H E L G O I ~ N D E R MEERESUNTERSUCHUNGEN
Helgol/inder Meeresunters. 36, 357-373 (1983)
T a x o n o m i c investigations of b a c t e r i o p h a g e sensitive
bacteria isolated from marine waters
K. Moebus & H. Nattkemper
Biologische Anstalt Helgoland (Meeresstation);
D-2192 Helgoland, Federal Republic of Germany
ABSTRACT: Based on 28 criteria the taxonomy of 366 phage sensitive bacterial strains isolated from
marine waters (Atlantic between European continental shelf and Sargasso Sea, Bay of Biscay, North
Sea near Helgoland) was investigated. Seventy-eight phage-intensity strains derived from the same
Atlantic Ocean regions as the sensitive ones were tested for comparison. While in the latter
considerable diversity was observed, the results obtained with the phage-sensitive bacteria are
characterized by stupendous uniformity. 362 of the 366 strains are assigned to the family Vibrionaceae, some 280 of which belong to the genus Vibrio. As discussed, this taxonomic uniformity
among the phage-sensitive bacteria is assumed to be an artifact mainly caused by the type of
enrichment culture employed for the isolation of all but a few bacteriophage strains used and, to a
lesser degree, by characteristics of the bacterial populations encountered.
INTRODUCTION
A l t h o u g h s o m e p r o g r e s s h a s b e e n m a d e s i n c e a b o u t 1970, t h e i n f o r m a t i o n a v a i l a b l e
on m a r i n e b a c t e r i o p h a g e s a n d t h e i r h o s t b a c t e r i a is still scant. S p e n c e r (1955, 1960) w a s
t h e first w h o r e p o r t e d t h e i s o l a t i o n of 7 t r u l y m a r i n e p h a g e s f r o m N o r t h S e a w a t e r t a k e n
10 m i l e s off t h e S c o t t i s h c o a s t w h i c h a r e a c t i v e on strains of Photobacterium phosphoreum (i), Pseudomonas sp. (3), Cjrtophaga sp. (1), a n d Flavobacterium sp. (2). O n e of
t h e l a s t m e n t i o n e d strains w a s l a t e r i d e n t i f i e d as a n o t h e r Cytophaga sp. ( C o l w e l l et al.,
1966). C h a i n a (1965) i s o l a t e d 629 b a c t e r i a l strains f r o m w a t e r a n d m u d c o l l e c t e d at
v a r i o u s d e p t h s in t h e I n d i a n O c e a n 10 of w h i c h w e r e s e n s i t i v e to b a c t e r i o p h a g e s d e r i v e d
f r o m t h e s a m e s a m p l e s : Pseudomonas sp. (5), Flavobacterium sp. (2), Achromobactersp.
(2), a n d Vibrio sp. (1). J o h n s o n (1968) i s o l a t e d a n o t h e r Vibrio s p . - p h a g e s y s t e m from a n
I n d i a n O c e a n m u d s a m p l e c o l l e c t e d at a d e p t h of m o r e t h a n 3000 m. In t h e s a m e y e a r ,
%~iebe & Liston (1968) d e s c r i b e d a p h a g e i s o l a t e d f r o m N o r t h Pacific O c e a n s e d i m e n t
( d e p t h 825 In) a c t i v e o n Aeromonas sp., a n d E s p e j o & C a n e l o (1968) r e p o r t e d t h e
c h a r a c t e r i s t i c s of a l i p i d - c o n t a i n i n g p h a g e i s o l a t e d f r o m w a t e r t a k e n 1 m i l e off t h e
C h i l e a n c o a s t w h i c h i n f e c t s b a c t e r i a t e n t a t i v e l y c l a s s i f i e d as Pseudomonadae. H i d a k a
(1971, 1973) a n d H i d a k a & F u j i m u r a (197I) d e s c r i b e d p h a g e s i s o l a t e d f r o m s e d i m e n t a n d
w a t e r s a m p l e s c o l l e c t e d f r o m t h e P a c i f i c s o u t h of K y u s h u , J a p a n , w h i c h i n f e c t strains of
Vibrio sp. (14), Pseudomonas sp. (9), Flavobacterium sp. (4), Achromobacter sp. (3), a n d
© Biologische Anstalt Helgoland, Hamburg
358
K. M o e b u s & H. N a t t k e m p e r
Aeromonas sp. (2). In 1977, H i d a k a reported the isolation of 7 p h a g e s from w a t e r s a m p l e s
collected i n the s o u t h w e s t e r n Pacific at depths r a n g i n g from 1 to 200 m w h i c h are lyric
for Vibrio sp. (3), Pseudomonas sp. (2), Photobacterium sp. (1), a n d Lucibacterium sp. (1).
Other reports are c o n c e r n e d with p h a g e - h o s t systems isolated from inshore or
e s t u a r i n e e n v i r o n m e n t s . Smith & KAiger (1955) derived a Vibrio sp.-phage system from a
m u d s a m p l e t a k e n i n S a n Francisco Bay. Carlucci & Pramer (1960) u s e d Serratia
marinorubra to e n r i c h p h a g e s from seawater collected about 200 m offshore i n N e w
Jersey. Both the p h a g e s a n d their host bacteria did not require sea salts, A h r e n s (1971)
i n v e s t i g a t e d the d i s t r i b u t i o n of p h a g e s infective for the g e n u s Agrobacterium i n the
Baltic Sea a n d o b s e r v e d a decrease i n p h a g e n u m b e r s at salinities b e l o w 8%0. In 1972,
S t e v e n s o n & Albright characterized a p h a g e attacking Cytophaga sp. w h i c h was isolated
from water t a k e n i n the i n t e r t i d a l zone i n British Columbia. Phages infecting l u m i n o u s
bacteria (Photobacterium fischeri, Beneckea harveyO were isolated by Hastings et al.
(1961) and Keynan et al. (1974), respectively, from samples collected at Woods Hole,
Mass. (USA). Kakimoto & Nagatomi (1972) isolated 6 phages attacking Viblio sp. (2),
Pseudomonas sp. (3), and one unidentified species from water taken in Kinko Bay,
Japan. Zachary (1974) described several morphologically different phages infecting
Beneckea natrieffens which were limited to brackish and marine waters collected in
coastal salt marshes. According to Baumann et al. (1980), the genus Beneckea is
abolished and its members are assigned to the genus Vibrio. Reports on phages lytic for
Vibrio parahaemolyticus were published by Nakanishi et al. (1966), Sklarow et al.
(1973), a n d Baross et aL (1978).
M o e b u s (1980) reported the isolation of some 250 b a c t e r i o p h a g e strains from
Atlantic O c e a n water samples, a n d i n 1981 M o e b u s & N a t t k e m p e r described the results
of p h a g e - h o s t cross-reaction tests performed with 774 bacterial a n d 298 b a c t e r i o p h a g e
strains derived from w a t e r samples collected from the Atlantic b e t w e e n the E u r o p e a n
c o n t i n e n t a l shelf a n d the Sargasso Sea as w e e as from the North Sea n e a r H e l g o l a n d . As
r e v e a l e d b y p h a g e sensitivy patterns, the majority of the Atlantic O c e a n bacteria could
be g r o u p e d into 14 clusters of related strains (Moebus & Nattkemper, 1981; Moebus,
1983). To c o m p l e m e n t these f i n d i n g s with basic information o n the t a x o n o m y of the
p h a g e - s e n s i t i v e bacteria, 444 strains were i n v e s t i g a t e d for a l i m i t e d n u m b e r of traits as
e m p l o y e d with the d e t e r m i n a t i v e scheme of S h e w a n et al. (1960).
MATERIALS AND METHODS
Bacteria and bacteriophages
All bacterial strains except one (H96, derived from a p h a g e e n r i c h m e n t culture)
were p i c k e d from seawater agar plates i n o c u l a t e d with freshly s a m p l e d seawater. Aseries bacteria were isolated i n 1979 from Atlantic water s a m p l e s (Moebus, 1980).
"Eastern" a n d " w e s t e r n " strains were derived from s a m p l e s t a k e n east or west, respectively, of the Azores. B-series b a c t e r i a were isolated from w a t e r samples collected i n
1978 from the Bay of Biscay. H-series bacteria were d e r i v e d from North Sea water
s a m p l e s t a k e n n e a r H e l g o l a n d b e t w e e n 1969 and 1978. The term " o r i g i n a l host" refers
to strains initially u s e d to isolate a n d purify bacteriophages.
T a x o n o m y of p h a g e sensitive b a c t e r i a
359
The b a c t e r i a w e r e m a i n t a i n e d on s e a w a t e r a g a r slants in t h e refrigerator a n d
c h e c k e d for p u r i t y before use. Strains of k n o w n sensitivity to a n y of the b a c t e r i o p h a g e
strains a v a i l a b l e w e r e also t e s t e d for p e r s i s t e n c e of their p h a g e sensitivity p a t t e r n s b y
s p o t t i n g u n d i l u t e d lysates. M e t h o d s a n d m e d i a as g i v e n b y M o e b u s & N a t t k e m p e r
(1981).
l~scherichia coli B, Staphylococcus aureus P209, and Pseudomonas fluorescens
( N C M B 129) were used as reference strains.
Most phages were derived from the s a m e water sample as their original host
bacteria. T h e m e t h o d for bacteriophage isolation w a s described by M o e b u s (1980). In
principle, it employs enrichment cultures set u p with organic nutrients-enriched raw
seawater w i t h o u t addition of prospective host bacteria. Phages infective for strains
H4, H7, H17, H54, H84, H85, and H 8 6 were isolated from enrichment cultures separately
inoculated with about 5 × 107 cells m l -~ of these strains before incubation.
Media and methods
S e a w a t e r a g a r (SWA), c o n t a i n i n g 5 g p e p t o n e (Difco), 1 g y e a s t extract (Difco), 0.1 g
FePO4, a n d 15 g a g a r (Difco) p e r liter of " s e a w a t e r m i x t u r e " (75 % a g e d North Sea w a t e r
a n d 25 % d i s t i l l e d water). The p H w a s a b o u t 7.6 after a u t o c l a v i n g for 20 rain at 121 °C.
Bacteria w e r e g r o w n in P e t r i - d i s h e s (90 m m ~D) c o n t a i n i n g 10 ml S W A for d e t e r m i n a t i o n of the f o l l o w i n g criteria: (a) C o l o n y form a n d size (read after 2 days of incubation).
(b) P i g m e n t p r o d u c t i o n : n o n - d i f f u s i b l e a n d diffusible p i g m e n t s as w e l l as fluorescent
ones (tested u n d e r UVqight), after 1 to 3 da~s. (c) Luminosity, after 1 to 4 days. (d) G r a m
stain, a c c o r d i n g to Benson (1973). (e) O x i d a s e production, a c c o r d i n g to Steel (1961). (f)
F l a g e l l a t i o n , a c c o r d i n g to M a y f i e l d & Inniss (1977) w i t h sterile s e a w a t e r m i x t u r e u s e d to
p r e p a r e celt suspensions.
S e a w a t e r b o u i l l o n (SWB) w a s of t h e s a m e c o m p o s i t i o n as S W A e x c e p t for the a g a r
content. SWB w a s u s e d (in still culture) to g r o w b a c t e r i a for or prior to d e t e r m i n a t i o n of
the following criteria:
(a) Cell m o r p h o l o g y : size, shape, a r r a n g e m e n t (phase contrast microscopy, × 1250),
after 1 to 2 days.
(b) M o t i l i t y (phase contrast microscopy, x 800), after 1 to 2 days.
(c) C a t a l a s e production, t e s t e d w i t h 10 % H202 after 2 days.
(d) Diffusible p i g m e n t s , after u p to 4 days.
(e) G e l a t i n e liquefaction: stab culture in s c r e w - c a p t u b e s w i t h 10 ml of m e d i u m
(15 % g e l a t i n e in s e a w a t e r mixture), i n c u b a t e d for 4 weeks.
(f) Starch d e c o m p o s i t i o n : stab c u l t u r e in P e t r i - d i s h e s (60 m m O) c o n t a i n i n g 6 ml of
s t a r c h - S W A w h i c h differs from S W A b y e n r i c h m e n t with 0.5 % s o l u b l e starch. After
2 d a y s t h e p l a t e s w e r e f l o o d e d w i t h L u g o l ' s solution.
(g) S e n s i t i v i t y to v i b r i o s t a t i c c o m p o u n d 0/129 (2.4-Diamino-6.7-diisopropyl
p t e r i d i n e ) : 6 m / o f a n t i b i o t i c - S W A (same c o m p o s i t i o n as S W A e x c e p t for t h e a g a r content
w h i c h is 5 g O x o i d a g a r No. 1 p e r liter) w e r e m i x e d w i t h 0.15 ml of b a c t e r i a l culture in
Petri-dishes (60 m m ~). After g e l l i n g of t h e agar, a n t i b i o t i c disks w e r e p l a c e d on the
surface. I n c u b a t i o n for 2 days. The a n t i b i o t i c disks (Schleicher & Schiill, No. 22, 9 m m {D)
w e r e s o a k e d w i t h 0 . 1 % (w/v) solution of 0/129 in a c e t o n e a n d d r i e d at 37 °C for 2 h
before u s e (Bain & S h e w a n , 1968).
360
K. M o e b u s & H. N a t t k e m p e r
(h) Glucose dissimilation: M O F m e d i u m after Leifson (1963), modified according to
BSlter (1977), was composed of 1 g p e p t o n e (Difco), 0.5 g a m m o n i u m s u l f a t e , 0.5 g TRISbuffer, a n d 0.01 g p h e n o l red per liter of seawater mixture. A 10 % solution (w/v, i n
distilled water) of glucose was autoclaved separately a n d a d d e d to a final glucose
c o n c e n t r a t i o n of 1 % after cooling. M O F m e d i u m was u s e d for aerobic a n d a n a e r o b i c
cultures, the latter overlayed with paraffin (about 2.5 cm thick). I n c u b a t i o n for up to 14
days.
(i) Methyl red test, after Pfister & Burkholder (1965), as modified b y W e y l a n d et al.
(1970): 5 g p e p t o n e (Difco) a n d 0.05 g K2HPO 4 per liter of s e a w a t e r mixture, with 5 g
glucose a d d e d after a d j u s t i n g p H 7.5. Test with methyl red after 4 days.
(j) Nitrate reduction: SWB e n r i c h e d with 0 . 1 % KNO 3. Test for NH3-production (with
Nessler's reagent) a n d NO2-production (with L u n g e ' s reagent) after 2 a n d 4 days. Gas
p r o d u c t i o n was tested with D u r h a m vials.
(k) A m m o n i a formation from p e p t o n e : SWB, tested with Nessler's r e a g e n t after 2 a n d
4 days.
(l) Salt requirements, tested with 3 m e d i a (incubation for up to 7 days): (1)5g
p e p t o n e (Difco), 1 g yeast extract (Difco), a n d 75 g NaC1 per liter of distilled water, p H
7.6; (2) 5 g p e p t o n e (Difco) a n d 1 g yeast extract (Difco) i n mixture of 150 ml a g e d North
Sea water a n d 850 ml distilled water (salinity about 5 %o), p H 7.6; (3) SWB.
(m) T e m p e r a t u r e sensitivity: tested i n SWB i n c u b a t e d at 37 °C for u p to 7 days.
Doubtful results were controlled b y s t r e a k i n g cultures on SWA a n d i n c u b a t i o n of plates
at the same temperature.
If not otherwise stated, all i n c u b a t i o n s were performed at 20 °C. Liquid m e d i a were
i n o c u l a t e d with 1 drop of freshly grown bacterial culture i n SWB.
The vibriostatic c o m p o u n d 0/129 was p u r c h a s e d from C a l b i o c h e m C o m p a n y , S a n
Diego, Cal. (USA) a n d all other chemicals from Merck C o m p a n y , Darmstadt (FRG).
RESULTS
This i n v e s t i g a t i o n e m p l o y e d 444 bacterial strains of m a r i n e origin i n c l u d i n g 366
p h a g e - s e n s i t i v e ones. As c o n c l u d e d from p h a g e sensitivity patterns, 259 of the latter
strains b e l o n g to 14 clusters of r e l a t e d b a c t e r i a which were isolated from Atlantic O c e a n
water samples in 1979. A d d i t i o n a l 29 strains of the same origin do not fit into a n y of these
clusters. The c o m b i n e d 288 A-series strains are sensitive to Atlantic O c e a n p h a g e s only.
The r e m a i n i n g 78 strains are sensitive to p h a g e s isolated from the North Sea or the Bay
of Biscay a n d originate either from these regions (31 H-series a n d 11 B-series strains,
respectively) or from the Atlantic (36 strains). S e v e n t y - e i g h t A-series bacteria w h i c h are
i n s e n s i t i v e to a n y of the p h a g e strains a v a i l a b l e (some 290) were i n v e s t i g a t e d for
comparison. They were r a n d o m l y selected from our stock culture collection to i n c l u d e 1
to 2 strains isolated from each of 48 water samples t a k e n b e t w e e n the E u r o p e a n
c o n t i n e n t a l shelf a n d the Sargasso Sea (Moebus, 1980). The results will b e p r e s e n t e d
according to this g r o u p i n g of the bacteria.
F l a g e l l a t i o n was i n v e s t i g a t e d with all original hosts a n d some of their doubles. From
the r e m a i n i n g bacteria, r a n d o m l y selected strains were c h e c k e d for flagella: 18 of the 36
A-series strains sensitive to p h a g e s from the North Sea a n d the Bay of Biscay, a n d 20 rods h a p e d strains of the 78 p h a g e - i n s e n s i t i v e A-series bacteria.
T a x o n o m y of p h a g e sensitive b a c t e r i a
361
G r o u p I: A - s e r i e s c o m p i l e d i n c l u s t e r s 1 to 13
The 13 clusters comprise 250 A t l a n t i c O c e a n bacteria i n c l u d i n g 136 "eastern" a n d
114 " w e s t e r n " strains. (Six a d d i t i o n a l d o u b l e t s b e l o n g i n g to cluster i a n d one of cluster 2
w e r e e x c l u d e d from this investigation.) It should b e n o t e d that formerly clusters 11 to 13
w e r e i n c l u d e d i n cluster 1 (Moebus & Nattkemper, 1981) b u t h a d to b e set apart
according to r e c e n t f i n d i n g s (Moebus, 1983).
Traits c o m m o n to (almost) all strains: G r a m n e g a t i v e rods, motile (3 exceptions i n
cluster 2), oxidase positive, glucose f e r m e n t e d (within I day!), colonies u n p i g m e n t e d , no
diffusible p i g m e n t s (1 exception i n cluster 2), no fluorescent p i g m e n t s , n o n - l u m i n o u s ,
N}-Is p r o d u c e d from peptone, N H s p r o d u c e d from nitrate (1 exception i n cluster 5), n o gas
from nitrate, positive in methyl red test (2 exceptions in cluster 2 a n d one i n cluster 4), no
growth at 37 °C (1 exception i n cluster 1). The results o b t a i n e d for other traits are
compiled i n T a b l e 1.
144 of the 150 strains tested were f o u n d to have single w a v y polar flagella. In 2
m o d e r a t e l y motile strains (clusters 2 a n d 3), no flagella were observed, a n d in 4 strains
(cluster 4), u n i q u e observations were made. In 3 of these strains (2 of t h e m h i g h l y motile),
no or only few flagellated ceils were f o u n d b u t m a n y cells with polar stumps. In the
fourth strain (highly motile), cells with u p to 3 long b u t straight polar a p p e n d i c e s were
observed.
As for the m e t a b o l i s m of glucose the bacteria of group I differ from most m e m b e r s of
the other groups b y their rapid reaction i n M O F m e d i u m . With no exception the c h a n g e
i n colour from red to yellow occurred w i t h i n 1 day of i n c u b a t i o n u n d e r both aerobic a n d
a n a e r o b i c conditions.
Diffusible p i g m e n t was o b s e r v e d with strain A64, one of 10 d o u b l e t strains of cluster
2 (see Moebus, 1983; Fig. 2). Its colourless colonies caused strong b r o w n n o n - f l u o r e s c e n t
colouration of the agar. Strain A734 (cluster 1) grows, t h o u g h poorly, in SWB at 37 °C,
p r o d u c i n g tiny colonies a n SWA at. this temperature.
S e v e n t e e n strains of cluster 1 are u n a b l e to reduce nitrate to nitrite. Fifteen of them
i n c l u d e a group of 14 doublets plus o n e other closely related strain (see Moebus, 1983;
Fig. 1, group II + strain A699). T h i r t e e n of these doublets also p r o d u c e d less N H 3 from
p e p t o n e t h a n o b s e r v e d i n most m e m b e r s of cluster 1.
R e g a r d i n g d u s t e r s 2, observations were m a d e w h i c h s o m e w h a t separate its m e m bers from the bacteria c o m p i l e d i n clusters 1 a n d 3 to 13. The majority of the cluster
2 bacteria gave a c o m p a r a t i v e l y w e a k oxidase reaction a n d 3 strains w e r e f o u n d to b e
oxidase positive only w h e n g r o w n at 25 °C. Furthermore, i n most m e m b e r s of this cluster
no or w e a k g e l a t i n e liquefaction w a s observed. It should be n o t i c e d that 51 strains of
cluster 2 are of w e s t e r n origin (see below).
Strong positive correlation was f o u n d b e t w e e n catalase p r o d u c t i o n a n d starch
digestion. Of the 250 strains, 192 (76.4 %) proved to b e either positive (55) or n e g a t i v e
(137) for both traits (Table 1). In some cases, correlations b e t w e e n traits were observed i n
d e p e n d e n c e of the origin of the bacteria. As c o m p i l e d i n T a b l e 2, a m o n g "eastern"
strains the n u m b e r s of starch digesters (71) a n d n o n - d i g e s t e r s (65) are similar, however,
a m o n g "western" b a c t e r i a n o n - d i g e s t e r s occurred m u c h more often (89) t h a n digesters
(25). O r i g i n - d e p e n d e n t correlations are also i n d i c a t e d b e t w e e n the traits " g e l a t i n e
liquefaction" on the one h a n d a n d "starch digestion" as well as "nitrate r e d u c e d to
nitrite" on the other h a n d (Table 2).
362
K. Moebus
& H. Nattkemper
T a b l e 1. D i f f e r e n t l y e x h i b i t e d
traits observed
Characters
Cluster
2
3
4
5
6
7
8
9
10
11
12
13
122
69
53
63
12
51
6
8
8
3
3
8
8
4
4
2
2
4
4
3
3
9
9
7
7
6
11
7
4
single polar
other
not observed
70
-
30
3
3
4
5
3
6
4
2
3
3
7
5
1
1
not tested
52
32
2
4
3
-
2
1
-
2
2
108
14
58
5
6
-
11
8
3
-
7
1
4
3
9
7
69
4
24
58
1
3
-
1
9
-
+:--- : +
1
4
2
-
5
-
+:+
25
no
weak
moderate
strong
Sensitivity
to 0/129
Catalase
of g r o u p I
1
No. of strains tested
"eastern" strains
"western" strains
Flagella:
in bacteria
+
--
versus
starch digestion:
Gelatine
liquefaction* :
Nitrite from
nitrate
-- : --
+
--
4
2
2
1
11
1
3
-
8
2
4
4
2
4
4
11
61
46
25
28
I0
4
1
1
1
5
5
1
-
5
3
2
1
8
-
3
1
2
3
1
2
9
105
17
20
43
10
1
7
1
3
8
3
1
2
4
3
9
6
1
2
-
2
6
2
4
1
-
1
5
--
2
-
-
1
1
-
6
-
2
1
6
Growth in salts media* *
7.5 %
NaC1
5 %0
sea
salts
SWB
+
--
--
--
+ +
+
+
-+
+ +
++
+
1
6
27
1
+ +
--
+ +
--
--
+
--
+++
+++
--
+
+ + +
+
+
+++
++
--
+++
6
++
+
++
+
++
++
+++
+++
+++
4
1
3
3
2
3
17
-
3
-
-
13
47
1
2
7
2
2
2
-
11
28
-
1
-
1
-
1
1
3
-
2
4
1
* weak = up to 10%, moderate
= u p t o 5 0 % , a n d s t r o n g ----- m o r e t h a n 5 0 % o f m e d i u m
liquefied after 4 weeks of incubation
* * + = w e a k g r o w t h , + + = m o d e r a t e g r o w t h , a n d + + + = p r o f u s e g r o w t h a f t e r 4 d a y s of
incubation
T a x o n o m y of p h a g e s e n s i t i v e b a c t e r i a
363
Table 2. Frequency distribution of selected traits among bacteria of group I d e p e n d i n g on origin of
bacteria. Presented are the n u m b e r of strains exhibiting the respective traits
Characters
"Eastern" strains
"Western'" strains
Gelatine liquefaction
--
--
+
8
128
+
25
89
Starch
digestion
+
7
1
58
70
19
6
70
19
Nitrite
from nitrate
-+
6
2
9
119
12
13
37
52
Group
II: A - s e r i e s
bacteria
o f c l u s t e r 14
C l u s t e r 14 c o m b i n e s 9 s t r a i n s , 8 of w h i c h a r e " w e s t e r n " o n e s . T r a i t s c o m m o n t o
( a l m o s t ) all s t r a i n s : G r a m n e g a t i v e r o d s , m o t i l e , s i n g l e p o l a r f l a g e l l a (1 e x c e p t i o n ) ,
o x i d a s e p o s i t i v e , g l u c o s e f e r m e n t e d ( w i t h i n 2 to 7 d a y s ) , i n s e n s i t i v e t o 0/129, c o l o n i e s
unpigmented, no diffusible pigments, no fluorescent pigments, non-luminous, starch not
h y d r o l y s e d , g e l a t i n e l i q u e f i e d (1 e x c e p t i o n ) , N H 3 p r o d u c e d f r o m p e p t o n e , n o g a s f r o m
nitrate, n e g a t i v e in m e t h y l r e d test. Results r e g a r d i n g other traits are c o m p i l e d in
T a b l e 3,
T h i s g r o u p is s e t a p a r t f r o m g r o u p I m a i n l y b y t h e s l o w e r r e a c t i o n o n g l u c o s e a n d b y
t h e i n s e n s i t i v i t y of t h e b a c t e r i a to t h e v i b r i o s t a t i c p t e r i d i n e 0/129. B e s i d e t h i s s o m e o t h e r
p e c u l i a r traits w e r e o b s e r v e d .
Table 3. Differently exhibited traits observed in bacteria of group II. Salt- and temperatured e p e n d e n t growth: + = weak, + + -- moderate, and + + + = profuse. F refers to flake formation
Designation
of strain
A185
A335
A398
Catalase
+
+
-
N H 3 from nitrate
+
+
.
Nitrite
+
+
--
Growth with
7.5 % NaC1
+ +
+ +
Growth with
5 %0 sea salts
+++
+++
Growth in SWB
at 20 °C
+++
F
+++
F
+ + +
+ + +
from
A400
+
.
.
A432
A897
+
-
A929
A1212
+
-
+
--
--
+
--
+ + +
--
.
A1409
+
+
+
+ +
F
+ +
F
+
+
+++
F
++
F
++
+++
++
++
+++
F
++
F
+++
++
F
++
+++
F
+ + +
+ + +
+ + +
--
nitrate
Growth i n S W B
at 37 °C
F
F
+++
--
F
F
--
F
364
K. M o e b u s & H. N a t t k e m p e r
Five strains grow p r o f u s e l y in SWB at 37 °C forming l a r g e flakes w h i c h also o c c u r r e d
in SWB at 20 °C and, w i t h 2 of the 5 strains, in 7.5 % NaC1- a n d 5 %0 sea salts-media.
Furthermore, the 8 " w e s t e r n " strains could be l y s o g e n i z e d w i t h 1 to 6 b a c t e r i o p h a g e
strains, w h e r e a s the " e a s t e r n " o n e (A1409) is i n h i b i t e d by 4 of the t e m p e r a t e p h a g e s .
Details r e g a r d i n g i n t e r a c t i o n s b e t w e e n t h e s e bacteria a n d the p h a g e s are r e p o r t e d by
M o e b u s (1983).
G r o u p III: P h a g e - s e n s i t i v e A - s e r i e s b a c t e r i a w h i c h d o n o t fit i n t o c l u s t e r s 1 to 14
This group c o m b i n e s 29 b a c t e r i a which, e x c e p t for 7 d o u b l e t strains, differ from e a c h
other in sensitivity to a n y of t h e p h a g e strains tested. N i n e t e e n are " e a s t e r n " strains a n d
10 are " w e s t e r n " ones. Traits c o m m o n to (almost) all strains: G r a m n e g a t i v e rods, motile,
s i n g l e polar f l a g e l l a (1 strain uncertain, 1 d o u b l e t strain not tested), o x i d a s e positive,
g l u c o s e f e r m e n t e d (within I to 7 days), colonies u n p i g m e n t e d , no diffusible p i g m e n t s , no
f l o u r e s c e n t pigments, g e l a t i n e l i q u e f i e d (1 exception), N H 3 p r o d u c e d from p e p t o n e , no
gas from nitrate. T h e f i n d i n g s for other traits are s u m m a r i z e d in T a b l e 4.
Table 4. Differently exhibited traits observed in bacteria of group III
"Eastern" strains
"Western" strains
Sensitivity
to 0/129
Catalase
Starch
digestion
NH 3 from nitrate
Nitrite from nitrate
Methyl red test
"*
12"
1
1
2
1
1
1
1
2
1
1
1 **
2
+
--
+
+
--
--
+
+
.
+
+
-
--
+
-
-
--
+
--
--
+
+
--
+
+
--
-F
--
+
--
+
--
+
+
+
+
+
+
4+
+
÷
+
+
+
+
+
+
+
--
+
.
--
+
+
-.
_
--
.
+
.
.
.
.
1
+
.
.
1
.
.
.
.
* two strains luminous
grows at 37 °C, forms flakes under any condition tested
Significant differences b e t w e e n " e a s t e r n " a n d " w e s t e r n " strains w e r e o b s e r v e d in
r e g a r d to their activity in M O F m e d i u m . G l u c o s e w a s f e r m e n t e d after only I d a y b y 16
" e a s t e r n " and 3 " w e s t e r n " strains but after 2 to 7 days by 3 " e a s t e r n " a n d 7 " w e s t e r n "
bacteria.
Salt r e q u i r e m e n t s are as v a r i a b l e as o b s e r v e d in the m e m b e r s of g r o u p I (Table 1).
O n e f l a k e - f o r m i n g strain grows w e l l in SWB at 37 °C. T w o d o u b l e t strains are luminous.
Both d i g e s t starch a n d g e l a t i n e a n d g r o w w e a k l y with 7.5 % NaC1.
G r o u p IV: H - s e r i e s b a c t e r i a
This group c o m p r i s e s 31 strains isolated from t h e North S e a n e a r H e l g o l a n d
b e t w e e n 1969 a n d 1978. T h e i r p h a g e sensitivity patterns w e r e r e p o r t e d by M o e b u s &
N a t t k e m p e r (I981; Fig. 4). Traits c o m m o n to (almost) all strains: G r a m n e g a t i v e rods,
T a x o n o m y of p h a g e s e n s i t i v e b a c t e r i a
365
o x i d a s e p o s i t i v e , c o l o n i e s u n p i g m e n t e d ( e x c e p t s t r a i n H85), n o d i f f u s i b l e p i g m e n t s , n o
fluorescent pigments, non-luminous, NH 3 produced from peptone, no gas from nitrate.
T a b l e 5 s u m m a r i z e s t h e f i n d i n g s o b t a i n e d for o t h e r traits. A c c o r d i n g to t h e s e r e s u l t s
t h e m e m b e r s of g r o u p I V c a n b e a r r a n g e d i n 3 s u b g r o u p s .
Table 5. Selected traits as observed in bacteria of group IV. mt = monotrichous (single polar), pt =
peritrichous, F = fermentative, O = oxidative
No. of strains
Subgroup
IVb
IVa
Motility
Flagella
2
1
1
t
2
5
13"
1
1
1"*
÷
mt
+
nat
+
mt
+
mt
+
mt
+
mt
+
mt
+
mt
+
mt
+
rat
---
+
mt
+
pt
F
F
F
F
F
F
O
F
+
--
-+
+
+
+
+
+
+
Glucose metabolism
F
F
F
Sensitivity to 0/129
Catalase
Starch digestion
Gelatine liquefaction
NH a from nitrate
Nitrite from nitrate
Methyl red test
Growth at 37 °C
+
+
+
+
+
+
+
+
+
+
+
+
+
+
--+
+
+
+
*
IVc
1
F
F
.
+
+
+
+
+
+
+
+
+
+
.
-+
+
.
.
--+
.
.
.
+
+
+
--
.
.
.
.
.
.
.
.
.
.
.
-.
.
.
.
.
.
.
.
.
1 + 1 ++
+
.
+
.
.
+
* see text, compare Table 6
Strain H85, forms yellow-orange colonies on SWA; + Strain H86; ++ Strain H96
*
S u b g r o u p I V a i n c l u d e s 4 s t r a i n s w h i c h a r e s e n s i t i v e to t h e p t e r i d i n e 0/129, p r o d u c e
N H 3 a n d N O 2 f r o m n i t r a t e , a n d r e a c t p o s i t i v e l y i n t h e m e t h y l r e d test. S u b g r o u p I V b
c o m b i n e s 24 s t r a i n s w h i c h a r e i n s e n s i t i v e to 0/129. M o s t of t h e s e s t r a i n s d o n o t p r o d u c e
N H a or N O 2 f r o m n i t r a t e , a n d o n l y o n e is p o s i t i v e i n t h e m e t h y l r e d test. In 18 b a c t e r i a of
t h i s s u b g r o u p , s i m i l a r i t i e s i n s e n s i t i v i t y to 3 H - s e r i e s b a c t e r i o p h a g e s w e r e f o u n d
( M o e b u s & N a t t k e m p e r , 1981}.
All m e m b e r s of s u b g r o u p s IVa a n d I V b f e r m e n t g l u c o s e , In m o s t s t r a i n s f e r m e n t a t i o n
w a s i n d i c a t e d a f t e r 2 to 8 d a y s , i n 2 s t r a i n s ( s u b g r o u p IVa) a f t e r o n l y 1 d a y . G r o w t h i n
7.5 % NaC1 m e d i u m w a s s l i g h t (23 strains} or m o d e r a t e (3 strains}. In 5 %o s e a s a l t s
m e d i u m g r o w t h g e n e r a l l y w a s e n h a n c e d : s l i g h t l y i n 7, m o d e r a t e l y i n 18, a n d p r o f u s e l y
i n 11 s t r a i n s . G r o w t h i n SWB a t 37 °C d i d n o t occur.
S u b g r o u p IVc c o m p r i s e s 3 s t r a i n s w h i c h a r e d i f f e r e n t f r o m o n e a n o t h e r . S t r a i n H85
p r o d u c e d y e l l o w - o r a n g e c o l o n i e s a n d its c e l l s a r e n o n - m o t i l e s l e n d e r r o d s w i t h o u t
f l a g e l l a . It f e r m e n t s g l u c o s e ( w i t h i n 7 days} a n d r e q u i r e s s e a s a l t s for g r o w t h w h i c h w a s
o b s e r v e d a t 20 °C o n l y . S t r a i n H 8 6 u s e s g l u c o s e o x i d a t i v e l y . Its m o t i l e c e l l s h a v e p o l a r
f l a g e l l a a n d t h e c o l o n i e s a r e c o l o u r l e s s . T h i s s t r a i n g r o w s p r o f u s e l y i n S W B a t 20 ° a n d
37 °C a n d m o d e r a t e l y i n m e d i a w i t h 7.5 % NaC1 or 5 %o s e a salts. T h e l a s t s t r a i n (H96)
m e t a b o l i z e s g l u c o s e f e r m e n t a t i v e l y a n d g r o w s p r o f u s e l y at all s a l t c o n c e n t r a t i o n s a n d
t e m p e r a t u r e s t e s t e d . Its c e l l s a r e p e r i t r i c h o u s l y f l a g e l l a t e d a n d s p r e a d v e r y r a p i d l y o n
S W A . T h e p r o g e n y of 2 or 3 c e l l s w i l l c o v e r t h e w h o l e a g a r s u r f a c e i n a P e t r i - d i s h of
90 r n m i n d i a m e t e r a f t e r l e s s t h a n 1 d a y of i n c u b a t i o n at 25 °C.
366
K. M o e b u s & H. N a t t k e m p e r
Group
V: B-series bacteria
T h e 11 strains of t h i s g r o u p w e r e i s o l a t e d i n 1978 f r o m w a t e r s a m p l e s t a k e n i n t h e
Bay of Biscay. W i t h 10 strains r a t h e r u n i f o r m results w e r e o b t a i n e d . Traits c o m m o n to
(almost) all strains: G r a m n e g a t i v e rods, o x i d a s e p o s i t i v e , c o l o n i e s u n p i g m e n t e d , n o
diffusible pigments, no fluorescent pigments, non-luminous, NH 3 produced from pept o n e ( e x c e p t strain B17), no g a s f r o m nitrate. T h e results for o t h e r traits a r e c o m p i l e d i n
T a b l e 6.
Table 6. Selected traits as observed in bacteria of group V. rot, F, O: see Table 5
No. of strains
Motility
Flagella
Glucose metabolism
4
1
1
1
1
2"
1 **
+
+
+
+
+
+
--
mt
F
mt
F
mt
F
mt
F
mt
F
mt
F
-O
Sensitivity to 0/129
+
+
+
.
.
.
.
Catalase
+
+
+
.
.
.
.
Starch digestion
--
+
+
-
+
+
--
G e l a t i n e liquefaction
+
+
+
+
+
+
--
NH 3 from nitrate
+
+
+
+
+
--
--
Nitrite from nitrate
Methyl red test
Growth at 37 °C
+
+
.
+
+
+
-.
+
+
+
+
---
+
-+
.
.
.
.
* see text, compare Table 5
* * Strain B17 produces no NH 3 from peptone
It s h o u l d b e n o t i c e d t h a t 2 strains ( m a r k e d b y a n a s t e r i s k in T a b l e 6) a r e s e n s i t i v e to
t h e 3 H - s e r i e s b a c t e r i o p h a g e strains r e f e r r e d to in s e c t i o n " G r o u p IV: H - s e r i e s b a c t e r i a " .
T h e r e s u l t s o b t a i n e d w i t h t h e s e 2 strains c o r r e s p o n d w i t h t h o s e f o u n d w i t h 13 H - s e r i e s
b a c t e r i a ( m a r k e d b y a n a s t e r i s k in T a b l e 5) 12 of w h i c h a r e s e n s i t i v e to t h e s a m e p h a g e s .
S t r a i n B17 differs m a r k e d l y f r o m t h e 10 o t h e r b a c t e r i a of this g r o u p . Its c e l l s a r e also
o x i d a s e p o s i t i v e , G r a m n e g a t i v e r o d s b u t f e w if a n y w e r e m o t i l e a n d no f l a g e l l a w e r e
d e t e c t e d . F u r t h e r m o r e , B17 u s e s g l u c o s e o x i d a t i v e l y , d o e s n o t p r o d u c e N H 3 f r o m p e p t o n e n o r l i q u e f y g e l a t i n e , a n d g r o w s b e s t i n SWB at 37 °C.
Group VI: A-series bacteria
sensitive to bacteriophages
of H - a n d B - s e r i e s o n l y
T h i s g r o u p c o m b i n e s 36 strains, 34 of w h i c h a r e s e n s i t i v e to 7 p h a g e strains of t h e H series. T h e r e m a i n i n g 2 strains a r e i n f e c t e d b y p h a g e B14/2. N u m e r o u s b a c t e r i a l strains
i n c l u d e d i n g r o u p s a l r e a d y d e s c r i b e d a r e also s e n s i t i v e to t h e s e p h a g e s . T r a i t s c o m m o n
to (almost) all strains: G r a m n e g a t i v e rods, m o t i l e (1 e x c e p t i o n , its cells, h o w e v e r , a r e
f l a g e l l a t e d ) , o x i d a s e p o s i t i v e (1 e x c e p t i o n ) , g l u c o s e f e r m e n t e d ( w i t h i n 1 to 7 days), N H 3
p r o d u c e d f r o m p e p t o n e , no g a s f r o m nitrate.
T h e f i n d i n g s r e g a r d i n g o t h e r traits a r e c o m p i l e d in T a b l e 7. O b s e r v a t i o n s m a d e w i t h
strains w h i c h b e l o n g to o t h e r g r o u p s b u t a r e s e n s i t i v e to t h e s a m e p h a g e s a r e i n c l u d e d i n
T a b l e 7 for c o m p a r i s o n ,
---
--
-
--
--
--
4
7
7
4
7
1
6
H4
2
H17
1
H27
1
H31
.
--
2-4
4
1
1
I
1
1
2
1
2
(1)
(4)
BI4
(15)
+
---
--
+
--
--
--
+
+
.
--
+
+
.
--
--
--
--
--
+
+
+
+
+
-
--
---
--
--
-
+
Catalase
.
--
+
--
+
+
+
--
--
+
+
+
--
--
--
--
+
.
--
+
+
+
+
+
+
--
+
+
-
.
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
liquefaction
Gelatine
in bacteria
digestion
Starch
traits observed
+
--
--
--
4
2
1
--
--
--
1
7
4
4
--
7
4
(I), H7
H54, H84
1
1
-
--
2-7
2-7
7
8
1
--
2-7
2
--
2
4
I
2
--
--
7
--
-
2
4
vity to
0/I 29
Sensiti-
exhibited
1
(1)
2
Glucose
fermented
in days
No. or
7. D i f f e r e n t l y
designation
of strains
Table
.
.
+
+
+
+
+
--
--
--
--
--
+
+
.
.
.
.
.
.
+
+
.
.
.
+
.
.
.
.
from
nitrate
NH 3
of group
.
.
.
.
.
.
.
.
.
.
.
.
.
.
+
--
+
+
+
--
--
--
--
--
--
--
+
+
--
.
.
.
.
.
.
.
.
.
.
.
.
.
from
nitrate
Nitrite
.
VI. B a c t e r i a
.
.
.
.
.
.
.
.
.
.
.
.
.
+
+
+
+
+
--
--
--
--
--
--
--
+
+
--
red
test
Methyl
+
---
--
--
--
+
+ + +
+ + +
+
+
--
--
--
--
--
+
at
37 °C
B14/2
I-I84/1
and/or
H54/1
and/or
H7/2
H31/1
H27/I
H17/1
H4/4
to
phage
I, d u s t e r
negative
1
Group V
G r o u p I, c l u s t e r 1
G r o u p I, c l u s t e r 1
Group
oxidase
Group IV
G r o u p III, I V
Group IV
luminous
Group IV
diffusible pigment
Group IV
Group IV
G r o u p III
Remarks
for comparison
Sensitive
are included
Growth
of other groups
3.
¢%
ar
~J
~r
¢u
o
o
p~
368
K. M o e b u s & H. N a t t k e m p e r
Polar flagella were observed with 18 strains r a n d o m l y selected from the m e m b e r s of
this group. All b u t 2 strains (sensitive to p h a g e B14/2) are i n s e n s i t i v e to the p t e r i d i n e
0/129. F a i n t p i g m e n t a t i o n (shades of y e l l o w a n d cream) was observed with 10 strains.
Two other strains p r o d u c e d n o n - f l u o r e s c e n t diffusible p i g m e n t s , a n d a n o t h e r is
luminous.
Slight or moderate growth i n 7.5 % NaC1 m e d i u m was observed i n 33 strains. With
5 %0 sea salts growth of most strains was as profuse as i n SWB at 20 °C. O n l y 2 strains,
both g r o w i n g m o d e r a t e l y i n 7.5 % NaC1 m e d i u m , did not grow at all with 5 %0 sea salts.
Growth at 37 °C was o b s e r v e d i n 6 strains.
G r o u p VII: A - s e r i e s b a c t e r i a i n s e n s i t i v e to a n y of t h e
bacteriophage strains tested
In the 78 strains of this group m u c h greater diversity was observed t h a n i n a n y group
of p h a g e - s e n s i t i v e bacteria. O n l y the most conspicuous f i n d i n g s will be reported shortly.
T e n strains a r e cocci o n e of w h i c h is G r a m negative. It forms p i n k colonies on SWA
a n d is u n a b l e to grow i n SWB at 37 °C. Of the 9 G r a m positive cocci, 8 strains form yellow
colonies a n d only 1 strain does not grow at 37 °C.
A m o n g the r o d - s h a p e d b a c t e r i a 3 G r a m positive a n d 2 G r a m v a r i a b l e strains were
found. T h e G r a m positive ones f e r m e n t glucose w h i l e the G r a m v a r i a b l e s are u n a b l e to
m e t a b o l i z e it.
The r e m a i n i n g 63 r o d - s h a p e d bacteria are G r a m n e g a t i v e a n d 52 of t h e m are
motile. Sixteen motile strains were c h e c k e d for, a n d found to possess, flagella. In 4 n o n motile strains no flagella were observed. Glucose is fermented b y 58 strains, 43 of w h i c h
are oxidase positive. 20 of the latter bacteria are sensitive to the vibriostafic c o m p o u n d
0/129. They most closely r e s e m b l e the p h a g e sensitive bacteria, which is also true i n
regard to other traits. The other 5 strains use glucose oxidatively. Four of t h e m are
oxidase positive a n d all are i n s e n s i t i v e to 0/129.
F o u r t e e n r o d - s h a p e d bacteria produce coloured colonies 'with yellow shades
d o m i n a t i n g . Two strains f o r m i n g white a n d p i n k colonies, respectively, p r o d u c e fluorescent p i g m e n t , a n d a n o t h e r strain is luminous. These 3 strains are motile, oxidase
positive, a n d able to f e r m e n t glucose.
Growth at 37 °C w a s observed i n 15 of the 63 G r a m n e g a t i v e rods w h i c h is a m u c h
larger proportion t h a n f o u n d with p h a g e - s e n s i t i v e bacteria. Finally, o n e of these 63
strains p r o d u c e d gas from nitrate. It is the only one observed to do so of the 444 strains
tested i n this investigation.
DISCUSSION
M o e b u s & N a t t k e m p e r (1981) reported 250 different p h a g e sensitivity patterns
observed i n 326 Atlantic O c e a n b a c t e r i a susceptible to p h a g e s of the same origin. O n the
one hand, these f i n d i n g s i n d i c a t e d c o n s i d e r a b l e genetic differentiation a m o n g bacteria
and p h a g e s as well. O n the other hand, similarities in the p h a g e sensitivity patterns due
to w h i c h the majority of the 326 bacterial strains were p l a c e d into 2 large a n d 8 small
clusters indicated little difference a m o n g the bacteria i n terms of their taxonomic
position.
T a x o n o m y of p h a g e sensitive bacteria
369
Similar observations were m a d e with bacteria a n d p h a g e s isolated from the North
Sea. Of 31 bacterial strains 18 were f o u n d to b e r e l a t e d to each other according to their
sensitivity to 3 b a c t e r i o p h a g e strains. Furthermore, 34 Atlantic O c e a n bacteria were
observed to b e sensitive to the 3 North Sea p h a g e s m e n t i o n e d , i n d i c a t i n g close relationship e v e n a m o n g bacteria isolated from far distant regions.
T a k i n g these observations into a c c o u n t the chances of f i n d i n g bacteria of different
g e n e r a a m o n g the p h a g e - s e n s i t i v e strains i n v e s t i g a t e d were expected to be limited.
However, they existed, at least a m o n g some of the 14 clusters of groups I a n d II as well as
a m o n g the m e m b e r s of groups III to VL
As p r e s e n t e d i n the section "Results" s t u p e n d o u s u n i f o r m i t y w a s f o u n d i n the 250
bacteria p l a c e d i n group L These strains are a s s i g n e d to the g e n u s Vibrio since for the
most important traits (Gram n e g a t i v e rods, motile b y polar flagella, oxidase positive,
f e r m e n t a t i v e on glucose, a n d sensitive to vibriostatic p t e r i d i n e 0/129) only a few
exceptional strains were observed. T h e s e c o n c e r n motility (3), flagellation (6), a n d 0/129
sensitivity (20). T h e y can safely b e i n c l u d e d i n the g e n u s Vibrio due to their relationship
to other strains (as i n d i c a t e d b y p h a g e sensitivity patterns) w h i c h on their part perfectly
correspond with the majority of strains i n this group. R e g a r d i n g 0/129 sensitivity,
c o n t r a d i c t o r y results were o b t a i n e d several times with d o u b l e t strains w h i c h differed
only i n this trait, i n d i c a t i n g the l i m i t e d s t r i n g e n c y of this character (see also below).
A high degree of uniformity was also f o u n d i n most strains c o m b i n e d i n groups II to
VI. These groups comprise 116 bacteria only 4 of w h i c h do not fit the patterns of the most
i m p o r t a n t traits c o m m o n to the majority of 112 strains. T h e latter all are G r a m n e g a t i v e
rods, motile (1 strain n o n - m o t i l e b u t flagellated), oxidase positive (1 exception i n group
VI, T a b l e 7), a n d a b l e to use glucose fermentatively. However, m a n y of the 112 strains
differ from the b a c t e r i a c o m b i n e d i n group I i n the following characters: insensitivity to
0/129, no p r o d u c t i o n of N H 3 a n d nitrite from nitrate, a n d n e g a t i v e m e t h y l red test. In 62
strains, strict correlations were found b e t w e e n these traits, 52 of w h i c h are sensitive to Hseries b a c t e r i o p h a g e s (20 strains of group IV a n d 32 of group VI).
These observations complicate the decision r e g a r d i n g the taxonomic position of the
112 strains i n question. A b o u t 30 of t h e m c a n b e a s s i g n e d safely to the g e n u s Vibrio. The
r e m a i n i n g ones m a y b e l o n g to the g e n u s A e r o m o n a s since no d o u b t is cast on their
i n s e n s i t i v i t y to 0/129 b y contradictory results as observed with d o u b l e t strains ~a m o n g the
m e m b e r s of group I. However, according to Bergey's M a n u a l (Buchanan & Gibbons,
1974) m e m b e r s of the g e n u s A e r o m o n a s are k n o w n to produce nitrite from nitrate w h i c h
was not observed i n the strains i n question. Furthermore, m e m b e r s of the g e n u s Vibrio
are stated to b e " u s u a l l y sensitive to" 0/129 ( B u c h a n a n & Gibbons, 1974, p. 340). This
s t a t e m e n t ist supported b y the results o b t a i n e d with bacteria of group I. Therefore, the
possibility c a n n o t b e ruled out that the 0/129 i n s e n s i t i v e strains also b e l o n g to the g e n u s
Vibrio.
W h e t h e r or not this is the case, there is no doubt that with the exception of 4 strains
all p h a g e - s e n s i t i v e bacteria i n v e s t i g a t e d b e l o n g to the family V i b r i o n a c e a e a n d most of
t h e m (ca. 280 of 362 strains) to the g e n u s Vibrio. The 4 exceptions are strains isolated
from the North Sea (3 strains) or from the Bay of Biscay (groups IV a n d V, respectively).
Of the H-series bacteria (Table 6), strain H85 is tentatively a s s i g n e d to the g e n u s
Flavobacterium. It is the only truly coloured strain a m o n g the p h a g e - s e n s i t i v e bacteria
i n v e s t i g a t e d a n d b e l o n g s to the few b a c t e r i a w h i c h were not d e r i v e d from the same
370
K. M o e b u s & H. N a t t k e m p e r
w a t e r s a m p l e as the i n f e c t i n g bacteriophage. Strain H86 is a s s i g n e d to the g e n u s
P s e u d o m o n a s (group tI of S h e w a n et al., 1960). Strain H96 m a y b e l o n g to the g e n u s
P r o t e u s . It is the only strain ever isolated from a b a c t e r i o p h a g e e n r i c h m e n t culture.
U n c e r t a i n is the t a x o n o m i c position of strain B17 of group V.
The taxonomic u n i f o r m i t y a m o n g the p h a g e - s e n s i t i v e b a c t e r i a raises the q u e s t i o n
w h e t h e r it m a y b e a n artifact c a u s e d b y the methods u s e d to isolate a n d m a i n t a i n the
bacteria a n d bacteriophages.
All b u t one (H96) of the bacterial strains were p i c k e d from plates i n o c u l a t e d with
seawater before cultures for p h a g e e n r i c h m e n t were started with the respective w a t e r
sample. Since as m a n y a p p a r e n t l y different colonies as possible were isolated, the
choice of bacteria basically should have b e e n u n b i a s e d . However, selection certainly
occurred d u r i n g further h a n d l i n g , especially d u r i n g storage, of the isolates.
Of the 1382 strains isolated from Atlantic water samples 451 did not survive u n t i l
soon after return to H e l g o l a n d a n d about 200 a d d i t i o n a l strains were lost d u r i n g the
following months. A m o n g the lost strains, coloured ones h a v e a disproportionately large
share (Moebus, 1980).
Furthermore, i n b a c t e r i a p l a c e d in cluster 3 extraordinarily fast die-off was
observed. W h e n M o e b u s & N a t t k e m p e r (1981) i n v e s t i g a t e d these bacteria, cluster 3
c o m b i n e d 17 strains. O n l y one year later their n u m b e r was r e d u c e d to 10, a n d i n spite of
c o n s i d e r a b l e effort m a d e to m a i n t a i n all strains of this cluster their n u m b e r has s h r u n k
m e a n w h i l e to 6. This observation, u n i q u e a m o n g the various clusters of p h a g e - s e n s i t i v e
bacteria, indicates differences i n the ability to survive d u r i n g storage e v e n a m o n g
closely related bacteria.
R e g a r d i n g the methods u s e d to isolate bacteriophages, selection pressure on populations of bacteria a n d p h a g e s as well is a matter of fact. Phage e n r i c h m e n t requires the
p r e s e n c e of fairly large n u m b e r s of suitable host cells. This condition is u s u a l l y a t t a i n e d
b y i n o c u l a t i n g prospective host b a c t e r i a at the b e g i n n i n g of the e n r i c h m e n t p r o c e d u r e to
a n initial titer greatly e x c e e d i n g that of the n a t u r a l bacterial population. The host
bacteria m a y b e isolated b e f o r e h a n d from the respective s a m p l e of w a t e r or s e d i m e n t
w h i c h is stored at low t e m p e r a t u r e u n t i l the e n r i c h m e n t culture is started. For t e c h n i c a l
reasons this procedure could not b e e m p l o y e d d u r i n g the e x p e d i t i o n to the Sargasso Sea.
Instead, e n r i c h m e n t cultures h a d to b e started i m m e d i a t e l y after collecting the water
s a m p l e s u s i n g seawater s u p p l e m e n t e d with n u t r i e n t s only to support growth of i n d i g e n ous bacteria (Moebus, 1980). Most of the H-series a n d all B-series p h a g e s were d e r i v e d
i n the same way. U n d e r such conditions the selection pressure a m o n g b a c t e r i a is h i g h
a n d fast growing species will b e favoured (Jannasch, 1968). Therefore, o n e has to expect
that preferably p h a g e s will b e e n r i c h e d that infect b a c t e r i a able to outgrow others.
Since most of the b a c t e r i o p h a g e strains tested b y M o e b u s & N a t t k e m p e r {1981) are
infective for bacteria all b u t a few of w h i c h are a s s i g n e d to the family Vibrionaceae, one
has to conclude that such bacteria were vastly favoured i n the e n r i c h m e n t cultures, t n
theory there are two possibilities for this to h a p p e n . V i b r i o n a c e a e either o u t n u m b e r e d
other types of bacteria i n the i n d i g e n o u s populations from the b e g i n n i n g or they a t t a i n e d
this status d u r i n g the course of e n r i c h m e n t culture due to superior growth characteristics.
Some clues can b e d r a w n from the following facts. Of 733 Atlantic O c e a n bacteria
i n v e s t i g a t e d by M o e b u s & N a t t k e m p e r (1981), 326 are sensitive a n d 407 are i n s e n s i t i v e
T a x o n o m y of p h a g e sensitive b a c t e r i a
371
to the b a c t e r i o p h a g e s employed. A m o n g the 78 r a n d o m l y selected p h a g e - i n s e n s i t i v e
strains (group VIII), 37 ( = 47 %) are motile G r a m n e g a t i v e rods, oxidase positive, a n d
f e r m e n t a t i v e on glucose, 17 of w h i c h are also sensitive to 0/129. The 37 strains, therefore,
are closely related to the p h a g e - s e n s i t i v e A-series bacteria of groups I to III a n d VI.
Provided that the 78 strains of group VII are a r e p r e s e n t a t i v e s a m p l e of the 407
p h a g e - i n s e n s i t i v e bacteria, 191 of the latter w o u l d be related to the sensitive strains. In
sum, 517 of the 733 strains tested b y M o e b u s & N a t t k e m p e r (1981) w o u l d b e r e l a t e d
ones, a m o u n t i n g to 70 %. E v e n a s s u m i n g that n o n e of the b a c t e r i a lost d u r i n g storage are
r e l a t e d to p h a g e - s e n s i t i v e strains, the 517 strains w o u l d r e p r e s e n t 37 % of the 1382
isolates collected from Atlantic w a t e r samples.
This c a l c u l a t i o n i n d i c a t e s that, i n the Atlantic O c e a n samples, m e m b e r s of the g e n u s
VibIio or, at least, of the family V i b r i o n a c e a e were a b u n d a n t . However, to e x p l a i n the
fact that 100 % of the p h a g e - s e n s i t i v e A-series bacteria b e l o n g to this family o n e has to
a s s u m e that these b a c t e r i a were favoured d u r i n g e n r i c h m e n t culture b y their growth
characteristics. Therefore, the c o n c l u s i o n is that the taxonomic u n i f o r m i t y a m o n g the
p h a g e - s e n s i t i v e b a c t e r i a observed i n this i n v e s t i g a t i o n is a n artifact.
C e r t a i n l y the same holds true i n r e g a r d to the bacteriophages. T h e r e is n o reason to
a s s u m e that in the Atlantic only p h a g e s exist the hosts of w h i c h b e l o n g to the taxonomic
group e n c o u n t e r e d i n this investigation. As can b e s e e n from the literature cited i n the
"Introduction", b a c t e r i o p h a g e s active o n m e m b e r s of 10 different g e n e r a have so far
b e e n isolated from m a r i n e a n d e s t u a r i n e e n v i r o n m e n t s . However, it m u s t be p o i n t e d out
that the n u m b e r of reports on p h a g e s infective for m e m b e r s of the V i b r i o n a c e a e
( i n c l u d i n g the g e n e r a Vibrio/Beneckea, Aeromonas, Photobacterium, a n d Lucibacterium) is c o n s p i c u o u s l y large.
Finally, a n o t h e r striking result of this i n v e s t i g a t i o n must be considered. A m o n g the
H-series bacteria (group IV, T a b l e 5) as well as the A~series bacteria sensitive to H-series
p h a g e s (group VI, T a b l e 7) all b u t 4 strains are i n s e n s i t i v e to the p t e r i d i n e 0/129, a n d
most of t h e m are n e g a t i v e i n p r o d u c t i o n of N H 3 a n d nitrite from nitrate a n d i n the methyl
red test. This c o m b i n a t i o n of traits was rarely observed i n the other bacteria (4 of 9 i n
group II, T a b l e 3; 4 of 29 i n group III, T a b l e 4; 2 of 11 i n group V, T a b l e 6).
It is not s u r p r i s i n g that H-series p h a g e s are active preferentially on bacteria from the
Atlantic w h i c h r e g a r d i n g the respective traits r e s e m b l e the p h a g e s ' original hosts.
However, it is s u r p r i s i n g that 20 of the 28 North Sea bacteria a s s i g n a b l e to the family
V i b r i o n a c e a e e x h i b i t exactly these traits w h i c h separate t h e m from the vast majority of
Atlantic O c e a n bacteria.
These f i n d i n g s m a y b e the expression of differences i n the composition of bacterial
a n d b a c t e r i o p h a g e p o p u l a t i o n s typical for the North Sea n e a r H e l g o l a n d a n d the o p e n
Atlantic. It c a n n o t b e r u l e d out, however, that also the c o n s i d e r a b l e differences i n the
n u m b e r s of (colony forming) b a c t e r i a p e r u n i t v o l u m e i n water s a m p l e s from both
regions g r a v e l y affect the outcome of p h a g e e n r i c h m e n t cultures started w i t h o u t prospective host b a c t e r i a added.
Acknowledgements. The authors are indebted to Prof. G. Rheinheimer, Dr. M. B6Iter and Mrs. R.
Kreibich of the Institut ffir Meereskunde, Kiel University, for hospitality and technical advice given
to Miss H. Nattkemper during 2 weeks training in marine bacterial taxonomy.
372
K. M o e b u s & H. N a t t k e m p e r
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sensitive bacteria
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