United States
                          Environmental Protection
                          Agency
      National Risk Management
      Research Laboratory
      Ada, OK 74820
                          Research and Development
      EPA/600/S-98/002
April 1998
 &EPA       ENVIRONMENTAL
                         RESEARCH   BRIEF
    The Importance of the Dynamics of Bacteriophage-Host Interactions to
      Bacterial Abundance and Genetic Diversity in Aquatic Environments

                                        mobert V. Miller, Ph.D.
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 DMA in freshwater microbial popula-
tions. These investigations have predicted that the most likely
reservoir for environmental bacteriophages capable of trans-
duction is the lysogenized members of the natural microbial
population. However, both primary infection of non-lysogens
and prophage activation from lysogens can generate signifi-
cant numbers of transducing particles. These studies have led
us to formulate a model for the dispersement of genetic mate-
rial 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-stimu-
lated 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  recipi-
ents for transduced DNA.

The potential for this model  to lead to genetic diversity in
natural bacterial communities has been demonstrated in con-
tinuous culture studies designed to determine the evolution of

''Miller, Robert V., Department of Microbiology and Molecular Genetics,
Oklahoma State University, Stillwater, OK 74078-0289.
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 disal-
lowed. 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 influ-
encing 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 dynam-
ics of phage-host interaction and to identify the  reservoirs  of
environmentally  observed bacteriophage particles.

Our preliminary studies investigating phage-host dynamics un-
der 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 im-
paired, 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-to-
bacterium ratio (PER) developed in the microbial consortium.
The PER is, in turn, proportional to the extent of starvation  of
the host organism. (Ill) The virulence of lytic viruses  is reduced
in starved hosts potentially allowing alternative  outcomes  of
infection.
                                                                               Printed on Recycled Paper

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Also, particulate matter, whether present as suspended mate-
rial 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 bacterioph-
age-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 bind-
ing 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 <|>X-174 infectivity of Escherichia coll threefold
at low concentrations of suspended particulates. Optimum  in-
fectivity occurred at kaolinite concentrations of 0.14-0.50 mg/ml
and at montmorillonite concentrations of 0.01-2 mg/ml.  In-
creased infectivity at higher clay concentrations was not seen,
suggesting  that the  cell may become enveloped by clay par-
ticles at these higher concentrations, thus essentially forming a
barrier against phage attack.

E. co//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 urn. This may be
due to the  ineffectiveness of large particles in forming a con-
tinuous barrier around the cell. Gaps thus allow for phage
contact and subsequent cell infection.

Alternatively, suspended particulates may act to alter phage-
host interactions by providing  a surface which facilitates con-
tact 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 interac-
tion 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 pro-
duce 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 fresh-
water 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 environ-
mental factors, such as particulate  matter, on these micro-
cosms significantly enhanced our understanding of host-phage
interactions and, hence, phage-mediated horizontal gene trans-
fer in natural aquatic ecosystems.  Since half-lives of phage
particles in these  environments are only 12-24 h, the mecha-
nisms  by which the  observed levels of phage  particles are
maintained in natural aquatic ecosystems  are currently unde-
fined. 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 obser-
vations.


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 pro-
totrophic  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 (j>F116. PA0303 is an argB2l non-lysogen.
4>F116 is a generalized transducing phage  and D3 is a spe-
cialized transducing phage. (|>UTI 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, Gm',  Su1, Sm1). This plasmid is Tra-
Mob-, which renders it incapable of being transferred by conju-
gation, thus ensuring that  only transduction can occur in our
system. The  genetic  recipient  was  RM296. It  is an  <|>FI16
lysogen of PA05-15  whose genotype  is  met-9011 nalAS
amiE200.  Bacteriophage c|>F116 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 Arbo-
retum, 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 immu-
nity 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 50°C  until three similar consecutive weights
were acquired.

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In Situ  incubated Microcosms
Microcosms were prepared using one-liter Lifecell tissue cul-
ture  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 wa-
ter were  determined to be approximately 0.005 mg/ml. Water
obtained at the 6 m-depth contained particulate  matter ap-
proaching 0.07 mg/ml.

Microcosm Sampling Procedure
Briefly, 60-ml samples were aseptically removed from the cham-
bers. 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 (0.45^) 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 ng/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 CPU concentrations by plating
dilutions prepared in saline on Luria agar, Luria agar containing
500  u.g/ml  carbenicillin, and Luria agar containing 500 ng/ml
nalidixic acid, respectively. A portion of this sample was  filtered
through a 0.45 n 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 tilers.
                               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). Elec-
                               trophoresis 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 l%  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 con-
                               tained at least one  phage-specific DNA sequence (Table 2).
Table 1.  Abundance of bacteria and P. aeruginosa-specffic bacteriophages in a freshwater lake during a nine-month period.
  Month
 Sampled
      Total
Recoverable CPU
 (X 10" CPU ml-1)
 Pseudomonas
  spp. Specific
(X 103CFUml-1)
P. aeruginosa-
    Phages
X lO'PFUml-1)
October
November
December
January
March
April
May
June
      20
      20
       9
      20
      10
      20
      40
      20
     10
      0.6
      0.5
      2
     10
     20
     10
      9
     10
     10
      1
      0.5
      0.003
      1
      1
      0.9

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Table 2. Relative abundance of sequences related to Pseudomonas phage DMAs in bacterial colonies recovered from a freshwater habitat.
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 incu-
bated 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 incu-
bated in the lake over a period of three weeks (Figure 4). A 10-
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 simula-
tions, the PER was also significantly higher in the bottom water
than in the  surface water microcosms.

The experiments described here indicate that suspended par-
ticles significantly influence cell-phage interactions in aquatic
milieus.  Enhanced infection probably occurs because aggre-
gates 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. Simi-
larly, In situ incubated locator microcosms exhibited a  higher
number of transductants in bottom water (Figure 4).
                        At high clay concentrations, phage-host interactions were re-
                        duced (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 interac-
                        tion. 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-inter-
                        active 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 environ-
                        mental 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
• Mackaloid
& Montmorillonite
D Barite
               0.01  0.02  0.04  0.06  0.08   0.1  0.15  0.2

                        Clay concentration (mg/ml)
                         x
                                                                        1.5
 Figure 3. Effect of clay concentration on <(>F116 plaguing efficiency.
                         Figure 4.  Comparison of transductant concentrations in situ incu-
                                  bated  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 seques-
tered in the host cytoplasm in a non-replicative form or achieved
in the host chromosome as described for <|>P22.

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. Men-
tion 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 partici-
pate 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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