FEMS Microbiology Letters 79 (1991) 57-60
© 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50
Published by Elsevier
A D O N I S 037810979100165Q
57
FEMSLE 04364
A new value for mol percent guanine + cytosine of D N A
for the salmonid fish pathogen Renibacterium salmoninarum
C.R. B a n n e r 1, J.S. R o h o v e c 2 a n d J.L. F r y e r 2
J Oregon Department of Fish and Wildlife, Oregon State University, and e Department of Microbiology, Oregon State University,
Corvallis, OR, U.S.A.
Received 16 November 1990
Revision received and accepted 23 November 1990
Key words: Renibacterium salmoninarum; G + C content; Taxonomy; Phylogeny
1. S U M M A R Y
The mol% G + C of D N A extracted from seven
different isolates of Renibacterium salmoninarum
was determined. Organisms studied were from
selected geographical areas (U.S.A., Canada, England and France) and were isolated from five
different species of salmonid fish. The mol% G + C
was determined to be 55.5, higher than the currently reported value of 53.
2. I N T R O D U C T I O N
The biology, pathophysiology, and phylogenetic position of the causative organism of
bacterial kidney disease have been of interest since
the disease was first described in 1930 [1,2]. Ordal
and Earp, observing characteristics of the organism
in culture and morphology in Gram stain, suggested it belonged to the genus Corynebacteria [3].
Correspondence to." C.R. Banner, Oregon Department of Fish
and Wildlife, Oregon State University, Department of Microbiology, Corvallis, OR 97331-3804, U.S.A.
Research by Sanders and Fryer [4] on the chemical composition of the cell wall and determination of a mol% G + C value of 53, prompted them
to place the bacterium in a new genus, Renibacterium, and give it the species name salmoninarum
[5]. Collins examined the polar lipids of Renibacterium and compared these data to selected members of the genus Corynebacteria. He found no
similarities in polar lipids between the two genera
Renibacterium and Corynebacteria [6]. Kusser and
Fiedler [7] described the primary structure of the
murein of Renibacterium and the polysaccharide
composition of the cell wall. Although there was
no evaluation of the taxonomic position of the
organism, they did note some similarities in murein
structure with the genus Arthrobacter and the
bacterium Peptostreptococcus evolutus. Fiedler and
Draxl [8] examined biochemical properties of the
cell surface of several R. salmoninarum isolates,
including the type strain, and found a high degree
of similarity between strains, including a cell wall
polysaccharide believed to be unique to Renibacterium. Stackebrandt et al. [9], using 16S r R N A
cataloguing, examined the genus, Renibacterium.
The relatively low tool% G + C reported [5] for
Renibacterium seemed to be inconsistent with
58
Stackebrandt's data which indicated a close relationship between Renibacterium and a group of
organisms having a mol% G + C content greater
than 55. Despite the discrepancy in G + C value
the genus fits well in a subdivision of the
actinomycetes that includes Arthrobacter and Micrococcus. Gutenberger et al. [14] determined 97%
of the sequence of the 16S ribosomal R N A of
Renibacterium and compared it with sequences of
16S r R N A from bacteria representing 17 different
genera. Their results were in agreement with those
of Stackebrandt et al. Renibacterium appeared to
be related to the Micrococcus/Arthrobacter subline of actinomycetes, organisms that would be
expected to have a mol% G + C of 55 or greater.
The work described here was initiated to confirm
the reported G + C content of R. salmoninarum or
to establish a new, correct value.
3. M A T E R I A L S A N D M E T H O D S
Seven isolates of R. salrnoninarum were obtained and the D N A extracted (Table 1). The
bacterium was cultivated in K D M 2 broth [10],
harvested by centrifugation, washed twice in distilled water and frozen at - 2 0 ° C for a minimum
of 12 h. One gram of cells was thawed and 4.5 ml
sterile distilled water added along with 6 mg of
crystalline lysozyme (Sigma Chemical C o m p a n y ,
St, Louis, MO) and 0.25 ml Pronase-CB (Calbiochem, LaJolla, CA) at a concentration of 40 m g / m l
in 0.15 M N a C I and 0.1 M EDTA, p H 7.0. The
mixture was incubated at 40 ° C for 4 h and 0.5 ml
of SDS (20% w / v in distilled water) added. The
solution was passed through a French pressure cell
four times at 1100 k g / c m 2 and 0.5 mg ribonuclease
dissolved in 0.15 M NaC1 (pH 5.0) was added. The
mixture was incubated for 2 h at 60 ° C and then
brought to a volume of 25 ml with saline-EDTA
buffer (0.15 M NaC1. 0.1 M EDTA. p H 8.0).
Redistilled phenol saturated with sterile distilled
water was used to deproteinize the cell lysate and
D N A was purified by the method of Johnson [11],
using hydroxylapatite (Bio-Gel-HPT, D N A grade,
BioRad Laboratories, Pdchmond CA). Purified
D N A was dialysed against a minimum of 400
volumes of saline-Hepes buffer (0.02 M NaC1.
0.001 M N-2-hydroxy-ethylpiperazine-N'-ethanesulfonic acid, sodium salt, p H 7.0) for 12 h and
frozen at - 7 0 ° C . Mole percent guanine plus
cytosine was determined by thermal denaturation
in 0.1 x SSC (0.015 M NaC1, 0.0015 M trisodium
citrate, p H 7.0). Absorbance at 260 nm was recorded with each temperature rise of 1 ° C between
50 and 90 ° C using a Beckman model DU-8 computing spectrophotometer equipped with a T m
compuset module. The mol% G + C was calculated by the equation of Mandel et al. [12]. A
reference D N A was purified from Escherichia coli
WP-2, 51 mol% G + C [13].
4. R E S U L T S A N D D I S C U S S I O N
An average mol% G ~ C for R. salrnonmarum
of 55.5 +__43 was determined by thermal denaturation of D N A from seven different isolates (Table
1). The new or higher value of 55.5 seems to be
supported by 16S ribosomal R N A cataloguing data
of Stackebrandt et al. [9]. He noted that the mol%
G + C reported for Renibacterium [4,5] was puzzling in light of the high degree of similarity between Renibacteriurn and the actinomycetes. The
Table 1
Mol% G + C of Renibacterium salmoninarum isolates
Isolate
Origin
Host species
mol% G + C
A T C C 33209 T
McKenzie 80
K-70
KDV-10
Margaree
K-28
3784-L
Oregon, U.S.A.
Oregon, U.S.A.
England
Oregon, U.S.A.
Eastern C a n a d a
France
Western C a n a d a
Oncorhynchus tshawytscha
Oncorhynchus tshawytscha
Oncorhynchus mykiss
Oncorhynchus tshawytscha
Salmo salar
Oncorhynchus kisutch
Onchorhynchus nerka
56.0
55.3
55.0
55.8
55.8
55.9
55.0
59
new or higher value reported here (55.5%) also
seems consistent with the 16S ribosomal R N A
sequencing data of Gutenburger et al. [14] who
also found R. salmoninarum to be closely related
to the actinomycetes, a group whose members all
have mol% G + C of 55 or greater.
ACKNOWLEDGEMENTS
This research was supported by the Oregon
Department of Fish and Wildlife under PL 89304,
Anadromous Fish Act. This is Oregon Agricultural Experiment Station Technical Paper No. 9426.
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