System. App!. Microbiol.

14, 386-388 (1991)

© Gustav Fischer Verlag, StuttgartlNew York

Short communication

Revival of the Species Streptococcus thermophilus

(ex OrIa-Jensen, 1919) nom. rev.

KARL HEINZ SCHLEIFER!, MATHIAS EHRMANN\ ULI KRUSCH 2 , and HORST NEVE 2

1 Lehrstuhl fur Mikrobiologie, Technische Universitat Miinchen, 8000 Miinchen 2, Germany

2 Institut fiit Mikrobiologie, Bundesanstalt fur Milchforschung, 2300 Kiel 1, Germany

Received March 18, 1991

Summary
DNA-DNA hybridization studies under stringent conditions and physiological data provide sufficient
evidence for conferring species rank on Streptococcus saliuarius subsp. thermophilus.

Key words: Streptococcus salivarius subsp. thermotJhilus - DNA-DNA hybridization - Streptococcus

thermophilus

Previous DNA-DNA hybridization experiments using
optimum or relaxed hybridization conditions have indi-
cated a close genetic relatedness between Streptococcus
salivarius and Streptococcus thermophilus (Ottogalli et
all., 1979; Garvie and Farrow, 1981; Kilpper-Balz et al.,
1982). Farrow and Collins (1984) performed DNA-DNA
hybridizations under optimum conditions on a larger
number of S. salivarius and S. thermophilus strains and
confirmed the close relatedness of both taxa. They prop-
osed to reclassify S. thermophilus as S. salivarius subsp.
thermophilus (Farrow and Collins, 1984). However, op-
timum hybridization conditions are not always sufficient
to distinguish closely related species (Schleifer and Stack-
ebrandt, 1983). Preliminary studies by Kilper-Balz (cited
in Schleifer and Kilpper-Balz, 1987) on the type strains of
S. salivarius subsp. salivarius and S. salivarius subsp. ther-
mophilus have yielded DNA similarity values of 30%
under stringent hybridization conditions. The distinct
species status of S. salivarius subsp. thermophilus is also
supported by numerical taxonomic studies using phenetic
characters (Carlson, 1968; Bridge and Sneath, 1982).
In the present paper DNA of a large number of S.
salivarius subsp. thermophilustrains were hybridized
with labelled DNA of the type strains of S. salivarius
subsp. salivarius and S. salivarius subsp. thermophilus.
Twenty four strains of S. saliuarius subsp. thermophilus have
been isolated from cheese, voghurt '1I1d corresponding starter cul-
tures (Krusch et al., 1987; Neue et al., 1989). They are listed in
Table 1.
The strains were grown at 37°C in 100 ml M17 broth (Ter-
zaghi and Sandine, 1975) modified according to Krusch et al.
(1987). The cells were harvested by centrifugation in the late
logarithmic growth phase. The sediment was resuspended in 6 ml
1 X SSC (standard saline citrate buffer; 0.15 M NaCl, 0.015 M
sodium citrate, pH 7.0) containing per ml: 5 mg lysozyme, 50
units mutanolysin and 0.6 mg RNase. The mixture was incubated
at O°C for 30 min and then at 37°C for 15 min. 150 J.ll of 20%
sodium dodecylsulfate were added and the temperature was
raised to 60°C for 10 min. Then 60 J.ll of proteinase K (20 mg/ml)
were added and the mixture was incubated at 50°C for 1 h.
Extraction and purification of DNA followed the procedure de-
scribed by Marmur (1961).
Chromosomal DNA from S. saliuarius subsp. saliuarius DSM
20560 T and from S. saliuarius subsp. thern10philus DSM 20617T
were labelled by nick translation, using y_ 32 p ATP (nick transla-
tion Kit, BRL, Eggenstein). Unlabelled DNA from all strains
studied were applied to a Zeta Probe nylon membrane (Biorad"
Munchen) and a dot blot hybridization was performed as previ-
ously described in detail (Liebl et al., 1991). Stringent hybridiza-
tion conditions (15°C below the melting point of the native
DNA, Schleifer and Stackebrandt, 1983) were applied.
The results are shown in Table 1. It is evident that dena-
tured DNA of S. salivarius subsp. thermophilus strains
formed rather extensive and stable hybrids with labelled
DNA of the type strain of S. salivarius subsp. ther-
mophilus. On the other hand, DNA similarity between
 Streptococcus thermophilus nom. rev. 387

Table 1. Origin of strains and results of

Strains Origin % similarity to labelled DNA from
DNA-DNA hybridization between type
strains of S. salivarius subsp. salivarius
S. salivarius S. salivarius
subsp. salivarius subsp. thermophilus
and S. salivarius subsp. thermophilus
DSM 20560 T DSM 20617T

S. salivarius DSM 20560 T 100 35

subsp. salivarius
S. salivarius DSM 20617T 40 100
subsp. thermophilus
SO 37 82
S3 39 89
S4 36 91
S6 33 91
SIS 38 89
St 37 95
St13 38 90
St22 40 89
ERI 32 74
50 29 78
55a 20 81
55n 46 94
71 43 98
124 42 82
3069 42 92
3070 31 95
3071 36 94
3072 26 96
J31 25 94
t1 28 92
ER2 29 94
BuJon4 30 94
J34-2 43 99
J02/1-1 32 98

these strains and the type strain of S. salivarius subsp.
salivarius was distinctly lower and the formed hybrids
were rather unstable under stringent hybridizahon condi-
tions.
On the basis of phenetic data (Carlson, 1968; Teuber
and Geis, 1981; Bridge and Sneath, 1982; Farrow and
Collins, 1984; Hardie, 1986) and the present DNA-DNA
hybridization studies it is proposed that Streptococcus
salivarius subsp. thermophilus is raised to species rank as
Streptococcus thermophilus (ex Oda-Jensen, 1919) nom.
rev. S. thermophilus can be readily distinguished from S.
salivarius.
Description of Streptococcus thennophilus (ex Orla-
Jensen, 1919) nom. rev. ther. mo' phil. us. Gr. n. therme
heat; Gr. adj. philus loving. M. L. ad. thermophilus heat
loving.
Spherical or ovoid cells 0.7-1.0 [tm in diameter, in pairs
to long chains. Irregular segments and cells can occur at
45 DC. Alpha-hemolysis or none on blood agar. Chemoor-
ganotrophic. Catalase negative. Facultatively anaerobic.
Grows at 45 DC, most strains also grow at 50 DC. No
growth at 15 DC. Survives heating to 60 DC for 30 min.
Does not grow at pH 9.6 or in the presence of 0.1 %
methylene blue. Variable growth in broth with 2% NaCl,
no growth in 3%. B-vitamins and some amino acids are
required. Acid produced from fructose, glucose, lactose,
mannose and sucrose. Acid not produced from N-acetyl-
glucosamine, adonitol, amygdalin, arabinose, cellobiose,
dulcitol, erythritol, gluconate, glycerol, glycogen, inulin,
maltose, mannitol, methyl D-glucoside, methyl D-man-
noside, methyl D-xyloside, rhamnose, salicin, sorbitol, tre-
halose and xylose. Variable results may be obtained for
arbutin, galactose, melizitose, melibiose, raffinose and rib-
ose. Aesculin, casein, gelatine and hippurate are not hy-
drolysed. Starch hydrolysis is variable. DNase and urease
are not produced. Ammonia is not produced from ar-
ginine. This is in accordance with Teuber and Geis (1981)
but in contrast to Hardie (1986). However, all S. ther-
mop hilus strains used in the present study did not produce
ammonia from arginine. Acid phosphatase, alkaline phos-
phatase, alpha-fucosidase, alpha-galactosidase, alpha-
glucosidase, beta-glucosidase, beta-glucuronidase, cysteine
arylamidase, N-acetyl-beta-glucosaminidase, pyrrolido-
nylarylamidase and valine arylamidase negative. Beta-
galactosidase and leucine arylamidase positive. Peptidog-
lycan type: Lys-Ala2_3'
No gr~mp specific antigen has been demonstrated.
Acknowledgement. This work was supported by Bundesmini-
sterium fUr Forschung und Technologie grant 0319274A and the
BRIDGE programme of the European Communities.
388 K. H. Schleifer, M. Ehrmann, U. Krusch, and H. Neve
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