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Applied and Environmental Microbiology, November 1998, p. 4128-4133, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Bacteriophage Diversity in the North Sea
Antje
Wichels,1,*
Stefan S.
Biel,2
Hans R.
Gelderblom,2
Thorsten
Brinkhoff,3
Gerard
Muyzer,3,
and
Christian
Schütt1
Biologische Anstalt Helgoland, D-27498
Helgoland,1
Robert-Koch Institut,
D-13353 Berlin,2 and
Max Planck
Institute for Marine Microbiology, D-28359
Bremen,3 Germany
Received 29 June 1998/Accepted 13 August 1998
 |
ABSTRACT |
In recent years interest in bacteriophages in aquatic environments
has increased. Electron microscopy studies have revealed high numbers
of phage particles (104 to 107 particles per
ml) in the marine environment. However, the ecological role of these
bacteriophages is still unknown, and the role of the phages in the
control of bacterioplankton by lysis and the potential for gene
transfer are disputed. Even the basic questions of the genetic
relationships of the phages and the diversity of phage-host systems in
aquatic environments have not been answered. We investigated the
diversity of 22 phage-host systems after 85 phages were collected at
one station near a German island, Helgoland, located in the North Sea.
The relationships among the phages were determined by electron
microscopy, DNA-DNA hybridization, and host range studies. On the basis
of morphology, 11 phages were assigned to the virus family
Myoviridae, 7 phages were assigned to the family
Siphoviridae, and 4 phages were assigned to the family
Podoviridae. DNA-DNA hybridization confirmed that there was
no DNA homology between phages belonging to different families. We
found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and
physiological tests and by 16S ribosomal DNA sequencing. All of the
bacteria were gram negative, facultatively anaerobic, motile, and
coccoid. The 16S rRNA sequences of the bacteria exhibited high levels
of similarity (98 to 99%) with the sequences of organisms belonging to
the genus Pseudoalteromonas, which belongs to the 
subdivision of the class Proteobacteria.
| |
INTRODUCTION |
The marine bacterial community is
responsible for a considerable portion of primary production and
regeneration of nutrients in the microbial loop and is associated with
a great variety of marine bacteriophages (5, 12). These
phages are capable of infecting a large portion of the bacterioplankton
(32, 34). It is assumed that as part of the marine food web,
bacteriophages play important quantitative and qualitative roles in
controlling marine bacterial populations (8, 24, 34, 39,
45). The phenotypic diversity and genotypic diversity of the
phage populations are related to the interaction between phages and
their host organisms, which provides a tool for understanding the
interaction itself (13). To estimate the influence of marine
bacteriophages on the diversity of bacterioplankton, we investigated
phage diversity. The virus species concept proposed by Murphy et al.
(37) delineates seven different families of bacteriophages
based on morphological criteria and provides criteria for new phage
species based on several traits, such as DNA homologies, serological
data, protein profiles, and host ranges.
In this paper, we describe the diversity and genetic relationships of
marine phages based on investigations of 22 representatives from 85 phage-host systems (35, 36) collected between 1988 and 1992 from waters around an island, Helgoland, located in the North Sea. All
of the phages were virulent and formed plaques on their host bacteria.
We assigned the phages to different virus families, species, and
strains based on morphology, DNA homology, and host range. Furthermore,
we characterized the phenotypic and genotypic features of the host bacteria.
| |
MATERIALS AND METHODS |
Bacterial strains, phages, and media.
Bacterial strains and
bacteriophages were kindly provided by K. Moebus, Biologische Anstalt
Helgoland, Helgoland, Germany. They were isolated from North Sea water
collected at one location near Helgoland, an island in the North Sea
belonging to Germany. The bacterial strains were grown as described by
Moebus and Nattkemper (33). Phage lysates were prepared by
the overlay agar technique (31). Confluent lysis was
generated, and the phages were eluted with 10 ml of SM buffer per plate
after incubation for 1 h at room temperature (47).
Phage stocks were stored at 4°C.
Phage-host cross-reaction test.
Two-layer agar plates
containing a 10-ml bottom layer and a 3-ml soft agar upper layer were
used for the phage-host cross-reaction test; the soft agar layer
contained ca. 108 bacteria (34). Phage lysates
were dotted in a dilution series from 100 to
106 onto the upper layer immediately after solidification
in order to distinguish between a clear lysis reaction caused by plaque formation and inhibition of the bacterial lawn. After incubation overnight at 18°C in the dark, plaque formation was evaluated.
Electron microscopy of phages.
High-titer phage stocks
(lysates) were prepared for electron microscopy. Lysates were allowed
to adsorb for 1 min to pioloform- and carbon-coated 400-mesh wide
copper grids. Then the grids with the adhering phage lysates were
washed three times with distilled water. Negative staining was
performed with 2% (wt/vol) uranyl acetate for 40 s (19,
20). Micrographs were obtained at a primary magnification of
×40,000 by electron microscopy (Zeiss model EM 10 A microscope). The
dimensions of phages were estimated by determining the mean values for
30 particles of each phage; catalase was used as an internal length
calibration standard (46).
Buoyant density of phage particles.
Phage buoyant density
was determined by CsCl2 gradient centrifugation by using
the method of Espejo and Canelo (15).
Isolation of phage DNAs and labeling of DNA probes.
Stocks
(200 ml) of phages were prepared as described above. DNAs were isolated
by using the general methods described by Sambrook et al.
(42). Purified phage DNAs were labeled with a nonradioactive digoxigenin labeling kit (Boehringer, Mannheim, Germany) as recommended by the manufacturer and were used as probes in subsequent DNA-DNA hybridization experiments.
DNA-DNA hybridization on nylon membranes.
For dot blot
hybridization whole-phage DNA (5 to 10 µl) was dotted onto a nylon
membrane and fixed with UV light (wavelength, 312 nm; 7 min). For
restriction fragment DNA-DNA hybridization the phage DNA was digested
with restriction enzyme HindIII (Boehringer) for 90 min
at 37°C. After gel electrophoresis in 0.8% (wt/vol) agarose gels,
the DNA pattern was transferred to nylon membranes (Hybond N; Amersham,
Braunschweig, Germany) by Southern blotting (43), and the
DNA was used as target DNA. DNA-DNA hybridization was carried out at
68°C for 16 h. Hybridization was detected as recommended by the
manufacturer (Boehringer). Positive hybridization signals occurred
after 1 to 4 h of incubation with
5-bromo-4-chloro-3-indolylphosphate toluidinium (salt) and nitroblue
tetrazolium (salt) (70%). The levels of DNA homology between
previously described phages and phages used in this work were not determined.
Determination of GC content of phage DNA.
The GC content of
phage DNA was determined as described by Marmur and Doty
(29).
Morphology and physiology of host bacteria.
Morphological
and physiological tests were performed as described by Moaledj
(30). Cell morphology (size, shape, arrangement) was
determined by phase-contrast microscopy (magnification, ×1,250) after
1 to 2 days of incubation at 18°C. The media used for the physiological tests were adapted so that they fulfilled the salt requirements of marine bacteria. Gram reaction, catalase and oxidase production, and motility tests were performed with freshly prepared liquid cultures, while the cultures used to test oxidation and fermentation of glucose, saccharose, and lactose (1%, wt/vol) were
incubated for up to 14 days before analysis.
PCR amplification of the 16S rRNA gene.
Bacterial DNA was
isolated by the method of Anderson and McKay (2), modified
for genomic DNA by omitting the NaOH step. The extracted DNA was used
as target DNA in PCR (41) to amplify the 16S ribosomal RNA
coding regions. The primers used for the 500-bp fragment examined were
27f (5'-AGAGTTTGATC[A/T]TGGCTCAG-3') and 519r
(5'-G[A/T]ATTACCGCGGC[G/T]GCTG-3' (26). The
sequences of the primers used for the nearly complete 16S rRNA gene
(GM3F and GM4R; Escherichia coli positions 8 to 1507) have
been published by Muyzer et al. (38). PCR amplification was
performed with a model 480 DNA thermal cycler (Perkin-Elmer Cetus) as
described by Muyzer et al. (38). To increase the specificity
of amplification and to reduce the formation of spurious by-products, a
"touch-down" PCR (14) was performed (65 to 55°C, 20 cycles). Aliquots (5 µl) of the amplification products were analyzed
by electrophoresis in 2% (wt/vol) agarose gels, which were stained
with ethidium bromide (0.5 µg/ml).
DNA sequencing of PCR products and comparative sequence
analysis.
PCR products that were 500 bp long were purified with
glasmilk (Bio-Rad). DNA sequencing was performed with a model ABI 377 sequencer by using a PRISM Ready Dye Deoxy terminator kit and Perkin-Elmer Taq polymerase according to the instructions of
the manufacturer (ABI, Foster City, Calif.). The sequences of whole 16S
ribosomal DNA fragments were determined by the method described by
Buchholz-Cleven et al. (10). All sequences were aligned with sequences obtained from the Ribosomal Database Project (27) or GenBank (3). Sequence alignment was performed with the
sequence editor SEQAPP (21). A phylogenetic tree was created
by using the neighbor-joining algorithm and maximum likelihood as a
model for evolution (PAUP test, version 6.3, developed by David
Swofford). A bootstrap analysis (100 replicates) was used to validate
the reproducibility of the branching pattern of the tree.
Nucleotide sequence accession numbers.
The sequences
obtained in this study have been deposited in the GenBank database
under accession no. AF069653 through AF060667.
| |
RESULTS |
Selection of phages for detailed investigation.
To select a
group of phages that were representative of the 85 phages isolated,
phage-host cross-reaction tests were performed with the phages and 70 bacterial isolates obtained from North Sea water. The phages were
assigned to sensitivity group I (SG I), SG II, and SG III on the basis
of their hosts. A total of 62 (73%) of the 85 bacteriophages were
highly host specific and members of SG I; these phages were found to be
reproduced only by their original hosts. Sixteen phages (19%) had host
ranges consisting of 2 to 10 bacteria and were members of SG II, and seven phages (8%) had broad host ranges consisting of 11 to 36 bacterial isolates and were members of SG III. A total of 22 bacteriophages were selected for further investigation. Seven of these
phages belonged to SG I, nine belonged to SG II, and six were assigned to SG III.
Morphological diversity.
The phenotypic diversity of the 22 bacteriophages was examined by electron microscopy. The phages were
identified by using morphological criteria outlined by the
International Committee of Taxonomy of Viruses (37) and the
species concept of Ackermann et al. (1). Morphological
studies revealed that all of the phages examined had tails and thus
belong to the order Caudovirales. The icosahedral heads of
the phages had diameters between 50.2 and 99.3 nm. The phages could be
assigned to three virus families. Eleven of the phages belonged to the
family Myoviridae, which contains phages that have
icosahedral heads and long contractile tails; seven phages were
assigned to the family Siphoviridae, which contains phages
that have icosahedral heads and long flexible tails; and four phages,
which had icosahedral heads and short tails, belonged to the family
Podoviridae. The phages belonging to the
Myoviridae were further divided into two different
morphotypes on the basis of different appendages, such as collars,
antennae, or tail fibers; the four morphotype 1 phages had a collarlike structure between the head and the tail (Fig.
1B), whereas the seven morphotype 2 phages had no special appendages (Fig. 1A).
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FIG. 1.
Phages belonging to three different families and their
morphotypes. (A) Myoviridae, morphotype 1: head without
antennae and short appendages on the tail (phage H106/1). (B)
Myoviridae, morphotype 2: collarlike structure between the
head and the tail and short appendages on the tail (phage H7/2; 15).
(C) Siphoviridae, morphotype 1: head and tail without
appendages (phage 10-77a). (D) Siphoviridae, morphotype 2:
knoblike appendages on the head and tail with a hook at the end (phage
11 68c). (E) Siphoviridae, morphotype 3: knoblike appendages
on the head and tail with short appendages (phage H105/1). (F)
Podoviridae, morphotype 1 (phage H100/1). Bar = 100 nm.
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|
Similarly, the seven bacteriophages belonging to the
Siphoviridae were subdivided into three different
morphotypes. The single morphotype 1 phage had no additional appendages
on its head or tail (Fig. 1C). The single morphotype 2 phage was
particularly striking because it had a hook at the end of its tail
(Fig. 1D). The remaining five phages, which had knoblike appendages on
their heads, belonged to morphotype 3 (Fig. 1E). All four phages
belonging to the Podoviridae were morphotype 1 phages with
no special appendages (Fig. 1F). The buoyant densities of the phages
investigated were between 1.49 and 1.54 g · cm
3.
Bacteriophage host ranges.
The results of our morphological
characterization of the phages were related to phage host ranges (Fig.
2). Phages were arranged according to
family. The following three observations were remarkable: (i) all of
the phages belonging to the Myoviridae had very broad host
ranges; (ii) three of the phages belonging to the
Siphoviridae had identical host ranges and belonged to the
same morphotype; and (iii) all four phages belonging to the
Podoviridae exhibited high specificity for their host
bacteria.
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FIG. 2.
Bacteriophage host ranges and families.  , phage
produces plaques;  , phage inhibits growth of bacterial lawn (no
plaques).
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|
GC contents of marine bacteriophages.
All of the phages
contained double-stranded DNA. The GC contents of the DNAs ranged from
33.7 to 64.7%. The GC contents of the phages belonging to the
Myoviridae ranged from 33.7 to 64.7%, whereas there was
less variation in the GC contents of members of the
Siphoviridae (GC contents, 40.1 to 51%). The GC contents of
the DNAs of members of the Podoviridae were between 38.4 and 57.6%.
DNA homologies of marine bacteriophages.
Dot blot
hybridizations revealed that phages belonging to different families did
not exhibit DNA homology. To obtain more detailed information, specific
HindIII endonuclease restriction digestion of the DNAs
and subsequent Southern hybridization were carried out with the phages
that exhibited DNA homology in dot blot hybridization experiments.
These experiments showed clearly that either there was a high level of
homology between the DNAs of two phages belonging to the same family
(i.e., DNA homology occurred in every DNA fragment) transferred to a
nylon membrane or no homology was detected. Besides the fact that there
was no homology between bacteriophages belonging to different families, an overview of all of the hybridization experiments revealed that within the families not all of the phages were genetically related (Fig. 3). Seven phages exhibited no DNA
homology to the other phages investigated. The other 15 phages
exhibited DNA homology to one or more phages belonging to the same
family. In the Myoviridae, DNA homology was observed between
phages 12-13a and 13-15b, as well as between phages 6-42c and 6-62c. No
Southern hybridizations were performed with phages H71/1, H7/2, H71/5,
and 12-41b; the nondigested DNAs of these four phages exhibited
reproducible homologies. Dot blot hybridization revealed that the DNA
of phage 12-41b exhibited a weak hybridization reaction to DNAs of
phages H71/1 and H71/5, while there was no DNA homology between phages
H7/2 and 12-41b (Fig. 3). In the Siphoviridae, phages
H103/1, H105/1, and H108/1 exhibited DNA homology, while phages H118/1
and H120/1 were homologous to each other. The DNAs of two of the phages
(10-33b and 10-94a) in the Podoviridae were homologous. No
homology was observed for any of the other phages. According to the
species concept of Ackermann et al. (1), phages belonging to
the same family that exhibit DNA homology should be assigned to the
same species. Within the Myoviridae six species could be
differentiated; four species were differentiated within the
Siphoviridae, and three species were differentiated in the
Podoviridae. Altogether, 13 species were identified among
the 22 phages tested.
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FIG. 3.
DNA-DNA hybridization of 22 marine bacteriophages. ,
strong hybridization signal; , weak hybridization signal. The
families and morphotypes of the phages and DNA probes are indicated.
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|
Morphology, physiology, and phylogenetic analysis of host
bacteria.
All of the bacteria examined were gram negative, motile,
catalase and oxidase positive, and facultatively anaerobic. A
comparative analysis of partial sequences (ca. 500 bp) of all of the
isolates revealed that they belong to the subdivision of the class
Proteobacteria and that Pseudoalteromonas species
are the closest relatives (Fig. 4). The
levels of sequence similarity for the host bacteria were more than
99%. The levels of similarity between the isolates and their closest
known relatives were around 98%. A phylogenetic analysis performed
with nearly complete 16S rRNA sequences of three isolates (H7, 12-13, and H120) gave a similar tree, which had a slightly different branching
order.
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FIG. 4.
Phylogenetic affiliations of host bacteria: distance
tree based on the first 500 bp of 16S ribosomal DNA. The bootstrap
values obtained with 100 replicates are shown. Only bootstrap values
equal to or greater than 75% are shown.
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| |
DISCUSSION |
Bacteriophages are ubiquitous, very abundant, and morphologically
diverse in marine environments (4, 9, 16, 17). The present
study provides data which reveal the great genetic diversity of marine
bacteriophages, which exceeds the morphological diversity. The great
genetic diversity represents a large gene pool for horizontal and
vertical gene transfer, which supports the view that marine
bacteriophages play an important role in controlling bacterioplankton
both quantitatively and qualitatively.
Classification of marine phages.
Criteria given by the
International Committee of Taxonomy of Viruses were used for taxonomic
classification of the 22 marine bacteriophages investigated. According
to these criteria, all of the phages belong to the order
Caudovirales (tailed bacteriophages), a very common, widely
distributed group. Up to 1993, 4,007 phages had been described in
detail; 96% of these phages had tails and thus belonged to the
Caudovirales, while only 4% had filamentous, isometric, or
pleomorphic morphology (28). About 150 marine bacteriophages
have been isolated and described, and only one phage, PM2
(15), has been classified as a member of the family Corticoviridae (37). The other 149 phages belong
to families of tailed phages (16).
The phages investigated in this study were found to belong to all three
families in the
Caudovirales (
Myoviridae,
Siphoviridae,
and
Podoviridae). These phages have
icosahedral heads with diameters
between 50.2 and 99.3 nm. The majority
of cultured marine phages
studied so far have the same characteristics
(
6); therefore,
the phages investigated belong to this major
group. In contrast,
estimates of diameters of phage heads based on
direct counting
of marine phages by electron microscopy revealed that
most phages
in marine environments are smaller (diameters, 30 to 60 nm)
(
6,
11).
Almost all of the phages isolated near Helgoland had morphological
structures that have been described previously (
16).
The
only exception was phage 11-68c, which belongs to the
Siphoviridae and has a hook at the end of its tail; this
morphology has not
been reported previously for the marine environment.
Phage H7/2
has been described previously (
16), and three
other phages belonging
to the
Myoviridae had the same
collarlike structure between the
head and the tail. Interestingly, the
morphology of the members
of the
Podoviridae was less
diverse than the morphology of the
members of the
Myoviridae
and
Siphoviridae.
The species to which phages belong were determined in this study by
using the concept of Ackermann et al. (
1), which is
based on
the occurrence of DNA homologies in phages belonging
to the same
family. Like Jarvis (
23) and Krylov et al. (
25),
Ackermann et al. did not observe DNA homology between phages belonging
to different families. The present study confirmed the findings
of
Ackermann et al. since there was no DNA homology between phages
belonging to different families, while several species were found
in
each family. For example, four phages belonging to the
Myoviridae that had the collarlike structure were found to
belong to the
same species by DNA homology studies. Different phages
belonging
to the same species were also characterized by using several
strain-specific
markers (
1,
28,
37), including host range,
density of phage
particles, and GC content of the phage DNA (Fig.
5).
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FIG. 5.
Arrangement of 22 phages as members of an order,
families, morphotypes, and species. Phage density data, GC contents of
DNAs, and host range groups are also shown.
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|
A total of 22 marine bacteriophages were tested, and 13 new species
based on the absence of DNA homology and 22 different
phage strains
were differentiated, which revealed that the genetic
diversity was
high, much higher than the morphological diversity
(Fig.
5). The very
broad host ranges of phages belonging to the
Myoviridae
compared to the host ranges of phages belonging to
the
Siphoviridae and
Podoviridae supports the
findings of Suttle
and Chan (
44). In a study of cyanophages
these authors found
that phages belonging to the latter two families
were more host
specific than phages belonging to the
Myoviridae.
Classification of host bacteria.
The majority of all marine
bacteriophages are highly host specific (6, 12, 32), and
73% lyse only the original host bacterium. However, a few phages were
able to form plaques on more than 50% of the 36 bacterial strains
investigated here. Therefore, the relationships of the bacterial
isolates can be deduced from their phage sensitivity patterns (22,
35, 36). Moebus and Nattkemper (33) studied the
taxonomy of 31 bacterial isolates obtained from phage-host systems
isolated near Helgoland. These authors reported that all of their
strains were members of the family Vibrionaceae and most
were members of the genus Vibrio. All of the host bacteria
investigated here belong to the subdivision of the class
Proteobacteria, with levels of DNA sequence homology of more
than 99%. They are all closely related to the genus
Pseudoalteromonas. No phages for this genus have been
described until now. However, the genus was described recently, and
some species formerly classified as members of the genus
Vibrio (e.g., Vibrio marinus) (18, 40) are now thought to be closely related to the
Pseudoalteromonas group.
Although we investigated only a small part of the marine bacterial
community, we established that there is great genetic variation
in the
infectious marine bacteriophages, which leads to high levels
of species
and strain diversity. It is likely that with in future
studies
increased genetic diversity among phages will be discovered,
especially
in groups of bacteria belonging to the "silent majority"
of marine
bacteria that have not been cultured
yet.
| |
ACKNOWLEDGMENTS |
We are very grateful to K. Moebus (Biologische Anstalt Helgoland)
for his very generous gift of the phage-host systems which we
investigated in this study. We thank Bärbel Jungnickl for her
help with the preparation of the photographic prints.
This investigation was supported by a grant from the Biologische
Anstalt Helgoland.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biologische
Anstalt Helgoland, D-27483 Helgoland, Germany. E-mail:
AWichels{at}AWI-Bremerhaven.de.
Present address: Netherlands Institute for Sea Research, NL-1790 AB
Den Burg, Texel, The Netherlands.
| |
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Applied and Environmental Microbiology, November 1998, p. 4128-4133, Vol. 64, No. 11
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
