ENVIRONMENTAL

RESEARCH BRIEF

The Importance of the Dynamics of Bacteriophage-Host Interactions to

Bacterial Abundance and Genetic Diversity in Aquatic Environments

Research and Development EPA/600/S-98/002 April 1998

United States National Health and Environmental

Environmental Protection Effects Research Laboratory

Agency Gulf Breeze, FL 32561-5299

Introduction

Using Pseudomonas aeruginosa and its bacteriophages as a

model system, we have clearly demonstrated a significant

potential for viral-mediated gene transfer (transduction) of both

plasmid and chromosomal DNA in freshwater microbial populations. These investigations have predicted that the most likely

reservoir for environmental bacteriophages capable of transduction is the lysogenized members of the natural microbial

population. However, both primary infection of non-lysogens

and prophage activation from lysogens can generate significant numbers of transducing particles. These studies have led

us to formulate a model for the dispersement of genetic material from an introduced organism to related members of the

autochthonous microbial community.

This model requires a unique sequence of events: (a) Phage

virions must be produced through spontaneous or stress-stimulated activation of prophages from environmental lysogens.

These viral particles must (b) infect, propagate, and (c) lyse the

introduced DNA donor. (d) Transducing particles produced

during this lytic infection must absorb and transfer DNA to the

remaining lysogens where it is established either as a plasmid

(e) or, if chromosomal in origin, is recombined into the host

chromosome. Hence, environmental lysogens serve as both

efficient sources of transducing phages and as viable recipients for transduced DNA.

The potential for this model to lead to genetic diversity in

natural bacterial communities has been demonstrated in continuous culture studies designed to determine the evolution of

mixed genetic populations as described in the model. We

found that at cell densities and generation time (hydraulic

residence times) similar to those predicted to occur in the

environment, transduction acted to stabilize and even increase

the frequency of genotypes which were lost through negative

selection in continuous cultures where gene transfer was disallowed. This is an exciting and significant result as transduction

has often been dismissed as an important evolutionary process

due to its perceived reductive nature.

These studies indicate that our transduction model has the

potential of occurring in natural habitats and significantly influencing the genetic makeup and diversity of natural bacterial

populations. To determine the true significance of the model in

natural ecosystems, it is imperative to understand the dynamics of phage-host interaction and to identify the reservoirs of

environmentally observed bacteriophage particles.

Our preliminary studies investigating phage-host dynamics under natural conditions indicated that a primary effector of the

outcome of environmental phage-host interaction is nutrient

availability: (I) While attachment to starved cells is not impaired, the replication of bacteriophages is significantly altered

in starved cells. In primary infection, the latency period is

lengthened and the burst size reduced when compared to fed

cells. (II) Continuous culture studies have demonstrated that

the rate of transduction is directly proportional to the phage-tobacterium ratio (PER) developed in the microbial consortium.

The PER is, in turn, proportional to the extent of starvation of

the host organism. (III) The virulence of lytic viruses is reduced

in starved hosts potentially allowing alternative outcomes of

infection.

†Robert V. Miller, Ph.D.

†

Miller, Robert V., Department of Microbiology and Molecular Genetics,

 Oklahoma State University, Stillwater, OK 74078-0289.
2

Also, particulate matter, whether present as suspended material or as sediments, is perhaps one of the most important of

the physical factors. Several observations have been made

which indicate that particulates significantly alter bacteriophage-host interactions and thus have the potential for affecting

transduction potentials in natural environments. It has been

demonstrated that bacteriophages adhere to clays and that

such adhesion protects phages against inactivation. The binding of clay to bacteria to block sites for phage attachment and

reduce interaction between the host and parasite has also

been hypothesized.

However, homoionic kaolinite and montmorillonite clays were

able to enhance φX-174 infectivity of Escherichia coli threefold

at low concentrations of suspended particulates. Optimum infectivity occurred at kaolinite concentrations of 0.14-0.50 mg/ml

and at montmorillonite concentrations of 0.01-2 mg/ml. Increased infectivity at higher clay concentrations was not seen,

suggesting that the cell may become enveloped by clay particles at these higher concentrations, thus essentially forming a

barrier against phage attack.

E. coli is protected from phage infection in the presence of fine

clay particulates, but this protection is decreased when the

particle size is increased to greater that 0.6 μm. This may be

due to the ineffectiveness of large particles in forming a continuous barrier around the cell. Gaps thus allow for phage

contact and subsequent cell infection.

Alternatively, suspended particulates may act to alter phagehost interactions by providing a surface which facilitates contact between the virus and its prey. The most effective source

of virus particles in the environment is lysogenized bacteria

present in the microbial community. Therefore, mixed

microcolony formation on suspended particle surfaces may act

to place the source of phages (i.e., lysogens) in close proximity

to the prospective hosts, thus forming centers of phage activity

within the aquatic environment. In this light, results which

indicate that the stimulation of clays on phage infection is

dependent on both the concentrations and average particle

size, can be interpreted as resulting from altered phage-host

interactions by altering the total surface area on which interaction can take place. As the concentration of clay increases or

the average diameter of the clay particle decreases, the total

surface area increases. In turn, increased surface area will

reduce the probability that hosts and viruses will be in close

enough proximity to interact.

Several studies have found that attached microorganisms are

more metabolically active than unattached cells. Since both the

length of the latency period and the size of the burst of progeny

virus particles have been shown to be dependent on the

metabolic rate of the host, it is likely that attached cells produce more phage than unattached microorganisms. This, too,

would increase phage-host activity in environments containing

suspended particulate surfaces as compared to purely liquid

phase habitats.

These observations encouraged us to attempt to determine the

effects of suspended particulates on transduction rates in freshwater lake microbial communities. We have previously used in

situ incubated lake water microcosms to study transduction in

freshwater habitats and we employed this model system for

these studies as well. Data concerning the effects of environmental factors, such as particulate matter, on these microcosms significantly enhanced our understanding of host-phage

interactions and, hence, phage-mediated horizontal gene transfer in natural aquatic ecosystems. Since half-lives of phage

particles in these environments are only 12-24 h, the mechanisms by which the observed levels of phage particles are

maintained in natural aquatic ecosystems are currently undefined. We initiated studies to investigate the natural reservoirs

of bacteriophages and the dynamics of phage-host interactions

in the aquatic environment. This report summarizes our observations.

Methods

Bacterial Strains and Bacteriophages

The bacterial strains used in this study are all derivatives of P.

aeruginosa (PAO) or P. aeruginosa (PAT). PAO1 is the prototrophic parental strain of all PAO derivatives. PAT2 is a

prototrophic PAT derivative which carries the P. aeruginosa

fertility factor FP2. RM242 is a non-lysogenic PAO strain with a

nal-933 met-1011 genotype, and RM132 is a prototrophic PAO

that is lysogenic for φF116. PA0303 is an argB2I non-lysogen.

φF116 is a generalized transducing phage and φD3 is a specialized transducing phage. φUTI is a generalized transducing

phage isolated from Fort Loudoun Lake near Knoxville, TN.

In the experiments on suspended particulates, the genetic

donor used for these studies was RM2140, a P. aeruginosa

derivative of the prototrophic strain PAO1 which contains the

plasmid Rms 149 (Cb1

, Gm1, Su1, Sm1). This plasmid is TraMob-, which renders it incapable of being transferred by conjugation, thus ensuring that only transduction can occur in our

system. The genetic recipient was RM296. It is an φFI16

lysogen of PA05-15 whose genotype is met-9011 nalAS

amiE200. Bacteriophage φF116 is a temperate, generalized

transducing phage of P. aeruginosa.

Environmental Sampling and Analysis of

Bacteriophage, Bacterial, and Lysogenic

Populations

 Samples were collected from Fisher Pond in the Morton Arboretum, Downers Grove, IL. P. aeruginosa strains PAO1 and

PAT2 were used as indicators for the enumeration of phage

titers.

Identification of Lysogens in Laboratory and

Microcosm Studies

Lysogeny was identified by demonstrating super-infection immunity and spontaneous release of bacteriophages. P. aeruginosa

PAO1 was used as the indicator strain for enumerating release

of bacteriophages from lysogens.

Clay

Mackaloid, montmorillonite, and barite clays were made

homoionic. Dry weights of each clay type were obtained by

oven drying at 50o

C until three similar consecutive weights

were acquired.
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In Situ incubated Microcosms

Microcosms were prepared using one-liter Lifecell tissue culture chambers (Fenwal Laboratories, Deerfield, IL). These

chambers are both gas and ultraviolet (UV) light permeable.

Chambers were filled with sterilized lake water collected from

either the surface or at a depth of 6 m from Lake Carl

Blackwell, a large freshwater reservoir in Stillwater, OK, and

inoculated with the donor and recipient strains, previously

washed with sterilized lakewater, at approximately 1 x 106

cells/mi. The chambers were then suspended in Lake Carl

Blackwell. Concentrations of particulate matter in surface water were determined to be approximately 0.005 mg/ml. Water

obtained at the 6 m-depth contained particulate matter approaching 0.07 mg/ml.

Microcosm Sampling Procedure

Briefly, 60-ml samples were aseptically removed from the chambers. Nalidixic acid was added to 50 ml of this sample to kill

genetic donor cells and inhibit further phage and transducing

particle formation. This sample was then filtered (0.45μ) and

extensively washed to remove all unabsorbed phages and

transducing particles. This procedure has previously been shown

to be effective in ensuring that transduction does not take

place in the sample during workup. These filters were placed

on Luria agar plates containing 500 μg/ml each of carbenicillin

and nalidixic acid to select for transductants.

The remaining 10 ml of the sample were used for determining

total viable, donor, and recipient CFU concentrations by plating

dilutions prepared in saline on Luria agar, Luria agar containing

500 μg/ml carbenicillin, and Luria agar containing 500 μg/ml

nalidixic acid, respectively. A portion of this sample was filtered

through a 0.45 μ syringe filter to remove bacteria, mixed with a

phage sensitive strain (PAO1), and plated in a lambda top agar

overlay on a Lucia agar plate to determine free phage titers.

Confirmation of Transductants by Genetic and

Molecular Methods

To confirm that Rms 149 was present in the transductants,

plasmid DNA was isolated by rapid alkaline lysis, and digested

with EcoRI according to the manufacturer’s directions

(Boehringer Mannheim Biochemicals, Indianapolis, IN). Electrophoresis was then performed on 0.7% agarose gels and

digestion patterns were compared to those of EcoRI digested

plasmid DNA from the parental strain, RM2140.

Results and Discussion

Abundance of Phages and Bacteria in a

Freshwater Lake

To begin our investigations, we monitored the occurrence of

bacteriophages, potential host bacteria, and lysogens at a

freshwater lake over a nine month period (Table 1). This study

indicated that not only are phage-like particles observed at

significant concentrations by transmission electron microscopy

in aquatic environments but that phages capable of producing

plaques on an environmentally prominent bacterial host are

present in significant titers. When lysogeny was evaluated,

between I% and 7% of the isolates tested positive by the

criterion of release of virions infectious on P. aeruginosa.

Colony hybridization studies using various DNA probes specific

for P. aeruginosa phages of environmental origin revealed that

a much larger fraction (70%) of Pseudomonas isolates contained at least one phage-specific DNA sequence (Table 2).

Total Pseudomonas P. aeruginosaMonth Recoverable CFU spp. Specific Phages

Sampled (X 104

 CFU ml-1) (X 103 CFU ml-1) X 103 PFU ml-1)

October 20 10 10

November 20 0.6 10

December 9 0.5 1

January 20 2 0.5

March 10 10 0.003

April 20 20 1

May 40 10 1

June 20 9 0.9

Table 1. Abundance of bacteria and P. aeruginosa-specific bacteriophages in a freshwater lake during a nine-month period.
4

These data indicate that the frequencies of Pseudomonas host

bacteria, phages, and lysogens are high in natural freshwater

ecosystems and reaffirms the viability of our model for environmental transduction.

Establishment of Lysogeny in Starved

P. Aeruginosa

 In order to evaluate the validity of our hypothesis that lysogeny or some other currently uncharacterized symbiotic relationship may provide a significant reservoir of phage genomes, we

initiated an investigation to compare the ability of fed and

starved cells to become lysogenized following primary infection. Two temperate phages were used for these studies.

Phage φD3 is a lambdoid-like phage of P. aeruginosa whose

prophage integrates into the P. aeruginosa genome during

lysogeny. Phage φF116 is a PI-like phage whose prophage is

carried as a plasmid in lysogenized hosts. By studying these

two phages, the effects of starvation on the two known mechanisms of lysogeny maintenance can be explored.

Preliminary results indicated that the effects of starvation on

lysogeny establishment appear to be phage specific. While

both phages plated with equal efficiency on fed and starved

cells (data not shown), the frequency of lysogeny establishment by φF116 was reduced in hosts that have been starved

for as little as 6 h. As the starvation period becomes longer, the

reduction in lysogeny establishment became more pronounced.

In contrast, establishment of φD3 lysogeny was stimulated in

acutely starved cells (6 h), while severely starved cells (48 h)

supported lysogeny establishment only at reduced levels. (Figure 1). It is possible that φD3, like the Escherichia coli bacteriophage 1, can respond molecularly to the energy state of the

acutely starved host and increase the frequency of infections

which lead to lysogenization. However, if this is the case, it

appears that φD3 cannot identify chronically starved hosts by

this molecular mechanism. Thus, the lysogenization coefficient

(the percent of survivors llysogenized at a multiplicity of infection of one) is first stimulated and then decreased for φD3,

while it is simply reduced for φF116.

Establishment of Lysogeny in a Riverwater

Microcosm

To assess the establishment and maintenance of lysogeny

under conditions found in an aquatic environment, microcosms

were established using water from the Des Plains River in

Maywood, IL. These microcosms were inoculated with equal

concentrations (104

-105 CFU/ml) of an F116 lysogen and a

non-lysogenic, sensitive strain of P. aeruginosa. In preliminary

studies, we found that no significant phage production or establishment of lysogen was observed until we added a low

level of nutrient (1.0-5% yeast extract). This, in light of the

other studies reported here, led us to ask if nutrients were

necessary for the establishment of lysogeny in an aquatic

population of bacteria. We therefore set up duplicate pairs of

microcosms and introduced nutrient into one on a weekly

basis.

The establishment of lysogeny in the population of nonlysogenic, phage-sensitive bacteria was found to be dependent

on the addition of nutrient (Figure 2). While no new lysogens

were observed in freshwater microcosms containing this mixed

population of P. aeruginosa under conditions of low nutrient

availability, periodic spiking of the microcosm with nutrient (1.0-

5% yeast extract) produced new lysogens within 12 h following

each spike. The lack of establishment of lysogeny was not due

to the inability of the bacteria to be infected as the frequency of

resistant cells among the non-lysogens remained undetectable

during the incubation period.

When we investigated the ability of the bacteria present in

these microcosms to produce phage particles, we found that

little phage production occurred except when the microcosms

were spiked with nutrient (data not shown). In fact, the phageto-bacterium ratio diminished during the periods between nutrient spikes due to severe reductions in PFU titers. These data

reflect the short half-life of virions (12-24 h) under these incubation conditions. The observations made in these preliminary

studies suggest that nutrient availability dramatically affects

temperate phage-host interactions. Both the establishment of

true lysogeny and the production of virions are reduced under

Percent Hybridizing

φUT1 φF116 φD3 Mixed Phage

Host Population Probe Probe Probe Population

Total CFU 2 3 1 1

Pseudomonas spp. CFU 45 13 5 7

Table 2. Relative abundance of sequences related to Pseudomonas phage DNAs in bacterial colonies recovered from a freshwater habitat.
5

starvation conditions, but increase following episodic addition

of nutrients into the cell’s milieu. However, it is not clear from

these studies whether acute infection coincident with nutrient

spiking leads to the increase in lysogeny or if infection during

the starvation period establishes an uncharacterized symbiotic

relationship, termed pseudolysogeny, which is transformed into

true lysogeny or lytic growth upon introduction of nutrient.

While we are currently conducting experiments designed to

distinguish between these two alternatives, some of the data

we have collected in an earlier study are germane to this

question. We studied the interaction of the newly isolated

virulent bacteriophage φUT1 and newly isolated P. aeruginosa

hosts indigenous to a freshwater environment over a 45-day

incubation in locator microcosms. Temporal changes in host

density, phage-to-bacterium ratio, and the appearance of apparent prophage carriers within the host bacterial population

were monitored. Decay rates of phage virions ranged from -

0.054 h-1 in natural water to -0.027 h-1 in filter-sterilized lake

water. While bacterial densities stabilized at significantly lower

densities when φUT1 was present (104 CFU/ml) than when it

was absent (107

CFU/ml) about 45% of the sensitive bacteria

incubated with φUTI were found to hybridize to probes of

φUT1-specific DNA sequences within 72 h of incubation in

sterile lake water and within 12 h in microcosms containing

natural lake water. Plaques formed by this phage on hosts

grown under conditions simulating the natural aquatic habitat

were turbid, but only clear plaques were observed when the

phage was plated on hosts grown in rich laboratory medium.

Effect of Clay Concentration on Plaquing

Efficiency

We wished to determine if suspensions of clay particles would

stimulate P. aeruginosa-φF116 interactions as had been found

for φX-174 and E. coli. Three types of clay (mackaloid, montmorillonite, and barite) were prepared as described in the

methods section and added at concentrations between 0.01-

0.2 mg/ml to a mixture of φFI16 at 10 phage/ml and PA0515

grown to mid-exponential phase. Three milliliters lambda top

agar was then added and the mixture poured on a Lucia agar

plate. Resulting plaque forming units were scored (Figure 3).

At low clay concentrations, plaquing efficiency was significantly

enhanced. As clay concentrations increased, the rate of phage

infection steadily decreased. These observations are consistent with the effects of clay on the interaction between φX-174

and its host. E.coli was concentration dependent.

Laboratory Simulations

Before actually performing experiments in situ, trials were first

attempted in the laboratory under more highly controlled conditions. The donor and recipient strains were inoculated at approximately 1 x 106

 cells/ml into 500 ml of Pseudomonas

Minimal Media containing 4 x 10-5 % yeast extract Mackaloid

clay was added to one flask at a final concentration of 0.01 mg/

ml. No clay was added to the second flask. These flasks were

incubated at room temperature with gentle shaking (75 rpm)

and the frequency of transduction in the two cultures compared. Genetic donor and recipient concentrations were unaffected by the presence of clay and remained essentially the

same in the two chambers. However, there was a significant

increase (100 fold after 24 h of incubation) in the number of

transductants when clay was present in the culture. As might

be predicted from the previous experiments, there was also an

increase in the concentration of phage present per bacterial

cell (the PER ratio) when clay was present. These simulations

demonstrated that increases in phage production due to the

presence of suspended clay particles can lead to an increased

frequency of transductants in an aquatic microbial population.

6

5

4

3

2

1

0

Lysogenization coefficient

0 6 24 48

Hour of starvation of host

D3

F116

Days of incubation in microcosm

120

100

80

60

40

20

0

0 10 20 30 40

Percent of population lysogenized

Figure 1. Effects of starvation on the Lysogenization Coefficient for

bacteriophages φD3 and φF116.

Figure 2. Establishment of lysogeny in a freshwater microcosm.
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In Situ Incubated Lakewater Microcosms

We next asked if water collected from near the bottom of a

freshwater lake which was enriched for suspended particulates

would support higher levels of transduction than surface water

that was relatively depleted of suspended particulates. These

experiments were performed in microcosm chambers incubated in situ at our freshwater-lake field site. The chambers

were filled with sterilized lakewater obtained either from the

extreme surface of the lake or from a depth of 6 m using a

LaMotte water sampler (LaMotte Chemical, Chestertown, MD).

These chambers were inoculated with the genetic donor and

recipient strains at approximately 1 x 106

 cells/ml and incubated in the lake over a period of three weeks (Figure 4). A l0-

fold increase in transduction frequency was observed in the

bottom water microcosms as compared to the surface water

microcosms. As had been observed in the laboratory simulations, the PER was also significantly higher in the bottom water

than in the surface water microcosms.

The experiments described here indicate that suspended particles significantly influence cell-phage interactions in aquatic

milieus. Enhanced infection probably occurs because aggregates consisting of cells, phage, and particulates form, thereby

allowing for progeny phage released from one infected cell to

readily interact with a neighboring host cell. Consequently,

greater numbers of transductants are seen when particulates

are present in aqueous environments. In the case of the

laboratory simulations, an almost 100-fold increase was seen

in the numbers of transductants when clay was added. Similarly, in situ incubated locator microcosms exhibited a higher

number of transductants in bottom water (Figure 4).

At high clay concentrations, phage-host interactions were reduced (Figure 3), this may be due to the production of a

continuous clay envelope surrounding the cell and acting as a

barrier to phage infection or simply to a dilution effect caused

by the increased particle surface area to which bacteria and

bacteriophage may become attached. Factors other than simple

interaction with the particle may influence phage-host interaction. These may include the increased metabolism and, hence,

phage production, of attached organisms due to increased

availability of nutrients. In addition, association with particles

may protect phages by inactivating environmental stresses

such as UV radiation or changes in pH.

In our previous work, we have shown that transduction is a

viable means of gene exchange in freshwater ecosystems. It is

evident from the study reported here that physical factors such

as particulate matter that can vary significantly among aquatic

habitats, can significantly alter transduction potentials in these

ecosystems.

Conclusion

The observations reported here suggest that the evaluation of

phage-host relationships will provide important and sometimes

unsuspected insights into microbial population ecology. They

suggest that pseudolysogeny may be an important host-interactive state for bacteriophages in aquatic environments. Data

from this and other studies suggest that both temperate and

virulent DNA phages and RNA phages establish

pseudolysogenic relationships with their hosts under environmental conditions. If pseudolysogeny does indeed occur in

aquatic environments, it would allow phage genomes to coexist

150

140

130

120

110

100

90

80

70

0 0.01 0.02 0.04 0.06 0.08 0.1 0.15 0.2

Clay concentration (mg/ml)

PFU as a percentage of control

Mackaloid

Montmorillonite

Barite

Figure 3. Effect of clay concentration on φF116 plaquing efficiency.

14

12

10

8

6

4

2

0

Surface

Bottom

1.5 2 3 6 13 20

Days

X 10 Transductants/ml -8

Figure 4. Comparison of transductant concentrations in situ incubated lakewater microcosms.
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with their starved host bacteria in a relatively protected state

during prolonged periods of starvation. When nutrients become

available, pseudolysogens may be transformed into true

lysogens, or the lytic response may be induced. Although the

physical and molecular manifestations of the pseudolysogenic

phage genome are not currently understood, it may be sequestered in the host cytoplasm in a non-replicative form or achieved

in the host chromosome as described for φP22.

Disclaimer

The U.S. Environmental Protection Agency through its Office of

Research and Development partially funded and collaborated

in the research described here under Cooperative Agreement

No. CR819630 to North Carolina State University. It has been

subjected to the Agency’s peer and administrative review and

has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute

endorsement or recommendation for use.

Quality Assurance Statement

All research projects making conclusions or recommendations

based on environmentally related measurements and funded

by the Environmental Protection Agency are required to participate in the Agency Quality Assurance Program. This project

was conducted under an approved Quality Assurance Program

Plan. The procedures specified in the plan were used without

exception. Information on the plan and documentation of the

quality assurance activities and results are available from the

Principal Investigator.

Research Products Cited

Miller, R. V. 1996. Genetic Stability of Genetically

Engineered Microorganisms in the Aquatic Environment.

In: T. E. Ford (ed.), Aquatic Microbiology - An Ecological

Approach —1997. Blackwell Scientific Publications,

Boston.

Kidamba, S. P., M. G. Booth, T. A. Kokjohn, and R. V.

Miller. 1996. RecA-dependence of the Response of

Pseudomonas aeroginosa to UVA and UVB Irradiation.

Microbiology 142:1033-1040.

Ripp, S., and R. V. Miller. 1995. Effects of Suspended

Particulates on the Frequency of Transduction among

Pseudomonas aeruginosa in a Freshwater Environment.

Appl. Environ. Microbiol. 61:1214-1219.

Replicon, J., Frankfater, A., and R. V. Miller. 1995. A

Continuous Culture Model to Examine Factors that Affect

Transduction among Pseudomonas aeruginosa Strains

in Freshwater Environments. Appl. Environ. Microbiol.

61:3359-3366.

Kidambi, S. P., S. Ripp, and R. V. Miller. 1994. Evidence

for PhageMediated Gene Transfer among Pseudomonas

aeruginosa Strains on the Phylloplane. Appl. Environ.

Microbiol. 60:496-500.

Kidambi, S. P., S. Ripp, and R. V. Miller. 1994. Evidence

of PhageMediated Gene Transfer among Pseudomonas

aeruginosa Strains on the Phylloplane. Appl. Environ.

Microbiol. 60:496-500.
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