Download Phage-host interactions in soil

FEMS MicrobiologyEcology 15 (1994) 99-108
© 1994 Federation of European Microbiological Societies 0168-6496/94/$07.00
Published by Elsevier
99
FEMSEC 00576
MiniReview
Phage-host interactions in soil
P. M a r s h * a n d E . M . H . W e l l i n g t o n
Department of Biological Sciences, University of Warwick, Coventry CF7 4AL, UK
(Received 10 March 1994; revision received 18 April 1994; accepted 25 April 1994)
Abstract: Phages are abundant and ubiquitous in nature, and are therefore important components of microbial communities. They
can impact on host populations in several ways, including predation and alteration of host phenotype by genetic interactions. The
dynamic survival of phage populations in soil requires infective interactions with host populations which must be undergoing
growth. Hence survivalis limited by the activityof soil bacteria, and phage populations must adopt strategies to overcome periods
of inactivity. One of the most effective strategies is the lysogeniccycle of temperate phages. It is argued here that lysogenyin soil
has a distinct advantage over virulence for phage and host survival, as opposed to aquatic ecosystemswhere virulence seems a more
successful strategy for phage populations.
Key words: Phage ecology; Soil streptomycetes; Phage-host interaction; Lysogeny
Introduction
The ecology of bacteriophages has received
comparatively little attention despite their abundance in natural environments. Phages can usually be isolated from all habitats where their hosts
exist, and are ubiquitous components of bacterial
communities [1]. Ecosystems which allow active
bacterial growth have the potential to support
multiplication of phage populations which require metabolically active hosts. The presence of
phage populations in these ecosystems therefore
presents a potential impact on bacterial populations. Our aim is to review the importance of
phages in natural environments with particular
regard to soil. This will involve consideration of
* Corresponding author.
SSDI 0168-6496(94)00059-X
the abundance of phages in nature, their survival
strategies, their impact on host populations, their
role in genetic interactions and the importance of
lysogeny.
Phages are predators or parasites of host bacteria which although they are able to persist in
the environment independently of their hosts,
they have the absolute requirement to infect host
cells in order to multiply and survive as dynamic
populations [2]. For multiplication to occur in the
environment a phage particle must be able to
protect its genome from adverse conditions, gain
access to infect a susceptible host and change the
host cell into a unit for producing phage progeny
which are finally released. Free phage particles
have to protect the genome from environmental
damage in a protein capsule known as the capsid
[3]. The most common phage morphology is that
of an isometric protein head classified as A and B
100
types, which have sheathed contractile or unsheathed noncontractile hollow tails respectively
[4,5]. Head size is generally between 50 and 100
nm whereas tail length varies greatly between
types and can be up to 200 nm [3,6]. A tail plate
is usually situated at the end of the tail, and this
apparatus is responsible for mediating specific
adsorption of phages to host cell surfaces prior to
infection.
Phages are categorized into two main types
depending on the infection cycle they may follow
on delivery of the genome into a host cell. Phages
which can only lyse the infected host cell to
produce daughter progeny follow the lytic cycle
of infection and are termed virulent [7]. Those
which follow either the lytic cycle or the lysogenic
cycle, where the phage genome is retained as a
replicable entity within the host without causing
lysis, are termed temperate phages [7]. Another
type of phage multiplication which cannot be
characterized as either the lytic or lysogenic cycle
is that of the filamentous phages, which attach to
the sex pili of F + Gram-negative bacteria and
mediate production of progeny particles from the
growing cell without causing lysis [7].
Contact between the host and phage may result in reversible binding of the phage end plate
to the host cell surface which often requires the
presence of divalent cations such as Ca 2+ and
Mg 2+. Pins on the tail plate allow irreversible
binding with specific receptors on the cell surface, and enzymes from the tail pin alter this site
to enable entry of the phage genome into the cell
lumen. Phage-encoded signals then cause shutdown or alteration of the cell's RNA and protein
synthesis, and the conversion of the cell into a
factory producing copies of the progenitor phage
particle. Phage-induced lysozyme causes rupture
of the cell wall followed by release of the daughter progeny into the environment. At this point,
large amounts of lysozyme are also liberated
[2,3,7].
The lysogenic cycle of temperate phages is
initiated after entry of the genome into the cell
cytoplasm, and the choice of this cycle rather
than the lytic cycle is dependant upon the expression of host and phage genes as well as environmental conditions which also influence this ex-
pression [8-10]. Generally, environmental conditions which are unfavourable for host proliferation will cause increased frequencies of lysogeny.
For example, there is evidence [9] that production
of cellular cAMP-CAP due to starvation conditions leads to the inhibition of host Hfl (high
frequency of lysogenization) protein which in turn
allows the establishment of lysogeny by phage ,~.
High multiplicities of infection (m.o.i.) lead to
excess amounts of phage-encoded protein cIII in
cells which also antagonizes Hfl and leads to
increased frequencies of lysogeny [9]. These influences lead to the suppression of lytic cycle genes,
and allow the expression of lysogenic cycle genes
which cause integration of the phage genome into
the host chromosome, and subsequent expression
of repressor protein(s) which prevent regression
into the lytic cycle [11]. The integrated phage
genome is known as the prophage, and is replicated with the host genome at cell division without lysing the host. Some temperate phages such
as coliphage P1 do not integrate into the host
chromosome, but exist in a repressed replicable
extra-chromosomal state. The host is now termed
lysogenic [12], and is immune to superinfecting
phages with genome sites homologous to the
binding site of the repressor proteins being produced by the prophage. Reversion to the lytic
cycle occurs at a certain frequency in growing
lysogenic populations, although prophage induction is usually caused by cellular damage, especially that which affects the host DNA. This
causes loss of expression of the repressor protein
and therefore a switching on of the lyric cycle.
Certain virulent phages can exist inside dormant
hosts in an arrested stage of the lytic cycle, which
can resume when the host recommences vegetative growth. This state is known as pseudolysogeny [13].
Around 90% of all phages isolated from natural environments have proved to be temperate [2],
so it may be postulated that temperate phages
have an advantage over virulent phages which
require actively growing hosts to multiply. Temperate phages can exist as prophages in hosts
through long periods of inactivity when free virions in the environment may become inactivated.
When considering phage populations in a particu-
101
lar ecosystem, one question to be addressed concerns mode of cell-to-cell transmission. In virulent populations the only available route is horizontal transmission, where the lytic cycle is a
rapid form of multiplication, but entails a high
risk in that the infected cell is destroyed leaving
the progeny particles with the possibility that new
susceptible host cells may not be encountered.
Temperate phage populations can follow the
lysogenic cycle, during which the prophage is
replicated along with the host chromosome. This
produces far less copies of the original prophage
at a slower rate than the lytic cycle, but has the
advantage of greater security in that the host is
maintained in a replicable state, protecting the
phage [14].
The abundance of phages in natural environments
Early studies have shown that phages were
present in very low numbers in natural environments, so it was assumed that they were of little
ecological importance. This assumption was based
on counts made by plating phages on indicator
host strains. More recent studies have used transmission electron microscopy (TEM) to make direct counts of viruses in aquatic samples (Table
1). These figures indicate that phages can be
present at very high densities. Direct phage counts
have so far not been made from soil, due in part
Table 2
Abundance of phages in bulk soil
Phage type
pfu a on indicator Reference
host/g soil
4.0 x 1 0 7
3.4 x 103
Actinomycete(temperate) 3.6 x 104
Actinomycete(virulent)
3.6 x 102
Actinomycete
2.0 × 104
Arthrobacter
7.1 × 102
22
23
24
24
25
26
Bacillus
Ensifer adhaerans
a pfu, plaque forming units.
to the difficulty in preparing samples suitable for
TEM. Counts of soil phages have been made
using indicator strains, and as in aquatic systems
the total phage densities may be much higher
than shown in Table 2. This abundance in ecosystems makes phage populations important in terms
of their influences on host population levels and
activity, as well as host population evolution due
to the intimacy of phage and host genomes. This
last point is emphasised by the suggestion that
phages, in conditions beneficial to their multiplication, are the most numerous self-perpetuating
genetic objects in soil [22].
Phage survival in nature
The main requirement for the survival of a
parasite or predator population is that the host or
Table 1
The abundance of phages in aquatic environments
Sample
Marine
(reefwater)
(sediment)
Marine
Marine
Marine
Marine
Freshwater
Freshwater
Activated
sludge
Phage
type
pill a on indicator
host (ml - 1)
Direct counts
(TEM, ml- 1)
Reference
mixed b
0.19
8.8
1.5 x 103
3.0
1.2 x 104
5.3 × 10s
3.5 X 10 7
1.3 x 1 0 7
1.5 X 107
2.5 x 10s
-
15
15
16
17
18
19
19
20
1.7 × 102/ml
-
21
mixed
mixed
mixed
mixed
mixed
coliphages
coliphages
a pfu, plaque forming units.
b Includes vibrio- and coliphages.
-
102
prey population must not be made extinct by
predation. This is often ensured by density dependent regulation of interacting phage and host
populations, and is manifested as alternating oscillations of phage and host populations where
both exist in a state of equilibrium [27,28]. Such
oscillations have been observed in the marine
environment [18]. The persistence of steady state
requires the continual multiplication of host populations and hence requires sustained nutrient
input.
Phages in aquatic ecosystems are subject to
continual movement and therefore have a relatively high probability of encountering susceptible
hosts [29]. The fluxing nature of aquatic ecosystems also facilitates the delivery of nutrients via
primary production, for example during spring
blooms in marine ecosystems. This in turn allows
bacterial blooms and consequent peaks in phage
populations. These peaks may be responsible for
controlling bacterial populations as they are
quickly followed by a drop in host numbers
[16,18,30]. This type of environment would seem
to favour the virulent phages which may move
freely in the water and have access to active
bacterial populations.
The rapid generation times of phage and bacterial populations facilitates the development of
new phenotypes. This is well illustrated by mutations to infection immunity in bacteria, and subsequent counter-mutations by phages to overcome host immunity [31]. This leads to polymorphic host and phage populations where there
originally existed one clone each of host and
phage [32].
In soil, free phages are in close contact with
soil components, and physical interactions between such particles and phages greatly influence
their survival. This can be beneficial or deleterious to the phage population depending on phage
and clay types. For example, the pH optima of
actinophages may be expanded or reduced by
binding to different types of clays [33]. Clay particles provide a highly active medium on which
phage-host interactions may take place. Cations
around clays will bind negatively charged phages
and bacteria, as well as proteins and other nutrient molecules providing substrates for host
-]
--'1
~
4
o
2
._/
~0
-~
0
i
t
I
I
5
I
I
I
k
~
10
I
I
I
I
]
15
Time (days)
Fig. 1. Growth of indigenous streptomycetes and increase in
free phage titre in pre-dried nonsterile amended (2% w / w
chitin) soil wetted (to 15% moisture) at day 0. Growth of hosts
is shown by increases in total ( n ) and spore (in) counts.
Temperate (*) and virulent ( o ) phages were detected 24 h
after initial wetting of soil (Marsh and Wellington, 1993.
Unpublished data),
growth, although there can be competition between phages and proteins for binding sites
[34,35]. Phages will often bind strongly to clays
[33] and infectivity can be prolonged according to
phage and clay type [36]. Infective free phages
can persist in soil for long periods of time [1]. It
could therefore be argued that virulent phages
reside in the free state in soil for prolonged
periods of host inactivity, and resume multiplication as the host grows. Virulent actinophages can
be readily isolated from soil which has been previously air-dried for several months, and then
re-wetted with sterile distilled water (Fig. 1). The
exact origin of these phages is unclear as neither
virulent nor temperate actinophages were detected at time 0 of incubation. The initial burst in
indigenous phage numbers after 24 h incubation
could either represent gradual elution of phages
tightly bound to clay particles, or the release of
daughter progeny from germinating lysogens and
pseudolysogens, or both. Phages producing turbid
103
plaques on the indicator strain (temperate) were
repeatably more numerous than those producing
clear plaques. This, and the fact that free phages
are susceptible to unfavourable environmental
changes, such as a drop in pH [37], whilst in an
unprotected state outside the host, suggest that
the lysogenic life-cycle is the more effective mode
of survival in soil.
Pseudolysogeny certainly ensures the survival
of some virulent phages in natural environments,
but the occurrence of true lysogeny in temperate
phages appears to be a more stable evolutionary
progression of this strategy.
10
M.S.D. (P<0.05):
-E-I
[] z
0
"0
o
_J
i
The impact of phages on host populations
0
The most obvious impact phages can have on
host populations is that of reducing population
density by direct lysis. One-step growth experiments in flasks readily demonstrate the crash of a
host population by lysis [3]. This destruction of a
host population is rarely seen in natural environments, and probably only occurs in restricted
areas of host growth such as inside soil aggregates. Observations of the epidemiology of Drosophila C virus (DCV) showed that susceptible fly
populations were completely infected when one
infected fly was added to laboratory vials [38].
Similar epidemics have never been reported for
DCV in natural environments, and indeed no
viral epidemic has been known to wipe out its
host population on a global scale. This is probably a result of the combined effects of density
dependent regulation and high mutational rate of
virus and phage populations.
Peaks in marine bacterial numbers during a
spring bloom of diatoms in a Norwegian fjord
were closely followed by peaks in virus numbers
[18]. The peak in viral numbers was closely followed by a drop in bacterial numbers, and was
subsequently followed by a drop in viral numbers,
illustrating the predator-prey pattern of alternating, oscillating peaks of host and predator population numbers. It may be concluded that the
marine bacteria in this ecosystem were regulated
in part by the presence of viral populations, although other factors such as temperature and
h I
0
I
I
I
I
I
5
I
I
I
I
I
I
I
10
I
t
15
Time (days)
Fig. 2. Survival of S. lividans TK24 in sterile soil in the
presence of actinophage KC301 (2.0×105 p f u / g dry soil).
Vegetative host growth (total viable count) between days 1
and 2 was retarded when the efficiency of infection was
1.4x 10 -5 (11), whereas no effect was seen at an efficiency of
infection of 1.1 x 10 -6 (ID). Following sporulation, host populations reached a density of 5.0 x 107 cfu/g dry soil regardless
of efficiency of infection (Marsh and Wellington, 1993. Unpublished data).
nutrient status would also play a part in host
population regulation.
Little information is available concerning the
effects of in situ lysis by phages in soil. The
results of Pantastico-Caldas et al. [39] suggested
that the presence of the virulent Bacillus subtilis
phage, SP10C, in soil resulted in a decrease in
the host population level by one order of magnitude. The lytic action of the temperate actinophage KC301 appeared to have no numerical
impact on streptomycete populations in sterile
and nonsterile soil [40-42]. Where the infection
efficiency of KC301 was sufficiently high to retard
vegetative growth of Streptomyces lividans in sterile soil, sporulation compensated for the reduction in propagule numbers (Fig. 2).
Other impacts on host populations can be both
adverse and beneficial. Many effects relate to the
104
states of lysogeny and transduction where the
expression of phage-borne genes and maintenance of lysogeny burden the host cells.
tions, whereby released lysozyme would attack
bacteria sensitive to both the phage and lysozyme
in a particular niche and provide additional resources to the lysogens in the form of liberated
nutrients.
The importance of lysogeny in nature
Lysogenic infections can result in lysogenic
conversion of the host where the expression of
genes on the phage genome alter the host phenotype [43]. Superinfection immunity is the most
common form of lysogenic conversion, whereby
the expression of the phage-encoded repressor
protein(s) inhibits further infections by the same
phage type or different phages with specific binding sites for the repressor protein. Superinfection
immunity in addition to changes in phage specificity can also be caused by prophage-encoded
alterations to attachment sites on the cell surface,
a feature seen in some lysogenic strains of E. coli
and Salmonella species [7]. Lysogenic conversion
can also confer pathogenicity, for example the
prophage of lysogenic Corynebacterium diptheriae
strains encodes the toxins enabling establishment
of bacterial infection in hosts [7]. Engineered
phages containing antibiotic and heavy metal resistance determinants [40,41,44-46] may be advantageous to lysogenic hosts in selective environments [42].
Conversely, the need to synthesise extra foreign DNA and express prophage genes exerts a
burden on the host cell by utilising extra resources, and results in a metabolic burden on the
host [32]. Therefore the lysogenic relationship
between temperate phage and host can be seen
as a situation of compromise on both parts. The
host population is burdened by the presence of
extra DNA and induction, whilst the rate of phage
multiplication is restricted by the low level of
prophage induction and the particular rate of
host multiplication at a given time.
In natural environments, the rates of prophage
induction are usually very low [42,47] probably
resulting in a negligible impact on host populations. This and the large amounts of lysozyme
released at prophage induction [47] means that
reversion to the lytic cycle by prophages constitutes a potential 'weapon' for lysogenic popula-
The role of phages in genetic interactions
Phage-mediated transfer of genetic elements
(transduction) between bacterial cells [43] has
been well documented, particularly with regard to
the use of phages in molecular biology [2]. Transduction in natural environments may be a rare
event, as generalised transduction has only been
demonstrated once in freshwater ecosystems [48].
The potential for transduction in soil was assessed by adding coliphage transducing lysates to
soil containing recipients which were subsequently transduced [44]. The general inactivity of
soil microbial populations suggests that transduction in soil occurs at a low frequency, although
this frequency may be comparable to those of
conjugation and transformation in soil which also
require recipients to be present in an active state.
Lysogenic conversion as shown by lysogenic
infections of inoculated hosts in soil by temperate
phages containing genetic markers [40-42,45,46]
is likely to be a far more frequent occurrence in
soil in comparison to transduction. This is because all of the daughter progeny of a lysing cell
will contain the genetic information to be expressed by the prophages of subsequent lysogenic
infections. Hence genes on temperate phages
which can be expressed by lysogenic hosts may be
spread through soil by the movement of phage
particles through soil water via channels in and
between aggregates [49]. These genes will have a
higher likelihood of surviving than genes on
transducing particles.
The potential for an engineered actinophage
to establish itself in indigenous host populations
in soil was assessed by Marsh and Wellington
[41]. Lysates of KC301 (a derivative of ~bC31)
carrying the thiostrepton resistance gene tsr were
added to nonsterile soil. Thiostrepton resistant
streptomycete spores were extracted from this
soil and were found to contain KC301 DNA by
105
123456
5.7kb
.....
2.5kb
Fig. 3. Southern blot of DNA from putative streptomycete
lysogens isolated from soil inoculated with actinophage KC301.
The KC301 junction fragment of S. lividans TK24 (KC301)
and the indigenous lysogens can be seen at 5.7 kb (lane 3) and
2.5 kb (lanes 4, 5 and 6) resepectively. Lanes: 1, KC301; 2, S.
lividans TK24; 3, S. lividans TK24 (KC301); 4, 5 and 6,
indigenous lysogens (Marsh and Wellington, 1993. Unpublished data).
colony blot analysis with radiolabelled KC301
DNA. Most of the putative lysogens were stable,
and a sample of the stable lysogens proved to
contain the KC301 prophage integrated into the
indigenous host's chromosomes, as shown by
Southern analysis of ClaI digested host DNA
using nonradioactive DIG-labelled KC301 DNA
(Fig. 3). Thus the release of an engineered phage
such as KC301 into the environment where susceptible hosts were present could lead to the
phage's persistence in natural habitats, particularly when the host concerned survives well in the
soil.
Conclusions
Phages are abundant and widely distributed
components of natural habitats, with several ef-
fective survival strategies which ensure the persistence of phage DNA both inside and outside host
cells. However, the lysogenic lifestyle is a distinct
advantage in soil where long periods of host
inactivity can be survived by residence within host
populations. Phages may regulate host populations by lysis, but this regulation is probably extremely limited in discontinuously active ecosystems such as soil when host populations have
periods of inactivity. Differentially growing hosts
such as streptomycetes may escape the long-term
deleterious effects of lytic attack by phages by
only growing vegetatively for short periods of
time, and existing as resistant spores for long
periods of time. Lysogeny represents a compromise between hosts and phage where both parties
are granted advantages in terms of improved survival capabilities in return for reduced abundance
or multiplication potential. Phages are obviously
active participants in genetic interactions between bacteria in nature, and therefore must
contribute to evolutionary development of host
populations.
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