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INTERNATIONAL JOURNAL
OF
SYSTEMATIC BACTERIOLOGY, Jan.
1996,
p.
128-148
Copyright
0
1996,
International Union
of
Microbiological Societies
0020-7713/96/$04.00+0
Vol.
46,
No.
1
Cutting a Gordian Knot: Emended Classification and Description
of
the Genus
Flavobacterium,
Emended Description
of
the Family
Flavobacteriaceae,
and Proposal
of
Flavobacterium hydatis
norn. nov. (Basonym,
Cytophaga aquatilis
Strohl and Tait 1978)
J.-F. BERNARDET,'*
P.
SEGERS,*
M.
VANCA"EYT,2
F.
BERTHE,'
K.
KERSTERS,'
AND
P.
VANDAMME2
Unitk de Virologie et Immunologie Moliculaires, Institut National de la Recherche Agronomique,
78352
Jouy-en-Josas Cedex, France,' and Laboratorium voor Microbiologie,
Faculteit Wetenschappen, University
of
Ghent, B-9000 Ghent, Belgium2
The phylogenetic positions and
G+C
contents of most species belonging to the genera
Flavobacterium,
Cytophaga,
and
Flexibacter
and several related taxa were determined. Most of the strains included in this study
belong to rRNA superfamily
V,
as shown by DNA-rRNA hybridization data, but the three main genera are
highly polyphyletic. Several so-called
Cytophaga
and
Flexibacter
species isolated from soil and freshwater
cluster with the type species of the genus
Flavobacterium, Flavobacterium aquatile,
and with
Flavobacterium
branchiophilum.
The fatty acid and protein profiles of members of this group of organisms were determined. We
provide an emended description of the genus
Flavobacterium
and propose new combinations for the following
7
of the
10
validly described species included in this genus:
Flavobacterium columnare, Flavobacterium JEevense,
Flavobacterium johnsoniae
(we also correct the specific epithet of this taxon),
Flavobacterium pectinovorum,
Flavobacteriurn psychrophilum, Flavobacterium saccharophilum,
and
Flavobacterium succinicans.
A new name,
Flavobacterium hy&tis,
is proposed for
[Cytophaga] aquatilis
Strohl and Tait 1978. The emended genus
FZa-
vobucterium
contains bacteria that have the following main characteristics: gram-negative rods that are motile
by gliding, produce yellow colonies on agar, are chemoorganotrophs and aerobes, decompose several polysac-
charides but not cellulose, and are widely distributed in soil and freshwater habitats. Three
Flavobacterium
species are pathogenic for fish. The
G+C
contents of
Flavobacterium
DNAs range from 32 to 37 mol%. An
emended description of the family
Flavobacteriaceae
is also provided.
The long and complex history of the genera
Flavobacterium,
Cytophaga,
and
Flexibacter
and the heterogeneity of these gen-
era have been well documented. The most recent reviews of
the taxonomy of these organisms were published in
Bergey's
Manual
of
Systematic Bacteriology
(40, 68), in
The Prokaryotes
2nd ed. (37, 69), and in
Advances
in
the Taxonomy and Signif-
icance
of
Flavobacterium, Cytophaga and Related Bacteria
(38,
70). Because of the numerous phenotypic similarities of
Fla-
vobacterium, Cytophaga,
and
Flexibacter
strains, for a long time
differentiation of these genera has been based on the presence
(in the genera
Cytophaga
and
Flexibacter)
or absence (in the
genus
Flavobacterium)
of gliding motility. This characteristic,
whose relevance for genus delineation has been questioned, is
probably an ancestral property of this bacterial group that was
lost by some organisms in the course
of
evolution
(68,
101).
The genus
Flavobacterium
was created in 1923 (7) to accom-
modate gram-negative, non-spore-forming, yellow-pigmented
rods that produce acid from carbohydrates weakly
(40).
Be-
cause of this limited definition, the genus rapidly acquired
many poorly defined species and consequently became very
heterogeneous. However, through successive emendations, the
genus
Flavobacterium
was restricted to nonmotile and nonglid-
ing species and thus achieved what could be considered rea-
sonable homogeneity in
Bergey
's
Manual
of
Systematic Bacteri-
ologv
(40). The acceptability
of
Flavobacterium aquatile
as
the
*Corresponding author. Phone:
(33)
1
34-65-25-87.
Fax:
(33)
1
34-65-25-91.
type species of the genus
Flavobacterium
has been discussed
repeatedly (37, 38, 40). This species is represented by a single
strain, which is not the strain that was described originally, and
several studies have demonstrated that
Flavobacterium aquatile
is
indeed more closely related to certain
Cytophaga
species than
to other
Flavobacterium
species;
Flavobacterium aquatile
exhib-
its swarming and gliding motility under certain conditions, and
the structure of its cell wall is similar to that
of
[Cytophaga]
johnsonae
(29) (brackets indicate generically misclassified bac-
teria). As
Flavobacterium aquatile
was not considered repre-
sentative of the genus, Holmes and Owen requested that this
name be rejected as a nomen dubium and the species be
replaced with
Flavobacterium breve
as the type species
of
the
genus (39). Because this request was denied by the Judicial
Commission
(96),
Flavobacterium aquatile
must be retained as
the type species. Several species previously placed in the genus
Flavobacterium
have been reclassified and placed in new or
different genera, including the genera
Beigeyella
(85),
Cyto-
phaga
(68),
Empedobacter
(85),
Sphingobacterium
(37,
102),
and
Weeksella
(37, 43, 44). Recently, the genus
Chryseobac-
teriuin
has been proposed for several species previously in-
cluded in the genus
Flavobacterium;
some of these species are
found in aquatic environments, while others are human or fish
pathogens
(85).
Several new
Flavobacterium
species have also
been described recently (24, 54).
The genus
Cytophaga,
which was created in
1929
by Wino-
gradsky for aerobic cellulolytic gliding soil bacteria, was
sub-
sequently expanded to include many environmental gliding
128
VOL.
46, 1996 CLASSIFICATION OF THE GENUS
FLA
VOBACTERIUM
129
organisms that degrade several polysaccharides (e.g., agar,
chitin, pectin, heparin, and carboxyme thy1 cellulose) but form
neither microcysts nor fruiting bodies
(68).
As a consequence,
this genus also became very heterogeneous. The same is true
for the genus
Flexibacter,
which was created by Soriano in 1945
for soil and freshwater bacteria that were phenotypically sim-
ilar to
Cytophagu
strains but were not able to degrade cellu-
lose (77). The description of the genus
Flexibacter
was later
emended
so
that it included organisms that produced long,
slender, thread-like cells in young cultures, did not degrade any
polysaccharide, and produced yellow, red, or pink pigments
(50).
Because these criteria were not sound and failed to deal
with the obvious heterogeneity of the genera
Cytophaga
and
Flexibacter,
Reichenbach proposed that these
two
genera
should be distinguished on the basis of changes in cell mor-
phology in aging cultures, G+C content ranges, and habitats
(68).
The bacterial species that were similar to
Flexibacter
spe-
cies but were isolated from marine environments were placed
in the genus
Microscilla,
which was described by Pringsheim in
1951
(66)
and was emended by Lewin
(50,
68).
Since the mid-l980s, 16s rRNA oligonucleotide catalog
(63)
and sequence
(30,
45, 56, 59,
100,
101) data and DNA-rRNA
hybridization data
(6,
72, 73,
85)
have shown that the genera
Flavobacterium, Cytophaga,
and
Flexibacter
belong to one of
the 10 main phylogenetic branches of the
Bacteria.
Depending
on the authors, this branch is called the
Cytophaga-Flavobac-
terium-Bacteroides
group, rRNA superfamily
V
(73), or the
“flavobacter-bacteroides”
phylum
(30).
More surprising is the
fact that this group, which is composed of aerobic bacteria for
the most part, also includes the capnophilic genera
Capnocy-
tophaga, Riemerella,
and
Omithobacterium
(58,
72,
88)
and the
obligately anaerobic genera
Bacteroides
(63),
Mitsuokella
(36),
Prevotella, Porphyromonas,
and
Rikenella
(62). Additional data
have revealed that the following other taxa also belong to the
Cytophaga -Flavobacterium -Ba cteroides
group: the family
Spiroso
-
maceae
(including the genera
Spirosoma, Runella, Flectobacil-
lus,
and
Cyclobacterium)
(67, 98), the recently described genus
“Tuxeobacter”
(names in quotation marks have not been validly
published) (71), and the genera
Saprospira, Haliscomenobacter,
Microscilla, Flexithrrjc, Sporocytophaga,
and
Chitinophaga
(68).
In addition, phylogenetic analyses have revealed that
two
ther-
mophilic organisms,
Thermonema lapscim
(64) and
Rhodother-
mus marinus
(5),
are also included in the
Cytophaga-Flavobac-
terium-Bacteroides
group. Recent data from 23s rRNA
sequencing studies have suggested that the closest relatives of
the
Flavobacterium-Bacteroides-Cytophaga
group are the green
sulfur bacteria (99).
The following branches
of
rRNA superfamily
V
have been
studied previously in detail by the DNA-rRNA hybridization
technique: the
Chryseobacterium-Bergeyella-Riemerella
branch
(72,
85);
the
Weeksella-Empedobacter
branch
(85);
and the
Omi-
thobacterium, Capnoqtophaga,
and
Sphingobacterium
branches
(72,
80,
81,
88).
In order to determine the phylogenetic rela-
t ions hips within the
Cytophaga -Fluvobackv-ium -Ba cteroides
group more precisely, we performed extensive DNA-rRNA
hybridization experiments and determined guanine-plus-cy-
tosine (G+C) contents; in this study we used the type strains
and well-characterized isolates
of
most valid and invalid spe-
cies belonging to the genera
Cytophaga
and
Flexibacter,
as well
as several members
of
the genera
Flavobacterium
and
Sphin-
gobacterium
and related taxa. One major rRNA cluster, which
contained the type species
of
the genus
Flavobacterium, Fla-
vobacterium aquatile,
was also studied by using a polyphasic
approach that included fatty acid analysis and sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of
whole-cell proteins in order to determine the relationships at
the generic level. Because
of
the phenotypic similarity between
[
Cytophaga] johnsonae
and
[Flexibacter] aurantiacus,
DNA-
DNA hybridization experiments were performed with four
[Cytophaga] johnsonae
strains and the only two
[Flexibacter]
aurantiacus
strains available in order to determine the levels of
DNA relatedness of these organisms.
Our results showed that the genera
Cytophuga
and
Flexi-
bacter
are highly polyphyletic and that most
Cytophaga
and
Flexibacter
species are only distantly related to their respective
type species (i.e.,
Cytophaga hutchinsonii
and
Flexibucterflexilis,
respectively). Several
of
these generically misnamed species,
including soil and freshwater isolates, are close relatives
of
Flavobacterium aquatile
and have a considerable number of
phenotypic and chemotaxonomic characteristics in common.
Therefore, we propose that these species should be transferred
to an emended genus
Flavobacterium
that has
Flavobacterium
aquatile
as its type species. Because of the high levels of DNA
relatedness between the
[Cytophaga] johnsonae
type strain and
the two
[Flexibacter] aurantiacus
strains, we also propose that
[Flexibacter] aurantiacus
strains should be transferred to the
species
[Cytophaga] johnsonae.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
The strains included in this study are
shown in Table
1
along with their sources and the growth media used for them.
Most of the bacteria were grown at
25°C;
the exceptions were
[Flexibacter]
ovolyticus
and
[Flexibacter] psychrophilus,
which were grown at
19”C,
and
[Flec-
tobacillus] glorneratus,
which was grown at 15°C. The following media were used
to grow the bacteria: Dubos mineral medium
(69)
supplemented with
1%
(wt/
vol) D-cellobiose; marine medium
2216E
(Difco Laboratories, Detroit, Mich.);
Microcyclus-Spirosoma
medium (medium
81
[57a]);
modified Shieh medium
(76);
and Trypticase soy medium (BBL, Becton Dickinson Microbiology Systems,
Cockeysville, Md.). Most of the bacteria were grown on agar plates; the only
exceptions were the
[Flexibacter] columnaris
strains, which were grown in liquid
medium because their colonies adhered strongly to agar.
Preparation
of
DNA.
Previously described methods
(12)
were used to extract
and purify high-molecular-weight native DNAs.
DNA base composition.
The G+C contents
of
the DNAs were determined by
the thermal denaturation method and were calculated by using the equation
of
Marmur and Doty
(51),
as modified by De Ley
(21).
DNA-DNA hybridization experiments.
A previously described procedure was
used for in vitro labeling of bacterial DNA (nick translation) with tritium-labeled
nucleotides (Amersham International, Amersham, England)
(31);
the only mod-
ification was that a nick translation kit (Amersham International) was used in
this study. Previously described methods were used for hybridization experiments
(the
S1
nuclease-DE81 method)
(65)
and for determining the temperature
(T,)
at which
50%
of
a reassociated DNA became hydrolyzable by
Sl
nuclease
(17).
The difference between the
T,,,
of
a homologous reaction and the
T,,,
of a
heterologous reaction
(ATJ
was an estimate
of
the level of divergence between
two
DNAs.
Preparation
of
rRNA.
The rRNAs
of
[Flexibacter]
colurnnaris
NCIMB
224BT
(T
=
type strain),
[Flexibacter] maritirnus
NCIMB
2154T,
Cytophaga hutchinsonii
LMG
10844T,
and
Flexibacterflexilis
NCIMB
12853T
were radioactively labeled
in vivo by adding 3H-labeled adenine to early-log-phase broth cultures as de-
scribed by De Ley and De Smedt
(23).
The labeled rRNAs were extracted by the
method
of
Aiba et al.
(2)
(slightly modified as described by Vandamme et al.
[88])
and were separated into
23s
and
16s
rRNA fractions by ultracentrifuga-
tion on a sucrose gradient
(23).
Labeled
Flavobacteriurn aquatile
LMG 400gT,
Sphingobacteriurn heparinurn
LMG
10339T,
and
Sphingobacteriurn spiritivorum
LMG
8347T
rRNAs were prepared by the same method during a previous study
DNA-rRNA hybridization experiments.
Previously described methods
(91)
were used for fixation of single-stranded DNAs on cellulose nitrate filters, de-
termination
of
the amount
of
DNA fixed on filters, saturation hybridization with
labeled rRNAs, RNase treatment, and determination of the thermostability of
DNA-rRNA hybrids.
FAME
analysis.
We determined fatty acid methyl ester (FAME) profiles of
strains belonging to the
Flavobacterium aquatile
rRNA cluster (see below). Most
of the bacteria were grown on modified Shieh agar for
48
h at
25°C
(the only
exception was
[Flexibacter] psychrophilus,
which was grown at
19’C),
and the cells
were harvested and used for FAME extraction. The FAMEs were then separated
by gas-liquid chromatography, and the FAME profiles obtained were compared
by performing a numerical analysis
(93).
PAGE
of
whole-cell proteins.
Protein patterns were determined for the same
taxa that were used in the FAME analysis, which were grown under the condi-
(72).
130 BERNARDET ET
AL.
TABLE
1.
Bacterial strains used in this study
INT. J.
SYST.
BACTERIOL.
Name
as
received Mediumb
Strain designation
LMG
no.a
as
received“ Source
Chitinophaga pinensis
Chitinophaga pinensis
Cyclobacterium marinus
[Cytophaga] agarovorans
“[
Cytophaga] allerginae”
[Cytophaga] aprica
[Cytophaga] aprica
[Cytophaga] aquatilis
[Cytophaga] awensicola
Cytophaga aurantiaca
[Cytophaga] difluens
[Cytophaga] fermentans
[Cytophaga] flevensis
Cytophaga hutchinsonii
Cytophaga hutchinsonii
[Cytophaga] johnsonae
[Cytophaga] johnsonae
[Cytophaga] johnsonae
[Cytophaga] johnsonae
[
Cytophaga] johnsonae
[
Cytophaga] johnsonae
[Cytophaga] johnsonae
“[Cytophaga] keratolytica”
[Cytophaga] latercula
[Cytophaga] lytica
[Cytophaga] lytica
[Cytophaga] marinoflava
[Cytophaga] pectinovora
[Cytophaga] saccharophila
[
Cytophaga] salmonicolor
[
Cytophaga] succinicans
[
Cytophaga] succinicans
[
Cytophaga] succinicans
[Cytophaga] uliinosa
“[Cytophaga] xantha”
Flavobacterium aquatile
Flavobacterium branchiophilum
Flavobacterium branchiophilum
Flavobacterium branchiophilum
Flavobacterium branchiophilum
Flavobacterium branchiophilum
Flavobacterium branchiophilum
Flavobacterium branchiophilum
[Flavobacterium] ferrugineum
[Flavobacterium] gondwanense
[Flavobacterium] gondwanense
[Flavobacterium] odoratum
[Flavobacterium] odoratum
[Flavobacterium] odoratum
[Flavobacterium] salegens
[Flavobacterium] salegens
“[Flavobacterium] tirrenicum”
[Flectobacillus] glomeratus
Flectobacillus major
[Flexibacter] aurantiacus
[Flexibacter] aurantiacus
“[Flexibacter] aurantiacus
subsp.
copepodarum”
“[Flexibacter] aurantiacus
subsp.
excathedrus”
[Flexibacter] canadensis
[Flexibacter] columnaris
[Flexibacter] columnaris
[Flexibacter] columnaris
[Flexibacter] columnaris
NCIMB 2217T
ATCC 35408
ATCC 23126T
NCIMB 1411
DSM 2063T
JCM 2836T
NCIMB 8628T
NCIMB 1402T
NCIMB 2218T
DSM 1076T
NCIMB 10782
DSM 2064T
ATCC 29585
ATCC 29586
NCIMB 11391
UASM 405d
UASM 444d
NCIMB 1399T
NCIMB 1423T
DSM 2040“
NCIMB 397T
NCIMB 9059T
NCIMB 2072T
NCIMB 2216T
NCIMB 2277T
NCIMB 2278
NCIMB 2279
NCIMB 1863T
Cy
jlc
ATCC 35035T
NCIMB 2219
BGD 77365
FL-W
THP-~~
FDL-
if
BV-d
ACAM 4@
ACAM 52g
Fv
tlTc
NCIMB 1382T
NCIMB 1455
NCIMB 2248=
DD3-69h
JIP 44/87
CRT
LMG 13176T
LMG 13042
LMG 13164T
LMG 13037T
LMG 8385T
LMG 8359T
LMG 1337T
LMG 13036T
LMG 1338T
LMG 8328T
LMG 10844T
LMG 13160
LMG 1341T
LMG 1342
LMG 13142
LMG 13161
LMG 11610
LMG 1343T
LMG 1344T
LMG 13155
LMG 1345T
LMG 4031T
LMG 8384T
LMG 1346T
LMG 10402T
LMG 3809T
LMG 8372T
LMG 4008T
LMG 13707T
LMG 10403T
LMG 13192T
LMG 1233T
LMG 4028
LMG 4029
LMG 13193T
LMG 4037T
LMG 13858T
LMG 13163T
LMG 3987T
LMG 10404
LMG 10405T
LMG 3986T
LMG 8368T
LMG 13035T
S
S
M
M
S
M
M
S
S
C
M
M
S
C
C
S
S
S
S
S
S
S
S
M
M
M
M
S
S
M
S
S
S
M
S
S
S
S
S
S
S
S
S
T
M
M
S
S
S
M
M
M
M
S
S
S
M
S
T
S
S
S
S
Pine litter, Brisbane, Australia
Freshwater, Queensland, Australia
Coelomic fluid of sand dollar, California
Marine mud, California
Water in air-cooling unit, Florida
Rocky sand, Kailua, Hawaii
Mud, Dubrovnik, Yugoslavia
Gills of diseased salmon, Michigan
Soil, Osaka, Japan
Swampy soil, Germany
Beach mud, Bombay, India
Marine mud, California
Lake Ijssel, The Netherlands
Soil
Soil
Soil or mud, Rothamsted or Cambridge, England
Diseased freshwater fish, Manitoba, Canada
Diseased freshwater fish, Manitoba, Canada
Root surface
of
grass, Scotland
Soil
Soil, Ottawa, Ontario, Canada
Unknown
Unknown
Outflow of marine aquarium, La Jolla, Calif.
Beach mud, Limon, Costa Rica
Outflow of marine aquarium, La Jolla, Calif.
Seawater
off
Aberdeen, Scotland
Soil, England
River Wey, Surrey, England
Marine mud, California
Eroded fin of salmon, Washington
Lesion of salmon, Snake River, Idaho
Water from a fish tank, Washington
Marine sediment
Showa Station, Antarctica
Deep well, Kent, England
Diseased gills of fish, Gumma, Japan
Diseased gills of fish, Bonneville Hatchery,
Diseased gills of fish, Gumma, Japan
Diseased gills of sheatfish, Hungary
Diseased gills of fish, Tokushima, Japan
Diseased gills
of
fish, South Santiam, Oreg.
Diseased gills of fish, Bonneville Hatchery,
Oregon
Unknown
Water, Organic Lake, Antarctica
Water, Organic Lake, Antarctica
Unknown
Urine, England
Wound swab, England
Water, Organic Lake, Antarctica
Water, Organic Lake, Antarctica
Seawater, Gulf of Naples, Italy
Water, Burton Lake, Antarctica
Algal culture, Russia
Garden soil, Minneapolis, Minn.
Unknown
Offshore copepod, La Jolla, Calif.
Pool in cathedral, Cartago, Costa Rica
Soil, Canada
Kidney of diseased salmon, Snake River,
Gill lesion of salmon, Dexter Dam, Oregon
Skin lesion of brown trout, Basse Normandie,
Jaw erosion
of
trout, Finland
Oregon
Washington
France
Continued
on
following page
VOL.
46, 1996 CLASSIFICATION OF THE GENUS
FLA VOBACTERIUM
13
1
TABLE
1-Continued
Strain designation
Name as received
as
receivedo LMG no.“ Mediumb Source
[Flexibacter] colurnnaris
[Flexibacter] elegans
[Flexibacter] fiIiformis
Flexibacter jlexilis
“[Flexibacter] jlexilis
subsp.
algavorum”
“[Flexibacter] JEexilis
subsp.
pelliculosus”
[Flexibacter] litoralis
[Flexibacter] rnaritirnus
[Flexibacter] rnaritirnus
[FIexibacter] maritimus
[Flexibacter] ovolyticus
[Flexibacter] ovolyticus
[Flexibacter] psychrophilus
[Flexibacter] psychrophilus
[Flexibacter] psychrophilus
[Flexibacter] psychrophilus
[Flexibacter] psychrophilus
[Flexibacter] roseolus
[Flexibacter] ruber
[Flexibacter] sancti
Flexithrix dorotheae
Haliscomenobacter hydrossis
“Microscilla aggregans”
“Microscilla aggregans”
“Microscilla arenaria”
“Microscilla furvescens”
Microscilla marina
“Microscilla sericea
”
“Microscilla tractuosa
”
“
[Prornyxoba cteriurn
]
jla vum
”
Runelia slithyforrnis
Saprospira grandis
Sphingobacterium heparinum
Sphingobacterium rnizutae
Sphingobacteriurn rnultivorum
Sphingobacterium spiritivorum
Sphingobacteriurn thalpophilurn
Spirosorna linguale
“[Sporocytophaga] caulifomis”
type
1
“[Sporocytophaga] caulifomis”
type 2
Sporocytophaga myxococcoides
Sporocytophaga rnyxococcoides
“Taxeobacter gelupurpurascens”
CR8’
ATCC 2949ST
NCIMB 12853T
DSM 4510Te
NCIMB 1366T
NCIMB 2154T
NCIMB 2153
NCIMB 2158
EKD 002?
LMG 10750T
LMG 10391T
LMG 3989=
LMG 13158T
LMG 3991T
LMG 3992T
LMG 11612T
LMG 13038
LMG 13026T
VKB
0041 LMG 13027
NCIMB 1947T LMG 13179T
LMG 10400
SH3-81h
FPC 836 LMG 13183
JIP 22/90
ATCC 23088T LMG 13507T
ATCC 23103T LMG 1350gT
NCIMB 1379T LMG 8377T
Ft
dlT‘ LMG 8379T
LMG 10767T
NCIMB 1443T LMG 8376T
LMG 13137
NCIMB 1413T LMG 13024T
NCIMB 1419T
NCIMB 1400T
NCIMB 1403T
NCIMB 1408T
DSM 74T‘
ATCC
100IOT
DSM 1813‘
Tx glT‘
LMG 13023T
LMG 13022=
LMG 13021T
LMG 837gT
LMG 10389T
LMG 11500T
LMG 10407T
LMG 10339T
LMG 8340T
LMG 8342T
LMG 8347T
LMG 11520T
LMG 13140T
LMG 8362
LMG 8363T
LMG 8393=
LMG 13345
LMG 13512T
S
T
T
S
S
S
M
M
M
M
M
M
S
S
S
S
S
S
S
S
M
N
M
M
M
M
M
M
M
S
N
M
T
T
T
T
T
N
S
S
C
C
S
Tail lesion
of
salmon, Finland
Hot spring, Rotorua, New Zealand
Soil, Upolu, Apia, Samoa
Lily pond, San Jose, Costa Rica
Pond, Saint Petersburg, Russia
Shore of Birch Lake, Minnesota
Outflow
of
marine aquarium, La Jolla, Calif.
Kidney of diseased sea bream, Hiroshima, Japan
Kidney of diseased sea bream, Hiroshima, Japan
Skin lesion
of
Dover sole, Hunterston, Scotland
Adherent epiflora of halibut eggs, Austevoll,
Norway
Water in halibut egg incubator, Austevoll,
Norway
Kidney of coho salmon, Washington
Salmon, Minter Creek Hatchery, Washington
Kidney
of
coho salmon, Oregon
Coho salmon, Migayi, Japan
Skin lesion of brown trout, Nord-Pas-de-Calais,
France
Hot spring, Agua Caliente, Costa Rica
Hot spring, Geysir, Iceland
Soil, Buenos Aires, Argentina
Beach silt, Ernakulum, Kerala, India
Activated sludge,
Oss,
The Netherlands
Sand, Canoe Beach, Tema, Ghana
Sand, Ernakulum, Kerala, India
Sand, Norse Beach, Puerto Peiiasco, Sonora,
Mexico
Sand, Samoa
Outflow
of
marine aquarium, La Jolla, Calif.
Outflow
of
marine aquarium, La Jolla, Calif.
Sand, Nhatrang, Vietnam
Rhizosphere of tomato plant, Russia
Freshwater lake near Baton Rouge, La.
Rock pool, upper littoral, Woods Hole, Mass.
Dry soil
Ventricular fluid
of
fetus, Japan
Spleen, Washington
Uterus, Kansas
Wound swab, New York, N.Y.
Laboratory water bath
Water, Lake Constance, Germany
Water, Lake Constance, Germany
Soil, Quebec, Canada
Sewage water, Germany
Soil, Alberta, Canada
‘‘
ACAM, Australian Collection of Antarctic Microorganisms, University of Tasmania, Hobart, Australia; ATCC, American Type Culture Collection, Rockville, Md.;
DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany; JCM, Japanese Collection of Microorganisms, Tokyo, Japan; JIP, Culture Collection of
the Unit6 de Virologie et Imrnunologie Moltculaires, Institut National de la Recherche Agronomique, Jouy-en-Josas, France; LMG, Culture Collection of the
Laboratorium voor Microbiologie, University
of
Ghent, Ghent, Belgium; NCIMB, National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom;
UASM, Soil Microbiology Laboratory, University of Alberta, Edmonton, Canada.
’
Strains were grown on Dubos agar supplemented with
1%
cellobiose (C), Difco marine agar
2216E
(M), National Collection of Industrial and Marine Bacteria
medium
81
(N), modified Shieh agar
(S),
or Trypticase soy agar (T).
Strain provided by H. Reichenbach, Gesellschaft fur Biotechnologische Forschung, Braunschweig, Germany.
Strain provided by
R.
P. Burchard, Department of Biological Sciences, University of Maryland, Baltimore.
Strain provided by
K.
A.
Malik, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany.
f
Strain provided by H. Wakabayashi, Laboratory of Aquaculture Biology, Department of Fisheries, University of Tokyo, Tokyo, Japan.
R
Strain provided by C. A. Mancuso, Department of Agriculture Science, University of Tasmania, Hobart, Tasmania, Australia.
I’
Strain provided by R. A. Holt, Department of Microbiology, Oregon State University, Corvallis.
’
Strain provided by P. Rintamaki, Department
of
Zoology, University
of
Oulu, Oulu, Finland.
Strain provided by G. H. Hansen, Department
of
Microbiology and Plant Physiology, University
of
Bergen, Bergen, Norway.
tions described above. Previously described methods were used to prepare
of the scanned protein gel electropherograms with the GelCompar software
package (Applied Maths, Kortrijk, Belgium)
(92).
RESULTS
DNA
base compositions.
The
DNA
base compositions
of
the
strains which we studied are shown in Table
2.
Differences
of
whole-cell protein extracts, for SDS-PAGE, and to perform a numerical analysis
TABLE
2.
DNA base
compositions
of
strains
and
T,+)
values
of
DNA-rRNA hybrids
h,
i‘*,(=,
(“C)
of hybrid with rRNA from:
Taxon
I,
td
m
Sphingobacterium Sphingobacterium
F1exibacter
Cytophaga
w
spj,.itivorurn
LMG
heparinurn
LMG
flexiris
z
LMG LMC
>
3989=
10844T
E
G+C
[Flexibacter] [Ftexibacter]
Straina
content
Flavobacterium
[Cytophagal
columnaris
man’timus
NCIMB
8347T
10339T
(mol%)
aquatile
LMG
johnsonae
400ST
LMG
1341T
Giy
2154=
U
Chitinophaga pinensis
Chitinophaga pinensis
Cyclobacterium marinus
[Cytophaga
]
agarovorans
‘
‘
[
Cy tophaga
]
allerginae
”
[Cytophaga] aprica
[Cytophaga] aprica
[Cytophaga] aquatilis
[Cytophaga] arvensicola
Cytophaga aurantiaca
[Cytophaga] difluens
[Cytophaga] fermentans
[Cytophaga] flevensis
Cytophaga hutchinsonii
Cytophaga hutchinsonii
[Cytophaga] johnsonae
[Cytophaga] johnsonae
[Cytophaga] johnsonae
“[
Cytophaga] keratolytica”
[Cytophaga] latercula
[Cytophaga] lytica
[Cytophaga] lytica
[Cytophaga] marinojlava
[
Cytophaga] pectinovora
[
Cytophaga] saccharophila
[Cytophaga] salmonicolor
[Cytophaga] succinicans
[Cytophaga] succinicans
[Cytophaga] uliginosa
“
[
Cytophaga
]
xan tha
”
Flavobacterium aquatile
Flavobacterium branchiophilum
Flavobacterium branchiophilum
[Flavobacterium] ferrugineum
[Flavobacterium] gondwanense
[Flavobacterium] gondwanense
[Flavobacterium] odoratum
[Flavobacterium] odoratum
[Flavobacterium] odoratum
[Flavobacterium] salegens
[Flavobacterium] salegens
“[Flavobacterium] tirrenicum”
[Flectobacillus] glomeratus
Flectobacillus major
[Flexibacter] aurantiacus
[Flexibacter] aurantiacus
LMG
13176T
LMG
13042
NCIMB
1802T
NCIMB
2217T
ATCC
35408
ATCC
23126T
NCIMB
1411
DSM
2063T
JCM
2836T
NCIMB
8628T
NCIMB
1402T
NCIMB
2218T
DSM
1076T
LMG
10844T
NCIMB
10782
DSM
2064T
ATCC
29585
ATCC
29586
LMG
11610
NCIMB
1399T
NCIMB
1423T
DSM
2040
NCIMB
397T
NCIMB
9059T
NCMB
2072T
NCIMB
2216T
NCIMB
2277T
NCIMB
2278
NCIMB
1863T
LMG
8372T
LMG
400gT
ATCC
35035T
BGD
7736
LMG
10403T
LMG
13192T
ACAM
46
LMG
1233T
LMG
4028
LMG
4029
LMG
13193T
ACAM
52
Fv
tlT
LMG
13858T
NCIMB
11363T
NCIMB
1382T
NCIMB
1455
45.2
45
.O
38.4
41.8
34.0
33.3
34.5
ND~
46.9
ND
41.3
35.7
ND
39.6
39.4
35.2
35.5
35.2
40.3
31.8
33.0
33.3
37.0
35.2
35.7
40.8
36.7
35.2
43.0
35.9
33.0
34.0
33.3
48.9
35.7
36.2
36.7
ND
ND
37.4
37.7
ND
31.3
38.9
35.5
ND
71.8
75.5
71.8
62.2
71.4
73.2
73.4
70.0
68.3
71.0
70.9
72.3
74.2
72.4
67.8
71.9
76.3
72.5
72.5
58.0
68.3
69.6
68.0
72.9
76.9
75.1
70.3
71.6
74.7
71.4
68.0
67.8
73.1
71.5
72.3
71.3
72.0
71.3
76.2
76.2
54.0
52.4
55.6
70.8
57.2
71.6
51.4
55.5
70.5
71.2
71
66.1
66.3
65.9
69
70.3
58.1
70.6
70.9
67.8
67.9
71.6
71.1
70.5
66.2
65.5
70.5
66.6
66.4
55.2
65.1
71
72
58.6
55.9
67.3
67.4
67.5
67
58.7
66.5
67.2
67.7
67.8
64.1
67.7
68.1
71.2
m
58.0 56.8 55.3
cl
61.5
59.4
59.6
58.6
61.1
60.6
58.5
72.8
59.9
55.7
59.6
60.4
56.0
57.1
54.6
65.1
57.8
56.1
60.4 62.5
63.4
58.1
M
61.5
cl
56.5
60.1
59
57.1
78.1
61.4
57.1
78.7
80.1
p
55.7 54.6 57.1
62.3 61.6
r
9
rn
VOL.
46.
1996
CLASSIFICATION OF THE GENUS
FLA
VOBACTERIUM
133
v?
cn
a
".
00
v,
'4
N
b
r?
a
a
4
dX
ab
?"!
03
x
bb
vi
F-?
Ir,
3 3
v,Fb
W
v,
v?
fi
a
2
b
134
BERNARDET ET
AL.
INT.
J.
SYST.
BACTERIOL.
rRNA
superfamily
t
to
Vt
:
Fa.
J
ddonlum
7
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I I
-
Emended genus
Fla
vo
bacte
rium
;
[Fa.J
salegsna
7
Sp. heparinurn
Emended family
Flavobacteriaceae
FIG.
1.
Simplified rRNA cistron similarity dendrogram for rRNA superfamily
V.
Data from this study and references
72,
73,
80,
85,
and
88.
Abbreviations:
Fa.,
Flavobactenum; Cy., Cytophaga; Fx., Flexibacter; Sp., Sphingobactenum; Mi., Microscilla.
2 to 4 mol% G+C with previously published data
(68,71)
were
observed for the following strains:
[Cytophugu] fernentuns
NCIMB 22MT,
"[Cytophugu] xuntha"
LMG 8372T,
[Flexi-
bucter] uuruntiucus
NCIMB
1382T,
"Tuxeobucter gelupztpuras-
cens"
TxglT, and
"[Sporocytophugu] caulifomis"
type
1
strain
LMG 8362. The greatest difference was observed with
[Cyto-
phuga]
sulrnonicolor
NCIMB 2216T; the
two
different DNA
batches which we studied had DNA G+C contents of 41 and
42 mol%, respectively, while the previously published value
was
37
mol% (68). All
of
other DNA base ratios determined in
this study were similar to the values published previously (35,
40, 48, 53, 68,
71,
95).
DNA-rRNA hybridization experiments.
Table 2 shows the
results of DNA-rRNA hybridizations between DNAs from
strains belonging to the genera
Fluvobuctenum, Cytophugu,
Flexibucter,
and
Microscilla
and related genera and radioac-
tively labeled rRNAs from several reference strains belonging
to rRNA superfamily V. These results are also shown in Fig.
1
as a dendrogram based on melting temperatures of elution
[T,,,,]
(T,,,,
is the temperature at which
50%
of
a DNA-
rRNA hybrid is denatured). The
T,(,)
values obtained from
the reciprocal hybridization experiments performed with all
of
the strains belonging to each rRNA homology cluster were
used to calculate the average levels
of
linkage between pairs of
rRNA clusters. The dendrogram in Fig.
1
also includes some
previously published DNA-rRNA hybridization data (73,
81,
85,
88).
DNA-DNA hybridization experiments.
The results of the
VOL.
46, 1996
CLASSIFICATION
OF
THE GENUS
FLA
VOBACTERZUM
135
TABLE
3.
Levels
of
DNA
relatedness
for
strains
of
[Cytophaga] johnsonae
and
[Flexibacter] aurantiacus
as determined
by
the
Sl
nuclease method at
60°C
~ ~~~ ~ ~
Source of unlabeled DNA
%
Reassociation with labeled DNA from:
[C’tophaga]
johnsome
[Cytophaga]
johnsonae
[Flexibacter] aurantiacus
DSM
2064T
ATCC
29585
NCIMB
1382T
Species Strain”
[Cytophaga] johnsonae
DSM
2064T
100‘7
[
Cytophaga
]
johnsonae
ATCC
29585 13
[
Cytophaga] jolznsonae
ATCC
29586
ND‘
’Cytophaga] johiisonae
NCIMB
11391 13
(Flexibacter] aurantiacus
NCIMB
1382T
64,
72,60 (1.2)
[Flexibacter] aurantiacus
NCIMB
1455 67,64
18
100
100
ND
20
22
60,
63
(0.5)
ND
ND
18
100
85
“See Table
1,
footnote
u.
’
The levels of DNA relatedness are expressed
as
percentages of relative binding; the results of one,
two, or
three experiments are shown. The values in parentheses
‘
ND, not determined.
are
AT,,,
values (thermal stabilities
of
heteroduplexes) (in degrees Celsius).
DNA-DNA hybridization experiments performed with the
[Cy-
tophaga] johnsonae
and
[Flexibacter] aurantiacus
strains are
shown in Table
3.
FAME
analysis.
The fatty acid profiles of
Flavobacterium
aquatile
and its closest phylogenetic relatives were determined,
and the results
of
the FAME analysis are shown in Table 4.
The predominant fatty acids
in
all of the taxa studied were
15:0, 15:O iso, 15:O anteiso, 15:O is0 30H, summed feature 4
(15:O
is0 20H, 16:l 07c, or 16:l w7t or any combination of
these fatty acids [Table 4]), 16:O is0 30H, 17:l is0 09c, and
17:O is0 30H. The organisms which we studied were differen-
tiated mainly on the basis of quantitative differences in these
major fatty acids. However, some minor qualitative differences
also occurred, and these differences were used to characterize
several taxa.
[Flexibacter] columnaris, [Flavobacterium] odora-
tum,
and
LLIFlexibacterJ aurantiacus
subsp.
excathedrus”
LMG
3986 did not contain significant amounts of 15:l w6c and 17:l
06c. In addition,
[Flavobacterium] odoratum
also did not con-
tain 15: 1 is0 G.
Flavobacterium branchiophilum
strains did not
contain 16:O iso. Intraspecific heterogeneity in fatty acid con-
tents was observed in the following four species:
[Cytophaga]
johnsonae, [Cytophaga] succinicans, “[Sporocytophaga]
caulifor-
mis,”
and
[Flavobacterium] odoratum.
In most cases this het-
erogeneity was based on quantitative differences; however,
[Flavobacterium] odoratum
LMG 4029 contained several fatty
acids that were not detected in the two other strains of this
species that were studied. When we compared our fatty acid
data with data for some other taxa belonging to rRNA super-
family
V
reported previously, we found both significant simi-
larities and diagnostic differences. The members of most tax-
onomic groups contained high levels
of
15:O is0 and 17:O is0
30H. The levels of other fatty acids varied in the different
genera and species (55,72,85,
88,
103). However, it should be
noted that the cultivation conditions were different in most
of
the previous studies, which may have significantly affected the
fatty acid compositions.
PAGE
of
whole-cell proteins.
Figure 2 shows the protein
profiles of strains belonging to the
Flavobacterium aquatile
rRNA cluster and the corresponding dendrogram obtained
after a numerical comparison. For two species,
[Flexibacter]
psychrophilus
and
[Flexibacter] columnaris,
the different strains
which we studied produced very similar protein profiles, and
thus these taxa could be identified easily on the basis
of
SDS-
PAGE results.
In
contrast, we observed intraspecific heteroge-
neity in the protein profiles obtained for several other species,
including
[Flavobacterium] odoratum, [Cytophaga] johnsonae,
[Cytophaga] succinicans,
and
Flavobacteriurn branchiophilum.
The last three species are discussed below. The heterogeneity
of the
[Flavobacterium
J
odoratum
strains, which was suspected
on the basis of the cellular fatty acid analysis results (Table
4)
and the genomic differences among the strains (60), was con-
firmed by differences between the protein profile of
[Flavobac-
terium] odoratum
LMG 4029 and the protein profiles of the
two other strains studied (LMG 1233T and
LMG
4028).
DISCUSSION
Taxonomic structure of
rRNA
superfamily
V.
At this time,
several major clusters of rRNA branches and a number of
solitary taxa that have very distinct positions (e.g., the genera
Ornithobacterium
and
Capnocytophaga)
can be distinguished in
rRNA superfamily
V
on the basis
of
DNA-rRNA hybridization
data (Fig.
1)
(73,
85,
88;
this study). Similar conclusions were
drawn previously on the basis of 16s rRNA sequence data
(30,
56, 101).
The genera
Chryseobacterium
(including
six
species previ-
ously considered
Flavobacterium
species
[Chryseobacterium
balustinum, Chryseobacterium gleum, Chiyseobacterium indolo-
genes, Chiyseobacterium indoltheticum, Chiyseobacterium me-
ningosepticum,
and
Chryseobacterium scophthalmum])
(85),
Bergeyella
(including only one species,
Beigeyella zoohelcum,
previously considered a
Weeksella
species)
(85),
and
Riemerella
(including a single species,
Riemerella anatipestifer,
long con-
sidered a
Moraxella
species) (72) make up an rRNA cluster
that comprises four different rRNA branches. The genera
Em-
pedobacter
(including
only
Empedobacter brevis,
formerly
[Fla-
vobacterium] breve)
and
Weeksella
(containing only one species,
Weeksella virosa)
(85)
belong to two different rRNA branches
in a second cluster. A third cluster consists
of
the
Flavobacte-
rium aquatile
and
[Flexibacter] columnaris
rRNA branches, and
13 other taxa (most of which are generically misclassified) are
located at the base level between these two rRNA branches.
This last rRNA cluster is referred to as the
Flavobacterium
aquatile
rRNA cluster and is discussed in detail below. The
four members
of
the
[Flexibacter] maritimus
rRNA branch (i.e.,
the marine organisms
“[Flexibacter] aurantiacus
subsp.
copepo-
darum,” [Flexibacter] maritimus, [Flexibacter] ovolyticus,
and
[Flectobacillus] glomeratus)
and several other marine species
located at the base level between
[Flexibacter] maritimus
and
the
Flavobacterium aquatile
rRNA cluster are the closest rela-
tives of the
Flavobacterium aquatile
rRNA cluster. The average
level of linkage between the rRNA clusters mentioned above
and the solitary taxa is a
Tm(.)
of 65
?
1.5”C. A separate rRNA
cluster is formed by the genus
Sphingobacterium,
in which
Sphingobacterium heparinum
occupies a rather distinct position
(72,73,80, 81). Finally,
Flexibacterflexilis
is the only member of
136 BERNARDET ET
AL.
INT.
J.
SYST.
BACTERIOL.
TABLE 4.
Fatty acid compositions
of
the taxa
studied”
%
of
15:l
15:O
15:O
15:O
15:1
o6c
30H is0 anteiso is0 G‘
15:O
Taxun’ 13:O 14:O
is0 is0
Flavobacterium aquatile
(1)
Flavobacterium branchiophilum
(7)
[Flexzbacter] columnaris
(5)
[
Cytophaga
1
flevensis
(1)
[Cytophaga] aquatilis
(1)
[Cytophaga] johnsonae
(1)’
[Cytophaga] johnsonae
(6)’
[Cytophaga] pectinovora
(1)
[Flexibacter] psychrophilus
(5)
[Cytophaga] saccharophila
(1)
[Cytophaga] succinicans
(2y
[Cytophaga] succinicans
(1)”
“[Cytophaga] allerginae”
(1)
“[Cytophaga] xantha”
(1)
[Flexibacter] aurantiacus
(2)
“[Flexibacter] aurantiacus
subsp.
“[Promyxobacterium] JEavum”
(1)
“[Sporocytophaga] caulifomis”
(1
)’
“[Sporocytophaga] caulifomis”
(1
)’
[Flavobacterium] odoraturn
(2)k
[Flavobacterium] odoraturn
(1)‘
excathedrus”
(1)
1.2
t
0.3
1.8
t
0.5
tr
1.3
IT
0.2
tr
tr
tr
3.3
1.1
tr
tr
2.2
1.2
1.5
1.3
?
0.7
1.2
2.0
t
0.7
1.2
1.6
2
0.1
tr
3.3
tr
tr
1.2
2.6
tr
2.7
12.7
11.0
?
3.0
4.4
?
0.9
7.6
9.6
6.1
4.5
&
1.1
6.7
6.0
5
1.0
8.5
11.8
?
2.1
7.6
7.9
10.9
6.5
?
2.0
6.4
6.9
7.8
8.3
tr
3.9
9.1
7.6
5
1.6
8.6
5.0
1.3
3.1
2
0.8
6.4
5.6
t
0.6
7.3
10.7
t
1.6
6.9
2.0
11.6
2.4
5
1.4
4.5
5.2
3.3
tr
1.5
1.8
?
2.3
tr
1.6
2.0
1.2
1.6
t
0.4
2.1
tr
2.5
3.3
5
1.2
tr
1.4
1.9
1.6
2
0.3
2.2
1.5
1.8
2.1
1.2
21.5
22.2
t
3.5
39.0
IT
4.0
14.5
17.6
24.9
20.1
5
3.0
24.1
19.7
?
1.4
9.5
16.8
t
0.3
30.0
27.3
9.0
29.0
?
3.6
25.6
28.0
17.0
30.0
53.5
?
5.2
39.0
1.6
2.5
2
1.3
1.4
t
0.8
10.4
tr
3.4
3.8
5
3.6
2.0
3.7
t
0.7
1.3
1.1
t
0.7
1
.o
1.2
3.9
1.3
&
0.1
tr
1.7
1.5
tr
1.8
t
1.2
1.3
9.0
10.9
t
2.2
13.1
t
1.7
5.7
3.8
4.9
5.2
5
1.7
8.0
11.7
t
3.5
6.9
9.2
t
1.9
9.0
3.6
3.9
6.9
2
0.6
17.1
7.3
6.1
4.2
In addition, small amounts (less than 2.5% of the total fatty acids) of one or more of the following fatty acids occur in the taxa studied: unknown 11.541 (fatty acid
whose identity is unknown; the number indicates the equivalent chain length), unknown 13.566, unknown 16.580, 14:0,
15:O
20H, 16:l
W~C,
16:l
is0 G, 17:l o8c, 17:O
30H. 18:l o5c, summed feature 3 (14:O 30H or 16:l
is0
I
or both), and summed feature 5
(17:l
is0 I or 17:l anteiso B or both).
’
The numbers in parentheses are the numbers
of
strains studied. Unless indicated otherwise below, the strains used were the strains shown in Table
1.
When two
or more strains were used, the mean
+-
standard deviation is shown.
The double bound position indicated by the capital letter is not known.
The fatty acids 15:0 is0 20H, 16:l
w7c,
and 16:l
07t
could not be separated from each other by gas chromatography by using the Microbial Identification System
[Cytophuga]
johnsonue
DSM 2064T.
(Microbial ID, Inc., Newark, Del.) software package and together were considered summed feature
4.
f
All
of the
[Cytophaga]
johnsonue
strains in Table
1
except DSM 2064T.
R
[Cyrophugu] succinicans
NCIMB 2277T and NCIMB 2279.
[Cytophaga]
succinicuns
NCIMB 2278.
“[Sporocytophugu]
caulifomis”
LMG 8362.
J
“[Sporocytophugu]
caulifomis”
LMG 8363T.
[Flavobacterium] odoraturn
LMG 1233T and LMG 4028.
‘
[Fluvobucterium] odoraturn
LMG 4029.
its rRNA branch, whereas the cellulolytic organisms
(Cyto-
phaga hutchinsonii, Cytophaga aurantiaca,
and
Sporocytophaga
nzyxococcoides)
make up the
Cytophaga hutchinsonii
rRNA
branch. The
Sphingobacterium
rRNA cluster and the
Cyto-
phaga hutchinsonii
and
Flexibacter jlexilis
rRNA branches are
linked to each other and to the other rRNA clusters in super-
family
V
at a
T+,,
of
60
*
2.0”C.
Several organisms, most of which are represented by a single
strain, do not belong to any
of
the rRNA clusters described
above. With average
T,
e)
values less than 60T, the following
organisms occupy peripfieral positions on the
Tm(e)
dendro-
gram:
Bacteroides fragilis
and
Bacteroides oralis
(88);
Chiti-
nophaga pinensis; Cyclobacterium marinus
(formerly
[Flectoba-
cillus] marinus)
(67);
[Cytophaga] agarovorans; [Cytophaga]
aprica; [Cytophaga] awensicola; [Cytophaga] difluens; [Cyto-
phaga] fermeritans; [Cytophaga] salmonicolor; [Flavobacterium]
fermgineum; Flectobacillus major; [Flexibacter] elegans; [Flexi-
bacter]
filiformis;
“[Flexibacter] flexilis
subsp.
algavorum”;
“
[
Flexiba
c
ter] jlexilis
sub sp
.
pelliculosus
”
;
[
Flexiba cter] litora lis
;
[Flexibacter] roseolus; [Flexibacter] ruber; [Flexibacter] sancti;
Flexithrix dorotheae; Haliscornenobacter hydrossis; bb[Microscilla]
aggregans”; “[Microscilla] arenaria”; “[Microscilla] fUwescens”;
[Microscilla] marina; “[Microscilla] sericea”; “[Microscilla] trac-
tuosa
”;
Runella slithyformis; Saprospira grandis; Spirosoma lin-
guale;
and
“Taxeobacter gelupurpurascens.”
rRNA sequencing
is a better method than DNA-rRNA hybridization for reveal-
ing deep phylogenetic relationships
(97),
and recent data ob-
tained by the former technique have indeed indicated that
most of these taxa should be assigned to the
Cytophaga-Fla-
vobacterium-Bacteroides
group (30,
56,
58,
98,
101).
Two ma-
rine facultatively anaerobic species,
[
Cytophaga] agarovorans
and
[Cytophaga] salmonicolor,
have recently been reclassified
in the new genus
“Marinolabilia”
(57).
“[Flavobacterium] tir-
renicum”
is
probably not related to superfamily
V,
as demon-
strated by its very low level of rRNA homology (Table
2)
and
its quite different polyamine distribution (34), but at this time
no rRNA sequence is available to determine the phylogenetic
relationships
of
this organism.
For technical reasons, the following species were not in-
cluded in this study:
[Cytophaga] xylanolytica, [Flexibacter] poly-
morphus, Sphingobacterium antarcticus, Sphingobacterium fae-
cium, Sphingobacterium piscium, “Taxeobacter ocellatus,”
and
“Taxeobacter chitinovorans.”
rRNA sequencing data have re-
vealed that
[Cytophaga] lcylanolytica
and
[Flexibacter] polymor-
phus
belong to the
Cytophaga-Flavobacteriurn-Bacteroides
group
(32,
58).
The three new
Sphingobacteriurn
species men-
tioned above have been clearly identified as members of this
genus on the basis
of
chemotaxonomic data and DNA-DNA
hybridization data (74,83,84).
No
rRNA sequences have been
published yet for members of the recently proposed genus
VOL.
46, 1996
CLASSIFICATION
OF
THE GENUS
FLA
VOBACTERIUM
137
TABLE
&Continued
p%
of:
Summed
16:O
I6:O
164
feature
4“
30H
iso
is0
30H
16:O
1s:o
is0
30H
6.6
14.3
t
2.7
8.7
t
2.5
4.9
8.9
6.6
8.8
5
1.7
7.9
8.5
i
2.2
5.9
8.6
t
3.7
10.7
6.7
5.2
9.2
i
0.7
7.5
8.7
7.9
8.7
5.7
2
1.6
4.0
tr
1.1
t
0.3
tr
1.4
1.3
2.7
1.6
t
0.5
tr
tr
tr
tr
tr
3.0
1 .0
1.4
t
0.3
tr
1.1
1.4
tr
tr
4.0
4.5
t
1.9
tr
18.4
13.4
12.8
18.2
t
1.6
5.3
2.3
2
0.5
12.3
7.5
5
6.3
5.8
14.5
5.0
10.1
2
0.8
5.4
6.6
11.8
12.4
8.3
1.6
t
2.3
tr
2.3
t
0.6
tr
4.1
4.5
4.7
3.7
t
1.2
tr
1.4
2.2
t
0.5
1.7
4.5
1.4
3.4
t
0.7
tr
tr
3.2
5.1
3.5
-t
1.5
5.4
2.4
2.0
t
1.2
1.4
2.0
3.4
1.3
?
0.5
I
.5
2.5
2
1.0
3.7
1.3
t
0.5
1.9
1.5
4.1
1.0
i
0.6
1.9
2.3
4.7
1.2
1.4
t
0.5
4.4
5.1
1.7
t
0.6
2.6
2
1.0
3.8
6.8
4.4
4.7
t
2.0
4.7
3.3
2
1.0
5.3
4.5
&
1.3
3.3
3.2
9.6
2.8
-+
1.3
4.3
3.1
8.2
4.1
tr
2.1
16:l
iso
H
17:l
17:l
17:O
W6C
is0
0%
iso
30H
2.2
tr
tr
1.4
tr
tr
4.2
t
2.0
1.8
t
1.7
2.6
tr
1.1
2.1
1.9
.8
.0
4.1
5.1
t
1.2
2.7
4.1
2.2
2.8
2
0.7
5.2
2.2
-+
0.6
7.6
4.0
5
1.0
3.1
2.2
12.5
2.2
2
0.9
tr
3.5
3.2
2.1
tr
4.8
tr
7.5
t
1.6
tr
2.5
2.4
2.7
t
0.7
5.1
12.2
5
1.4
5.6
2.4
t
1.7
3.3
3.1
2.1
3.8
-+
0.5
7.4
3.7
2.5
2.3
13.5
2
2.9
8.3
7.4
5.7
2
0.8
12.1
?
1.3
3.9
8.3
9.4
0.6
t
1.3
12.1
9.0
t
2.2
9.6
5.8
t
0.5
7.8
8.7
6.2
11.5
t
1.5
12.6
13.6
7.9
7.7
12.8
t
2.8
8.6
“Taxeobacter”
(71), but an oligonucleotide cataloging study
revealed that a
“Taxeobacter ocellatus”
strain is relatively
closely related to
Sphingobacterium heparinum
(63). In this
study, we observed only low levels of rRNA homology between
“Taxeobacter gelupurpurascens”
and the rRNA probes tested
(Table 2).
Genera
Cytophaga
and
Flexibacter.
Our DNA-rRNA hybrid-
ization data clearly show that the genera
Cytophaga
and
Flexi-
bacter
(as presently defined) are polyphyletic and thus confirm
and extend similar conclusions based on previous DNA-rRNA
hybridization results and on 16s rRNA sequence comparisons
(6,
30, 45, 46, 56, 58, 72, 73,
85,
101). The genus names
Cyto-
phaga
and
Flexibacter
must be preserved for members of the
Cytophaga hutchinsonii
and
Flexibacter flexilis
rRNA branches,
respectively. Other
Cytophaga
and
Flexibacter
species, which
belong to several rRNA branches and clusters in rRNA super-
family
V
or occupy peripheral positions on the
T,,,,,,
dendro-
gram, must be considered generically misclassified
(Fig.
1).
As
the type species
of
the genus
Flexibacter, Flexibacter flexilis,
is
the only member of its rRNA branch, the genus
Flexibacter
is
restricted to this species. All other
Flexibacter
species should be
reclassified. In the case of the genus
Cytophaga,
the type spe-
cies
(Cytophaga hutchinsonii)
and
Cytophaga uurantiaca
are the
only genuine
Cytophaga
species, and the members of the cel-
lulolytic microcyst-forming genus
Sporocytophuga (Sporocyto-
phugu myxococcoides
is the type and only valid species) are
their closest relatives. The difference in
T,,(,,
between
Sporo-
cytophaga myxococcoides
and
Cytophaga hutchinsonii
(
13.5”C)
is great enough to justify separate generic status for the former
taxon. The cellulolytic
Cytophaga
species can also be clearly
differentiated from the other
Cytophaga
and
Flexibacter
species
on the basis of their sulfonolipid contents (26). All other
Cy-
tophaga
species (i.e., noncellulolytic
Cytophaga
species) should
be reclassified. Such a restriction of the genera
Cytophaga
and
Flexibacter
to the type species and some close phylogenetic
relatives was suggested previously on the basis of
16s
rRNA
sequence data (56, 70).
As shown by DNA-rRNA hybridization data, several gener-
ically misclassified
Cytophaga
and
Flexibacter
species are
lo-
cated in the
Sphingobacterium
rRNA cluster (Fig.
1).
The ge-
neric status of
Sphingobacterium heparinum
has been discussed
repeatedly, and transfers of this organism to other or new
genera have been proposed (16, 68,
80,
84); however, the
specific status of
Sphingobacterium heparinum
has now been
established on the basis of genomic data (80). The results of
rRNA homology experiments also confirmed that all
Sphingo-
bacterium
species belong to a single rRNA branch (72). These
findings corroborate the proposals of Takeuchi and Yokota
(83, 84), who included
[Flavobacterium] yabuuchiae
in
Sphin-
gobacterium spiritivorum
and
[Cytophaga] keratolytica
in
Sphin-
gobacten’um multivorum. [Flexibacter] canadensis
is also a pe-
ripheral member of this cluster, a position confirmed by the
16s
rRNA sequence of this organism (56). Because
Sphingo-
bacterium thalpophilum
(formerly
[Flavobacterium] thalpophi-
lum)
was found to contain sphingophospholipids, transfer of
this species to the genus
Sphingobacterium
was proposed
(19,
84). As
[Flexibacter] canadensis
occurs at the same position on
the dendrogram as
Sphingobacterium thalpophilum,
the results
of lipid content analyses and the possible presence of sphin-
gophospholipids may be decisive
in
transferring
[Flexibacter]
canadensis
to the genus
Sphingobacterium
or in creating a new
genus
to
accommodate this species.
The
[Flexibacter] maritimus
rRNA branch includes several
species that were isolated from marine environments and are
generically misclassified. One of these is
[Flectobacillus] glom-
eratus,
since the type species of the genus
Flectobacillus, Flec-
tobacillus major,
is located at a rather low level on the
Cyto-
phaga hutchinsonii
rRNA branch as determined by
16s
rRNA
sequence data (24). Additional data will be necessary to de-
termine the generic relationships of members of the
[Flexi-
bacter] maritimus
rRNA branch, the
Flavobacterium aquatile
rRNA cluster, and the peripherally related taxa
[Cytophaga]
latercula, [Cytophaga] lytica, [Cytophaga] marinoflava, [Cyto-
phaga] uliginosa, [Flavobacterium] gondwanense,
and
[Fla-
vobacterium] salegens
(Fig. 1). The great genomic heterogene-
138
BERNARDET
ET
AL.
INT.
J.
SYST.
BACTERIOL.
I’
I
Fa.
branchrophrlurn
TH
P-
1
Fa. branchophdurn
FDL
1
Fa.
branchiophilurn
NCIMB
221
9
Fa.
branch/ophrlum
BV
6
Fa. branchtophhm
ATCC
35035T
Fa.
branchmphrlum
FL-
1
5
Fa.
branchmphrlurn
BGD
7736
[Fx]
columnaris
JIP
44-87
[Fx.]
col’urnnars
DD3-69
[Fx.]
columnairs
C
R
-7
[Fx
]
columnairs
C
R
-8
[Fx.)
columnarts
NCIMB
2248T
[Fa.]
odoraturn
LMG
I233T
[Fa.]
odoraturn
LMG
4028
‘~Sporocytop haga]
ca
utdormts
”
L
MG
8363T
’[Fx
]
aurantiacus
excathedrus”
LMG
3986T
[Cy.]
flevensls
DSM
1076T
’‘i[Promyxobacter/um] flavum”
LMG
1
0389
[Cy.]
saccharophda
NCIMB
2072T
’YCy.]
allergmae”
ATCC
35408
[Cy]
succrntcans
NCIMB
227JT
(Cy
J
succtnrcans
NClMB
2278
[Cy]phnsonae
NClMB
11
391
[Fx.]
auranhacus
NCIMB
1455
[Fx.]
aurantracus
NCIMB
1382T
[Cy]
johnsonae
UASM
405
[Cy
]
johnsonae
UASM
444
[Cy
J
phnsonae
LMG
1
34
1
T
[Cy.]
johnsonae
ATCC
29586
[Cy
]
johnsonae
ATCC
29585
[Cy
]
phnsonae
Cy
J
1
[Cy]
aquatrlrs
DSM
2063T
[Cy.]
pecfrnovora
NCIMB
9059T
Icy.]
Succinicans
NCIMB
2279
”[Sporocytophaga]
cauhlorrn~s”
LMG
8362
Fa.
aquahle
LMG
4008T
’YCy.]
xantha”
LMG
8372
[Fx.]
psychrophilus
S
H
3
-8
1
[Fx.]
psychrophrlus
LMG 10400
[Fx.]
psychrophlus
FPC
830
[Fx
]
psychrophilus
J
I
P
22
90
[Fx.]
psychroph//us
N
C
I
M
B
1
94
7T
[Fa.]
adoraturn
LMG
4029
FIG.
2.
Electrophoretic protein profiles and corresponding dendrogram for all
of
the strains studied in the
Fluvobacteriurn
aquatile
rRNA cluster. The dendrograrn
was constructed by using unweighted pair group average linkage of the correlation coefficients for pairs
of
protein patterns. The molecular weight markers used
(indicated at the bottom) were (from left to right) trypsin inhibitor (molecular weight,
20,100),
trypsinogen
(24,000),
carbonic anhydrase
(29,000),
glyceraldehyde-3-
phosphate dehydrogenase
(36,000),
egg albumin
(45,000),
and bovine albumin
(66,000).
For
abbreviations see the legend to Fig.
1.
ity in the
[Flexibacter] maritimus
rRNA branch was also
revealed by
16s
rRNA sequence data
(24,
30,
56,
88).
Genus Flavobacterium.
Since several species previously in-
cluded in the genus
Flavobacterium
have been reclassified as
members of new or other genera, at this time the only valid
Flavobacterium
species are
Flavobacterium aquatile
(the type
species),
Flavobacterium branchiophilum, [Flavobacterium] fer-
rugineum, [Flavobacterium] gondwanense, [Flavobacterium]
odoraturn,
and
[Flavobacterium] salegens
(24,
40,
85,
95).
The
large differences in
Tm(e>
between
[Flavobacterium]
ferrug-
ineum,
[Flavobacterium] gondwanense,
or
[Flavobacterium]
salegens and
Flavobacterium aquatile
(Table
2
and Fig.
1)
dem-
onstr ate that
[
Fluvobacterium] femgineum
,
[
Flavobacteriurn
]
gondwanense,
and
[Flavobacterium] salegens
do not belong
to
the genus
Flavobacterium
and should be reclassified. In con-
trast, phenotypic similarities and low differences in
Tm(el
be-
tween
Flavobacterium branchiophilum
and
Flavobacterium
aquatile
support inclusion of
Flavobacterium branchiophilum
in
the genus
Flavobacterium. [Flavobacterium] odoratum,
several
misclassified
Cytophaga
and
Flexibacter
species,
“[Promyxobac-
ten‘um] flavum,”
and
“[Sporocytophaga]
caulifomis”
all form a
single rRNA homology cluster.
So
far, this cluster includes two
rRNA branches; one of these branches includes
[Flexibacter]
columnaris
and
“[Flexibacter] aurantiacus
subsp.
excathedrus,”
and the other is composed of
Flavobacterium aquatile
and
[Cytophaga] aquatilis.
All other species
([Flavobacterium] odo-
ratum, “[Cytophaga] allerginae,” [Cytophaga] pevensis, [Cyto-
phaga] johnsonae, [Cytophaga] pectinovora, [Cytophaga]
sac-
charophila, [Cytophaga] succinicans, “[Cytophaga] xantha,”
Flavobacterium branchiophilum, [Flexibacter] aurantiacus, [Flexi-
bacter] psychrophilus, “[Promyxobacterium] flavum,”
and
“[Spo-
rocytophaga] caulifomis”)
are located at the base level be-
tween the two branches (Table
2
and Fig.
1).
The range of
levels of genomic divergence in this rRNA cluster
is
rather
wide, corresponding to a difference in
To,,,,
of about
8”C,
although several other genera, such as the genus
Sphingobac-
terium
(72),
have similar levels
of
genomic divergence.
In
such
cases, all species belonging to the rRNA cluster can be placed
in one genus or several genera can be created, depending on
the phenotypic characteristics
of
the species
(22,
85).
Besides their similar habitats (soil or freshwater or both)
most of the taxa included in this rRNA cluster have a consid-
VOL.
46,
1996
CLASSIFICATION
OF
THE
GENUS
FLA
VOBACTERIUM
139
erable number of characteristics in common. They contain
menaquinone 6 as the major respiratory quinone (56,61); their
G+C contents are in the range from 32 to 37 mol% (Table
1)
(40,68); they exhibit clear gliding motility (a possible exception
is
Flavobacterium branchiophilum,
but the motility of this or-
ganism may have been overlooked, as the motility of
Flavobac-
terium aquatile
was for a long time); they produce yellow
non-
diffusible pigments; and they have many classical phenotypic
characteristics in common (see below). In addition, the metab-
olism
of
most of these organisms is strictly aerobic (the excep-
tions are
[Cytophaga] aquatilis
and
[Cytophaga] succinicans,
which can also grow anaerobically when some growth factors
are provided [68]). Finally, these organisms have very similar
fatty acid profiles (see below) (Table
4).
[Flavobacterium] odo-
ratum
can be easily distinguished from the other members of
the cluster by its clinical origin, its lack of gliding motility, its
good growth at 37”C, its halotolerance (10,41, 61), and several
differences in its fatty acid profile (Table 4).
Therefore, both phenotypic and genotypic criteria justify
inclusion of most of the members of the
Flavobacterium aqua-
tile
rRNA cluster in a separate genus; the only exception is
[Flavobacterium] odoratum.
This conclusion is supported by
16s rRNA sequence data; the soil and freshwater organisms
cluster on a separate branch, while
[Flavobacterium] odoratum
occupies a clearly independent position (30,56,57). Moreover,
researchers have found small-subunit rRNA sequence signa-
tures which clearly differentiate
[Flavobacterium] odoratum
from its neighbors (30). Therefore, we propose that
Flavobac-
terium aquatile
should be the type species of an emended genus
Flavobacterium
and that all valid taxa isolated from soil and
freshwater belonging to the
Flavobacterium aquatile
rRNA
cluster should be placed in the emended genus
Flavobacterium.
Below, we present an emended description of the genus
Fla-
vobacterium
and propose new combinations for seven of its
members.
There is a nomenclatural problem concerning
[Cytophaga]
aquatilis.
This valid species (82), which at this time
is
repre-
sented by only one available strain, is indeed an independent
taxon, as demonstrated by DNA-DNA homology data. The
DNA of this organism does not exhibit significant levels of
homology with DNAs from
Flavobacterium aquatile
and sev-
eral other members of the
Flavobacterium aquatile
rRNA clus-
ter
(9).
The fatty acid and protein profiles of
[Cytophaga]
aquatilis
are also clearly different from the profiles of the other
organisms (see below) (Table 4 and Fig. 2). If the combination
[Cytophaga] aquatilis
(Strohl and Tait 1978) was modified in a
way that was consistent with its new generic status, it would
become a junior homonym of
Flavobacteriurn aquatile
(Frank-
land and Frankland 1889). Therefore, below we propose a new
name for this taxon,
Flavobacterium hydatis,
on
the basis of
Rule 12b
of
the
International Code
of
Nomenclature
of
Bacteria
(47a). The new specific epithet was chosen because its meaning
is similar to that of the former epithet.
The specific epithet of
[Cytophaga] johnsonae
is another
problem, as this epithet was incorrectly formed by Stanier in
1947 (78). In 1957 Stanier changed the epithet to
johnsonii,
which is also incorrect (79). As this epithet was created in
honor of the American microbiologist Delia
E.
Johnson, who
first isolated this species, it should be a genitive
noun
with a
feminine ending. Therefore, we propose that the new name for
this organism should be as
Flavobacterium johnsoniae.
Other problems concern the taxonomic status of the valid
species
[Flexibacter] aurantiacus
and the invalid taxa
“[Cyto-
phaga] allerg’nae,” “[Cytophaga] xantha,” “[Flexibacter] auran-
tiacus
subsp.
excathedms,” “[Promyxobacterium] fiavum,”
and
“[Sporocytophaga] caulformis.”
We propose that
[Flexibacter]
aurantiacus
strains should be included in
[Cytophaga] johnso-
nae
and that all of the invalid taxa should be referred to as
Flavobacterium
sp. (see below).
Consequently, the emended genus
Flavobacterium
contains
the following species:
Flavobacterium aquatile, Flavobacteri-
um branchiophilum, Flavobacterium columnare, Flavobacterium
flevense, Flavobacterium hydatis, Flavobacterium johnsoniae,
Flavobacterium pectinovomm, Flavobacterium psychrophilum,
Flavobacterium saccharophilum,
and
Flavobacterium succini-
cans.
All other
Flavobacterium
species are generically misclas-
sified.
Emended description
of
the genus
Flavobacterium
Bergey,
Harrison, Breed, Hammer, and Huntoon
1923.
Cells are rods
with parallel or slightly irregular sides and rounded or slightly
tapered ends and usually are 2 to
5
pm long and 0.3 to
0.5
pm
wide. Under certain growth conditions, some species may also
produce shorter (1-pm) or longer
(10-
to 40-pm) filamentous
cells. The longer rods are flexible. Motile by gliding (this char-
acteristic has not been observed in
Flavobacterium branchio-
philum).
Flagella are absent. Gram negative. Resting stages are
not known. Intracellular granules of poly-P-hydroxybutyrate
are absent. Colonies are circular, convex or low convex, and
shiny with entire or wavy edges (sometimes sunken into the
agar) on solid media containing high nutrient contents.
On
solid media containing low levels of nutrients most species also
produce flat or very thin, spreading, sometimes very adherent
swarms with uneven, rhizoid, or filamentous margins. Colonies
are typically yellow (they vary from cream to bright orange)
because of nondiffusible carotenoid or flexirubin types of pig-
ments or both, but nonpigmented strains do occur. Most spe-
cies do not grow on seawater-containing media; an exception
to this is
Flavobacteriumfievense.
Most species are able to grow
on
nutrient agar and
on
Trypticase soy agar. Chemoorganotro-
phic. Aerobic with a respiratory type of metabolism. When
certain growth factors are provided,
Flavobacterium hydatis
and
Flavobacterium succinicans
also grow anaerobically
(4,
14,
68, 82). Peptones are used as nitrogen sources, and NH, is
released from peptones; growth occurs
on
peptone alone. Acid
is produced from carbohydrates by all species except
Flavobac-
terium columnare
and
Flavobacterium psychrophilum
.
All spe-
cies except
Flavobacterium flevense
decompose gelatin and
casein, and several species also hydrolyze various polysaccha-
rides, including starch, chitin, pectin, and carboxymethyl cel-
lulose.
Flavobacterium fievense
and
Flavobacterium sacchar-
ophilum
are also agarolytic. Cellulose is never decomposed.
Tributyrin and Tween compounds are decomposed. Indole is
not produced. Catalase is produced. Cytochrome oxidase is
produced by all species except
Flavobacterium saccharophilum.
Menaquinone
6
is the only respiratory quinone. The predom-
inant fatty acids are
15:0,
15:O iso,
15:l
is0 G,
15:O
is0 30H,
summed feature
4
(15:O
is0 20H,
161
w7c, or 16:l w7t or any
combination of these fatty acids), 16:O is0 30H, 17:l is0 09c,
and 17:O is0 30H. Sphingophospholipids are absent. Homo-
spermidine is the major polyamine in all 10
Flavobacterium
species; all species except
Flavobacterium branchiophilum
and
Flavobacterium saccharophilum
also contain putrescine as
a
minor component (33, 34). Spermidine and spermine are also
minor components in
Flavobacterium branchiophilum,
while
Flavobacterium johnsoniae
is the only member of the genus
that contains minor amounts of agmatine and 2-hydroxypu-
trescine (34). The optimum temperature range for most spe-
cies is 20 to 30°C; the optimum temperature range for
Fla-
vobacterium psychrophilum
is
15
to 18°C.
These organisms are widely distributed in soil and freshwa-
ter habitats, where they decompose organic matter. Several
species are pathogenic for freshwater fish
(Flavobacterium
INT.
J.
SYST.
BACTERIOL.
140
BERNARDET ET AL.
TABLE
5.
Phenotypic characteristics that can be used to differentiate the
10
valid species belonging to the genus
Flavobacten'urn"
Flavohacteriurn Flavohacteriurn Flavohactrriurn
branchiophihrn columnare peverrse
Characteristic
Flavohacteriurn aquatile
Low convex, round, with Low convex, round, with
entire margins entire margins
-
d
-
-
-
Flat, rhizoid, strongly
adherent to agar Low convex, round,
sunken into agar
Morphology of colonies on AOA'
Congo red absorption'
Growth
on
seawater media
Growth
on
nutrient agar
Growth
on
Trypticase soy agar
Gliding motility
Flexirubin type of pigmentsf'
Glucose used as a sole carbon
and energy source
Acid produced aerobically from
carbohydrates
Degradation
of
Gelatin
Casein
Starch
Carboxymethyl cellulose
Agar
Alginate
Pectin
Chitin
Esculin
DNA
Tyrosine
Brown diffusible pigment
produced
on
tyrosine agar
Precipitate formed on egg yolk
agar
P-Galactosidase activity
Susceptibility to vibriostatic
compound 0/129
H,S
production
Production of cytochrome oxidase
Nitrate reduction
(+>
+
"
-
ND
ND
+
+
+
V
+
V
-
+
+
+
-
+
+
-
-
ND
ND
-
-
ND
ND
ND
ND
V
V
-
-
+ +
+
V
-
+
+
+
+
-
+
+
+
V
+
V
+
V
a
Data from references
1,
4, 8-10, 13,
15,
16, 40,
61,
68, 82, 90, and
95.
AOA,
hacker-Ordal agar (0.05% tryptone, 0.05% yeast extract, 0.02% beef extract, 0.02% sodium acetate) (3).
Production of an extracellular galactosamin glycan was revealed by flooding the colonies with a 0.01% aqueous solution of Congo red (52).
''
+,
all strains are positive;
-,
all strains are negative;
(+),
weakly positive; v, variable among strains;
V,
variable among references;
ND,
no data available.
''
Gliding motility was observed in the type and only strain of
Flavohacteriurn
aquatile
under certain conditions (39).
I
The presence
of
the flexirubin type of pigments was revealed by a distinct, reversible color shift of the colonies when they were flooded with a 20% (wt/vol)
KOH
aqueous solution (28).
Determined by the
o-nitrophenyl-P-D-galactopyranoside
test.
''
Determined by a diffusion method in which
500-pg
disks were used.
branchiophilum, Flavobacterium columnare, Flavobacteriumpsy-
chrophilum)
or occasionally are isolated from diseased fresh-
water fish
(Flavobacterium hydatis, Flavobacterium johnsoniae,
Flavobacterium succinicans).
The G+C contents of the DNAs
are
32
to 37 mol%. The type species is
Flavobacterium aquatile
(Frankland and Frankland 1889) Bergey, Harrison, Breed,
Hammer, and Huntoon 1923.
Description
of
Flavobacterium aquatile
(Frankland and
Frankland 1889) Bergey, Harrison, Breed, Hammer, and
Huntoon 1923.
The description of
Flavobacterium aquatile
is
the same as that given previously (40), with the following ad-
ditions and modifications: no flexirubin type of pigment is
produced; peptones are used as nitrogen sources, but urea and
Casamino Acids are not; gelatin, tyrosine, starch, esculin, trib-
utyrin, and lecithin are degraded, but carboxymethyl cellulose,
agar, and chitin are not; nitrate
is
reduced; and o-nitrophenyl-
P-D-galactopyranoside
is
hydrolyzed (10, 15, 61).
Description
of
Flavobacterium branchiophilum
Wakabayashi,
Huh,
and Kimura 1989.
Flavobacterium branchiophilum
was
originally described as
Flavobacterium branchiophila
(95),
but
the specific epithet was later corrected (94). The description of
this taxon is the same as that given previously (95), with the
following additions: tyrosine, tributyrin, lecithin, and Tween
compounds are degraded, but carboxymethyl cellulose is not;
hydrogen sulfide is not produced;
o-nitrophenyl-P-D-galacto-
pyranoside is hydrolyzed; and no growth occurs at 37°C (9).
This species
is
frequently isolated from diseased gills of fish,
and pathogenicity for fish has been demonstrated by experi-
mental infection tests (95).
Description
of
Flavobacterium columnare
comb. nov.
Flavo-
bacterium columnare
(basonym,
Flexibacter columnaris
(Davis
1922) Bernardet and Grimont 1989). The following combina-
tions have been used for this species:
Bacillus columnaris
Davis
1922;
Chondrococcus columnaris
Ordal and Rucker 1944;
Cy-
tophaga columnaris
Garnjobst 1945;
Cytophaga columnaris
Reichenbach 1989 (68);
Flexibacter columnaris
Leadbetter
1974 (49); and
Flexibacter columnaris
Bernardet and Grimont
1989 (10). The description
of
this taxon is the same as that
given previously (lo), except that tyrosine is not hydrolyzed.
This species is frequently isolated from superficial lesions on
VOL.
46, 1996 CLASSIFICATION OF
THE
GENUS
FLAVOBACTERIUM
141
TABLE
5-Continued
Flavobacterium Flavobacterium Flavobactenum Flavobactenum Flavobactenum
johnsoniae pectinovonim p.lychrophdum saccharophihim
succinicans
Flavobactenum hydatis
Flat, spreading, with Flat, spreading, with
filamentous margins filamentous margins Low convex, round,
with entire margins Low convex, round, Flat, spreading, sunken Flat, spreading, with
filamentous margins
with entire
or
into agar
uneven margins
-
-
-
(+I
+
+
+
+
+
+
+ +
+
ND
+
-
+
+
+
V
-
+
+
+
+
(+)
-
+
+
+
+
-
+
+
+
+
+
+
V
+
+
+
+
+
+
+
+
+
+
-
-
+
+
(+>
V
-
+
+
+
+
+
ND
+
+
+
-
-
-
(+I
+
+
ND
ND
ND
+
+
-
-
-
+
-
-
V
+
+
-
-
+
V
+
+
V
+
+
-
+
+
+
+
+
+
+
-
+
V
diseased fish and sometimes from internal organs. Pathogenic-
ity for fish has been demonstrated by experimental infection
tests (18).
Description
of
Flavobacterium flevense
comb. nov.
Flavobac-
terium flevense
(basonym,
Cytophaga flevensis
van der Meulen,
Harder, and Veldkamp 1974). The description of this taxon is
the same as that given previously (90), with the following ad-
ditions and modifications: starch, esculin, and Tween com-
pounds are degraded;
DNA,
carboxymethyl cellulose, tyrosine,
and lecithin are not degraded; catalase is produced; o-nitro-
phenyl-P-D-galactopyranoside
is hydrolyzed; and good growth
occurs on nutrient agar Trypticase soy agar (10).
Description
of
FZavobacterium hydatis
nom. nov.
Flavobacte-
rium hydatis
(basonym,
Cytophaga aquatilis
Strohl and Tait
1978) (hy’ da.tis. Gr. n.
hydor,
water;
N.
L.
gen. n.
hydatis,
from
water). The description of this taxon is the same as that given
previously for
[Cytophaga] aquatilis
(82), with the following
additions and modifications: lecithin is not degraded; cyto-
chrome oxidase is produced;
o-nitrophenyl-P-D-galactopyrano-
side
is
hydrolyzed; and
good
growth occurs on Trypticase soy
agar (10). The only currently available strain was isolated from
the gills of a diseased salmon, but the pathogenicity of this
organism has not been tested
(82).
Description
of
Flavobacterium johnsoniae
comb. nov. corrig.
Flavobacterium johnsoniae
(basonyms,
Cytophaga johnsonae
Stanier 1947,
Cytophaga johnsonii
Stanier 1957)
(jo
hn.so
’
ni.ae.
N.
L.
gen. fem. n.
johnsoniae,
of Johnson, named after D.
E.
Johnson [see above]). The description of this taxon is the same
as that given previously
(68),
with the following additions: good
growth occurs on nutrient agar and Trypticase soy agar; ty-
rosine, esculin, tributyrin, and Tween compounds are degrad-
ed; lecithin is not degraded; and o-nitrophenyl-P-D-galactopyr-
anoside is hydrolyzed (10, 61).
FZavobacterium johnsoniae
is
common in soil and freshwater, and strains are frequently
isolated from superficial lesions on diseased fish (9). Thus, for
a long time this species was considered opportunistic, but some
clues to its pathogenicity have been found recently (13).
FZa-
vobacterium johnsoniae
includes
two
strains previously known
as
[Flexibacter] aurantiacus
strains (strains NCIMB 1382* and
NCIMB 1455) (see below).
Description
of
Flavobacterium pectinovorum
comb. nov.
Fla-
vobacterium pectinovorum
(basonym,
Cytophaga pectinovora
(Dorey 1959) Reichenbach 1989) was described as
Flavobac-
terium pectinovorum
by
Dorey in 1959
(25),
but this name was
not included on the Approved Lists of Bacterial Names (75). It
was later reclassified
as
Cytophaga johnsonae
(15)
and then
restored as an independent species under the combination
Cytophaga pectinovora
(68).
The description of this taxon
is
the
same as that given previously
(689,
with the following addi-
tions: good growth occurs on nutrient agar and Trypticase soy
agar; esculin and tyrosine are degraded, but no pigment is
produced
on
tyrosine agar; no precipitate is formed on egg
yolk agar;
o-nitrophenyl-P-D-galactopyranoside
is hydrolyzed;
142
BERNARDET
ET
AL.
INT. J.
SYST.
BACTERIOL.
and the organism is susceptible to vibriostatic compound
0/129
(8).
Description
of
Flavobacterium
psychrophilum
comb. nov.
Fla-
vobacterium psychrophilum
(basonym,
Flexibacter psychrophilus
(Borg 1960) Bernardet and Grimont 1989) was described as
Cytophaga psychrophila
by Borg in 1960 (1 1); this combination
was also used in
Bergey’s
Manual
of
Systematic Bacteriology
(68), but in a 1989 study Bernardet and Grimont proposed that
this taxon should be transferred to the genus
Flexibucter
(10).
The description of this taxon is the same as that given previ-
ously (10, 68). This species is frequently isolated from internal
organs and superficial lesions of diseased fish, and pathogenic-
ity for fish has been demonstrated by experimental infection
tests (18).
Description
of
Flavobacterium saccharophilum
comb. nov.
Flavobacterium saccharophilum
(basonym,
Cytophaga sacchar-
ophila
Agbo and Moss 1979). The description of this taxon is
the same as that given previously
(1,
68). Catalase and cyto-
chrome oxidase activities were listed as positive in the original
description of the species
(1),
but they were not observed in a
later study (68). We clearly observed catalase activity, but no
cytochrome oxidase activity was detected when we used both
dimethylparaphenylene diamine (oxidase discs; bioMkrieux,
Marcy-l’Etoile, France) and
tetramethylparaphenylene
dia-
mine (oxidase liquid reagent; bioM6rieux). Good growth oc-
curs on nutrient agar and Trypticase soy agar; esculin and
tyrosine are degraded, but no pigment is produced on tyrosine
agar; no precipitate is formed on egg yolk agar; and o-nitro-
phenyl-6-D-galactopyranoside
is hydrolyzed
(8).
Description
of
Fluvobacterium succinicans
comb. nov.
Flavo-
bacterium succinicans
(basonym,
Cytophaga succinicuns
Ander-
son and Ordal 1961). The combination
Flexibacter succinicans
has also been used for this species (49). The description of this
taxon is the same as that given previously
(4,68).
Good growth
occurs on nutrient agar and Trypticase soy agar; esculin and
DNA are degraded; tyrosine is not degraded; no precipitate is
formed on egg yolk agar;
o-nitrophenyl-P-D-galactopyranoside
is hydrolyzed; the organism is susceptible to vibriostatic com-
pound 0/129; and H,S is not produced
(8).
The three
Fla-
vobacterium succinicans
strains currently available were iso-
lated from superficial lesions on diseased fish and from water
in a fish tank, but the pathogenicity of these strains was not
tested
(4).
Differentiation
of
Flavobacterium
species.
The main charac-
teristics that differentiate the
10
valid species that belong to the
emended genus
Flavobacterium
are shown in Table
5.
Since the
data in this table were collected from several references, it is
possible that different procedures used in the different studies
resulted in apparent phenotypic differences. Additional differ-
entiating characteristics for the
Fluvobacterium
species include
their API ZYM profiles (Table 6) and their polyamine distri-
bution patterns (see above).
We determined the fatty acid and protein profiles of all valid
species and invalid taxa belonging to the
Flavobacterium
aqua-
tile
rRNA cluster (Table
4
and Fig. 2). Some differences in the
fatty acid profiles
of
species are described above, and these
differences are valuable characteristics for differentiating sev-
eral species. From Fig.
2,
it is obvious that whole-cell protein
analysis combined with a computer-assisted numerical compar-
ison of the patterns is a useful technique for differentiating
Flavobacterium
strains. As mentioned above, the different
[Flaibacter] columnaris
and
[Flexibacter] psychrophilus
strains
which we studied are identical as determined by protein elec-
trophoresis; this finding confirms that these
two
species are
homogeneous genotypically, as determined by DNA-DNA hy-
bridization (10). In this study, several species were represented
by only one strain. However, other species exhibited various
levels of protein electrophoretic heterogeneity (Fig.
2),
as dis-
cussed below.
In the case of
Flavobacterium succinicuns,
the protein profile
of strain NCIMB 2279 is clearly different from the protein
profiles of the
two
other strains, whereas the results of the fatty
acid analysis can be used to differentiate strain NCIMB 2278
from the
two
other strains. These data confirm the previous
observations that there are phenotypic differences in this spe-
cies (68). The G+C contents of the three strains are very
similar (68) (Table 2), but no DNA-DNA hybridization study
has been performed yet.
The seven
Flavobacterium branchiophilum
strains which we
tested all produce very similar fatty acid profiles, but they can
be separated into
two
very distinct groups when their protein
profiles are compared. As there are no obvious differences
in the phenotypic characteristics of these strains and they
have very similar G+ C contents, additional studies (including
DNA-DNA hybridization analyses) will be necessary to inves-
tigate the taxonomic structure of
Flavobacterium branchiophi-
lum.
The
two
strains belonging to the invalid taxon
“[Sporocyto-
phaga]
caul if or mi^'^
clearly produce different fatty acid and
protein profiles, which is consistent with the differences in
G+C contents and phenotypic characteristics noticed previ-
ously (68).
[Flexibacter] aurantiacus
Lewin 1969 contains only two
strains
(SO).
Type strain NCIMB 1382 was previously consid-
ered a
Cytophaga aurantiaca
strain, while strain NCIMB 1455
was previously classified as a strain of
[
Cytophaga] psychrophila
(synonym,
[Flexibacter] psychrophilus
[see above]). Both strains
are phenotypically indistinguishable from the
Flavobacterium
johnsoniae
type strain but very clearly different from both bona
fide
Cytophaga aurantiaca
and
[Flexibacter] psychrophilus
strains (9, 10). In the 1990 National Collection of Industrial
and Marine Bacteria
Catalogue
of
Strains
(57a)
both strains are
listed as “possibly
[Cytophaga] johnsonae.”
Moreover, the two
[Flexibacter] aurantiacus
strains were the closest relatives
of
the
Flavobacterium johnsoniae
type strain when the fatty acid pro-
files were analyzed numerically by a principal-component anal-
ysis (data not shown). The data obtained from DNA-DNA
hybridization studies (Table 3) confirmed that the two
[Flexi-
bacter] aurantiacus
strains belong to the same species; their
high levels of DNA-DNA relatedness and low
AT,,,
with the
DNA
of
the
Flavobacterium johnsoniae
type strain showed that
these three strains form a tight genomic species. Thus,
[Flexi-
bacter] aurantiacus
Lewin 1969 appears to be a junior synonym
of
[Cytophaga] johnsonae
Stanier 1947, and we propose that
it should be included in
Flavobacterium johnsoniae.
In gen-
eral,
Flavobacterium johnsoniae
appears to be very heteroge-
neous. The type strain,
two
additional strains, and the former
[Flexibacter] aurantiacus
strains make up
two
separate electro-
phoretic subgroups. Similar intraspecific protein electro-
phoretic subgroups have been described for several other bac-
teria (87, 89). However, four additional strains, two of which
(ATCC 29585 and ATCC 29586) form a single cluster, occupy
distinct positions on the dendrogram (Fig. 2). DNA-DNA hy-
bridization experiments performed with strains ATCC 29585
and ATCC 29586 revealed that these organisms exhibit a high
level of DNA homology, which confirmed the general rule that
the results of whole-cell protein electrophoresis can be used to
group closely related strains. However, these strains and one of
the other strains (LMG 11391) did not exhibit significant levels
of DNA homology with other
Flavobacterium johnsoniae
strains. The fourth strain which occupied a separate position
on the dendrogram was not included in the DNA-DNA hy-
VOL.
46, 1996
CLASSIFICATION
OF
THE GENUS
FLA
VOBACTERZUM
143
TABLE
6.
API
ZYM
profiles
of
the type strains
of
the
10
valid
species belonging to the genus
Fluvobacrerium”
Hydrolysis of the following substrates‘
:
Strain’
a,
m
c
a
Y
B
9
s
2-
5
c
x
c
-
c-4
a,
m
-
g
f
0
-
x
c
a
s
2
r;
a,
m
c
4
9
-
x
c
s
2-
-
x
c,
c
?
B
A
a
-
>,
c
c
c.
?
PI
Flavobacterium aquatile
LMG
4008T
Flavobacterium branchiophilum
ATCC
35035T
Flavobacterium columnare
NCIMB
224gT
Flavobacterium flevense
DSM
1076T
Flavobacterium hydatis
DSM
2063T
Flavobacterium johnsoniae
LMG
1341T
Flavobacterium pectinovortlm
NCIMB
9059T
Flavobacterium psychrophilum
NCIMB
1947T
Flavobacterium saccharophilum
NCIMB
2072T
Flavobacterium succinicans
NCIMB
2277T
“
Data from references
8
and
10.
See Table
1,
footnote
u.
The values are API
ZYM
reaction scores.
424155200120005
5 23 05 42 20440001
523044131330000
512151100331503
524145200450004
513155211450304
533044200440214
5
2 31510
0 0
3
3
0 0
0
0
534044200532405
533044210550004
bridization analysis. Our results confirm the previous conclu-
sion concerning the phenotypic and genomic diversity of
Fla-
vobacterium johnsoniae
(68).
We concluded that clearly protein
electrophoretic heterogeneity occurs in this species, but that
most of the strains that produce aberrant protein patterns are
misidentified field isolates.
Finally, the taxa
“[Cytophaga] allerginae,” “[Cytophaga] xan-
tha,” “[Flexibacter] aurantiacus
subsp.
excathedrus,” “[Promyx-
obacterium] jlavum,”
and
“[Sporocytophaga] caulifomis”
clearly
belong to the emended genus
Flavobacterium,
as shown by
their phenotypic characteristics, rRNA homology data
(Fig.
1
and Table
2),
and fatty acid analysis data (Table 4). However,
these names have not been validly published, and each of them
except
“[Sporocytophaga] cauliformis”
is represented by only a
single strain;
two
rather different strains of
“[Sporocytophaga]
caulifomis”
are available
(68).
No
16s
rRNA sequence data
have been published for any of these organisms except one
“[Sporocytophaga] caulifomis”
strain
(30).
Thus, instead of
creating five new
Flavobacterium
species, each represented by
one or
two
poorly characterized strains, we decided to refer to
these isolates as
Flavobacterium
sp., pending isolation of new
strains. A larger collection of strains should enable researchers
to properly describe these five taxa and to determine
if
each of
them really represents a distinct species belonging to the genus
Flavobacterium.
Preliminary DNA-DNA hybridization studies
have shown that the DNAs from
“[Cytophaga] allerginae,”
“[Cytophaga] xantha,” “[Promyxobacterium] jfavum,”
and the
two
“
[Sporocytophaga
]
caulijfbmis”
strains do not exhi bit sig-
nificant levels of homology with DNAs from the
Flavobacte-
rium aquatile
and
Flavobacterium johnsoniae
type strains
(8).
Differentiation of
Flavobacteriurn
from related genera.
The
main characteristics that can be used to differentiate the
emended genus
Flavobacterium
from several related taxa be-
longing to rRNA superfamily
V
are shown in Table
7.
The
0
0
0
0
0
1
5
0
0
2
-
000
0
0
0
0
0
0
4 0
0
2
0
0
4
0 0
3
0
0
0
0 0
4 0
0
4
I)
0
following taxa were included in Table
7:
(i) the closest phylo-
genetic relative
of
the
Flavobacteriurn
rRNA branch, as deter-
mined by DNA-rRNA hybridization (i,e.,
[Flavobacterium]
odoratum)
(Fig.
1);
(ii) the genera to which the valid species
included in the emended genus
Flavobacterium
were previously
assigned (i.e., the genera
Cytophaga
and
Flexibacter;
these gen-
era are represented by their type species,
Cytophaga hutchin-
sonii
and
Flexibacter jfexilis,
respectively); (iii) the genera that
now contain species that previously were considered
Flavobac-
terium
species (i.e., the genera
Bergeyella, Chryseobacterium,
Empedobacter, Sphingobacterium,
and
Weeksella;
the genera
Bergeyella, Empedobacter,
and
Weeksella
are represented by
their type and only species,
Bergeyella zoohelcurn, Empe-
dobacter brevis,
and
Weeksella virosa,
respectively); and (iv)
other taxa belonging to the emended family
Flavobacteriaceae
(see below).
Delineation of the families belonging to the
Cytophaga-Ffa-
vobacteriurn-~ac~ero~~e~
rRNA
homology group.
In
Bergey
’s
Manual
of
Systematic Bacteriology,
the genera
Flavobacterium,
Cytophaga,
and
Flexibacter
were placed in the order
Cytophu-
gales
along with several other related taxa
(68).
This definition
of the order took into account classical phenotypic character-
istics, recent data obtained from chemotaxonomic investiga-
tions, and the first results of rRNA studies. The following two
families were included
in
the order
Cytophagales:
the family
Cytophagaceae
Stanier 1940 (including the genera
Cytophaga,
Capnocytophaga, Flexithrix,
and
Sporocytophaga;
the genera
Flexibacter, Microscilla,
and
Chitinophaga
were considered
close relatives of this family) and the proposed family
“Fla-
vobacterzaceae.”
The name of the latter family, which con-
tained strictly aerobic, nonmotile, nongliding, free-living or
parasitic organisms, was subsequently validated (44a). The fol-
lowing other taxa were considered rather close relatives of the
order
Cytophagales:
the family
Bacteroidaceae
Pribram 1933
INT.
J.
SYST.
BACTERIOL.
144 BERNARDET ET
AL.
TABLE
7.
Characteristics that can be used to differentiate the emended genus
Flavobacterium,
related taxa belonging to rRNA superfamily
V,
and the taxa included in the emended family
Flavobacten'aceae"
Characteristic
Flavobacterium
[Flavobacterium] Cytophaga
F1exibacter
Sphingobacterium Chryseobactenum
odoratum hutchinsonii
flailis
Habitat
Pigmented colonies
Gliding motility
Menaquinone"
Capnophilic metabolism
Presence of sphingophospholipids
Growth at 37°C
Growth at 42°C
Growth
on
MacConkey agar
Growth on P-hydroxybutyrate
Acid production from glucose
Acid production from sucrose
DNase activity
Urease activity
Catalase activity
Production of indole
Degradation of cellulose
Degradation of esculin
Degradation of gelatin
Resistance to penicillin G
G+C content (mol%)
Free living or
saprophytic
+h
+'
MK-6
-
Free living or
saprophytic
+
-
MK-6
-
ND
ND
V
V
V
V
+
-
-
V
+c
32-37
V
+
+
+
-
-
+
+
+
-
+
ND
37
Free living
(soil)
+
+
MK-7
-
ND
-
-
ND
ND
+
ND
+
+
+
ND
+
ND
40
-
-
Free living
(freshwater)
+
+
ND
ND
-
-
-
ND
ND
+
ND
ND
ND
-
-
-
ND
+
ND
40-43
Free living or
saprophytic
(+I
-
MK-7
+
V
-R
-
+
ND
+
+
V
V
+
-
-
+
ND
39-45
V
Free living or
parasitic
+
MK-6
-
-
-
4
+h
+
+
'
+
V
+
v'
+
+
+
V
-
-
33-38
Data from references
9,
10,
19, 20, 24, 37, 41-44, 45, 46, 61, 68,
72,
84,
85,
88,
and
102
and this study. Additional phenotypic characteristics that differentiate the
+,
positive reaction;
-,
negative reaction;
(+),
weak positive reaction; v, variable within and between species; ND, not determined or determined for some species
Capnocytophaga
species,
Omcthobacterium rhinotracheale,
and
Riemerella anatipestifer
are described in reference
88.
only.
'See exceptions in Table
5.
"
Positive for all members
of
the rRNA cluster except
[Flavobacterium] gondwanense
and
[Flavobacterium] salegens.
'
MK-6, menaquinone 6; MK-7, menaquinone 7.
1
Most strains are positive for this characteristic.
R
Negative for all
Sphingobacterium
species except
Sphingobacterium thalpophilum.
"
Strain dependent for
Chryseobactenum indologenes.
Positive for all other
Chtyseobacterium
species except
Chryseobacterium scophthalmum.
'
Strain dependent for
Chtyseobacterium meningosepticum.
Positive for all other
Chryseobacterium
species except
Chtyseobacterium scophthalrnum.
J
Positive for all
Capnocytophaga
species except
Capnocytophaga canimorsus.
(36)
and the ring-forming bacteria, which were later placed in
a fourth family, the
Spirusomaceae
Larkin and Borrall
1978
(48).
The general structure of the order
Cytophagales
and re-
lated taxa was later confirmed and completed by new data
obtained from rRNA studies
(30, 56, 58,
72,
101).
Thus, this
taxonomic entity seems to correspond to the phylogenetic en-
tity called superfamily
V
or the
"flavobacter-bacteroides"
phy-
lum depending on the authors and the method used for rRNA
homology studies
(30, 73).
Because of the taxonomic and nomenclatural changes pro-
posed in this study and in other recent publications
(%),
there
are many new problems concerning the definition of the fam-
ilies
Flavobacteriaceae
and
Cytophagaceae
(38).
An emended
genus
Cytophaga
(i.e., a genus restricted to
Cytophaga hutchin-
sonii
and a few other celluIolytic taxa) must remain the type
genus of the family
Cytophagaceae.
Most other misclassified
Cytophaga
and
Flexibacter
species have already been placed in
other genera or new genera. The emended genus
Flavobacte-
rium
proposed in this paper
is
the type genus of the family
Flavobacteriaceae,
but the current description of this genus is
indeed much more similar to the description of "noncellulo-
lytic cytophagas" than to its previous description.
The levels of genotypic divergence corresponding to differ-
ences in
T,(e)
of
14°C
or more (Fig.
1
and Table
2)
between
former members of the genus
Flavobacterium
are far too great
to include all
of
these bacteria in a single family. On the other
hand, from a phenotypic and chemotaxonomic point of view, it
would not be logical to separate some of the taxa belonging to
different major rRNA clusters or solitary rRNA branches, such
as the genera
Chryseobacteriurn
and
Empedobacter
or the gen-
era
Omithobacterium
and
Riemerella.
Three of the rRNA clus-
ters (the
Chryseobacterium-Bergeyella-Riemerella
rRNA cluster,
the
Empedobacter- Weeksella
rRNA cluster, and the
Flavobac-
terium aquatile
rRNA cluster) and
two
solitary rRNA branches
(the
Umithobacterium
and
Capnocytophaga
rRNA branches)
are linked at an average
T,(,,
of about
65"C,
which corre-
sponds to a difference in
Tm(e)
of about
14°C.
The differences
in
Tm(e)
within most well-characterized families of bacteria are
in the range from
8
to
12°C.
In our opinion, the phenotypic
similarities of all
of
the taxa that clustered above a yalue of
65°C
in this study overcame the obviously high levels of geno-
typic divergence. A major problem in the chemotaxonomic
description
of
this group was the previously reported presence
of menaquinone
7
as the sole respiratory quinone in
Omi-
thobacterium
and
Riemerella
strains
(27),
while menaquinone
6
is the only quinone found in members of the genera
Capnocy-
tophaga, Chryseobacterium, Empedobacter,
and
Flavobacterium
(as defined in this study),
[Flavobacterium] odoratum,
members
of the
[Flexibacter] maritimus
rRNA branch, and
Weeksella
virosa
(56,85)
(no data for
Bergeyella toohelcum
are available).
However, the menaquinone contents of
two
reference strains
of
Ornithobacterium rhinotracheale
and
two
reference strains
of
Riemerella anatipestifer
were determined by high-performance
liquid chromatography and mass spectrometry, and all four
strains were found to contain large amounts
of
menaquinone
6
and trace amounts of menaquinone
5
(47).
Thus, the taxa
VOL.
46, 1996
CLASSIFICATION
OF
THE
GENUS
FLA
VOBACTERIUM 145
TABLE
7-Continud
Bergeye
flu
Cupnocytophagu
Ornithobactenurn Riemerella [Flexibacter] niaritimus
zooiielcum rhin otruchea
le
anatipestifer
rRNA cluster
Weeksellu virosu
Empedobacter
brevis
Free living or
parasitic
+
MK-6
-
+
+
+’
t
+
+
+
+
31-33
Parasitic or
saprophytic
-
-
MK-6
__
-
+
+
+
+
-
-
-
-
+
+
-
-
+
35-38
-
Parasitic or
saprophytic
-
-
ND
-
-
+
-
+
+
+
-
+
35-37
-
Parasitic or
saprophytic
+
+
MK-6
+
ND
+
ND
ND
+
+;
ND
ND
-
V
-
-
+
V
-
34-40
Parasitic
-
-
MK-6
+
ND
+
+
ND
-
V
-
-
+
-
-
-
-
-
V
37-39
Parasitic
-
-
MK-6
+
ND
+
+’
ND
-
V
-
ND
V
+
-
-
ND
4
29-35
-
Free living (marine environ-
+
+
(‘
MK-6
ment) or saprophytic
-
ND
-
-
ND
ND
V
V
ND
V
V
-
-
ND
ND
V
33-42
belonging to each cluster and branch in this bacterial group
contain the same respiratory quinone. The members of three
genera (the genera
Capnocytophaga, Omithobacterium,
and
Ri-
emerella)
exhibit similar capnophilic metabolism
(88),
while
the other taxa are considered obligately aerobic
(68).
In fact,
within a genus whose members are for the most part aerobic,
such as the emended genus
Flavobacterium
proposed above,
some species may exhibit capnophilic behavior under certain
conditions (this is true of
Flavobacterium hydatis
and
Flavobac-
terium succinicans).
Moreover, other well-defined bacterial
families contain organisms that have rather different types of
metabolism
(86).
Thus, we believe that the phenotypic similar-
ities, as well as the genotypic similarities, of all of these organ-
isms justify their inclusion in a single family. The emended
description of the family
Flavobacteriaceae
below is a compi-
lation of previously published phenotypic data (43, 44,
72,
85,
88)
(Table
5).
Emended description
of
the family
Flavobacteriaceae
Reichenbach
1989. Cells are short to moderately long rods with
parallel or slightly irregular sides and rounded or slightly ta-
pered ends and are usually 0.3 to
0.6
pm wide and
1
to
10
pm
long, but
some
species may form filamentous flexible cells
under certain growth conditions. Cells in old cultures may form
spherical or coccoid bodies. Gram negative. Spores are not
formed. Flagella are absent. Nonmotile
(Bergqella, Chiyseo-
bacterium, Empedobacter, [Flavobacterium] odoratum, Omi-
thobactenum, Rietizerella,
and
Weeksella
strains) or motile by
gliding
(Capnocytophaga
and
Flavobacterium
strains and mem-
bers
of
the
[Flexibacter] maritimus
rRNA branch). Growth is
aerobic
(Bergeyella, Chiyseobacterium, Empedobacter, Fla-
vobacterium, [Flavobacterium] odoratum,
and
Weeksella
strains
and members of the
[Flexibacter] maritimus
rRNA branch) or
microaerobic to anaerobic
(Capnocytophaga, Omithobacte-
rium,
and
Riemerella
strains). The optimum temperature is
usually in the range from 25 to 35°C. Colonies are nonpig-
mented
(Bergeyella, Ornithobacterium, Riemerella,
and
Week-
sella
strains) or pigmented by carotenoid or flexirubin types of
pigments or both
(Capnocytophaga, Chiyseobacterium, Empedo-
bacter, Flavobacterium,
and
[Flavobacterium] odoratum
strains
and members of the
[Flexibacter] maritimus
rRNA branch).
Menaquinone
6
is the only respiratory quinone or the major
respiratory quinone. Chemoorganotrophic. Intracellular gran-
ules of poly-P-hydroxybutyrate are absent. Sphingophospholip-
ids are absent. Homospermidine is the major polyamine, and
agmatine and putrescine are frequently present as minor com-
ponents. Cellulose is not decomposed. The DNA base compo-
sition ranges from 29 to 45 mol%. Mostly saprophytic in ter-
restrial and aquatic habitats. Several members of the family are
commonly isolated from diseased humans or animals; some
species are considered true pathogens.
The type genus is
Flavobacteiium
Bergey, Harrison, Breed,
Hammer, and Huntoon 1923, as emended in this study. Other
taxa included in the family are the genera
Bergeyella, Capno-
cytophaga, Chiyseobacterium, Empedobacter, Omithobacterium,
Riemerella,
and
Weeksella, [Flavobacterium] odoratum,
and the
taxa belonging to the
[Flexibacter] maritimus
rRNA branch.
Differential characteristics for the taxa belonging to the family
Flavobacteriaceae
are shown in Table
7.
ACKNOWLEDGMENTS
We thank Brigitte Kerouault, Richard Tytgat, and Urbain Torck for
excellent technical assistance. We are very grateful
to
T.
0.
MacAdoo
(Department
of
Foreign Languages, Virginia Polytechnic Institute and
State University, Blacksburg) for his expert advice concerning bacterial
nomenclature and to B. Holmes (National Collection of Type Cul-
tures, London, United Kingdom) for kindly reviewing the manuscript.
We thank all of the individuals who provided strains (Table
1)
and our
colleagues on the International Committee
on
Systematic Bacteriology
Subcommittee on the Taxonomy of
Flavobacteriurn
and
Cytophaga-
Like Bacteria who kindly supported
our
work by providing free bac-
terial strains and advice. We are very grateful to Eiko Yabuuchi
(Osaka City University Medical School, Osaka, Japan) for kindly of-
fering to verify the menaquinone contents of
Ornithobacteriurn
rhino-
tracheale
and
Riernerella
anatipestifer
and to Yoshimasa Kosako (The
Institute of Physical and Chemical Research, Japan Collection of Mi-
croorganisms, Saitama, Japan) for kindly performing the menaquinone
analysis.
146
BERNARDET ET
AL.
INT. J.
SYST.
BACTERIOL.
J.-F.B. thanks the North American Treaty Organization for a re-
search grant. P.V. is indebted to the National Fund for Scientific
Research (Belgium) for
a
position as a postdoctoral research fellow,
K.K.
is
indebted
to
the Fund for Medical Scientific Research (Bel-
performed
in
the framework of CEC contract BIOT-CT91-0294.
sp. nov.,
two
new species from a hypersaline antarctic lake. Int.
J.
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25.
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19.59. Some properties
of
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Gen. Microbiol. 20:91-104.
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w-
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14.
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18.
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