゐ1cobacterium,the Cl2HKP entity provided the most powerful and discriminating dataset.

Key words: housekeeping proteins, mycobacterium, classification, identification, MLSA

INTRODUCTION

To date, 160 species have been classified into the

genus Mycobacterium (http:/ /www.bacterio.net/

mycobacterium.html); however, the interspecies

variation of the 16S rRNA gene sequence among

these 160 species is less than 6%. In this genus, 16S

rRNA gene sequence identities between several

closely related species are higher than 99%. Many

authors have used housekeeping genes (HKGs) such

as dna]l (Yamada-Noda et al., 2007), gyrB (Kasai et

al., 2000; Niemann et al., 2000), rρoB (Kim et al.,

1999), hsρ65 (Brunello et al., 2001; McNabb et al.,

2004; Ringuet et al., 1999), recA (Blackwood et al.,

2000), or sodA (Adekambi & Drancourt, 2004), or

some combination thereof. to discriminate between

closely related species. The 16S rRNA gene

*Corresponding author

E-mail: mizuno-takuya@pref.gifu担.jp

Accepted: January 29, 2016

sequence identity between Mycobacterium avium

and M. intracellulare is 100%, whereas that ofゆoB

is 95.8%. Therefore, rρoB seems to be more

powerful than the 16S rRNA gene for discriminating

among llの1cobacteriumspecies, but the data obtained

by using this single HKG are not variable enough to

fully evaluate phylogenetic relationships in this

genus (Kim et al., 1999).

More recently, multilocus sequence analysis

(MLSA) has been used as a powerful alternative for

identifying and classifying bacteria (Gevers et al.,

2005; Schleifer, 2009). This multigenic approach

fulfills the recommendation of the ad hoc committee

for the re田evaluationof the definition of bacterial

species (Stackebrandt et al., 2002). However,

researchers have used many different sets of genes

for MLSA (Adekambi & Drancourt, 2004; Dai et al.,

2011; Devulder et al., 2005). As a result, the

proposed phylogenetic relationships determined

among closely related species by using different

MLSA datasets differ substantially.

-25一

Core proteins for mycobacteria

Draft genome sequencing has become a relatively

easy and inexpensive procedure, and complete

protein sequences are available from these draft

sequences. In 2004, Cole et al. (1998) published a

complete genome of M. tuberculosis H37Rv; this was

the first such report for the genus Mycobacterium.

Recently, genome analysis of many mycobacterial

strains has been completed, but most of the

sequenced strains listed on the National Center for

Biotechnology Information (NCBI) home page are M.

tuberculosis strains.

Determination of average nucleotide identity

(ANI) with whole genome information is a powerful

method for analyzing the relationships among

strains within a species. ANI values of >95%

correspond to the traditional >70% DNA-DNA

reassociation standard that has been used to define

a species (Konstantinidis & Tiedje, 2005). ANI data

have been used for species identification (Cho et al.,

2013) and can distinguish species more clearly than

can MLSA data (Adekambi & Drancourt, 2004; Dai

et al., 2011; Devulder et al., 2005). However, ANI

values do not offer enough information to determine

interspecies re la tionshi ps.

Here, we generated draft genome sequences for

19 Mycobacterium species and then used these

genome data and publicly available data on other

JIゐ1cobacteriumspecies to analyze 107 housekeeping

proteins (HKPs) that are commonly found in, and

used to analyze, Mycobacterium species. We then

generated datasets based on two different

concatenated protein sequences. One concatenated

sequence was constructed by using 50 proteins from

SOS or 30S ribosomal proteins and designated

C50RP. The other was constructed from the

12 most variable HKPs and designated Cl2HKP;

these 12 HKPs were selected from among 107

proteins belonging to seven protein categories of

NCBI classification, namely ribosomal proteins (small

and large subunit), molecular chaperones, DNA/

RNA replication or modification enzymes, tRNA-

synthesis proteins, and cell division-associated

proteins.

Datasets generated by using either of the

concatenated protein sequences or the 16S rRNA

gene wer巴comparedwith regard to their usefulness

in identifying and differentiating species in the

genus Mycobacterium.

MATERIALS AND METHODS

Bacterial strains and DNA preparation

Mizuno et al.

Table 1 lists the 27 strains (9 of which were type

strains) that represented 19 non凶 tuberculosis

mycobacteria and were used to generate the draft

genome sequence. Strains were provided by Gifu

University School of Medicine as the Gifu Type

Culture Collection (GTC), these strains were

deposited in the Japan National Collection of

Bacterial Pathogen (JNBP) Data Base. Each strain

was cultured on Middlebrook 7Hll Agar

supplemented with OADC (BD Co., Franklin Lakes,

NJ, USA) and incubated at 30℃. EZ beads (AMR

Co., Ltd., Gifu, Japan) were used according to the

manufacturer’s instructions to disrupt bacterial cells,

and DNA purification was performed as described

previously (Boom et al., 1990).

Draft genome sequence

All genome sequences were determined by using

the Illumina HiSeq_ system (Illumina, Inc., San Diego,

CA). Each genome sequence was automatically

annotated with the Microbial Genome Annotation

Pipeline (MiGAP) ver. 2.18 (http:/www.migap.org/).

Start codon positions and any additional genes

missing from the MiGAP annotation were manually

inspected and corrected.

Sequence diversity among Mycobacterium species

was calculated for 30 species and 42 strains (Table

1). The DNA sequence of the 16S rRNA gene and

the amino acid sequences of 107 HKPs were used to

calculated percent diversities. Mycobacterium

tuberculosis H37Rv was used as a reference strain to

generate multiple sequence alignments and calculate

percent diversities for each of the other strains;

these multiple sequence alignments were generated

with Clustal W (Thompson et al., 2002), implemented

in DN ASIS Pro version 3.0 software (Hitachi

Software Engineering Co., Ltd., Tokyo, Japan).

Likewise, intraspecies diversities were calculated for

M. abscessus and 』1.tuberculosis. Strains for these

sequences were obtained from the Patric database

(http:/ /www.patricbrc.org).

The set of 107 HKP proteins (Fig. 1) comprised

SOS and 30S ribosome proteins (n=50), molecular

chaperones (n=7), proteins associated with

replication and modification (n=21), tRNA-synthesis

proteins (n=24), proteins associated with cell division

(n=4), and RecA.

-26一

Microb. Resour. Syst. June 2016 Vol. 32, No. 1

Table 1 List of J的1cobαcteriumspecies analyzed

Gen om巴 G+CStrain name JNBP No." History 1Mb 、 1% tRNA CDSb Reference

size 1 p1 mo1

M. abscessus JNBP _03165 くGTC15113くSputum.Osaka, Japan 5.1 64.1 47 5019 This study 1990

M. abscessus JNBP 03167 くGTC15115くSputum,Osaka, Japan 5.1 64.1 47 5032 This study 1990

M. avium JNBP 03535 くGTC15594<Sputum. Kyoto, Japan 5.2 69.1 47 4979 This study

1991 M. branderi JNBP _03209TくGTC00811く ATCC 51789 5.9 66.5 46 5800 This study M. chelonae JNBP 03213 くGTC12637くSputum,Mie, Japan 4.9 64.2 46 4746 This study

1990 M. chelonae JNBP _03238 くGTC15348くSputum,Osaka. Japan 5 63.9 47 4960 This study

1988 M. flavescens JNBP _03250 くGTC15352くSputum,Osaka, Japan 5.4 66.8 46 5154 This study

1988

M. fortuitum JNBP 03252 くGTC12780くSputum,Japan 1992 6.6 66.1 54 6545 This study M. fortuitum JNBP _03300TくGTC15396くKPM4015 6.3 66.2 54 6137 This study

M. gordonae JNBP 03328 くGTC15401くSputum.Kyoto. Japan 6.6 66.8 49 6348 This study 1990

M. kansasii JNBP 034 73 くGTC15535くSputum.Kyoto, Japan 6.5 66 43 6031 This study

1990 M. kansasii JNBP 03464 くGTC03844くSputum.Tokyo, Japan 6.4 66.1 46 5861 This study

2007 M. lent抑avum JNBP _03532 くGTC03852くSputum,Tokyo, Japan 6.5 65.8 48 6117 This study

1990 M. malmoense JNBP _03536TくGTC15595くATCC 29571 5.3 67 45 5001 This study M. mucogenicum JNBP _03551 TくGTC03155くCCUG47451 6.2 67.2 57 6052 This study

M. sρhagni JNBP _03654TくGTC01619くATCC 33027 5.5 66.7 46 5241 This study M.ρeregrinum JNBP _07211 TくGTC01725くATCC14467 7 66.3 48 6879 This study

M.ρeregrinum JNBP 03578 くGTC15642くSputum.Kyoto. Japan 7.4 66.1 72 7299 This study 1990

M.ρeregrinum JNBP 03577 くGTC15641くSputum,Kyoto. Japan 7 66.3 48 6902 This study 1991

M.ρeregrinum JNBP 03881 くGTC16404くAbscessleft leg, Japan 6.5 66.3 53 6340 This study 2010

M. interjectum JNBP 03801 くGTC12928くIWGMTstrain. USA 7 67.9 47 6846 This study 1992

M. scrofulaceum JNBP 03599 くGTC12927くIWGMT90529.USA 5.4 67.6 48 5149 This study 1992

M. senegalense JNBP _03637TくGTC01622くATCC35796 6.1 66.6 47 5832 This study

M. szulgai JNBP _03662TくGTC15712くJCM6383 6.8 65.6 49 6248 This study

M. triplex JNBP 03854 くGTC12949くIWGMT90553.USA 5.5 68.4 48 5284 This study 1992

M. vaccae JNBP 03729 くGTC15849くclinical.Osaka. Japan 6.2 68.5 49 6007 This study 1988

M. vaccae lNBP :::03730TくGTC15850くKPM4714 6.3 68.5 49 6040 This study ー．．．．．．．．．．．．．．．．．．．．．．．．．．． 干 4・・ー－－・ a・・・・ a・・・・ a・・・－－－・ー・ーーーー－－－－－－－－－－－－－－－－－－－－－－－－－－－－－－司－・・・・－－－－－－・－－－－－－－－－－－ーー－－－－－－－－－－－－－－圃』 d』守・・ 4』守．．．．『・・・・・ー・・・・・・・・・・・・・・－－－

NCBic Database

M. africanum GM041182 4.4 65.6 45 4038 Bentley, et al .. 2012

M. avium strain 104 5.5 69 46 5280 Horan, et al., 2006 M. a世iumsubsp. JNBP _07180T ATCC 25291 T 4.9 69.2 47 4792 Unpublished avium

M.σvium subsp. strain K-10 4.8 69.3 46 4624 Li, et al., 2005 ρaratuberculosis M. bovis subsp. AF2122/97 4.3 65.6 45 4004 Garnier. et al., 2003 bovis

27

Core proteins for mycobacteria Mizuno et al.

Table 1 Continued

Strain name

M. bovis BCG M. canettii M. gilvum

JNBP No."

M. intracellulare JNBP _03815T A主leρraeM. marinum M. smegmatis M. tuberculosis JNBP 03875T M. ulcerans M. 世 間bααlenii

History

strain Pasteur ll 73P2 CIPTl 40010059

PYR-GCK ATCC 13950T

Br4923 strain M

strain MC2 155 H37RvT Agy99 PYR-lT

G+C tRNA CDSb size (Mbp) mol%

Reference

4.4 65.6 47 4023 Brosch, et al.. 2007 4.5 65.6 43 4049 Supply, et al.. 2013 5.6 67.9 46 5429 Badejo, et al., 2013 5.4 68.1 46 5104 Kim, et al., 2012 3.3 57.8 45 3050 Monot. et al.. 2009 6.6 65.7 46 5600 Stinear. et al., 2008 7 67.4 46 6769 Gallien, et al.. 2009 4.4 65.6 45 4066 Cole. et al., 1998 5.6 65.4 46 5513 Stinear. et al., 2007 6.5 67.8 49 6218 Unpublished

aJNBP; Japan National Collection of Bacterial Pathogen. National Bioresource Project (http://www.nbrp.jp/) bCDS; coding sequence cNCBI; National Center for Biotechnology Information

Phylogenetic analysis

The amino acid sequences were edited with

DNASIS Pro ver. 3.0 Software. The phylogenetic

tree based on the amino acid sequence alignment

had higher bootstrap values than the values based

on the nucleotide sequence alignment. Therefore,

we used amino acid sequence alignment.

Phylogenetic analysis of the 16S rRNA gene, C50RP.

and Cl2HKP datasets were aligned with Clustal W

and the percent diversities calculated. Neighbor-

joining was performed with MEGA (Molecular

Evolutionary Genetics Analysis) 6 software (Tamura

et al .. 2013). Two different models were used for

neighbor-joining analysis, namely the Kimura

2-parameters model for nucleotide substitution and

the Poisson model for amino acid substitution. The

bootstrap values of the phylogenetic trees were

calculated on the basis of 1000 replicates.

RESULTS

The DDBJ accession numbers for the gene

sequences we determined are LC077734-LC077793,

LC079090-LC080649, and LC082309-LC82335.

Characteristics of the draft genomes are

summarized in Table 1. The Mycobacterium

genomes ranged in size from 4.9 to 7.4 Mbp; the %

G+C content ranged from 63.9% to 69.1%, and the

number of predicted coding sequences ranged from

4746 to 7299.

Protein diversities of l的1cobαcteriumspecies

Mycobacterium tuberculosis H37Rv NC 000962.3

data were used as reference sequences to calculate

protein sequence diversity values for each other

28

Mycobacterium species. The percentage amino acid

difference for each protein from each species was

calculated relative to the M. tuberculosis H37Rv

NC_000962.3 dataset, and each such value was

designated the “diversity value" for the respective

HKP in the respective species. The diversity values

for each protein are shown in Fig. 1. We assessed

the range of diversity values for seven groups of

proteins. Those from: (i) 50S ribosome proteins

(large subunit) ranged from 2.23% to 19.02%; （五） 30S

ribosome proteins (small subunit) ranged from 1.94%

to 12.71 %; (iii) molecular chaperones ranged from

1.86% to 23.53%, (iv) DNA/RNA modification

proteins ranged from 0.12% to 16.36%. (v) DNA/

RNA replication proteins ranged from 6.83% to

24.12%, (vi) tRNA-synthesis proteins ranged from

6.96% to 19.58%, and (vii) cell division目 associated

proteins ranged from 14.53% to 22.41 %.

The protein with the largest average interspecies

diversity value (24.12%) was DnaQ. The 12 most

variable HKPs (with regard to comparisons among

species within the genus Mycobacterium) were the

ribosomal proteins RplY (19.02%) and RplI (18.27%);

the modification enzymes GrpE (23.53%), DnaQ

(24.12%). DnaN (17.24%). HolA (17.05%). PheT

(19.58%). MiaA (18.35%). and MetG (17.89%); and the

FTS proteins FtsQ (22.41 %). Fts Y (21.43%). and FtsX

(18.21%). Notably, among the 107 HKPs. intraspecies

protein variation was small.

Analysis of a concatenated sequence comprising

50 ribosome proteins (C50RP)

The C50RP sequence of M. tuberculosis H37Rv

(the type strain) comprised 6802 residues. The

Microb. Resour. Syst. June 2016

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Fig. I Graphical depiction of mean diversity value for each of 107 Mycobαcterium housekeeping proteins (HKPs), with M. tuberculosis used as the reference species. HKPs are listed along the Y axis. The X-axis shows the

averag巴%amino acid differences between the M. tuberculosis H37Rv reference sequenc巴 andother

Mycobacterium sequences. The HKPs are classified into seven groups: Group l, 50S ribosome proteins; Group 2,

30S ribosome proteins; Group 3, molecular chaperones; Group 4, DNA/RNA modification proteins; Group 5, DNA/

RNA replication proteins; Group 6, tRNA-synthesis proteins; and Group 7, cell division proteins.

7

Diversi句I (%)

-29一

Core proteins for mycobacteria

C50RP diversity values among the 30 species

ranged from 0.06% to 14.65% (mean diversity 9.08%)

(Table 2).

Intraspecies diversity of the C50RP values ranged

from 0% to 1.26% (data not shown); these values

were about double those obtained with the 16S

rRNA gene data (Fig. 2a and Table 2). On the basis

of the C50RP sequence analysis, phylogenetic trees

were constructed by using a neighbor-joining

algorithm (Fig. 3b). The bootstrap values at each

node in the C50RP tree were higher than those for

any node in the 16S rRNA gene tree (Fig. 3a).

The phylogenetic relationships determined by

using the C50RP data corresponded well with those

determined by using the 16S rRNA gene tree. The

correlation coefficient between C50RP and 16S

rRNA gene sequence diversity was 0.75 (Fig. 4a).

Phylogenic analysis based on a concatenated

protein sequence (Cl2HKP) generated by using the

12 most variable HKPs

The Cl2HKP sequence obtained by using the M.

tuberculosis H37Rv data comprised 3928 residues.

With the Cl2HKP data from 30 species, the

diversity values ranged from 0.08% to 37.23% (mean

diversity 22.50%) (Tab！巴 2). The degree of diversity

with the Cl2HKP data was larger than that with

Mizuno et al.

the 16S rRNA gene, C50RP, Dna]l, RpoB, RecA, or

GyrB data (Fig. 2a and 2b and Table 2).

On the basis of the Cl2HKP sequence analysis,

phylogenetic trees were constructed by using a

neighbor-joining algorithm (Fig. 3c). The bootstrap

values of the Cl2HKP tree were similar to those of

the C50RP tree, but the diversity values calculated

with the Cl2HKP data were larger than those

calculated with the C50RP data (Fig. 2a and 2b and

Table 2).

The correlation coefficient between the Cl2HKP

and C50RP sequence diversities was 0.94 (Fig. 4b);

notably, this value was bigger than the correlation

coefficient between the Cl2HKP and 16S rRNA

gene sequence div巴rsities(0.66) (Fig. 4a).

Differentiation between closely related species

Data from individual HKPs, the 16S rRNA gene,

C50RP, or Cl2HKP were used to compare between

closely related, slow-growing Mycobacterium species;

we define d“closely relate d”as species that

exhibited >99% 16S rRNA gene sequence identity

(Table 2). Discrimination by using Cl2HKP was

better than that with C50RP or the 16S rRNA gene.

DISCUSSION

Differentiation among closely related species by

Table 2 ANI values and diversity values based on 16S rRNA gene, C50RP, Cl2HKP, DnaJl, RpoB, RecA, or GyrB data

Mycobacterium species ANP value (%)

Diversity (%)

16S rRNA C50RP

gene Cl2HKP RpoB 旬

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D

Rec A GyrB

Average diversities among 4.01 9.08 22.50 14.45 6.08 4.66 10.49 30 species (Range) (0.00-7.88) (0.06 14.65) (0.08-37.23) (0.00-26.84) (0.00 10.30) (0 10.08) (0.00 17.53)

M. tuberculosis complexb 0.00 0.00-0.19 0.00-0.36 0.00-0.51 0.00-0.37 0.00 0.00-0.15 99.86-99.96 M. tuberculosis-M.悦 α：rinu悦 0.79 5.48 16.84 11.03 4.29 2.82 8.86 80.1 M tuberculosis-M. ulcerans 0.93 5.76 17.44 11.53 4.38 3.23 9.01 80.12 M. marunum-M. ulcerans 0.14 0.98 1.14 0.51 0.09 0.40 0.15 98.97 M. avium complex' 0.00-0.36 0.09 1.27 1.09-3.95 0.00-2.81 0.00-1.10 0.00 0.00-0.15 98.64-99.23 M. avium-M.intracellulare 0.65-1.00 1.99-3.17 6.21-8.48 0.00-2.81 0.00-1.10 0.00 1.92-2.07 86.01-88.58 M. avium-M. szulgai 1.00 5.33-5.57 15.46-17.08 9.41-10.43 2.75-2.84 2.02 6.06-6.20 80.51-80.69 M. int:γαcellulare-M. szulgai 0.86 5.80 15.79 9.41 2.84 2.02 5.17 80.35 M. kansasii-M. malmoense 0.86 4.95-4.97 13.87 10.58 4.72 2.42 7.98 81.65-81.69 M. kansasii-M. szulgai 1.00 4.78-4.79 12.89 12.37 4.12 0.81 9.16 80.68

"ANI; Average nucleotide identity bDiversity values calculated based on comparisons among species within the M. tuberculosis complex (M. tuberculosis H37Rv, M. bovis AF2122/97, M. bovis BCG str. Pasteurll73P2, M. africanum GM041182 and M. canettii CIPTl 400100590). 'Diversity values calculated based on comparisons among M.。viumsubspecies (M. avium subsp. avium ATCC 25291, M. avium 104, M. avium JNBP _03535, and M. avium subsp.仰ratuberculosisK 10).

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Microb. Resour. Syst. June 2016 Vol. 32, No. 1

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b: 16S rRNA gene/Cl2HKP

Fig. 2 Comparison between 16S rRNA gene and C50RP or Cl2HKP sequences with regard to diversity values

X-axis indicates the percent difference (diversity) for each Mycobacterium sequence relative to the M. tuberculosis H37Rv

reference sequence. Y-axis shows each individual Mycobacterium strain. a, Comparison between 16S rRNA gene and

C50RP data. b, Comparison between 16S rRNA gene and Cl2HKP data. C50RP diversity was double the 16S rRNA

gene diversity. Cl2HKP diversity was four times the 16S rRNA gene diversity. Group 1: M. tuberculosis complex (M.

tuberculosis H37RvT, M. africanum GM041182, M. bovis subsp. bovis AF2122/97, M. bovis BCG str. Pasteur 1173, M.

canettii CIPT140010059); Group 2: M. kansasii (JNBP03473, JNBP03464); Group 3: M. marinum group (M. marinum M

strain M. ulcerans Agy99); Group 4: M. avium (M. avium 104, 11主 aviumJNBP03535, M. avium. subsp.ρaratuberculosis

KlO, M. avium A TCC25291 T): Group 5：・ M.ρeregrinum(JNBP 03881, JNBP 03577, JNBP 03578, JNBP03576T): Group 6: M.

Jortuitum (JNBP 03252, JNBP 03300T); Group 7: M. vaccae (JNBP 03730T, JNBP 03729); Group 8・M.abscessus (JNBP 03165,

JNBP 03167); Group 9: M. chelonae (JNBP 03238, JNBP 03213)

Core proteins for mycobact巴ria

a M C四oeUiiCIPT140010059 M q庁icanumGM04 l l 82 M 加berculosisH3 7豆VT

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Mizuno et al.

Fig. 3 Neighbor-joining phylogenetic trees based on 16S rRNA gene (a), C50RP (b), or Cl2HKP (c) sequences and

obtained by using 42 Mycobαcterium strams. Percentage at each node represents a bootstrap value (1000

trials). Scale bar represents 2% sequence divergence. Only bootstrap values >80% are shown.

-32一

Vol. 32. No. 1 Microb. Resour. Syst. June 2016

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（企） vs. C12HRP diversity (b). Each data point represents a pair of taxa and is plotted according to the pair

wise sequence di妊erence. Each solid line represents a regression line. Each r valu巴 representsthe correlation

coefficient.

Fig. 4

Core proteins for mycobacteria

using Cl2HKP

The phylogenetic trees constructed with either

the Cl2HKP data or the C50RP data had very high

bootstrap values; therefore, either of these trees was

presumably more r巴liablethan the 16S rRNA gene

tree. The correlation coefficient between the

Cl2HKP and C50RP sequence diversities was 0.94.

Nevertheless, the mean diversity (22.50%) and

diversity range (0.08% to 37.23%) calculated with the

Cl2HKP data were higher than the mean diversity

(9.08%) and range (0.06% to 14.65%) calculated with

the C50RP data. ANI can significantly distinguish

closely related species, but it cannot be used to

analyze phylogenetic relationships among distantly

related species. Cl2HKP could be used for both

types of analysis. Therefore, we used the Cl2HKP

data to analyze and compare three genetically

related species complexes, namely the M.

tuberculosis complex, the M. avium-intracellulare

complex, and the M. marinum-M. ulcerans group.

Within the M. tuberculosis complex, five

established species (M. tuberculosis, M. bovis, M.

africanum, M. cゆrae,and M. microtz) and a recently

described putative progenitor species "M. canettii”

have almost identical 16S rRNA gene sequences.

The ANI for four of the genomes analyzed (M.

tuberculosis, M. bovis, M. africanum, and “M.

canettii ) was higher than 99% (Fig. 2a and Table 2).

The Cl2HKP variation within the M. tuberculosis

complex was less than 0.36% (Fig. 2b and Table 2).

Therefore, these species could not be di妊erentiated

from one another, even with the Cl2HKP dataset.

ANI values of more than 95% correspond to the

traditional >70% DNA-DNA reassociation standard

currently used for definition of a species

(Konstantinidis & Tiedje, 2005). The close

relatedness between M. tuberculosis complex

bacteria has been established by DNA-DNA

hybridization, 16S rRNA gene analysis, and

housekeeping gene analysis (Baess, 1979; Feizabadi

et al., 1996). Mycobacterium tuberculosis complex

may be considered a subspecies of M. tuberculosis

(Tsukamura et al., 1985; Wayne and Kubica, 1986).

Our data supported the conclusion that the species

in the M. tuberculosis complex can be classified as a

single species.

The strains that constitute the M. avium M.

intracellulare complex have very similar 16S rRNA

gene sequences. Variation in the 16S rRNA gene

sequence ranged from 0.65% to 1.00% (Table 2), and

Mizuno et al.

it was difficult to differentiate between any two

species on the basis of the 16S rRNA gene data.

However, the ANI value for M. avium A TCC 25291

and M. intracellulare A TCC 13950 was 88.58%; this

value indicated that these two strains were

independent species; moreover, the Cl2HKP

variation between these strains was 6.21 %. Notably,

it was possible to differentiate between these two

species on the basis of the Cl2HKP-sequence

companson.

Another closely related pair of species, M.

marinum and M. ulcerans, had 99.86% identity in

16S rRNA gene sequences. The ANI value for

these two species was 98.97%. These data indicated

that the two species were identical. Similarly, the

Cl2HKP diversity between the two species was only

1.14%. Stinear et al. (2000, 2007) indicated that M.

marinum and M. ulcerans should be classified as a

single species. Our data, as in Table 2, supported

出isopinion.

Effective use of the three data sets

Polymorphism in the three data sets -16S rRNA

gene, Cl2HKP, and C50RP一hasdi佐 rentmeanings.

Current bacterial systematics is based on 16S rRNA

gene sequence diversity. Because the 16S rRNA

genes are shared among all prokaryotes and have

commonly shared conserved sequences, it is possible

to use them to compare all microorganisms.

The Cl2HKP data set was effective for

di妊erentiatingclosely related species within a genus.

However, because the types of genes held in

common differ at the taxonomic level of family or

higher, this data set cannot be applied at these

higher taxonomic levels. The diversity of the

C50RP data set was mid-way between those of the

16S rRNA gene and Cl2HKP data sets. Ribosomal

proteins in this data set are found in the higher

taxa, but each gene sequence polymorphism is

difficult to compare because the variations are too

big and conserved sequences are not found in each

ribosomal protein. Within the family

Corynebacteriaceae, however, members of the genus

Mycobacterium shared common amino acid

mutations in many ribosomal proteins (data not

shown). This suggests that C50RP might be helpful

in re目evaluatingthe taxonomic position of current

species, although genome-based genus criteria have

not yet been fully discussed.

-34-

Microb. Resour. Syst. June 2016

ACKNm司「LEDGMENTS

This work was supported mainly by the Japanese

National Bioresource Project and the NBRP Genome

Information Upgrading Program.

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抗酸菌の分類同定指標として有効なコアーハウスキーピング遺伝子

水野卓也 1.2），名取達矢 1)，金津泉 1），イブラヒム・エルデソウキー 3），福永肇 1），江崎孝行 1)

1)岐阜大学大学院再生医科学病原体制御分野， 2）岐阜県保健環境研究所，

3）カーフレルシェイク大学獣医学部細菌菌類免疫学講座

Mycobacterium属30菌種42株のドラフトゲノム解析を行い，分類と同定に有効な housekeepingprot巴in(HKP）を選択し

解析した NCBIの遺伝子の各分類区分から解析菌種に共通に保有され，多型の大きい 107タンパクを選択したそこから

さらに多型が大きく，大きな脱落と挿入がある遺伝子を除外し，菌種識別に有用な 12個の完全長の HKPを連結し Cl2HKP

として解析した. Cl2HKPは16SrRNA遺伝子との相関は 0.66で，比較に使用した 50個の ribosomalprot巴in(C50RP）と

は0.94と高い相関があった. Cl2HKPの解析菌種間での多型の大きさは 22.50%,C50RPでは 9.08%.16S rRNA遺伝子で

は4.01%であり， Cl2HKPが最も大きな多型を示した このことから Cl2HKPは類縁菌種の識別と同定に最も有用性が高

いことが証明された．

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