Jou nal
o
Gene al Mic obiology
(1983),
129,
2649-2657.
P in ed
in
G ea B i ain
2649
Nume ical Taxonomy
o
Mode a ely Halophilic G am-nega i e Bac e ia
om Hype saline Soils
By E. QUESADA,' A. VENTOSA,'?
F.
RODRIGUEZ-VALERA,*
L.
MEGIAS3
AND
A. RAMOS-CORMENZANA'*
I
Depa men
o
Mic obiology, Facul y
o
Pha macy, Uni e si y
o
G anada, G anada, Spain
Depa men o Mic obiology, Uni e si y o Alican e, Alican e, Spain
Depa men
o
Ana omy, Facul y
o
Medicine, Uni e si y
o
G anada, G anada, Spain
(Recei ed
15
No embe I982
;
e ised 26 Janua y 1983)
~ ~ ~ ~ ~
A o al o 132 mode a ely halophilic bac e ia we e isola ed om hype saline soils wi h a C1-
con en be ween 2-36 and 12.72% (w/ ) loca ed nea Alican e
(S.E.
Spain) and examined o 98
pheno ypic cha ac e is ics including hei esponse o cy ological, physiological, biochemical
and nu i ional es s. They we e submi ed o a nume ical analysis oge he wi h six e e ence
s ains using bo h simple ma ching
(SsM)
and Jacca d
(S,)
coe icien s, and clus e analysis was
ca ied ou by he unweigh ed pai g oup me hod o associa ion (UPGMA), single linkage and
comple e linkage. Wi h he
S,
coe icien and UPGMA clus e ing, eigh phenons we e ob ained
a he 65% simila i y le el. F om each phenon ep esen a i e s ains we e chosen o he
de e mina ion o DNA base composi ion and o elec on mic oscopy. Bac e ia belonging o
phenons
D,
E, and F we e assigned o he genus
Alcaligenes.
Phenon
G
included 27 s ains
assigned o
Acine obac e ,
bu he high
G
+
C composi ion (58.9 mol%) o a ep esen a i e
s ain o his phenon sugges s ha i may ep esen a new axon. Phenons
A,
B,
and
C
we e
designa ed
Fla obac e ium
and phenon
H
was
Pseudomonas.
The bac e ia ound in hese
en i onmen s a e no ela ed o hose om hype saline wa e s o no mal soils.
INTRODUCTION
Mode a ely halophilic bac e ia ha e been de ined as hose showing op imal g ow h be ween
3
and 15% (w/ ) NaCl (Kushne , 1978). Al hough e y ew g oups o mode a e halophiles ha e
p e iously been desc ibed (La sen, 1962; Kushne , 1978), we ecen ly showed ha a a ie y o
axonomic g oups had mode a ely halophilic ep esen a i es in hype saline wa e s (Ven osa
e
al.,
1982) and soils (Quesada
e al.,
1982). The lack o sys ema ic s udies o isola es om na u al
sou ces has esul ed in a sca ci y o axonomic in o ma ion conce ning mode a e halophiles.
Mos o he desc ip ions e e o o ganisms isola ed om sal ed ood (Buchanan
&
Gibbons,
1974) o ound as acciden al labo a o y con aminan s (No i sky
&
Kushne , 1976).
In hype saline wa e s we ound mos o he gene a ha can be isola ed om seawa e
:
Vib io
was he mos abundan , ollowed by
Fla obac e ium, Alcaligenes, Al e omonas
and
Ch omobac e -
ium
(Ven osa
e al.,
1982). All hese bac e ia we e mode a e halophiles ha ing, in addi ion o a
high (5-10%, w/ ) sal equi emen o op imal g ow h, a minimal equi emen
o
2-5% (w/ )
NaCl in mos cases. In hype saline soils, howe e , al hough many halo ole an G am-posi i e
bac e ia we e ound, mos o he mode a e halophiles we e G am-nega i e ods. These also
equi ed a high (abou
5-10%,
w/ ) sal concen a ion o op imal g ow h, bu a lowe minimal
sal concen a ion (0-5-2
%,
w/ ) suppo ed g ow h. These esul s p obably e lec he ecological
di e ences be ween wa e and soil en i onmen s, wa e being ela i ely homogeneous and
cons an whe eas soil is he e ogeneous and mo e a ec ed by ac o s such as ain all, which may
elimina e o e -specialized o ganisms (Quesada
e al.,
1982).
P esen add ess
:
Depa men
o
Mic obiology, Facul y
o
Pha macy, Uni e si y
o
Se illa, Se illa, Spain.
0022-1287/83/OOOl-0909 $02.00
0
1983
SGM
2650
E.
QUESADA AND OTHERS
The pu pose o his wo k was o compa e mode a e halophiles om soils wi h hose isola ed
om wa e s, and also o p o ide a mo e comple e axonomy o his ecological g oup. Using
nume ical me hods, we ha e s udied 132 andomly-chosen new isola es o mode a e halophiles,
oge he wi h some e e ence s ains om cul u e collec ions.
METHODS
Zsola ion and main enance
o
s ains.
The mic o-o ganisms we e isola ed om hype saline soils nea Alican e
(S.E.
Spain) which ha e been desc ibed p e iously in physical and chemical e ms (Quesada
e al.,
1982). Samples
we e aken as be o e a egula in e als om Janua y 1980 o Ap il 1981 (12 samplings om i e di e en si es).
The C1- con en o he soils (de e mined using AgNOJ was in he ange 2.36 o 12.72%.
Isola ion medium con ained
(%,
w/ ): yeas ex ac (Di co),
1
;
p o eose-pep one no. 3 (Di co), 0.5; glucose, 0.1
;
bac o-aga (Di co), 2. This medium was supplemen ed wi h a balanced mix u e o sea sal s gi ing inal
concen a ions o
0.5,
5, 10, 20,
o
25% (w/ ). The sal s and p opo ions used we e as in e apo a ed seawa e
acco ding o Subo (Quesada
e al.,
1982). One g am
o
each soil sample was suspended in 10 ml o a sal solu ion
simila o hose used o he cul u e media, en old dilu ions we e made (always keeping he same balanced sal
concen a ions) and 0.1 ml o each dilu ion was pla ed on he isola ion medium o he same sal concen a ion.
A e incuba ion a 32
"C
o 15 d in sealed plas ic bags, h ee o i e colonies pe pla e we e andomly selec ed and
subcul u ed on he same medium un il pu e. A o al o 132 isola es o G am-nega i e ods ha we e mode a ely
halophilic, i.e. able o g ow in media con aining 2-20% (w/ ) sal s ( he sal g ow h ange de e mined as
p e iously: Ven osa
e al.,
1982), we e selec ed o his s udy.
Re e ence s ains. 'Ch omobac e ium ma ismo ui'
ATCC 17056,
'Pseudomonas halosaccha oly ica'
CCM 285 1
and
Vib io cos icola
NCMB 701 we e included. Quo a ion ma ks indica e species no in he App o ed Lis s o
Bac e ial Names (Ske man
e al.,
1980). Re e ence s ains om a p e ious wo k on hype saline wa e s (Ven osa
e
al.,
1982) we e included:
Alcaligenes
sp. A-336,
Al e omonas
sp. A-387 and
Fla obac e ium
sp. A-101.
Main enance medium.
The s ains we e main ained on slan s o medium sui able o mode a e halophiles (MH
medium) con aining 10% (w/ ) ma ine s?l s. The inal composi ion o his medium was
(%,
w/ ): NaCl, 8-1
;
MgC12, 0.7; MgS04, 0.96; CaCl,, 0.036; KCI, 0-2; NaHCO,,
0.006;
NaB , 0.0026 (Rod iguez-Vale a
e al.,
1980)
supplemen ed wi h
0-5
%
(w/ ) p o eose-pep one no. 3 (Di co), 1
%
(w/ ) yeas ex ac (Di co) and 0.1
%
(w/ )
glucose. This was solidi ied wi h bac o-aga (Di co) 2.0% (w/ ) when necessa y.
Cha ac e iza ion
o
isola es.
The pheno ypic cha ac e is ics (98) examined a e gi en in Table 1. Mos o he es s
ha e been desc ibed elsewhe e (Ven osa
e al.,
1982); de ails
o
he emainde a e gi en below. Incuba ion was a
32
"C
o up o 7 d in sealed con aine s; all media con ained 10% (w/ ) ma ine sal s and had hei pH adjus ed o
7.2 wi h
1
M-KOH (Rod iguez-Vale a
e al.,
1980). The abili y o g ow anae obically was e alua ed on pla es o
MH medium which we e s eaked and incuba ed in ja s wi h he Gas Pak Anae obic Sys em (BBL). The
appea ance o g ow h was checked a e 15 d incuba ion. The abili y o g ow in na ow Weinbe g ubes o liquid
MH medium con aining glucose and phenol ed (0.001
%,
w/ ) was de e mined by apid inocula ion in o he boiled
and cooled medium. The ubes we e sealed wi h s e ile aga plugs and also co e ed wi h liquid pa a in.
Phospha ase ac i i y was es ed by adding
1
%
(w/ ) aqueous phenolph halein diphospha e solu ion o MH
medium.
Elec on mic oscopy.
Rep esen a i e s ains om each phenon we e g own o mid-exponen ial phase on su ace
MH pla e cul u es co e ed wi h liquid medium. Samples o he liquid cul u es we e nega i ely s ained wi h a 2%
(w/ ) solu ion
o
phospho ungs ic acid (pH 7.0) and examined in a Ca l Zeiss EM-9s-2 ansmission elec on
mic oscope.
DNA composi ion.
Exponen ial-phase cells o ep esen a i e s ains om each phenon we e up u ed, and he
DNA was pu i ied using he me hod o Ma mu (1961). The mol
%
G
+
C
con en was de e mined om he
midpoin o he he mal dena u a ion p o ile (Ma mu
&
Do y, 1962).
Nume ical analysis.
Taxonomic cha ac e s we e coded in a bina y o m o he p esence/absence ype. S ain
simila i ies we e es ima ed wi h bo h simple ma ching
(SsM)
(Sokal
&
Michene , 1958) and Jacca d
(S,)
(Jacca d,
1908) coe icien s, and clus e analysis was ca ied ou using he unweigh ed pai g oup me hod o associa ion
(UPGMA), single linkage and comple e linkage (Snea h
&
Sokal, 1973). Tes e o was es ima ed in eigh epea ed
s ains (Snea h
&
Johnson, 1972). Cophene ic co ela ion was also e alua ed (Snea h
&
Sokal, 1973). These
compu a ions we e pe o med using he MINT p og am o D
F.
J. Rol o he Depa men o Ecology and
E olu ion, S a e Uni e si y o New Yo k a S ony B ook, N.Y., U.S.A., using a Uni ac 1108 compu e in he
Compu e Cen e o he Uni e si y o G anada, Spain.
RESULTS
Mo phological
and
halophilic cha ac e is ics
All he s ains we e G am-nega i e ods and obliga e ae obes. Conspicuous polyhyd oxy-
bu y a e inclusions we e obse ed in he majo i y o s ains.
Taxonomy
o
bac e ia om hype saline soils
265
1
All 132 s ains g ew op imally in 5-10% (w/ ) sal s media and a e he e o e mode a e
halophiles (Kushne , 1978). Mos equi ed a leas 2% (w/ ) sal s o g ow h (Table 1). The
de ailed esul s
o
all he physiological, biochemical and nu i ional es s a e summa ized in
Table 1.
Nume ical analysis
The esul s o he nume ical s udy
o
he cha ac e is ics o he s ains g ouped by means o he
SJ
coe icien and UPGMA clus e ing yielded he dend og am shown in Fig. 1. Simple
ma ching coe icien
(SsM)
and single linkage and comple e linkage algo i hms we e also used,
bu clus e composi ion was no ma kedly a ec ed by ei he he coe icien o he clus e ing
me hod. Cophene ic alues we e 0.9726 and 0-8236 o he
SJ
and
S,,
coe icien s, espec i ely.
The es ima ed es e o was less han 3.0%, which would no signi ican ly a ec he clus e
analysis.
The majo i y o he s ains we e g ouped in o eigh phenons a a 65% simila i y le el. Ele en
s ains clus e ed sepa a ely, i e o which we e e e ence s ains. Table 1 shows he ea u es o
he eigh phenons, and Table 2 summa izes hei di e en ial cha ac e is ics.
Phenon A.
This g oup included nine s ains which we e non-mo ile ods and oxidase posi i e.
They educed ni a es bu no ni i es. Nei he p o eoly ic, lipoly ic no amyloly ic ac i i y was
shown. The G
+
C con en
o
s ain F8-11, chosen as ep esen a i e o his g oup, was
694 mol%. These s ains could be included in he genus
Fla obac e ium
(Buchanan
&
Gibbons,
1974).
Phenon
B.
This phenon, con aining only h ee s ains, was e y simila o phenon A (Table 1) bu
hese s ains educed ni i es. S ain F8-6, aken as ep esen a i e, had a G
+
C con en o
61.8 molx. This phenon could also be placed in he genus
Fla obac e ium
(Buchanan
&
Gibbons, 1974).
Phenon
C.
This g oup included h ee s ains which we e non-mo ile sho ods and oxidase
posi i e. They educed ni a e and ni i e and u ilized a na ow ange o compounds as sole
sou ce
o
ca bon and ene gy. S ain G-4 was selec ed as ep esen a i e o his g oup and had a
G
+
C con en o 65.9mol%. These s ains may be placed in he genus
Fla obac e ium
(Buchanan
&
Gibbons, 1974).
Phenon
D.
This comp ised 16 s ains ha clus e ed oge he because o hei simila biochemical
and nu i ional cha ac e is ics
;
howe e , some we e assigned o he genus
Fla obac e ium
and
o he s o
Alcaligenes.
S ains G-23 and
F8-10,
ep esen a i es
o
each g oup, espec i ely, had
G
+
C
con en s o 65.4 and 69.2
mol%.
Re e ence s ain
Alcaligenes
sp. A-336 appea ed in his
phenon.
Phenon
E.
Twen y- ou s ains clus e ed in his g oup; all we e s aigh , pe i ichously
lagella ed ods. Mos
o
he s ains we e oxidase and ni a e nega i e. None possessed
p o eoly ic o amyloly ic ac i i ies. Twel e o he s ains we e phospha ase posi i e, and his
ea u e di e en ia ed wo subphenons ela ed a di e en simila i y le els. The wo
ep esen a i e s ains selec ed, G-30 (phospha ase posi i e) and F9-4 (phospha ase nega i e),
had G
+
C con en s
o
66.7 and 67.4 mol%, espec i ely. Bo h subphenons can be assigned o
Alcaligenes
(Buchanan
&
Gibbons, 1974).
Phenon
F.
The 40 s ains included he e we e long, mo ile ods wi h pe i ichous lagella ion (Fig.
1). They we e oxidase posi i e bu no p o eoly ic no lipoly ic. Some (30%) o he s ains
hyd olysed u ea. S ain F5-7 had a
G
+
C con en o 66.7 mol%. All membe s
o
his phenon
we e assigned o he genus
Alcaligenes
(Buchanan
&
Gibbons, 1974).
Phenon
G.
This g oup comp ised 27 s ains ha clus e ed a he 66% simila i y le el and
cons i u ed a e y homogeneous g oup o p edominan ly sho ods, occasionally wi h la ge
cu ed cells and ilamen s (some imes a he nume ous). Mos we e non-mo ile, oxidase
nega i e,
H2S
p oduce s and educed ni a es bu no ni i es. All we e phospha ase posi i e.
2652
E. QUESADA AND OTHERS
Pe cen age simila i y Phenon No.
o
s ains/
0
20
40
60
80
100
I
I
I
I
I
I1
I
I
1
i
I
I
I
I
I
I
I
I
ni
I
11‘
I
I
1
I
I I
1
I
I1
1
I
I
I
A
B
C
D
E
F
G
s ain
designa ion
9
3
3
16
Fll-7
24
40
Flauobac e ium
sp. A-101
27
‘Ch o nobac e ium
,
ma ismo ui’
-
/
Al e omonas
sp.
A-387
-A
-
/
F8-2
H4
F9-
1
G-l
I
--
-
F
12-7
--
-
__z
-
F8-4
-1
Vib io cos icola
‘Pseudomonas
halosaccha oly ica’
Fig.
1.
Simpli ied dend og am showing he clus e ing
o
s ains in o eigh phenons based
on
he
S,
coe icien and unweigh ed a e age linkage clus e ing
(UPGMA),
o 132 mode a ely halophilic
G am-nega i e bac e ia isola ed
om
hype saline soils and six e e ence s ains.
'
Taxonomy
o
bac e ia
om
hype saline soils
2653
Table
1.
F equencies
o
posi i e cha ac e s ound
in
he eigh phenons, exp essed as a pe cen age
o
he o al sco ed o each g oup
o
he gi en es
Phenon
.
.
.
A
9
No.
o
s ains
. . .
Mo phology
Long ods
Coccobacilla y
Colony pigmen
C eam
Yellowish
O ange
Mo ili y
Sal s: g ow h a
%
(w/ )
0.5
2
15
20
25
30
PH
5
PH
8
PH
9
pH
10
5
"C
15
"C
45
"C
Galac ose
Glucose
Lac ose
Mal ose
Manni ol
Suc ose
Xylose
pH: g ow h a
Tempe a u e
:
g ow h a
Acid p oduc ion
om
Oxidase
H,S
p oduc ion
Ni a e educ ion
o
ni i e
Ni i e educ ion o gaseous compounds
Casein hyd olysis
Gela in hyd olysis
S a ch hyd olysis
Tween
80
hyd olysis
U ea hyd olysis
Me hyl ed
Voges-P oskaue
DNAase
Phospha ase
U iliza ion
o
o ganic compounds as sole sou ce
o
ca bon and ene gy:
Ca bohyd a es
Aesculin
L-A abinose
D-Cellobiose
D-F uc ose
D-Galac ose
Lac ose
Mal ose
D-Mannose
L-Ra inose
L-Rhamnose
Salicin
L-So bose
44
56
66
34
0
0
0
100
90
50
11
11
0
89
54
22
33
89
11
11
77
0
22
0
22
0
89
33
100
0
0
0
0
0
0
0
0
0
0
88
55
11
100
66
33
100
55
0
0
0
33
B
3
0
100
33
66
0
0
0
100
1
00
100
0
0
0
100
100
66
0
100
0
33
0
0
0
33
33
0
100
33
100
100
0
0
0
0
0
0
0
0
0
0
0
0
100
33
0
66
0
0
0
0
0
C
3
0
100
0
100
0
0
0
100
100
1
00
33
0
33
100
100
100
100
100
0
0
33
0
100
0
0
33
100
0
100
100
0
0
0
0
0
0
0
0
0
0
0
0
66
0
0
100
33
0
0
0
0
D
16
63
37
44
56
0
56
12
100
100
94
50
25
0
100
100
75
50
94
56
12
87
0
68
48
75
0
100
0
69
62
0
0
6
6
0
0
0
0
0
6
0
0
94
31
0
I00
50
0
12
0
0
E
24
96
4
88
12
0
96
0
100
96
96
83
40
0
100
96
88
50
100
50
75
50
0
25
4
16
12
20
76
20
0
0
0
0
0
0
4
4
0
54
16
80
20
100
100
0
92
92
0
54
20
4
F
40
100
0
78
22
0
95
10
100
100
1
00
93
93
47
100
93
88
10
100
25
93
93
2
83
10
88
22
80
80
65
2
0
0
2
0
30
0
0
0
0
78
98
90
100
98
10
90
90
2
15
85
0
G
27
4
96
67
7
26
22
85
96
100
96
89
89
41
100
100
96
70
100
48
4
7
0
0
7
0
4
4
89
93
0
4
26
0
0
0
0
0
4
100
100
93
1
00
100
100
100
100
100
93
89
96
37
H
4
100
0
0
100
0
100
50
100
75
50
0
0
0
100
100
25
0
100
0
75
100
0
75
0
0
75
100
50
100
25
0
0
100
100
0
0
0
0
0
0
0
0
100
0
0
100
75
0
0
0
0

2654
E. QUESADA
AND
OTHERS
Table
1
(con inued)
Phenon . .
.
No.
o
s ains
. .
.
S a ch
Suc ose
D-T ehalose
D-x ylose
Alcohols
Adoni ol
Dulci ol
DL-Glyce ol
meso-Inosi o1
D-Manni ol
D-So bi ol
Ace a e
Benzoa e
Cap yla e
Ci a e
Fo ma e
Fuma a e
D-Glucona e
Hippu a e
DL-Lac a e
DL-Maia e
Oxala e
Py u a e
P opiona e
Succina e
D-Ta a e
Ca boxylic acids
U iliza ion
o
amino acids as sole sou ce
o
ca bon, ni ogen and ene gy
:
L-Alanine
L-A ginine
L-
Aspa agine
L-Aspa ic acid
L-Glu amic acid
L-His idine
L-Isoleucine
L-Leucine
L-Lysine
L-O ni hine
L-Se ine
L-T yp ophan
L-Valine
A
9
55
77
77
88
88
0
88
88
44
88
88
0
0
88
100
100
100
66
100
100
0
100
88
100
11
100
88
100
100
100
88
33
33
11
11
100
0
0
B
3
0
100
66
100
0
0
66
66
33
100
100
0
0
100
100
100
100
66
100
100
66
100
100
100
66
100
66
100
100
100
0
66
100
0
0
100
33
33
C
3
0
66
100
33
0
0
100
0
0
66
66
0
0
100
100
100
66
0
66
66
0
66
66
100
0
100
66
100
100
100
0
0
0
0
66
100
0
100
D
16
0
100
100
31
0
0
87
31
44
44
100
6
0
94
100
100
75
56
94
100
100
100
94
87
19
100
94
100
94
100
81
56
81
56
81
100
6
25
E
24
32
92
100
60
0
0
96
64
50
54
96
28
0
100
100
100
100
66
88
100
8
100
1
00
100
16
100
100
100
100
100
96
96
80
80
96
100
24
0
F
40
0
98
95
85
2
2
90
0
35
32
95
95
2
93
95
95
93
90
98
98
10
90
98
98
5
95
95
98
93
95
15
20
25
75
57
98
27
2
G
27
4
100
100
100
44
33
100
96
74
89
96
0
18
100
100
100
100
15
15
15
11
100
100
100
89
100
100
67
100
100
100
44
52
67
33
100
7
7
H
4
100
25
25
25
0
0
75
25
0
0
75
0
0
100
100
100
100
25
100
100
0
50
100
100
0
50
25
100
50
100
0
0
0
25
25
25
25
0
All s ains we e G am-nega i e, o med ca alase, we e s ic ae obes, g ew a
5
and 10% (w/ ) o al sal s, g ew a
pH
6
and
7,
g ew a
25
and
32
"C,
and g ew on D-glucose as sole sou ce
o
ca bon and ene gy. None o med indol o
g ew on inulin o L-cys eine.
They we e e y e sa ile nu i ionally, and we e able
o
u ilize a e y high numbe
o
compounds
as sole ca bon and ene gy sou ces. The s ains in his phenon we e able o g ow a low sal
concen a ions
(0.5
%,
w/ ) al hough
o
op imal g ow h hey needed
5-10%
(w/ ) o al sal s.
S ain
F2-12
had a G
+
C
con en
o
58.9
mol%. Wi h he excep ion
o
G
+
C
alue hese
s ains could be assigned
o
he genus
Acine obac e
(Buchanan
&
Gibbons,
1974).
Phenon
H.
This phenon was composed
o
ou
s ains, which we e s aigh ods mo ile by one o
a ew pola lagella. Thin ilamen s we e common. They we e oxidase posi i e, educed ni a es
and showed amyloly ic and lipoly ic ac i i ies. Few o ganic compounds we e used as sole ca bon
and ene gy sou ces. The ep esen a i e s ain
F12-1
had a G
+
C
con en
o
58.7
mol%, These
ou
s ains can be included in he genus
Pseudomonas
(Buchanan
8z
Gibbons,
1974).
Taxonomy
o
bac e ia
om
hype saline soils
2655
Table
2.
Di e en ial cha ac e is ics
o
he eigh phenons
o
mode a ely halophilic G am-nega i e
bac e ia om hype saline soils
Phenon
...
A
B
C
D
E
F
G H
No.
o
s ains..
. 9
3 3
16 24 40 27 4
Mo phology
Long ods
Coccobacilla y
Mo ili y
Flagella
G ow h a
0.5%
(w/ ) sal concen a ion
G ow h a
5
"C
Oxidase
H,S p oduc ion
Ni a e educ ion o ni i e
Ni i e educ ion o gaseous compounds
S a ch hyd olysis
Tween
80
hyd olysis
Phospha ase
U iliza ion
o :
DCellobiose
Oxala e
L-His idine
L-Leucine
- - -
3-+
8++
3
1-
+++
-++
-
-
-
1-
-
1
2-
8-
-
3+-
-
10
23
+
1+
9 23
38
6
+
-/pe . pe . pe . -/pe . pol.
4 23 2 2-
8
12
4
19
-
+
5
-
32
1+
-
19 32 24 2
6 1-26-
-
-
-
1-
1-+
1-
-
-+
-13-+-
-
536+-
+
243-
-
13g6+-
13
10 14
-
-
+,
all s ains posi i e;
-,
all s ains nega i e. Numbe s indica e numbe
o
posi i e s ains; unde lined
numbe s indica e ha he numbe ep esen s
80%
o
mo e
o
he s ains. pe ., pe i ichous lagella;
pol.,
pola
lagella.
DISCUSSION
The e a e ew published s udies o bac e ia om saline soils (Ce an es
&
Oli a es, 1976;
Mahmoud
e al.,
1978), and axonomic desc ip ions o such o ganisms a e sca ce. Henis
&
E en
(1963), wo king on a saline soil nea he Red Sea, concluded ha mo e colonies de eloped on
media wi hou sal s han on media wi h 10% (w/ ) NaCl. These esul s di e om ou s,
p obably because hese au ho s used media wi h NaCl as sole sal , while o he ions such as Mg2+
and K+ a e also equi ed o good g ow h o mode a ely halophilic bac e ia (Kushne , 1978).
As
in ou p e ious s udy on bac e ia om hype saline soils (Quesada
e al.,
1982), mos o he
popula ion li ing in he soils s udied we e mode a e halophiles as de ined by Kushne (1978),
since hey g ew op imally be ween
5
and 10% (w/ ) sal s. This was also he case o bac e ia
om hype saline wa e s (Rod iguez-Vale a
e al.,
1981
;
Ven osa
e al.,
1982). Ne e heless,
many bac e ia isola ed om hype saline soils we e able o g ow a low sal concen a ions,
(abou
0.5%,
w/ ), while bac e ia om hype saline wa e s could no . This was a common
cha ac e o he G am-posi i e bac e ia desc ibed by Quesada
e al.
(1982); howe e , 31 o he
G am-nega i e s ains included in his s udy also showed ole ance o low sal concen a ions.
Al hough di e ences exis ed among he s ains included in phenons
A,
B and C, all can be
conside ed as ep esen a i es o he genus
FZa obac e ium,
and he sepa a ion among hem
p obably a ose om hei di e en capabili y o use o ganic compounds as sole ca bon and
ene gy sou ces. Wi hin he genus
Fla obac e ium,
he only mode a ely halophilic species
desc ibed o da e is
F. halmephilum
(Elaza i-Volcani, 1940). The s ains included in hese
phenons show some esemblance he desc ip ion o his species, al hough hey di e by
hei abili y o p oduce acids om suga s, hei p o eoly ic ac i i y and hei nu ien
equi emen s. Besides,
F. halmephilum
has a
G
+
C con en
o
45.6 mol
%
and he e o e belongs
o Sec ion
I
o his genus, while ou s ains, wi h a G
+
C
con en in he ange 61.8-69.4 mol%,
a e in Sec ion
11.
Among he ma ine bac e ia belonging o he genus
Fla obac e ium
and
included in he App o ed Lis s o Bac e ial Names (Ske man
e al.,
1980),
F.
oceanosedimen um
is he only one ha esembles he s ains ha appea in his s udy, and i di e s om hem only
by he p oduc ion o acids om some suga s, he abili y o educe ni a es, and sal esponse
;
he
2656
E. QUESADA AND
OTHERS
G
+
C
con en o
F.
oceanosedimen um
is also e y simila (67.5 mol%) o ha o he s ains in
phenons A, B and
C.
Holmes
&
Owen (1979, 1981) ha e p oposed a ede ini ion o he genus
Fla obac e ium,
es ic ing his genus o he species wi h low
G
+
C
con en (31-40 mol
%).
Since i is clea ha la obac e ia o high
G
+
C con en a e easily isola ed om a wide a ie y
o en i onmen s (Hayes
&
Mapp, 1981), mo e s udies a e necessa y o decide i hey could be
accommoda ed in he genus
Empedobac e
as p oposed by McMeekin
&
Shewan (1978).
The s ains included in phenons
E
and
F
we e assigned o he genus
Alcaligenes.
Phenon
E
was
e y simila o he desc ip ion o he ma ine bac e ium
A caligenes cupidus
(Baumann
e
al.,
1972) excep in he abili y o educe ni a e o ni i e and hei di e en sal ange o g ow h.
Howe e , phenon
F
was composed
o
a la ge g oup o bac e ia wi h p ope ies e y di e en
om hose o p e iously desc ibed species o
Alcaligenes.
Acco ding o he
G
+
C con en , hey
we e ela ed o he ma ine bac e ium
A.
paci icus
(Baumann
e al.,
1972) bu he e we e many
physiological, biochemical and nu i ional di e ences om his species. In
ou
opinion, hey
could cons i u e
a
new axon no p e iously desc ibed. Howe e , mo e gene ic and molecula
da a a e necessa y o suppo his sugges ion.
The 27 s ains ha clus e ed in phenon
G
had mo phological, physiological, biochemical and
nu i ional cha ac e is ics o he genus
Acine obac e
(Buchanan
&
Gibbons, 1974). Howe e ,
he
G
+
C
con en o he ep esen a i e s ain o his g oup was 58-9 mol%, whe eas he ange
accep ed o his genus is 42-47 mol
%
(Hen iksen, 1973). Mode a ely halophilic bac e ia ha e
no been desc ibed and accep ed as membe s o his genus. Onishi
&
Hidaka (1978) s udied he
p ope ies o an amylase p oduced by a mode a ely halophilic
Acine obac e
sp. bu
a
desc ip ion
o his s ain has no been published. I seems clea ha he s ains in phenon G ep esen
a
new
g oup o mic o-o ganisms ha could be included in
Acine obac e ,
bu he di e en
G
+
C
con en could indica e ha hey belong o no only a di e en species bu possibly a di e en
genus. S udies a e in p og ess o de e mine i his di e ence in he
G
+
C
con en
is
also
e lec ed by di e ences a he pheno ypic le el.
Phenon H was included in he genus
Pseudomonas,
bu hese s ains we e no ela ed o he
mode a e halophile
P.
halosaccha oly ica
used a ma ke in his s udy (Fig. 1). The e we e
di e ences in nu i ional e sa ili y as well
as
in amyloly ic, lipoly ic and p o eoly ic ac i i ies.
Wi h espec o ma ine species o he genus
Pseudomonas
included in he App o ed Lis s
(Ske man
e
al.,
1980), hey could be ela ed o
P.
doudo o ii
(Baumann
e
al.,
1972), he
di e ence being hei lipoly ic and amyloly ic ac i i ies, absen in his species.
Compa ison wi h p e ious s udies (Ven osa
e al.,
1982) sugges s ha he bac e ial
popula ions in he saline e es ial en i onmen s s udied he e show many di e ences om
bac e ia om hype saline wa e s. In ac , he e e ence s ains om hype saline wa e s did no
g oup wi h he s ains om he e es ial habi a . All he s ains s udied he e had a s ic ae obic
me abolism, a p edominan ea u e ha we had al eady epo ed o bac e ia om hype saline
soils: e y ew s ains we e acul a i ely anae obic (Quesada
e
al.,
1982). This poin has been
con i med by an independen s udy ca ied ou by D M.
E.
Rhodes-Robe s (pe sonal
communica ion). Howe e , in hype saline wa e s acul a i e anae obes ep esen a majo pa
o he popula ion (Ven osa
e
al.,
1982). In aqueous saline en i onmen s
Vib io
spp. we e he
mos abundan g oup
o
mode a ely halophilic bac e ia, whe eas his genus is e y sca ce in
hype saline soils, which a e domina ed by he gene a
Alcaligenes
and
Fla obac e ium.
Pseuda nonas
is a e y common inhabi an o no mal soils (Alexande , 1961), bu sca ce in he
hype saline soils we s udied. Ne e heless, he mic o lo a o hese hype saline soils is much
mo e simila axonomically o he mic o lo a o no mal soils (Alexande ,
1961)
han i
is
o he
mic o lo a o hype saline wa e s. This sugges s ha he gene al ea u es o he en i onmen may
be mo e impo an han indi idual ac o s such as high salini y in de e mining he mic o lo a o
a pa icula habi a .
The au ho s hank D
A.
Vila o he in aluable help wi h he compu a ion and he Compu e Cen e o he
Uni e si y o G anada, Spain, o he
use
o i s compu e and acili ies. We a e g a e ul o D
F.
Ruiz-Be aque o
o his ad ice du ing he cou se o his wo k, D C. Cal o o he G
+
C
con en de e mina ion,
J.
Muiioz o his
aid in he p epa a ion o he
EM
g ids, and especially o D
M.
E.
Rhodes-Robe s (Depa men o Bo any and
Taxonomy
o
bac e ia
om
hype saline
soils
2657
Mic obiology, Uni e si y College o Wales, Abe ys wy h,
U.K.),
who kindly con i med some o he esul s o his
wo k and ga e aluable ad ice and discussion.
E.
Q.
g a e ully acknowledges he eceip o a ellowship om he Plan de Fo macibn del Pe sonal in es igado .
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