Application of the variable region in 16S rDNA to create an index for rapid species identification in the genus Streptomyces

FEMS Microbiology Letters 151 (1997) 249^255

Application of the variable region in 16S rDNA to create an index for rapid species identi¢cation in the genus Streptomyces Masakazu Kataoka b

1 ;2 ;a

, Kumiko Ueda 3 a , Takuji Kudo b , Tatsuji Seki a *, Toshiomi Yoshida a ;

;

a The International Center for Biotechnology, Osaka University, Yamada-oka, Suita, Osaka 565, Japan Japan Collection of Microorganisms, Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako, Saitama 351-01, Japan

Received 10 April 1997 ; accepted 14 April 1997

Abstract

Partial nucleotide sequences (120 bp) of the 16S rRNA gene (rDNA) containing a variable K region were compared in 89 strains of the genus Streptomyces belonging to eight major clusters of category I in Bergey's Manual of Systematic Bacteriology. Fifty-seven kinds of partial 16S rDNA sequences were observed among the 89 strains. Forty-three of the strains were grouped into 11 `identity groups', based on the fact that the strains in each group shared an identical sequence in the 120bp region. The results of a phylogenetic analysis based on the 16S rDNA 120-bp sequences revealed that 60 of the 89 strains could be categorized into seven clusters, each consisting of four or more strains. Based on these observations it was concluded that short nucleotide sequences bearing the variable K region are useful for Streptomyces species identification. Keywords : Phylogenetic analysis; Streptomyces; 16S rRNA gene

1. Introduction

In the conventional taxonomy of Streptomyces, its systematics is, in practice, based on characteristics of mycelial morphology, pigment production, and cer* Corresponding author. Tel.: +81 (6) 8797453; fax: +81 (6) 8797454; e-mail: [email protected] 1

M. Kataoka and K. Ueda contributed equally to this work.

2 Present address: Mitsubishi-Kasei Institute of Life Sciences, 11 Minami-Ooya, Machida-shi, Tokyo 194, Japan. 3 Present address: Institute for Fermentation, Osaka, Juso-honmachi,Yodogawa-ku, Osaka 532, Japan.

tain physiological properties. In 1964, the International Streptomyces Project (ISP), a collaborative study group on Streptomyces taxonomy, proposed an elaborate description of criteria for some of the authentic and extant type strains of Streptomyces and their related taxa, which is dependent on a limited number of taxonomic characteristics selected from those accumulated in conventional taxonomic tests [1,2]. In 1983, a large-scale numerical phenetic survey of Streptomyces and related taxa was carried out by Williams et al. [3]; 394 strains of Streptomyces-type cultures were examined with respect to 139 taxonomic characteristics and the results were analyzed statistically. Based on the ¢ndings, Streptomy-

0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 1 6 9 - 9

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M. Kataoka et al. / FEMS Microbiology Letters 151 (1997) 249^255

-type strains were grouped into 23 `major clusters' each containing four or more strains, 20 `minor clusters' each containing two to three strains, and 25 `single member clusters' [3]. The results of the above work are compiled in a chapter of Bergey's Manual of Systematic Bacteriology, Vol. 4 [4]. The proposed classi¢cation, however, disagrees with that obtained by the conventional taxonomic methods in several respects. Recent progress in molecular biology and population genetics has dramatically increased the amount of evolutionary information available for species classi¢cation. For example, a database of ribosomal RNA genes, particularly of small subunits of ribosomal RNAs (16S rRNAs), has been compiled and successfully applied to determining phylogenetic relationships in bacteria [5]. In the case of Streptomyces, these studies have revealed that, in some cases, phenetic classi¢cation better re£ects the phylogenetic relationships between the strains examined [6,7]. Here, 89 ISP standard strains belonging to eight major clusters of category I de¢ned by numerical identi¢cation in Bergey's Manual of Systematic Bacteriology [4] were subjected to a sequence comparison of a part of their 16S rRNA gene containing a highly variable K region and to phylogenetic analysis. From the phylogenetic relationships of these strains, we propose the usefulness of a data base generated on the basis of partial rDNA sequences for the rapid identi¢cation of species of the genus Streptomyces. ces

2. Materials and methods

2.1. Strains and culture conditions

The 89 strains of the genus Streptomyces used in this study (see Fig. 3) were obtained from the Japan Culture Collection of Microorganisms (JCM; Riken, Wako, Saitama, Japan). All these strains belong to eight major clusters (species groups) of category I, de¢ned by numerical classi¢cation, in Bergey's Manual of Systematic Bacteriology [4]: S. albido£avus, S. anulatus, S. halstedii, S. exfoliatus, S. violaceus, S. fulvissimus, S. rochei and S. chromofuscus

. They were cultivated by the methods recommended in the JCM Strain Catalogue [8].

2.2. PCR ampli¢cation and sequencing of a part of 16S rDNA

To achieve the reliable sequencing of the partial 16S rDNA obtained by PCR ampli¢cation from various Streptomyces strains, direct sequencing of PCR products were carried out. Chromosomal DNA for the PCR template was prepared from cells in a single colony formed on a plate using an Insta Gene kit (Bio-Rad) according to the supplier's protocol. DNA fragments covering the variable region of 16S rDNA was ampli¢ed using phosphorylated sense and nonphosphorylated anti-sense primers [10]. The sense primer was phosphorylated using T4 polynucleotide kinase (Takara Shuzo) and ATP. PCR was performed in 50 Wl reaction mixture (10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2 ; 0.001%, w/v, glycerol) for 30 or 40 cycles of denaturation (for 30 s at 97³C), annealing (for 1 min at 50³C) and extension (for 1 min at 72³C) with Amplitaq DNA polymerase (Perkin Elmer). Whole samples were fractionated by agarose gel electrophoresis, and PCR products of 500 bp were recovered using a Gene Clean II kit (Bio 101). The phosphorylated sense strand was digested for 1 h at 37³C with V-exonuclease (BRL) in the recommended bu¡er (67 mM glycine-KOH, pH 9.4; 2.5 mM MgCl2 [9]). After phenol/chloroform extraction, the remaining antisense strand was used as a sequencing template. DNA sequences were determined using a 7-deaza dGTP Sequenase Version 2.0 kit (USB) according to the supplier's protocol, except that 1 Wg of singlestranded DNA binding protein (SSB, Stratagene) was added to 15 Wl of the reaction mixture. After termination of the reaction, SSB was digested with 20 Wg proteinase K (Sigma) for 30 min at 37³C in 6 Wl of the termination mixture. 2.3. Oligonucleotides

Nucleotide sequences of the synthesized oligonucleotides used were as follows. Sense primer for PCR: 5P-TCACGGAGAGTTTGATCCTG-3P ; anti-sense primer for PCR: 5P-GCGGCTGCTGGCACGTAGTT-3P ; sequencing primer: 5P-AGTAACACGTGGGCAATCTG-3P. The primer sequences were selected from the conserved region and corresponded to nucleotide positions 1^20, 481^500, and 105^124, re-

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M. Kataoka et al. / FEMS Microbiology Letters 151 (1997) 249^255

Fig. 1. Phylogenetic relationships among published nucleotide sequences of the 16S rRNA gene in seven

251

Streptomyces

strains. The phylo-

genetic trees were constructed by the neighbor-joining method using whole (left) and partial (right) sequences of the 16S rRNA gene. The bar labeled 0.05 indicates 5 base changes per 100 nucleotides.

spectively, according to the

ciens

Streptomyces ambofa-

rDNA sequence [11].

estimated by the method of Kimura [12]. The phylogenetic tree was constructed by the neighbor-joining method [13].

2.4. Analysis of rDNA sequences and construction of phylogenetic trees 3. Results and discussion

Nucleotide sequences of the ampli¢ed products were edited by the GENETYX program (Software Development)

on

a

PC9801

personal

computer

(NEC). Evolutionary analysis of nucleotide sequen-

3.1. Selection of a variable region suitable for phylogenetic analysis in the 16S rRNA gene of Streptomyces

ces was performed using the ODEN program (National Institute of Genetics, Japan). For construction

To identify the most informative region of 16S

Streptomyces

of the evolutionary genetic distance matrices, fre-

rDNAs for the phylogenetic study of

quencies of base substitutions per nucleotide were

species, the nucleotide sequences of 16S rDNAs of

Fig. 2. Sequence variation in the variable region of 16S rRNA among 17 corresponding to the 16S rRNA gene of

S. ambofaciens.

Streptomyces

strains. Numerals indicate the nucleotide positions

The region of nucleotide positions 172^202 is the most variable region, and is be-

lieved to form a stem and loop structure (see text). Asterisks represent perfectly conserved bases in the 17 species so far examined.

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M. Kataoka et al. / FEMS Microbiology Letters 151 (1997) 249^255

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M. Kataoka et al. / FEMS Microbiology Letters 151 (1997) 249^255 Fig. 3. Phylogenetic tree constructed from the 120-bp

K

region sequences of 89

Streptomyces

253

strains belonging to eight major clusters of

category I in Bergey's Manual of Systematic Bacteriology [4]. Strain names are followed by the description in JCM catalogue but are cited as subjective synonyms in Bergey's Manual except for the type strains of the major cluster. The number preceding the species name indicates a major cluster of category I used in Bergey's manual : 1,

olaceus ;

6,

S. fulvissimus ;

S. rochei ;

7,

cession numbers (D) for the 120-bp

6

K

8,

S. chromofuscus.

S. albido£avus ;

S. anulatus ;

2,

3,

S. halstedii;

4,

S. exfoliatus ;

5,

S. vi-

JCM strain numbers (J), ISP strain numbers (I) and DDBJ DNA sequence ac-

region sequences are given along with the strain names. The IG and PC group symbols are shown

in bold letters. The bar labeled 0.05 indicates 5 base changes per 100 nucleotides.

Streptomyces lividans S. griseus [16],

[15],

S. coelicolor A3(2) S. ambofaciens [11] were

TK21 [14],

nucleotides in the stem were changed together. The

and

GCAT sequence in the loop was found to be highly

aligned. This enabled the selection of a 120-bp region

conserved, indicating that it may have an important

from nucleotide positions 158^277 of the 16S rRNA

role in the ribosome function. Only three strains,

S. ambofaciens,

gene of iable

K

as it includes the most var-

region [17].

To test the verity of a phylogenetic tree based on partial sequences of the 16S rRNA gene, we compared

two

phylogenetic

trees

constructed

by

alboniger (Fig. spheroides (not having

a

S. niveus

2),

(not

shown),

and

S. S.

shown), di¡ered from the others in

TCCT

sequence

at

the

corresponding

GCAT site.

the

Among the 89 strains, 57 kinds of 120-bp nucleo-

neighbor-joining method using published total and

tide sequences could be identi¢ed. In particular, 11

Streptomyces

identical sequences were shared by 47 of the strains :

strains [11,14^16,18]. We found the general topolo-

each sequence was shared by two to eight strains.

gies of the trees from the partial and total sequences

The groups containing these strains were designated

to be similar (Fig. 1), but the genetic distance to be

as `identity groups' (IGs). It is apparent from Fig. 3

more emphasized in the partial-sequence tree. This

that the strains in each IG belong to the same re-

observation indicated that the variation in the partial

spective numerical major cluster, the only exception

16S rDNA sequences covering the variable region is

being IG-G which includes strains from the major

su¤cient for deducing phylogenetic relationships of

clusters of

Streptomyces

fact

partial 16S rRNA sequences of seven

strains

at

the

intra-species

level,

that

S. exfoliatus

K

the

region

and is

S. violaceus.

the

most

From the

variable,

it

is

although these segments are probably too variable

thought that strains labeled as various di¡erent spe-

for comparison at the generic level.

cies names but sharing an identical sequence may actually belong to a single species even though minor

3.2. DNA sequences of 89 independent Streptomyces strains A 500-bp DNA sequence, partially encompassing the

16S

sample

rRNA

gene,

prepared

was

from

a

ampli¢ed

single

from

colony

of

a

DNA

each

in

K region was determined (see Fig. 3 for the

nucleotide

assisted

the

hyper-variable

characteristics

re-

To clarify the phylogenetic relationships of the 89

Streptomyces the variable

of

phenotypic

3.3. Phylogenetic relationship through the partial sequence

DDBJ accession numbers of these sequences). The sequences

their

sulted in their being classi¢ed as di¡erent.

of

the 89 strains, and the 120-bp sequence bearing the variable

di¡erences

region

strains, the 120-bp sequences bearing

K

region were subjected to computer-

phylogenetic

analysis

for

the

construction

from nucleotide positions 172^202 in 17 out of the

of a phylogenetic tree (Fig. 3). This tree has seven

89 strains, as well as some variations outside of the

major

region, are shown in Fig. 2. It has been suggested

PC-G), each comprised of four or more strains ; 60

that the hyper-variable region forms a stem and loop

of the 89 strains were classi¢ed into these seven clus-

structure in vivo in

S. ambofaciens

16S rRNA [11].

phylogenetic

clusters

(classi¢ed

as

PC-A

to

ters. Williams et al. [3] have shown that the numer-

S. albido£avus, S. anulatus, and S.

The conservation of the secondary structure is par-

ical major clusters,

ticularly notable, but the sequences forming the stem

halstedii,

are the most variable : the most of complementing

to form a large `super cluster'. This is partially in line

FEMSLE 7597 2-9-97

are closely related, and can be considered

M. Kataoka et al. / FEMS Microbiology Letters 151 (1997) 249^255

254

with our results, the exception being the strains of the

S. albido£avus major cluster ;

according to the

phylogenetic tree generated (Fig. 3), these are not closely related to the strains belonging to the major

S. anulatus, and S. halstedii. In our tree, strains of the S. albido£avus major cluster are

clusters of all the

grouped

into

phylogenetic

cluster

PC-D

with

less

than three base substitutions. The homogeneity of these strains has also been suggested by the results

of type cultures of

Streptomyces. IV. Species descriptions from

the second, third and fourth studies. Int. J. Syst. Bacteriol. 19, 391^512. [2] Shirling, E.B. and Gottlieb, D. (1972) Cooperative description of type strains of

Streptomyces.

V. Additional descriptions.

Int. J. Syst. Bacteriol. 22, 265^394. [3] Williams,

S.T.,

Goodfellow,

M.,

Alderson,

G.,

Wellington,

E.M.H., Sneath, P.H.A. and Sackin, M.J. (1983) Numerical classi¢cation of

Streptomyces

and related genera. J. Gen. Mi-

crobiol. 129, 1743^1813. [4] Williams, S.T., Goodfellow, M. and Alderson, G. (1989) Ge-

Streptomyces

AL .

from whole 16S rRNA sequences [19], chromosomal

nus

DNA hybridization [20], pyrolysis mass spectrometry

Bergey's Manual of Systematic Bacteriology, Vol. 4 (S.T. Wil-

[21] and fatty acid analysis [22]. PC-B is the largest cluster in our tree, comprising 19 of the 25 strains of the

S. anulatus major cluster and ¢ve of the seven S. halstedii major cluster but it is not

strains of the

closely related to PC-D. Strains of other numerical cluster

were

found

to

be

distributed among

other

phylogenetic clusters or were independent. The identity groups IG-J and IG-K are closely related to PCG. The two strains belonging to IG-K,

S. £aviscle-

rotiacus and S. minutisclerotiacus, were formerly classi¢ed as Chainia species, but were later transferred to Streptomyces [23].

Waksman

and

Henrici,

1943,

339

In :

liams, M.E. Sharpe and J.G. Holt, Eds.), pp. 2452^2492. Williams and Wilkins, Baltimore, MD. [5] Woese, C.R. (1987) Bacterial evolution. Microbiol. Rev. 51, 221^271. [6] Gladek, A., Mordarski, A., Goodfellow, M. and Williams, S.T. (1985) Ribosomal ribonucleic acid similarities in the classi¢cation of

Streptomyces.

FEMS Microbiol. Lett. 26, 175^

180. [7] Witt, D. and Stackebrandt, E. (1990) Uni¢cation of the gen-

Streptoverticillum and Streptomyces, and amendation of Streptomyces Waksman and Henrici 1943, 339AL . Syst. era

Appl. Microbiol. 13, 361^371. [8] Nakase, T. (1992) In : JCM Catalogue of Strains, 5th edn. (T. Nakase, Ed.), RIKEN, Saitama, Japan. [9] Higuchi,

R.G.

and

Ochman,

H.

(1989)

Production

of

In conclusion, we believe that the phylogenetic tree

single-stranded DNA templates by exonuclease digestion fol-

presented here will serve as a useful tool for rapid

lowing the polymerase chain reaction. Nucleic Acids Res. 17,

identi¢cation

of

newly isolated

the

phylogenetic

Streptomyces

localization

of

strains, and that it is

likely to prove more e¡ective than the conventional methods. Additionally, it should provide a means for

5865. [10] Saiki, R.K., Gelfand, D.H., Sto¡e, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primerdirected enzymatic ampli¢cation of DNA with a thermostable DNA polymerase. Science 239, 487^491.

easy and appropriate identi¢cation, which is espe-

[11] Pernodet, L.L., Boccard, F., Alegre, M.T., Gagnat, J. and

cially important in the fermentation industry in or-

è rineau, M. (1989) Organization and nucleotide sequence Gue

der to avoid taxonomic confusion of strains dealt with in patent literature.

analysis of a ribosomal RNA gene cluster from

ambofaciens.

Streptomyces

Gene 79, 33^46.

[12] Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111^120.

Acknowledgments We

are

[13] Saitou, N. and Nei, M. (1987) The neighbor-joining method : a

grateful

to

Tobias

Kieser

(John

Innes

new

method

for

16S

partially

Acids Res. 16, 370.

T.Y.

under

the

by

Grant-in

International

Aid

07044149

Scienti¢c

to

Research

Project of the Japanese Ministry of Science, Educa-

phylogenetic

trees.

Mol.

[14] Suzuki, Y. and Yamada, T. (1988) The nucleotide sequence of

Centre, UK) for helpful discussions. This work was supported

reconstructing

Biol. Evol. 4, 406^425.

rRNA gene from

Streptomyces lividans

TK21. Nucleic

[15] Baylis, H.A. and Bibb, M.J. (1989) The nucleotide sequence of a 16S rRNA gene from

Streptomyces coelicolor

A3(2). Nucleic

Acids Res. 18, 831.

tion, Sports and Culture.

[16] Kim, E., Kim, H., Kang, K.H., Kho, Y.H. and Park, Y.-H. (1991) Complete nucleotide sequence of a 16S ribosomal RNA

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