Content uploaded by Antonio Ventosa
Author content
All content in this area was uploaded by Antonio Ventosa on Jan 06, 2015
Content may be subject to copyright.
INTERNATIONAL
JOURNAL
OF
SYSTEMATIC
BACTERIOLOGY,
July 1990,
p.
261-267
Copyright
0
1990,
International
Union
of
Microbiological Societies
0020-7713/90/070261-07$02.00/0
Vol.
40,
No.
3
Volcaniella eurihalina
gene
nov
sp.
nov.
a
Moderately Halophilic
Nonmotile Gram-Negative
Rod
E.
QUESADA,l* M. J. VALDERRAMA,l
V.
BEJAR,l A. VENTOSA,2
M.
C. GUTIERREZ,2
F.
RUIZ-BERRAQUERO,*
AND
A. RAMOS-CORMENZANAl
Department
of
Microbiology, Faculty
of
Pharmacy, University
of
Granada, Granada,’ and Department
of
Microbiology,
Faculty
of
Pharmacy, University
of
Seville, Seville,2 Spain
A comparison of 16 gram-negative moderately halophilic aerobic rod-shaped bacteria with other halophilic
and nonhalophilic gram-negative bacteria supported the establishment of
Volcaniella
eurihalina
gen. nov., sp.
nov. This comparison included phenotypic properties, salt requirements, and guanine-plus-cytosine contents of
the DNAs, as well as DNA-DNA homology studies. The distinguishing features of this new bacterial genus are
as
follows: the organisms are nonmotile short rods that are oxidase negative; they are aerobic with a strictly
respiratory type of metabolism; they are moderate halophiles, optimal growth occurs at a total salt
concentration of
7.5%
(wthol), and they exhibit a strongly euryhaline character; and they have a specific
requirement
for
Na+ ions (sodium can be supplied as NaCl, Na,S04,
or
NaBr). The minimum NaCl
concentration required is
1.5%
(wtlvol). The guanine-plus-cytosine content of the DNA
is
59.1 to 65.7 mol%.
This organism was isolated from hypersaline habitats, including saline soils and salt ponds, and from seawater.
The type strain is strain F9-6
(=
ATCC
49336).
Kushner and Kamekura
(13)
defined moderate halophiles
as those microorganisms that grow optimally at NaCl con-
centrations between 0.5 and
2.5
M. These bacteria have
a
specific requirement for Naf ions, which cannot be replaced
by other cations or nonionizable solutes. In hypersaline
habitats both heterotrophic and phototrophic moderately
halophilic eubacteria have been found. The first group is
constituted by a large variety of bacteria, including gram-
positive and gram-negative species
(30).
The gram-negative
aerobic or facultatively anaerobic heterotrophic rods-shaped
bacteria seem to be the most abundant type in natural
hypersaline habitats. The following species have been val-
idly published previously:
Vibrio costicola
(8),
Chromohalo-
bacter marismortui
(31),
Deleya halophila
(23),
Halomonas
elongata
(32),
and
Halomonas halmophila
(7).
However, there are moderately halophilic gram-negative
microorganisms that have been isolated from hypersaline
habitats and cannot be included in the species described
above. In
1987
Quesada et al.
(21)
described
a
group of
oxidase-negative nonmotile moderately halophilic strains
which had phenotypic characteristics similar to those of
members of the genus
Acinetobacter
(12).
However, these
organisms differed from the only species described up to that
time in this genus,
Acinetobacter calcoaceticus,
by some
characteristics which prompted us to think that our strains
could constitute a separate taxonomic entity.
The purpose
of
this study was to examine 16 of those
moderately halophilic strains more extensively. On the basis
of our results, we propose to name these organisms
Volca-
niella eurihalina
gen. nov., sp. nov.
MATERIALS
AND
METHODS
Bacterial strains.
The methods used for isolation and
further selection of the
16
moderately halophilic nonmotile
strains which we studied and some of the environmental
parameters associated with the sampling sites have been
described previously
(20,22).
The sources of the strains used
in this study were as follows:
14
strains were isolated from
*
Corresponding author.
saline soils located near Alicante, Spain;
1
strain was iso-
lated from the Mediterranean Sea by the Alicante Coast in
Spain; and
1
strain was isolated from the Laguna of Teven-
quiche in Salar of Atacama, Chile.
Reference strains.
The following gram-negative bacteria,
including nonhalophilic and halophilic microorganisms, were
used as reference strains:
Acinetobacter calcoaceticus
CCM
5581,
Alcaligenes faecalis
CCM 1052= (T
=
type strain),
Alteromonas luteoviolacea
ATCC
33492=,
Chromohalobac-
ter marismortui
ATCC
17056T,
Deleya aesta
NCMB
1980T,
Deleya cupida
NCMB
1978=,
Deleya halophila
CCM
3662T,
Deleya marina
ATCC
25374T,
Deleya pacijica
NCMB
1977T,
Deleya venusta
NCMB
1979T,
Flavobacterium men-
ingosepticum
CCM
2719T,
Halomonas elongata
ATCC
33 173=,
Halomonas halrnophila
CCM
2833=,
“Pseudomo-
nas halosaccharolytica”
CCM
2851,
Pseudomonas putida
CCM
1977T,
Vibrio alginolyticus
CECT
521T,
Vibrio costi-
cola
NCMB
701T,
and
Vibrio natriegens
CECT
526=.
(Names in quotation marks indicate that the names were not
included in the Approved Lists of Bacterial Names, nor have
they been included in the supplements to the Approved Lists
u7,
261.)
Maintenance media.
Halophilic strains (marine or moder-
ately halophilic bacteria) were maintained on agar slants of
MH medium
(22)
containing (per liter)
10
g of yeast extract
(Difco, Laboratories, Detroit, Mich.),
5
g of Proteose Pep-
tone no.
3
(Difco),
1
g of glucose, and
20
g of Bacto-Agar
(Difco); this medium was supplemented with a balanced
mixture of sea salts
(24)
to give a final salt concentration of
3
or
7.5%
(wthol). The salt solutions containing this sea salt
mixture are referred to below as total salts; a
10%
solution
contained
7.86%
(wthol) NaC1,
0.66%
(wtlvol) MgCl,,
0.96%
(wthol) MgS04,
0.0036%
(wtlvol) CaCl,,
0.2%
(wt/
vol) KC1,
0.006%
(wthol) NaHCO,, and
0.023%
(wthol)
NaBr. The pH was adjusted to
7.2
with
1
N NaOH.
Nonhalophilic strains were grown on Trypticase soy agar
(Difco). Cultures were incubated at
32°C
for
24
h
and then
stored at 15°C.
Phenotypic characterization.
To
complete our previous
phenotypic characterization of the strains
(21),
several addi-
tional features were studied; strain was chosen as a
261
262
QUESADA ET AL.
-
INT.
J.
SYST.
BACTERIOL.
-
-Pseudomonas Putida
CCM
1977T
-
Flavobacterium meningosepticum
CCM
27lgT
-
vmrio aiginotyticus
CECT
52iT
-VlbrlO natriegens
CECT
526T
-
Halomonas elongata
ATCC
33173T
representative of the group. The cellular morphology of
strain F9-6T was observed in order to determine its length
and shape and to look for the presence of slime, spores, and
a sheath. Encapsulation was observed by using the Muir
staining technique
(4).
Microscopic examinations were also
performed to determine whether morphological changes
occurred as a consequence of different salt concentrations in
the culture medium or different incubation times. In these
studies we used cultures grown in liquid MH medium and
phase-contrast and electron microscopy. For transmission
electron microscopy, samples of the cultures were stained
with a 2% (wthol) solution of phosphotungstic acid (pH 7.0)
and examined with a Zeiss model EM902 transmission
electron microscope. For scanning electron microscopy,
cells were fixed with a 2% (wthol) glutaraldehyde solution.
The fixed cells were dehydrated by washing them with
increasing concentrations of acetone. The samples were
critical point dried and sputter coated with gold (in a partial
argon atmosphere at
100
mtorr or a vacuum of 10 torr) (1 torr
=
133.3 Pa). Electron micrographs were obtained by using a
Zeiss model DSM950 scanning electron microsope operated
at 15 kV and having a beam specimen angle of
45".
Mid-log-phase cultures (20 h) and old cultures
(3
days)
grown on solid MH medium supplemented with 7.5% (wt/
vol) total salts were observed in order to describe colonial
morphology and pigmentation; the type
of
growth was also
observed in liquid cultures.
The growth rates of strain F9-6T at different salt concen-
trations were determined by using MH medium. The follow-
ing total salt concentrations were tested:
0.5,
2.5,
5,
7.5,
10,
15,20, and 25% (wt/vol). Inocula (0.1 ml; ca.
lo5
cells per ml)
from an appropriate dilution of an 18-h culture of strain
F9-ST grown in MH medium containing 7.5% (wthol) total
salts were added to 250-ml Erlenmeyer flasks containing
SO-ml portions of the different salt concentrations listed
above. The cultures were then incubated in a rotatory
shaker. Viable counts were determined by measuring plate
PERCENTAGE SIMILARITY
20 40
so
80
100
I
I
I
I
counts, using the method of Milles and Misra (16) and MH
medium. Five replicate drops (0.03 ml) were used for each
dilution, which was made at the appropriate salt concentra-
tion. These experiments were performed in triplicate at
22"C, 32°C (optimal temperature), and 42°C.
All
of
the reference strains were investigated by using the
same phenotypic tests used previously for the new isolates
(21). In these tests we used media containing salt solutions
that were adequate for optimal growth.
Numerical taxonomy.
Differential features were used for a
numerical analysis. Positive and negative results were coded
as
1
and
0,
respectively. Incomplete, missing, or intermedi-
ate susceptibility data were coded as 9. Levels of strain
similarity were estimated by using the simple matching
coefficient (29), and cluster analysis was carried out by using
the unweighted pair group method of association (28). The
test error was evaluated by examining five strains in dupli-
cate. A cophenetic value was also estimated (27). Computa-
tions were made with an Eclipse model MV/10000 computer
by using the MINT program (25) at the Computer Centre,
University of Granada, Granada, Spain.
DNA base composition.
Exponential-phase cells of some
representative strains were ruptured, and the DNAs were
purified by using the method
of
Marmur
(14).
The guanine-
plus-cytosine (G+C) content of each DNA was determined
from the midpoint
of
the thermal denaturation profile
(T,)
(15) obtained with a model UV-Vis 551s spectrophotometer
(The Perkin-Elmer Corp., Norwalk, Conn.) at 260 nm; this
instrument was programmed for temperature increases of
l.O"C/min. The
T,
was determined by the graphic method
described by Ferragut and Leclerc (6), and the G+C content
was calculated from this temperature by using
0.1
x
SSC
(1
x
SSC is 0.15 M NaCl plus
0.015
M
sodium citrate). The
T,
of
reference DNA from
Escherichia
coli
NCTC
9001
was
74.4"C in 0.1~ SSC (19).
Preparation
of
labeled DNA.
DNA was labeled by using the
multiprime system, a commercial kit (kit RPN 1601Y; Am-
STRAIN DESIGNATION
Volcanlella eurihallna
FIG.
1.
Simplified dendrogram showing clustering
of
the
16
strains
of
Volcaniella
eurihalina
gen.
nov.,
sp.
nov.,
and
18
reference strains
based
on
the simple matching coefficient and unweighted pair group method of association clustering.
VOL.
40,
1990
VOLCANIELLA EURIHALINA
GEN. NOV., SP. NOV.
263
ersham International, Amersham, England), and [1',2',5-
3H]dCTP (Amersham). The average specific activity ob-
tained with this procedure was 8.4 x
lo6
cpm/pg of DNA.
The labeled DNA was denatured before hybridization by
being heated at 100°C for
5
min and then placed on ice.
DNA
homology experiments.
DNA homology studies were
performed by using the competition procedure of the mem-
brane method described by Johnson (11). Competitor DNAs
were sonicated (Braun Melsungen, Melsungen, Federal Re-
public of Germany) at
50
W
for two 15s bursts. Membrane
filters (type HAHY; Millipore Corp., Bedford, Mass.) con-
taining reference DNA (ca. 25 pg/cm2) were placed in 5-ml
screw-cap vials which contained the labeled, sheared, dena-
tured DNA and the denatured, sheared competitor DNA.
The ratio of the concentration of competitor DNA to the
concentration of labeled DNA was at least 150:l. The final
volume was adjusted to 140
pl,
and the reaction mixture
contained final concentrations of 2X SSC and 30% formam-
ide solution. The hybridization temperature ranged between
56
and 58"C, which is below the upper limit permitted for the
validity of the filter method
(5).
The vials were shaken
slightly for 18 h in a water bath (Grant Instruments, Cam-
bridge, England); these procedures were done
in
triplicate.
After hybridization the filters were washed in 2X SSC at the
optimal renaturation temperature. The radioactivity bound
to the filters was measured with a liquid scintillation counter
(Beckman Instruments, Inc., Palo Alto, Calif.), and the
percentage of homology was calculated by using the method
of Johnson
(11).
At least two independent determinations
were performed for each experiment; the results reported
below are the mean values.
RESULTS
Figure
1
shows the results of the numerical analysis. At a
similarity level of SO%, the 16 moderately halophilic strains
grouped in a single phenon. The cophenetic value was
0.93424, and the estimated test error was less than 2%. None
of the reference strains clustered with
our
isolates, which is
in agreement with the numerous differences in the pheno-
typic characteristics of these organisms.
All of the strains were gram-negative, oxidase-negative,
nonmotile rods with poly-P-hydroxybutyrate inclusions,
formed catalase, and had a respiratory type
of
metabolism.
All
cultures grew at total salt concentrations between
5
and
20% (wthol), at pH
5
to 10, and at 15, 25, 32, 37,
40,
and
45°C.
No
strain produced pycocyanine or fluorescine, nor
did any strain produce acid from the following carbon
compounds: adonitol, L-arabinose, D-cellobiose, dulcitol,
ethanol, D-fructose, D-galactose, D-glucose, inulin, lactose,
maltose, D-mannitol, D-mannose, D-melezitose, melibiose,
salicin, D-sorbitol, sucrose, D-trehalose, and xylose. All of
the strains reduced nitrate to nitrite, hydrolyzed gelatin,
tyrosine, and urea, grew on MacConkey agar and cetrimide
agar and in the presence of KCN, and reduced selenite. All
of them were negative for the following tests: indole, methyl
red, Voges-Proskauer, casein hydrolysis, starch hydrolysis,
hemolysis, lecithovitellin, gluconate oxidation, and nitrite
reduction. These organisms were not capable of respiration
on nitrate, nitrite, or fumarate. All of the strains utilized the
following compounds: D-gluconate
,
DL-lactate
,
malonate
,
L-alanine, L-arginine, and L-serine. No strain utilized escu-
lin, benzoate, starch, or L-tryptophan. The strains were
susceptible to chloramphenicol (30 pg), colistin
(50
pg),
nalidixic acid (30 pg), polymyxin
B
(300 IU), rifampin
(5
pg),
sulfonamide (200 pg), and sulfonamide (23.75 pg)-trimetho-
TABLE
1.
Characteristics for which the
16
strains
of
Volcaniefla
eurihalina
differ from one another
Frequency
of
Reaction
of
strain
positive reaction
ATCC
49336T
Characteristic
Growth at a salt concn
of
0.5%
(wthol)
3%
(wt/vol)
25%
(wthol)
30%
(wthol)
Growth at
5°C
H,S
production
Phenylalanine dearninase
ONPG"
Tween
80
hydrolysis
Tween
20
hydrolysis
DNase
Phosphatase
Esculin hydrolysis
Utilization of the following or-
ganic compound as the sole
source of carbon and energy:
L-
Arabinose
D-Cellobiose
D-Fructose
D-Galactose
D-Glucose
Inulin
Lactose
Maltose
D-Mannose
L-Raffinose
L-Rharnnose
D-Salicin
L-Sorbose
Sucrose
D-Tre halose
D-Xylose
Acetate
Caprilate
Citrate
Formate
Fumarate
Hippurate
Oxalate
Pyruvate
Propionate
Succinate
D-Tartrate
P-H
ydrox ybutyrate
Adonitol
Dulcitol
DL-Glycerol
meso-Inositol
D-Mannitol
D-Sorbitol
Utilization of the following amino
acid as the sole source
of
carbon, nitrogen, and energy:
L-Aspartic acid
L-Glutamic acid
L-Histidine
DL-Isoleucine
L-Leucine
L-Ly sine
L-Ornithine
Antibiotic susceptibility
Arnikacin
(30
pg)
Carbenicillin
(100
pg)
Cephalothin
(30
pg)
Erythromycin
(15
pg)
Gentamicin
(10
IU)
44
94
75
38
75
94
7
46
42
94
44
88
81
88
73
73
56
38
31
88
93
81
62
69
94
6
75
69
75
88
25
93
27
75
50
7
94
60
27
12
81
44
38
56
81
88
50
94
94
88
87
81
94
81
14
7
17
50
6
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
-
-
-
+
+
+
+
+
+
+
+
+
+
+
-
-
-
-
+
+
+
+
+
+
+
+
+
-
-
-
-
+
+
+
+
+
+
+
-
-
-
+
-
a
ONPG,
o-Nitrophenyl-P-D-galactopyranoside.
264
QUESADA
ET
AL. INT.
J.
SYST.
BACTERIOL.
f
Y
FIG.
2.
Electron micrograph
of
Volcaniella eurihalina
F9-ST
after cells were stained with phosphotungstic acid. Bar
=
1
pm.
prim (1.25 pg). All of them were resistant to ampicillin
(100
pg), clindamycin (2
IU),
penicillin
G
(10
IU), streptomycin
(10
pg),
and tetracycline (30 pg).
Other characteristics of the strains are summarized in
Table
1.
The cellular morphology of strain F9-fjT grown in MH
medium containing 7.5% (wthol) total salts is shown in Fig.
2 and 3. The cells were short, straight, capsulated rods (2.0
to 2.5 by
0.8
to 1.0 pm) which did not produce endospores or
sheaths. The cells occurred singly, in pairs, and sometimes
in short chains. A number of longer forms and filaments were
also
produced (Fig.
3).
Neither salt concentration, incuba-
tion time, nor temperature influenced cellular length and
shape. The strains developed slightly opaque, cream,
smooth, circular, entire colonies on MH medium containing
7.5% (wthol) total salts. They were remarkably mucoid after
3
to
5
days
of
incubation. In liquid cultures they produced
homogeneous turbidity.
The results
of
growth measurements
of
strain F9-6T at
different salt concentrations are shown in Fig.
4.
This
microorganism exhibited optimal growth at a total salt con-
centration
of
7.5% (wthol) when it was incubated at 32°C
(p
=
0.77 h-'); lower or higher salt concentrations resulted in
decreases in the growth rate although the organism could
grow at total salt concentrations between 2.5 and 25%
FIG. 3.
Scanning electron micrograph
of
Volcaniella eurihalina
F9-6T,
showing that some long cells are produced. Bar
=
2
pm.
I..
1.
95
2,s
5
7,5
10
15
20
25
Salt
concentratiom
(OI.1
FIG. 4.
Growth rates
of
Volcaniella eurihalina
F9-6T
at different
salt concentrations
in
MH
medium and at various incubation tem-
peratures. Symbols:
a,
22°C;
0,
32°C;
0,
42°C.
(wthol). However, when strain F9-tjT was incubated at
temperatures lower or higher than the optimal temperature,
the results obtained were different. At 22"C, this organism
exhibited optimal growth at a total salt concentration of
5%
(wt/vol) and could grow at total salt concentrations ranging
from
2.5
to 25% (wtlvol). When it was incubated at 42"C, it
exhibited its highest growth rate at
a
total salt concentration
of
15%
(wthol) and could grow at total salt concentrations
between
5
and
15%
(wdvol).
Strain F9-6T had a specific requirement for Na+ cations,
and salts of
K+,
Mg2+, Li+, or NH4+ did not support
growth. Its minimum NaCl requirement was 1.5% (wthol).
C1- anions could be replaced by
SO,2-
or Br- but not by
NO,- or
S2032-
(M.
J.
Valderrama,
V.
Bejar,
E.
Quesada,
and
A.
Ramos-Cormenzana, unpublished data).
Table 2 shows the DNA base compositions of and the
results of DNA-DNA homology experiments carried out
with five representative strains. The
G+C
contents ranged
from 59.1 to 65.7 mol%. The levels
of
similarity between
DNAs from labeled strains F9-6= and F2-12 and DNAs from
the other isolates ranged from 72 to
88%.
Low levels of
relatedness between labeled DNA from type strain F9-6, and
TABLE
2.
G+C
contents and homology values
of
DNAs from
representative strains
of
Volcaniella eurihalina
%
Homology
with
3H-labeled
Strain
F9-6=
Strain
F2-12
Unlabeled
DNA
G+C
content
DNA
from:
from strain: (mol%)
~~ ~
F9-6T 65.7 100 72
F2-12 61.4
80
100
41a
59.1 85
88
B-1
61.2 77 72
F2-15 59.4 82 76
VOL.
40, 1990 VOLCANIELLA EURIHALINA
GEN. NOV.,
SP.
NOV.
265
TABLE
3.
DNA base compositions and levels
of
DNA-DNA
homology between
Volcaniella eurihalina
and
other gram-negative species
Unlabeled DNA from:
%
Homol-
G+C
ogy
with
content 3H-labeled
(mol%) DNA from
strain
F9-ST
Volcaniella eurihalina
ATCC
49336T
Acinetobacter calcoaceticus
CCM
5581
Alcaligenes faecalis
CCM
1052T
Alteromonas luteoviolacea
ATCC
33492T
Chromohalobacter marismortui
ATCC
17056T
Deleya aesta
NCMB
1980T
Deleya cupida
NCMB
197gT
Deleya halophila
CCM
3662T
Deleya marina
ATCC
25374T
Deleya pacifca
NCMB
1977=
Deleya venusta
NCMB
1979T
Flavobacterium meningosepticum
CCM
2719T
Halomonas elongata
ATCC
33173T
Halomonas halmophila
CCM
2833T
“Pseudomonas halosaccharolytica”
CCM
2851
Pseudomonas putida
CCM
1977T
Vibrio alginolyticus
CECT
521T
Vibrio costicola
NCMB
701T
Vibrio natriegens
CECT
52tjT
65.7
ND“
ND
42.1’
62.3’
57.4’
61
.Ob
66.7’
ND
ND
53.7’
ND
60.5’
62.9
ND
ND
47.8’
49.9’
47.2’
100
0
0
17
0
8
6
19
15
15
22
12
7
12
22
20
10
22
3
a
ND, Not determined.
Data from references
1,
3,
7,
8,
9,
23,
and
32.
DNAs from the reference strains used for comparison were
observed (Table 3).
DISCUSSION
In a previous work we described two new groups of
nonmotile moderate halophiles on the basis of a numerical
analysis of the results of 147 phenotypic tests and the G+C
contents of representative strains (21). One of these groups
was composed of
22
oxidase-negative strains which were
tentatively assigned to the genus
Acinetobacter.
However,
these strains exhibited many differences when they were
compared with the only species included at that time in this
genus,
Acinetobacter calcoaceticus
(12). Since then, the
following five new
Acinetobacter
species have been de-
scribed:
Acinetobacter baumannii, Acinetobacter hae-
moly ticus, Acinetobacter johnsonii, Acinetobacter junii,
and
Acinetobacter lwofii
(2).
These organisms have G+C con-
tents that range from 40 to 46 mol%. The strains which we
used in this study have higher G+C contents (between 59.1
and 65.7 mol%). In addition, there are some other pheno-
typic differences between these
Acinetobacter
species and
our strains, including salt requirement, nitrate reduction,
H2S production, and utilization of different sources of car-
bon, nitrogen, and energy.
Table 2 shows the results obtained in the DNA-DNA
homology experiments carried out with five strains chosen
as representatives of the group. The representatives which
we used constitute a very homogeneous group, since the
levels of homology between them ranged from 72 to 88%.
However, the levels of DNA relatedness to other gram-
negative bacteria, including nonhalophilic and halophilic
species, were very low, ranging from
0
to 22% (Table 3). Our
strains showed higher levels of DNA homology with some
halophilic species than with
Acinetobacter calcoaceticus.
The strains which we studied can be easily differentiated
from related genera and other gram-negative bacteria, such
as
Pseudomonas, Deleya, Alteromonas, Halomonas,
or
Chromohalobacter
species, by a great number of important
phenotypic markers (e.g., morphology, motility, oxidase
test, ability to produce acid from glucose and other carbo-
hydrates, and ability to grow on different substrates as sole
sources of carbon, nitrogen, and energy).
Very recently, a new species of halophilic bacteria,
Me-
sophilobacter marinus,
was described by Nishimura et al.
(18). This taxon, which includes aerobic, mesophilic, non-
motile rods, is not related to our strains because of different
G+C contents (44.0 to 46.9 mol%) and because of the results
of the following tests: oxidase, acid production from carbo-
hydrates, indole, methyl red, and
H,S
production.
On the basis of the genotypic and phenotypic characteris-
tics of the strains which we studied, we propose that they
should be included in a new genus,
Volcaniella,
with the
single species
Volcaniella eurihalina
sp. nov. Useful char-
acteristics for distinguishing
Volcaniella eurihalina
from
other aerobic, gram-negative
,
moderately halophilic rod-
shaped bacteria are shown in Table 4.
Description
of
Volcaniella
gen. nov.
Volcaniella
(Volxa.
niel’la. M.
L.
fem. n.
Volcaniella,
named for B. Elazari-
Volcani, the microbiologist who first described halophilic
microorganisms from the Dead Sea). Cells are capsulated
short straight rods that are 0.8 to 1.0 pm in diameter and 2.0
to 2.5 pm long. They commonly occur singly or in pairs.
Nonmotile. Gram-negative. The cells accumulate poly-p-
hydroxybutyrate as an intracellular reserve product. They
do not form endospores or sheaths. They have a respiratory
type of metabolism with oxygen as the terminal acceptor.
They are moderate halophiles that are capable of growth at
salt concentrations between
5
and 20% (wthol). Optimal
growth occurs at a salt concentration of
7.5%
(wthol).
Chemoorganotrophs. All strains grow well at pH
5
to 10 and
at 15 to
45°C.
Oxidase negative and catalase positive.
Isolated from hypersaline habitats (soils, salt ponds) and
from seawater. The G+C content of the DNA is
59.1
to 65.7
mol%
(T,
method).
The type species is
Volcaniella eurihalina.
Description
of
Volcaniella eurihalina
sp. nov.
Volcaniella
eurihalina
(eu.ri.ha.li‘na. Gr. adj.
euris,
wide, broad; Gr.
adj.
halinos,
salted;
M.
L.
adj.
eurihalina,
growing in the
presence
of
a wide range
of
salt concentrations). The char-
acteristics are as described above for the genus. Colonies on
MH medium containing 7.5% (wthol) salts are circular,
convex, smooth, slightly opaque, and cream with entire
margins. They are 2 to
3
mm in diameter after
20
h and 4 to
5
mm in diameter and remarkably mucoid after 72 h at 32°C.
In liquid cultures they grow uniformly. The type strain
shows a specific requirement for Na+ cations; sodium can be
supplied as NaC1, Na2S0,, or NaBr. All strains are capable
of growth on KCN and reduce nitrate to nitrite but not to
gas. They hydrolyze tyrosine, gelatin, and urea, grow
on
MacConkey agar or cetrimide agar, and reduce selenite. No
strain produces acid from carbohydrates, forms pyocyanine
(or fluoresces) or indole, is methyl red or Voges-Prokauer
positive, hydrolyzes starch, casein, blood, or lecithovitellin,
or oxidizes gluconate. Strains are not capable of respiration
on nitrate, nitrite, or fumarate. All strains are susceptible to
chloramphenicol (30 pg), colistin
(50
pg), nalidixic acid (30
pg), polymyxin B (300 IU), rifampin
(5
pg), sulfonamide
(200
pg), and sulfonamide (23.75 pg)-trimethoprim (1.25 pg), and
all strains are resistant to ampicillin (100 pg), clindamycin (2
IU), penicillin G (10 IU), streptomycin
(10
pg), and tetracy-
cline (30 pg).
Most of the strains utilize a great diversity of organic
266 QUESADA ET AL. INT.
J.
SYST. BACTERIOL.
TABLE 4. Differential characteristics of
Volcaniella eurihalina
ATCC 49336T and other aerobic, moderately halophilic,
gram-negative, rod-shaped bacteria“
Characteristic
Vibrio Halomonas Halomonas Deleya Chromohalobacter Volcaniella
costicola halmophila elongata halophila marismortui eurihalina
Morphology
Pigmentation
Motility
Anaerobe (facultative)
Oxidase
Acid production from:
Arabinose
Glucose
Lactose
Manitol
H,S production
Hydrolysis
of
Gelatin
Casein
Esculin
DNA
Nitrate reduction
Nitrite reduction
Phosphate
G+C content of type strain (mol%)
Curved rods
+
+
+
-
b
-
+
d
d
-
+
+
d
d
-
-
d
49.9
Rods
Yellow
-
-
+
+
+
+
+
-
+
ND
+
-
-
-
+
62.9
Rods
+
+
+
-
-
+
-
-
-
d
d
+
+
+
60.5
-
-
Rods
Violet
+
-
-
+
+
+
-
-
-
-
-
ND
d
-
-
62.3
~ ~~
a
Data from references
10,
30,
and
31
and from this
study.
+,
Positive;
-,
negative;
(+),
80%
or more
of
the strains are positive; d, differs among strains; ND, not determined.
compounds as sole sources of carbon and energy. These
include carbohydrates, organic acids, polyols, and amino
acids. The following compounds are utilized by at least
80%
of the strains: L-arabinose, lactose, maltose, D-mannose,
D-salicin, acetate, citrate, D-gluconate, DL-lactate,
DL-
malate, malonate, pyruvate, P-hydroxybutyrate, rneso-inosi-
tol, D-mannitol, L-alanine, L-arginine, L-aspartic acid,
L-
glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine,
L-ornithine, and L-serine. At least
80%
of the strains cannot
use esculin, L-sorbose, benzoate, oxalate, starch, D-tartrate,
or L-tryptophan.
Eighty percent or more of the strains give positive results
for the following tests:
H,S
and phosphatase production,
Tween
20
hydrolysis, esculin hydrolysis, and growth at a
total salt concentration of 3% (wt/vol).
Eighty percent or more of the strains give negative results
for the following tests: phenylalanine deaminase and suscep-
tibility to amikacin (30 pg), carbenicillin
(100
pg), cephalo-
thin (30 pg), and gentamicin
(10
IU).
The strains were isolated from hypersaline habitats (soils,
salt ponds) and from seawater and have DNA G+C contents
ranging from 59.1 to
65.7
mol%
(T,
method).
The type strain is strain F9-6
(=
ATCC 49336), which has
a DNA G+C content of 65.7 mol%
(T,
method). This strain
has all of the characteristics described above for the type
species, including those for which
80%
or more of strains are
positive or negative. It was isolated from hypersaline soil
located near Alicante in southern Spain.
ACKNOWLEDGMENTS
This investigation was partially supported by grant
PS88-0105
from the Ministerio de Educaci6n y Ciencia (DGICYT) and by
grants from the Comisi6n Asesora para el Desarrollo de la Investi-
gaci6n Cientifica y Ttcnica and from the Junta de Andalucia.
LITERATURE CITED
1.
Baumann, L.,
P.
Baumann,
M.
Mandel, and R. D. Allen.
1972.
Taxonomy of aerobic marine eubacteria. J. Bacteriol. 110:
402-429.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Bouvet,
P.
J. M., and
P.
A.
D.
Grimont.
1986. Taxonomy
of
the
genus
Acinetobacter
with recognition of
Acinetobacter bauma-
nii
sp. nov.,
Acinetobacter haemolyticus
sp. nov.,
Acinetobac-
ter jonhsonii
sp nov., and
Acinetobacter junii
sp. nov. and
emended descriptions of
Acinetobacter calcoaceticus
and
Aci-
netobacter lwofii.
Int. J. Syst. Bacteriol. 36:228-240.
Callies, E., and
W.
Mannheim.
1978. Classification of the
Flavobacterium-Cytophaga
complex on the basis of respiratory
quinones and fumarate respiration. Int. J. Syst. Bacteriol.
28: 14-19.
Cowan,
S.
T., and K. J. Steel.
1974. Manual for the identification
of medical bacteria. Cambridge University Press, Cambridge.
De Ley, J., and
R.
Tijtgat.
1970. Evaluation of membrane filter
methods
for
DNA-DNA hybridization. Antonie van Leeuwen-
hoek
J.
Microbiol. Serol. 36:461-474.
Ferragut, C., and
H.
Leclerc.
1976. Etude comparative des
mtthodes de dktermination du T, de I’ADN bacttrien. Ann.
Microbiol. (Paris) 127:223-235.
Franzmann,
P.
D.,
V. Wehmeyer, and E. Stackebrandt.
1988.
Halomonadaceae
fam. nov., a new family of the class
Proteo-
bacteria
to accomodate the genera
Halomonas
and
Deleya.
Syst. Appl. Microbiol. 11:16-19.
Garcia, M. T., A. Ventosa, F. Ruh-Berraquero, and M. Kokur.
1987. Taxonomic study and amended description
of
Vibrio
costicola.
Int.
J.
Syst. Bacteriol. 37:251-256.
Gauthier, M. J.
1976. Morphological, physiological, and bio-
chemical characteristics of some violet-pigmented bacteria iso-
lated from seawater. Can.
J.
Microbiol. 22:138-149.
Holmes, B., R. J. Owen, and T.
A.
McMeekin.
1984. Genus
Flavobacterium
Bergey 1923, p. 353-360.
In
N.
R. Krieg and
J. G. Holt (ed.), Bergey’s manual of systematic bacteriology,
vol.
1.
The Williams
&
Wilkins Co., Baltimore.
Johnson, J. L.
1981. Genetic characterization, p. 450-472.
In
P.
Gerhardt, R. G. E. Murray, R.
N.
Costilow, E.
W.
Nester,
W. A. Wood,
N.
R. Krieg, and
G.
B. Phillips (ed.), Manual of
methods for general bacteriology. American Society for Micro-
biology, Washington, D.C.
Juni,
E.
1984. Genus 111.
Acinetobacter
Brisou and PrCvot 1954,
p. 303-307.
In
N.
R.
Krieg and
J.
G. Holt (ed.), Bergey’s
manual of systematic bacteriology, vol.
1.
The Williams
&
Wilkins
Co.,
Baltimore.
Kushner, D. J., and M. Kamekura.
1988. Physiology
of
halo-
philic eubacteria, p. 109-141.
In
F.
Rodriguez-Valera (ed.),
VOL.
40, 1990
VOLCANIELLA EURIHALINA
GEN. NOV., SP. NOV. 267
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Halophilic bacteria. CRC Press, Inc., Boca Raton, Fla.
Marmur, J.
1961.
A
procedure for the isolation of deoxyribo-
nucleic acid from microorganisms. J. Mol. Biol. 3:208-218.
Marmur,
J.,
and
P.
Doty.
1962. Determination of the base
composition of deoxyribonucleic acid from its thermal denatur-
ation temperature. J. Mol. Biol. 5109-118.
Milles,
A.
A.,
and
S.
A.
Misra.
1938. The stimulation of the
bactericidal power of blood. J. Hyg. 38:732-749.
Moore,
W.
V.
H.,
E.
P.
Cato, and L. V.
H.
Moore.
1985. Index
of the bacterial and yeast nomenclature changes published in the
International Journal of Systematic Bacteriology since the 1980
Approved Lists of Bacterial Names
(1
January 1980 to
1
January
1985). Int. J. Syst. Bacteriol. 35382407.
Nishimura,
Y.,
M. Kinpara, and
H.
Iizuka.
1989.
Mesophilobac-
ter marinus
gen. nov., sp. nov.: an aerobic coccobacillus
isolated from seawater. Int. J. Syst. Bacteriol. 39:378-381.
Owen, R.
J.,
and
D.
Pitcher.
1985. Current methods for estimat-
ing DNA base composition and levels of DNA-DNA hybridiza-
tion, p. 67-93.
In
M. Goodfellow and E. Minnikin (ed.), Chem-
ical methods
in
bacterial systematics. Academic Press, Inc.
(London), Ltd., London.
Quesada,
E.,
V. Bdjar,
M.
J. Valderrama,
A.
Ventosa, and
A.
Ramos-Cormenzana.
1985. Isolation and characterization of
moderately halophilic rods from different saline habitats. Micro-
biologfa 1:89-96.
Quesada, E., M.
J.
Valderrama, V. Bbjar,
A.
Ventosa,
F.
Ruiz-Berraquero, and
A.
Ramos-Cormenzana.
1987. Numerical
taxonomy
of
moderately halophilic Gram-negative nonmotile
eubacteria. Syst. Appl. Microbiol. 9:132-137.
Quesada,
E.,
A.
Ventosa,
F.
Rodriguez-Valera, L. Megias, and
A.
Ramos-Cormenzana.
1983. Numerical taxonomy of moder-
ately halophilic Gram-negative bacteria from hypersaline soils.
J.
Gen. Microbiol. 129:2649-2657.
Quesada,
E.,
A.
Ventosa,
F.
Ruiz-Berraquero, and
A.
Ramos-
24.
25.
26.
27.
28.
29.
30.
31.
32.
Cormenzana.
1984.
Deleya halophila,
a new species of moder-
ately halophilic bacteria. Int. J. Syst. Bacteriol. 34:287-292.
Rodriguez-Valera,
F.,
F.
Ruiz-Berraquero, and
A.
Ramos-Cor-
menzana.
1981. Characteristics of the heterotrophic bacterial
populations in hypersaline environments of different salt con-
centrations. Microb. Ecol. 7:235-243.
Rohlf,
F.
J.
1985. Numerical taxonomy system of multivariate
statistical programs. State University of New York, Stony
Brook.
Skerman,
V.
B.
D.,
V. McGowan, and
P.
H.
A.
Sneath (ed.).
1980. Approved lists of bacterial names. Int.
J.
Syst. Bacteriol.
30:22H20.
Sneath,
P.
H.
A.,
and R. Johnson.
1972. The influence on
numerical taxonomic similarities
of
errors
in
microbiological
tests.
J.
Gen. Microbiol. 72:377-392.
Sneath,
P.
H.
A.,
and R. R. Sokal.
1973. Numerical taxonomy.
The principles and practice of numerical classification.
W.
H.
Freeman, San Francisco.
Sokal, R. R., and C.
D.
Michener.
1958.
A
statistical method for
evaluating systematic relationships. Univ. Kans. Sci. Bull.
38: 1409-1438.
Ventosa,
A.
1988. Taxonomy of moderately halophilic hetero-
trophic eubacteria, p. 71-83.
In
F.
Rodriguez-Valera (ed.),
Halophilic bacteria. CRC Press, Inc., Boca Raton, Fla.
Ventosa,
A.,
M.
C.
Gutierrez,
M.
T.
Garcia, and
F.
Ruiz-
Berraquero.
1989. Classification of
“Chromobacterium maris-
mortui”
in a new genus,
Chromohalobacter
gen. nov., as
Chromohalobacter marismortui
comb. nov., nom. rev. Int. J.
Syst. Bacteriol. 30:485495.
Vreeland, R.
H.,
C.
D.
Litchfield, E. L. Martin, and E. Elliot.
1980.
Halomonas
elongata,
a new genus and species of ex-
tremely salt-tolerant bacteria. Int. J. Syst. Bacteriol.
30:
485-495.
