7 Lysogeny and Transduction

John H Paul' and Sunny C Jiang2

~Department of Marine Science, University of South Florida, St. Petersburg, FL 33 70 I, USA
2School of Social Ecology, University of California, Irvine, Irvine, CA 92697, USA

CONTENTS

Introduction and background

c-
Screening marine bacteria for lysogeny O
Transduction assay
Future directions "O
e"

#
"O
INTRODUCTION AND BACKGROUND e-

C
Lysogeny and transduction describe a type of phage/host interaction and
tu~
a method of bacterial gene transfer (procaryotic sex), respectively. O

Although they are often reviewed together, these topics are linked only in -1
that one type of transduction (specialized) has an obligate requirement for
a lysogenic interaction. In this chapter we describe the background for
understanding both of these processes, and give methods that we have
found useful in studying lysogeny and transduction in the marine
environment.

Lysogeny and pseudolysogeny

Lysogeny occurs when a phage enters into a stable symbiosis with its host
(Ackermann and DuBow, 1987). The host (bacterium or algal cell) and
phage capable of entering into such a relationship are termed a lysogen
and temperate phage, respectively. The temperate phage genome
becomes integrated into one of the replicons of the cell (chromosome,
plasmid, or another temperate phage genome) and is termed a prophage
(Figure 7.1). The lysogenic state is a highly evolved state (Levin and
Lenski, 1983) requiring coordinated expression (and repression) of both
host and viral genes. There is a selective pressure to favor lysogeny, partic-
ularly at times of low host density, because a temperate phage is less likely
to drive its host to extinction (Levin and Lenski, 1983). Other advantages
of lysogeny include the expression of prophage encoded genes, termed
conversion. This is in contrast to transduction (see below), whereby the
genes imparted into an infected host were the result of a phage packaging
error during a prior infection cycle. That is, in transduction the genes
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 '"-'-tion to adhesion deficient

....

Lytic Pseudolysogenic Lysogenic

Figure 7.1. Cartoon depicting lytic, lysogenic, and pseudolysogenic phage/bac-

terial interactions. The oval ring in the bacterial cell is the bacterial chrocnosome.
After phage adsorption, the phage DNA is injected into the bacterium, and is
represented as a coiled molecule in the figure. This can become integrated into the
host chromosome as a prophage in lysogeny (indicated as a straight bar in the
bacterial chromosome in the right side of the figure). By the process of induction,
the prophage is excised, and can go into lytic phage replication (left side of the
figure). In pseudolysogeny, the host cell can mutate to an adhesion-impaired or
deficient state (depicted by a wavy cell surface), whereby collisions result in a low
success rate of infection. Another type of pseudolysogeny (termed carrier state)
can occur when the prophage does not integrate but is maintained as a plasmid
(central panel in the figure). Both types of pseudolysogeny result in a high abun-
dance of both phages and host cells simultaneously.

originated in a bacterial host and are not a normal part of the p h a g e

genome. Traits conferred to the host by conversion include i m m u n i t y to
superinfection, which m e a n s that the host becomes i m m u n e not only to
infection by that particular t e m p e r a t e phage, but to other closely related
phages. Other traits include antibiotic resistance, restriction modification
systems, and toxin a n d / o r bacterial virulence (as for diptheria toxin and
botulinus toxins C and D; A c k e r m a n n and DuBow, 1987).
In a lysogenic symbiosis, there is a low rate of s p o n t a n e o u s reversion to
virulence, in that, on average, 1 in 10 ~ produces a lyric event ( A c k e r m a n n
and DuBow, 1987). Thus, there is a constant production of t e m p e r a t e
p h a g e in a lysogenic interaction. However, other interactions exist where
there is a high-level p r o d u c t i o n of both host cells and viral particles in a
process termed ' p s e u d o l y s o g e n y ' (Figure 7.1). P s e u d o l y s o g e n y has been
used s y n o n y m o u s l y with 'carrier state' and 'chronic infection', yet distinc-
tions can be m a d e between these conditions. By A c k e r m a n n and D u B o w ' s
(1987) definition, p s e u d o l y s o g e n y is a p h e n o m e n o n caused by a mixture
of sensitive and resistant host cells, or a mixture of t e m p e r a t e and virulent

106
phage, that results in a constant supply of host and viral particles. This in
many ways resembles the marine environment, where sensitive and resis-
tant cells coexist with lyric and temperate phages of many differing
strains and species. We have indicated two such interactions that may
occur in pseudolysogeny in Figure 7.1. The first is a mutation to an adhe-
sion-impaired or deficient state, thereby limiting the number of successful
infections. Also shown is what has been termed the carrier state; a
pseudolysogenic-like relationship occurs characterized by plasrnid-like
prophages, which do not integrate into the host genome (Figure 7.1).
Chronic infection is the process whereby certain bacteria produce phage
without host lysis, by budding or extrusion, as in Pl or M13 (Dehardt et
al., 1978).
In true lysogeny, when a temperate phage infects a host, a 'lysogenic
decision' is made, as to whether a lytic or lysogenic interaction will ensue.
Factors affecting the lysogenic decision are the multiplicity of infection
(MOI; a high MOI favors lysogeny), host growth rate, and nutrient status
(Levin and Lenski, 1983). In fact, Wilson and co-workers (Scanlan and
Wilson, 1999; Wilson et al., 1998) have hypothesized that phosphate
concentrations influenced the lysogenic decision in cyanophage infecting
Synechococcus. When phosphate-limited microcosms containing a bloom
of Synechococcus were enriched with Pi (inorganic phosphate), there was a
dramatic increase in phage production concomitant with a crash of the
Synechococcus population (Wilson et al., 1998). Lysogeny in Synechococcus
populations would be consistent with the observation of high cyanophage
abundance yet resistance to infection (Waterbury and Valois, 1993).
Lysogeny is extremely common amongst bacteria, at least in cultivated
strains. Ackerman and DuBow (1987) indicated that among 1200 diverse
strains of bacteria, an average of 47~7, contained inducible prophage. Jiang
and Paul indicated that among 110 marine bacterial isolates, 40(7,, were
lysogenized (Jiang and Paul, 1998a).
The importance of lysogeny among natural populations of bacteria is a
topic of debate. Wilcox and Fuhrman (1994) concluded that lytic infection
was far more important than lysogeny in bacterial mortality or phage
production based upon studies with natural populations exposed to
sunlight. Weinbauer and Suttle (1996, 1999) also concluded that a small
proportion (1.5-11.4%) of the bacteria in marine samples from the Gulf of
Mexico were lysogenized, with the highest values occurring for offshore
populations. Tapper and Hicks (1997) estimated from 0.1 to 7.4% of the
bacteria in Lake Superior to be lysogens, in agreement with other studies.
Our lab has studied the distribution of lysogeny in various environments,
and found eight of ten eutrophic estuarine environments to contain
inducible prophage, whereas only three of eleven offshore environments
were positive for prophage induction. We have shown that a series of
environmentally relevant pollutants (polynuclear aromatic hydrocarbons,
polychlorinated biphenyls, and pesticides) can all cause induction of
natural populations of lysogens (Jiang and Paul, 1996; Cochran et al.,
1998), and that there was a seasonality in the detection of lysogeny, with
lysogens prevalent in the summer months, but absent in winter
(November to February; Cochran and Paul, 1998). Our estimates of the

107
 percentage of bacteria lysogenized are in agreement with others, ranging
from undetectable to 37%, averaging 6.9%, based upon an assumed burst
size of 30. If 8% of the population was lysogenized, and half of these were
induced by some environmental factor, this would produce 5 x 10'~
phage ml ', or nearly half of the phage present in Tampa Bay. If nutrients
and temperature can control prophage induction a n d / o r the lysogenic
decision, it seems reasonable that induced temperate phage may consti-
tute a significant amount, or perhaps the majority, of phage present in
many coastal environments.
The detection of lysogeny in cultures or natural populations is usually
through prophage induction by use of a mutagenic agent, usually mito-
mycin C. The methods described below are all based on some derivative
of this procedure.

Gene transfer by transduction

Bacteriophage-mediated transduction is one of three well-known mecha-
nisms, along with conjugation and transformation, of horizontal gene
transfer among prokaryotic organisms. In transduction, bacterial DNA or
plasmid DNA is encapsulated into phage particles during lytic replication
of the phage in the donor cell and is transferred to the recipient cell by
infection. This donor DNA either undergoes recombination with the host
chromosome to produce a stable transductant or remains extrachromo-
somal as a plasmid.
Based on the mechanism of production of transducing viral particles
and the means by which DNA is incorporated into the recipient cell chro-
mosome, transduction can be classified as one of two types, specialized or
generalized. In specialized transduction, only a restricted number of
genes within tile host may be transferred; namely, those which flank the
site of integration of the prophage. Specialized transducing particles are
produced only from the integrated prophage during induction (Buck and
Groman, 1981; Cavenagh and Miller, 1985). The DNA present in trans-
ducing phage particles is produced by aberrant excision of prophage
DNA (Sternberg and Maurer, 1991). When this DNA is injected into the
recipient host during infection, it is established and maintained as a
prophage independent of the host's general homologous recombination
system. In contrast, all regions of the chromosome or other genetic
elements present in the donor cell can be transferred with approximately
the same frequency in generalized transduction (Zinder and Lederberg,
1952). Following injection of this DNA into the recipient cell, stable trans-
ductants are generated by the replacement of the homologous cell DNA
with the transducing DNA. Therefore, generalized transduction is depen-
dent on the general recombination system of tile host (Sternberg and
Maurer, 1991). Virulent as well as temperate bacteriophages are capable of
generalized transduction under appropriate conditions.
Genomics studies have indicated that considerable horizontal gene
transfer has occurred between prokaryotes (Jain et al., 1999). The transfer
of genetic information between distantly or even unrelated organisms

108
during evolution has been inferred from nucleotide sequence compar-
isons (Dr6ge et al., 1998). Among the bacterial gene exchange mecha-
nisms, transformation and conjugation were identified as mechanisms
with potentially the broadest host range of transfer. However, sufficient
evidence has accumulated to indicate that transduction is a significant
mechanism of gene transfer, being more important in natural ecosystems
than originally thought (Novick et al., 1986; Kokjohn, 1989; Saye and
Miller, 1989; Stozky, 1989; Saye et al., 1990; Miller et al., 1992; Schichlmaier
and Schmierger, 1995). Because the packaging of nucleic acids in a phage
particle may represent an evolutionary survival strategy for the genetic
material, bacteriophages may serve as reservoirs for exogenous genes
(Zeph et al., 1988).
Transduction has now been shown to be an important mechanism of
gene transfer within several natural ecosystems, including soils (Germida
and Khachatourians, 1988; Zeph ct al., 1988; Stotzky, 1989; Zeph and
Stotzky, 1989), plant surfaces (Kidambi et al., 1993), freshwater environ- c-

ments (Morrison et al., 1978; Saye et al., 1987; 1990; Amin and Day, 1988; o

Miller, 1992; Ripp et al., 1994; Ripp and Miller, 1995) and animals
(Jarolmen et al., 1965; Novick and Morse, 1967; Baross et al., 1978; Novick
e-
et al., 1986). Both chromosome and plasmid transduction in Pseudomonas
aeruginosa were demonstrated during in situ incubation in a freshwater #
lake (Morrison et al., 1978; Saye et al., 1987; 1990) and on submerged river e-

stones (Amin and Day, 1988), with transduction frequencies ranging from ¢,.

1.4 x 10 ~to 8.3 × 10 ~/recipient. Ripp and Miller (1995) also suggested that
the presence of suspended particulates in the water column facilitates 0
transduction by bringing the host and phage into close contact with each
other.
Compared with freshwater environments, less is known about trans-
duction in marine waters even though a transducing marine bacterio-
phage was isolated more than 15 years ago (Keynan et al., 1974). Over the
past ten years, bacteriophages were found to be the most numerous
microorganisms in the ocean. In addition, bacteriophages may have a
broader host range than previously expected. Jensen et al. (1998) have
demonstrated the prevalence of broad-host-range lyric bacteriophages
(90%) in both a freshwater pond and sewage waters. They also suggest
that standard bacteriophage enrichment using a single bacterial host is
unavoidably biased against the development of viruses with a broad host
range, and this bias may partially explain the general view that bacterio-
phages are restricted in their interactive host range. Wichels et al. (1999)
found that 8% of 62 marine bacteriophage isolates examined were capable
of infecting a variety of hosts. The host ranges consist of 11 to 36 unique
bacterial isolates. The prevalence of broad-host-range lytic bacteriophages
has profound ecological significance, especially with regard to natural
mechanisms for gene transfer.
Jiang and Paul (1998b) described a plasmid transduction system using
a temperate marine virus and host isolate (Figure 7.2). Transfer of an
antibiotic resistant plasmid by this phage was detected at a frequency of
10 ~-10" per pfu (plaque forming unit). Interestingly, all transductants
were also lysogenized with the temperate phage genome. To investigate

109
 H©PE -1 (~HSIC

Kan + Strep Plasmid

Hybridization

Treatment
Lysate

Control
+@ Kan + Strep

Figure 7.2. Cartoon depicting a plasmid transduction experiment using a plasmid

containing donor (indicated as the HOPE-1 bacterium; Jiang and Paul, 1998b) and
the phage 0HSIC. A lysate is made from the plasmid-containing host and used to
infect the wild-type host. Survivors are plated on media containing antibiotics, the
resistances for which were encoded in the transducing plasmid. The appearance of
antibiotic-resistant bacteria that hybridize to a probe for the transducing DNA
indicates that transduction has occurred. A no-Iysate control is included, which
yields no colonies, and does not hybridize to the gent probe specific for the
plasmid.

transduction to the indigenous marine bacterial community, Jiang and

Paul (1998b) used the concentrated marine bacterial c o m m u n i t y from
various environments as recipients (Figure 7.3). Transduction was found
in two sampling sites at a frequency of 10 ~ per pfu. The transductants
were confirmed by PCR amplification of plasmid-specific sequences.
Chiura (1997) reported the first intergeneric phage-mediated g e n t
transfer between marine bacteria and enteric bacteria. He demonstrated
that five marine bacteria isolated from seawater were capable of sponta-
neous induction of temperate phages after a prolonged incubation.
Although these phages did not form plaques on an E. coli bacterial lawn,
they were capable of generalized transduction of genes to repair amino
acid deficiencies in E. coll. The five marine isolates were not phylogeneti-
cally closely related and none of them were closely related to E. coll.
Auxotrophic markers on the E. coli c h r o m o s o m e exhibited gene transfer
frequencies ranging between 10 ~and 10 ~ per viral particle. Therefore, the
transducing frequencies of these viral particles spontaneously induced
from marine bacteria, were four to seven orders of m a g n i t u d e higher than
those of transducing phages isolated from freshwaters (Saye et el., 1990;
Ripp et el., 1994). Intergeneric transduction was also demonstrated in
another startling report in which viral-like particles (VLP) produced by a
hot-spring natural bacterial c o m m u n i t y (predominated by hyperthermo-
philic chemolithotrophic sulfur bacteria) were capable of transducing loci

II0
 to repair auxotrophic E. coli and Bacillus subtilis to prototrophy with an
average efficiency of 10 ~'per VLP (Chiura et al., 1998). These results indi-
cate that spontaneous viral production by marine bacteria may be an
important mechanism of generalized horizontal gene transfer involving a
broad range of bacterial hosts in the marine environment.

4,e4,ee4, S C R E E N I N G M A R I N E B A C T E R I A F O R
LYSOGENY
Isolates in c u l t u r e
The protocol that follows has been used to screen marine bacterial isolates
for inducible prophage a n d / o r bacteriocin-like particles (Jiang and Paul,
1994; 1998a). The protocol was developed for rapidly growing cultures in
flasks but has been readily adapted to microtiter plates. We have used
it only with our formulation of marine bacterial growth medium
(ASWJP+PY; Paul and Myers, 1982) but any heterotrophic bacterial
medium should work equally as well. Bacteria are grown into exponential
phase in batch culture and then exposed to mitomycin C (or another
mutagen such as UV light). The growth of the culture is followed by
optical density (absorbance) and prophage induction is detected by a
decrease or stasis in absorbance compared to a control (unamended)
culture. Viral counts are made (either by TEM or epifluorescence
microscopy) for both treated and control cultures. A significant increase in
viral particles over the control is indicative of lysogeny.

Materials and supplies

• Marine bacterial isolate(s) as frozen glycerol stock or from agar plate
(<1 month old)
• ASWJP+PY medium (Paul and Myers, 1982), Zobell's 2216 Marine Media
(Difco), or other marine bacterial medium
• 0.02qam-flltered DI water
• 0.02-1urn-filtered formalin
• All materials for SYBR Green or SYBR Gold viral direct counts (see
Chapter 3 by Noble)

Assay
1. Grow the selected culture of marine bacteria overnight in rich
medium. This can be accomplished by using 0.5 ml of a frozen
stock in 5 ml media in a sterile 15 cc centrifuge with shaking (200
rpm).
2. In the morning, add 0.5-2.0 ml of the culture to 50 ml sterile marine
bacterial media, incubating at the correct temperature, again with
shaking.

III
 3. Take 1 ml samples every hour and monitor absorbance at 600 nm.
4. When absorbance reaches between 0.4 and 0.6, immediately take a
1.0 ml sample and centrifuge in a microcentrifuge for 5 min at
14 000 rpm. Remove 0.5 ml of the supernatant, add it to 4.5 ml of
the 0.02-Mm-filtered DI water and 125 ~1 of the 0.02-~tm-filtered
formalin (final concentration -1%). This can be stained directly for
SYBR Green or Gold viral counts (500 B1 aliquots or diluted an
additional 1:10 with 0.02-~tm-filtered DI for counting).
5. Split the culture in two equal aliquots (20 ml each). Add
Mitomycin C to one (final concentration 0.5 Mg ml ~). Continue
incubating with shaking and take an absorbance reading every
hour. Sample again at 3 h, 8 h (optional) and overnight (-16 h) for
viral counts as in step 4.
6. Compare viral counts in the Mitomycin C treatment to the t = 0
and control at equal time points.

Screening for lysogens in natural populations

Questions that arise often in the study of lysogeny in the marine environ-
ment include 'How common an event is lysogeny? What proportion of the
bacterial population contain inducible prophage (are lysogens)? What
proportion of the viral population is temperate, or is the result of a
prophage induction event?' These questions cannot be unequivocally
answered with current technology. Using the methods below, it is possible
to determine how common lysogeny is when comparing different marine
or estuarine environments or different times of the year at the same envi-
ronment. Making a few assumptions, it is possible to estimate the propor-
tion of the bacteria which contain inducible prophage (and therefore are
by definition lysogenic). With speculation, one might be able to arrive at
an estimate of the proportion of the phage population which is the result
of prophage induction events. The techniques described below have been
successfully used with natural populations to detect the occurrence of
lysogeny (Jiang and Paul, 1994; Cochran and Paul, 1998), or to determine
if certain environmental pollutants can cause prophage induction by
natural populations (Jiang and Paul, 1996; Cochran et al., 1998). The
inducing agent has been Mitomycin C unless other pollutants were inves-
tigated. We have examined the capability of natural populations to be
induced by mutagens either with samples concentrated by Membrex
ultrafiltration (Jiang and Paul, 1996) or with unconcentrated natural
populations. The former method has been employed in oligotrophic,
offshore environments where bacterial populations were well less than
10" ml '. However, use of Membrex concentration to determine tile
number of lysogens present is probably not quantitative because of the
potential for bacterial growth during the concentration process. Both
methods have been used in conjunction with TEM for viral enumeration,
although we have almost exclusively changed to SYBR Gold enumeration
using epifluorescence microscopy.

112
Equipment and supplies
• Membrex Rotary Biofiltration Device, equipped with 100 KD filter (note
that this is necessary only for offshore samples and use withTEM enumer-
ation
• Mitomycin C (Sigma) or other mutagens to be tested
• Sterile 15 or 60 ml conical centrifuge tubes
• TEM-grade glutaraldehyde (forTEM enumeration only)
• 0.02-pm-filtered formalin (for epifluorescence viral enumeration only)

Assay
1. For Membrex concentration of the ambient microbial populations,
10-100 1 of sample are concentrated using the rotary biofiltration
device as described in the manufacturer's instructions. The
e,.
concentrate (termed retentate) usually has a v o l u m e of 35-60 ml. O
2. For concentrated samples, place 1 ml in a sterile 1.5 ml microcen- u
trifuge tube or 5.0 ml of the retentate in a 15 ml conical centrifuge "o

tube. Do this both for control (unamended) and treatment e-

(mutagen a m e n d e d ) samples. #
3. For unconcentrated samples, add 25 ml each to a control or treat- "0
ment, 50 ml sterile, conical centrifuge tubes. If several mutagens
>.,
are to be investigated, increase the n u m b e r of treatment tubes C
accordingly. ~0
0
4. Take an additional sample (1-5 ml for Membrex Retentate, 25 ml
for unconcentrated) and fix with glutaraldehyde (2% final concen-
tration) as a T = 0 control. If epifluorescence microscopy is to be
used for enumeration, fix sample with 1% 0.02-~tm-filtered
formalin.
5. For the treatment samples, add 0.5-1 Hg ml ' Mitomycin C. If other
mutagens are to be used, it is a good idea to include a Mitomycin
C treatment as a positive control. Mutagens can be added at any
concentration desired, but this can be limited by the solubility of
the m u t a g e n (e.g. Polynuclear aromatic hydrocarbons; Jiang and
Paul, 1996).
6. The samples are incubated for 16-24 h at room temperature and
either fixed with 2% glutaraldehyde (for TEM) or 1°/, formalin
(epifluorescence microscopy).
7. Samples for enumeration by epifluorescence microscopy should
be counted within 24 h of collection. For TEM samples, if the
sample has not been concentrated by Membrex ultrafiltration, use
ultracentrifugation (160000g) to impinge viral particles onto
Formvar-coated TEM grids (Borsheim et al., 1990). If the samples
have been concentrated by ultrafiltration, it may be necessary to
dilute the sample 1:10 with DI water before spotting 1 H1 onto a
Formvar grid. Count both bacteria and viruses in control and
treated samples. For induction to have occurred, viral counts in the
treatment must exceed those in the control.

113
 8. Calculate the percentage lysogenic bacteria as follows:

% lysogens = [(VDC, - VDC¢)/B~]/BDC, ,~

where VDC: is the viral direct counts (in viruses ml ') in the treat-
ment, VDCc is the viral direct counts in the control, B~ is the
average burst size, and BDC, ,, is the bacterial counts at the set up
of the experiment (T = 0). The average burst size can be derived by
TEM observation of bacterial bursts. We have found an average for
our samples from the Gulf of Mexico of 30, whereas taking an
average of the literature from a recent review (Wommack and
Colwell, 2000) indicates a value of 53.5 _+48.

P r o p h a g e i n d u c t i o n in n a t u r a l p o p u l a t i o n s m v i r a l r e d u c e d m e t h o d
The m e t h o d described above for detection of lysogeny in natural popula-
tions has the least a m o u n t of manipulation of the sample. However, the
ambient levels of viruses will confound detection of small increases in
viral counts because of prophage induction. To obviate this problem,
Weinbauer and Suttle (1996) used a technique to reduce the level of
ambient viruses by filtration of the ambient c o m m u n i t y through a 0.2 btm
filter and washing the c o m m u n i t y in viral-free water. In a seasonal study
of lysogeny currently u n d e r w a y in our laboratory, this procedure reduced
viral direct counts by 62% while decreasing bacterial direct counts by 35%.
In this study over five samplings, prophage induction was detected only
by the viral reduced method.

Materials and supplies

• All items listed in the section on Screening for lysogens in natural popula-
tions
• Sterile 47 mm polycarbonate filtration devices with reservoirs
• Sterile 47 mm Anodisc filters, 0.02 ~tm
• Sterile 47 rnm Nuclepore or Poretics filters, 0.2 ~m
• Sterile 47 mm Nuclepore or Poretics filters, 1.0 pm

Protocol
1. The water sample (300 to 1500 ml) is first filtered through a 1 ~m
filter to remove protozoan grazers. We often omit this step in estu-
arine waters because of the n u m b e r of bacteria which are greater
than 1 gm in size.
2. Prepare 0.02-btm-filtered water using one of the sterile polycar-
bonate filtration devices and the 47 m m 0.02 btm Anodisc filters.

114
 3. Set up a second sterile polycarbonate filtration device with a
47 m m 0.2 btm filter and gently filter the water sample (we typi-
cally use 60 ml), turning off the v a c u u m w h e n the v o l u m e is
reduced to about 5 ml, making sure not to filter to dryness.
4. A d d 40 ml of the virus-free sample water to the u p p e r reservoir of
the filtration device containing the 5 ml of filter-concentrated
sample. Again filter until the v o l u m e is reduced to 5.0 ml.
5. Using a sterile 10 ml pipette, collect the concentrated water sample
and place it into a sterile 125 ml p o l y m e t h y l p e n t e n e flask. Using
sterile forceps, remove the 0.02 btm filter and add it to the flask,
along with 40 ml of additional 0.02-btm-filtered water.
6. Vortex for 30 s, then remove the filter with sterile forceps.
7. Bring the v o l u m e to 60 ml with 0.02-~m-filtered sample water. At
this point we typically fix 10 ml for T -- 0 viral and bacterial counts,
and use 25 ml each for treatment (i.e. Mitomycin C) and control
prophage induction assay. Samples are then counted as described
in the protocol above.

Marine prophage induction assay

This protocol uses cultures of marine bacteria in an assay that can either
be used to detect mutagenic activity of samples or c o m p o u n d s w h e n
using a k n o w n lysogen, or used to detect lysogeny in marine bacterial
isolates. It is a rapid way of also detecting the sensitivity of the bacteria to
mutagens because it uses a range of concentration of mutagen. A disad-
vantage is that the level of induction m a y not be as great in the microtiter
plate format because of limited aeration compared to rapidly shaking,
well-aerated flasks.

Materials and supplies

• 96-well microtiter plate with lid (i.e. Costar 3799 96-well Cell Culture
Cluster)
• Stock solutions of mitomycin C: 2.5~g ml' and 0.5~g ml' in Marine
Nutrient Broth (ASWJP+PY)
• Marine Nutrient Broth (ASWJP+PY)
• Overnight marine bacterial culture

Assay
1. Depending on the n u m b e r of bacteria to be assayed, designate four
rows of the microtiter plate per strain, two for Mitomycin C, two
for control.

115
 2. Inoculate a fresh flask (i.e. 10-25 ml) with the overnight culture.
Monitor growth as A~,~,,,and w h e n the absorbance reaches 0.4-0.6,
use the cells in the assay.
3. Determine which rows are to be used for Mitomycin C, and add
55 ~tl of 2.5 gg ml ~Mitomycin C to the first well in those rows, and
55 gl of 0.5 ~g ml ' to the second well in those rows. Add 55 ~i of
Marine nutrient broth to the first and second wells in the control
rows. For the u n k n o w n (treatment rows) add 55 gl of the appro-
priate u n k n o w n sample to the first well, and then 55 gl of a 1:5
dilution of the u n k n o w n to the second row.
4. Using an Octapipette, pipette 50 gl of nutrient broth into all the
other wells. Note: it is probably necessary to go to only six or eight
columns (final conc. 0.5-1 ng ml ').
5. Using a multichannel pipettor (i.e. Octapipette) set for 5 ~1,
transfer 5 ~1 from the first column to the third column for a 1:10
dilution. Triturate to mix. Then proceed to the fifth and the
seventh, if necessary, performing 1:10 dilutions with trituration.
6. Using an Octapipette set for 5 gl, transfer 5 gl from the second
column to the fourth column, for a 1:10 dilution. Triturate to mix.
Then proceed to the sixth and repeat. This will result in a dilution
series starting with 0.5 gg ml ', and including 0.1, 0.05, 0.01, etc.
until 0.001 at the sixth column.
7. A d d 200 gl of the exponentially growing cells to all wells.
8. A d d the lid to the microtiter plate and rock very gently (no
sloshing) overnight (16 h) at the correct temperature for growth.
9. At the end of the experiment, add 6.7 gl of 0.02-~tm-filtered
formalin to each well. Pipette the contents of the wells into micro-
centrifuge tubes. Centrifuge the bacteria at 14 000 rpm in a micro-
centrifuge for 5 rain. Collect 200 gl of the supernatant and dilute
appropriately for SYBR Gold counts. Positive induction is deter-
mined as a significant increase in viral counts over controls.

,,~,,t TRANSDUCTION ASSAY

Transduction c o m p o n e n t s and conditions

Three basic components are required for a transduction system: a donor,
transducing phage and a recipient. In most transduction assays, both the
d o n o r and recipient should be sensitive to infection by the same bacterio-
phage.
Both cell-free phage lysates resulting from lyric phage production and
phage particles produced by spontaneous induction of lysogens can
mediate transduction. Also, both lysogenic and non-lysogenic bacteria
can serve as recipients, but lysogenic recipients have higher transduction
frequencies, possibly due to the lysogenic protection (homoimmunity)
from lytic attack (Miller et al., 1992).

116
 In theory, all bacteriophages are capable of generalized transduction at
various frequencies because mistakes in packaging of DNA within the
bacterial host always occur (Ackermann and DuBow, 1987). However, to
effectively detect or demonstrate the process of transducfion, phenotyp-
ical or genotypical markers are necessary to monitor the acquisition and
expression of transduced genes. Auxotrophic mutants with specific amino
acid requirements for growth or plasmids encoding for antibiotic resis-
tance are often the biomarker of choice. Both types of markers allow selec-
tion of transductants from recipients by plating on selective medium and
therefore reducing the background growth of recipient bacteria. Proper
control experiments are critical to subtract the rate of spontaneous rever-
tants from transduction (Levisohn et al., 1987). Cotransduction of closely
linked loci will allow a more definitive identification of a unique trans-
duced phenotype and reduce the background of revertants produced by
spontaneous mutation (Miller, 1992).
Compared to transduction of chromosomal markers for which a
good gene probe does not exist, transduction of antibiotic resistant plas-
mids is more easily confirmed, either by plasmid DNA extraction from
transductants followed by restriction enzyme profile analysis (Ripp et
al., 1994), or by colony hybridization with specific gene probes (Jiang
and Paul, 1998b; Figure 2). Control experiments are also necessary to
correct for rates of spontaneous mutation to resistance. If it is necessary
to further confirm the transfer event, plasmid extraction, Southern
hybridization or PCR techniques can also be used to verify the exis-
tence of the original plasmid in the transductants. However, rearrange-
ment or recombination of the plasmid DNA can occur, particularly
when natural populations are employed as recipients (Jiang and Paul,
1998b).
Another problem in the use of antibiotic resistance plasmids for use in
transfer to indigenous recipients is the high degree of antibiotic resistance
found in natural populations. In our studies of transfer of the plasmid
pQSRS0, which encodes kanamycin resistance on the transposon Tn5 as
well as streptomycin, a high level of resistance was often found to both
kanamycin and streptomycin in marine bacterial populations.
Additionally, some of the resistant colonies in the 'no plasmid control'
hybridized with a gene probe derived from the kan resistance gene of Tn5
(Figure 7.3). When such results were obtained, the results were discarded,
and only environments lacking Tn5-1ike kanamycin resistance were
studied further (Jiang and Paul, 1998b).
The size of the plasmid used in a transduction assay should be consid-
ered. Saye et al. (1987) found that transduction of plasmids was more effi-
cient if the molecular weight of the plasmid was similar to that of the
phage genome, favoring packaging errors.
The frequency of transduction varies with different phage-host
systems. However, exposing transducing particles to UV radiation is
generally known to increase transduction frequency (Miller, 1992). It has
been suggested that this treatment stimulates recombination within the
host cell leading to increased incorporation of the transduced DNA into
the recipient genome (Benzinger and Hartman, 1962). Secondly, it may

117
 HOPE -2 (1)DIB

Kan + Strep Plasmid

Hybridization
}. -I-
Treatment
Lysate
Kan + Strep

+ ) or

Control

Figure 7.3. Cartoon depicting plasmid transduction as in Figure 2 but using the
ambient microbial population (depicted as a tank containing copedods and fish)
as recipients. Unlike transduction with a known recipient, there is an indigenous
level of antibiotic resistance in the natural population, which yields colonies from
the no lysate control when plated upon kanamycin/streptomycin media. In
certain cases, some of these hybridize to the probe, showing that the indigenous
population contains some genes similar to those chosen for transduction. Only
when tile no-lysate controls contain no positive hybridizing colonies can trans-
duction be inferred.

reduce the infectivity (or virulence) of the phage, such that all putative
transductants are not lysed as the result of lytic infections.
The MOI (multiplicity of infection) is another factor influencing the
frequency of transduction (Keynan et al., 1974; Morgan, 1979). In general,
the optimal MOI range is between 0.1 and 1. It is thought that low MOIs
produce higher transduction frequencies by reducing the possibility of a
recipient cell simultaneously encountering both a transducing particle
and a lytic infectious phage particle (Miller, 1992). For transduction in
environmental chambers, Saye et al. (1990) reported optimal MOIs for
phage Fl16 and DS1 transduction in P. aeruginosa to be 0.02. Jiang and Paul
(1998b) found marine transduction occurred only w h e n the MOI was less
than 0.05.

Transduction in c u l t u r e d isolates
To assay transduction in a cultured p h a g e - h o s t system, the following
steps can be followed to establish a transduction system. However, the
m e t h o d presented here is highly generalized and can be adapted to
various plasmids and p h a g e - h o s t systems.

118
 Materials and supplies
• Marine bacteria and bacteriophage isolates
• Plasmids (preferably broad host range with two selectable markers and
accompanying gene probes)
• Antibiotics
• Marine broth and other nutrient medium (Difco)
• Bacto agar (Difco)
• Petri dishes
• Culture tubes
• 0.5 MTris.HCI buffer pH 8.0
• N-methyI-N'-nitro-N-nitrosoguanidine (Sigma)
• 0.2 lure membranes (Millipore)
• Chloroform
• E~Nase I (Sigma)
• UV lamp c-
• ~/ater bath O

U
"O
e-

Methods #
C
Construction of gen etically marked donor
C
QJ
The protocol below describes transferring an antibiotic resistant plasmid
0
to a donor strain by triparental mating. Alternative methods of plasmid
.J
transier, i.e. artificial transformation, can also be used to achieve the same
goal.
Protocol
1. Mix log-phase cultures of the following strains, a plasmid donor, a
helper strain containing conjugative helper plasmid and the plasmid
recipient, at approximately equal cell numbers.
2. Fi ter the mixture onto a sterile, 0.2 gm membrane filter and incubate
ox ernight on nonselective medium to allow conjugation.
3. The next morning, re-suspend cells in 5 ml of nutrient medium, then
plate onto selective medium to select for the traits of plasmid and
recipients.

Production of transducing phage particles from a lytic infection of donor cells

Protocol
1. Mix midlog donor bacteria with phages at a MOI of -0.1 in 3 ml of
melted 1% agar medium kept at 45-47°C in a water bath.
2. Pour the soft agar mixture over a 1.5% agar plate containing the proper
growth medium. For some marine phage-host systems that are sensi-
tive to a brief exposure to higher than ambient temperature, the soft agar
should be taken out of the water bath just before adding the phage-host
mixture then poured immediately to prevent heat inactivation.

119
 3. Incubate overnight for phage amplification, harvest phages by
flooding the top agar with 0.5 M Tris buffer (EH 8.0), using 5 ml for
each 110 mm diameter plate.
4. Remove cell debris or residual bacteria by low speed centrifugation
followed by filtration through a sterile 0.2 ~m filter. Alternatively, a
drop of chloroform is added to kill residual bacteria.
5. Repeat steps 1 to 4 for a second round of infection to ensure that the
transducing lysate contains markers derived from the donor host.
6. If desired, treat transducing lysates by ultraviolet radiation using a
lamp with a peak wavelength at 256 nm to reduce the infective phage
titer to 1% of the original.
7. Digest transducing lysates with 50 units ml ~of DNase I before use in
the transduction assay to reduce the chance of transformation.

Titering of the transducing lysate

Protocol

1. Prepare a culture of the indicator strain in nutrient medium and grow

to midlog phase.
2. Make a serial dilution of the phage lysate in 0.5 M rris buffer just before
use.
3. Mix 100 ~Jl of each phage dilution with 1 ml of indicator bacteria in soft
agar and pour immediately onto an agar plate as described for produc-
tion of phage particles.
4. Enumerate plaque forming units after overnight incubation.

Transduction

Protocol
1. Mix 10-100 ml of log-phase recipient cell culture with transducing
phage particles at MOIs ranging from 0.01 to 5.
2. Incubate the mixture at room temperature for 10 min to allow phage
adsorption.
3. Remove the unabsorbed phages by three rounds of centrifugation and
washes with artificial seawater.
4. Resuspend the final cell pellet in 0.5-1.5 ml of nutrient broth, and
allow the cells to recover in this nonselective medium for 10-20 min
before plating onto nutrient plates selective for the genetic determi-
nants serving as markers for transduction.
5. The transducing lysate (containing no recipient) and recipient only
should also be plated onto the same selective plates as controls.
6. Incubate the plates for two to six days before counting colonies of
transductants. Longer incubation periods may be needed for slow-
growing marine bacteria. Extended incubation is often necessary to
allow for the phenotypic expression of the transduced gene.
7. The frequency of transduction can be expressed as transductants per
transducing phage or per recipient.

120
Transduction in natural populations
Natural marine bacterial populations can be used directly as recipients for
transduction if proper genetic markers (i.e. an unique plasmid) are used
for the donor bacteria (Figure 7.3). The production of transducing lysates
should be the same as for transduction in the cultured system described
above. Compared to recipients in culture, bacteria in natural seawater are
much less abundant. Therefore, the bacterial community should be
concentrated to allow detection of transduction.

Equipment and supplies

• Membrex Rotary Biofiltration Device, equipped with 100 KD filter, or
other cell concentrating device (tangential flow, etc.)
• All materials and supplies for transduction assay in the cultured bacterio-
c-
phage and host systems O

Protocol
1. Concentrate 20 to 100 1 of seawater from offshore environments e,,

using a Membrex vortex flow filtration system (Jiang et al., 1992) to #

"O
50 ml. e"
2. Mix 10 ml of this concentrate with various concentrations of trans-
>,,
ducing particles and allow phage to adsorb onto the bacteria at e"

room temperature for 10 min.

O
3. Filter the mixture onto a 0.2 ~m filter and rinse with sterile artificial
..1
seawater to wash off the unadsorbed phages.
4. Resuspend cells collected on the filter in 2 ml of nutrient broth and
plate 100 ~tl on selective medium plates.
5. Plate an equal volume of concentrated sample without addition of
transducing phages and transducing lysate alone as negative
controls.
6. Incubate for at least 4 days before enumeration of transductants.
Many indigenous marine bacteria are resistant to various anti-
biotics. If an antibiotic resistant plasmid is used as a genetic
marker for transduction, colony hybridization with a plasmid
specific probe can be performed to distinguish the transductants
from indigenous resistant bacteria.
7. The frequency of transduction can be expressed as transductants
per transducing phage particle.

FUTURE DIRECTIONS
In terms of lysogeny, the factors which control the regulation of this
phenomenon in the environment will hopefully be determined by future
research. That is, what environmental conditions control the lysogenic
decision upon infection with a temperate phage? Does pseudolysogeny

121
 play a role in production of phage in the marine environment? And, do
p h y t o p l a n k t o n blooms crash because of induction of temperate algal
viruses? Some of these questions are experimentally difficult to answer
with current technology, while others have yet to be investigated,
In comparison with freshwater environments, m u c h less effort has
been directed at the investigation of transduction in marine waters. Many
methods that were designed for freshwater habitats are also suitable for
the investigation of in situ transduction in marine environments.
Examples of these methods include: (1) transduction assays in flow
through environmental chambers that are incubated at ambient tempera-
ture; (2) transduction with spontaneous induced phage without separa-
tion and purification from d o n o r bacteria (i.e. mixing the d o n o r and
recipient in an environmental chamber). In addition, several new
approaches that m a y extend our current understanding of transduction in
the marine environment are also w o r t h y of investigation.
First, the native marine bacteriophages can now be easily concentrated
and purified from seawater. It should be interesting to investigate the
frequency of transduction by these indigenous marine bacteriophages. In
this transduction system, auxotrophic bacteria can be used as recipients as
per Chiura (1997).
Secondly, all transduction assays to date have been designed to detect
the gene transfer event in cultivable marine bacteria. Since less than 1% of
marine bacteria are culturable by current methods, it is important to
develop strategies to detect transduction events in non-cultivable marine
bacteria. One of the strategies is to use a fluorescent in situ hybridization
technique (FISH) to trace the uptake of genetic markers in a single cell
without cultivation. Alternatively, in situ PCR can also be used to increase
the detection sensitivity. Additionally, plasmids containing the green fluo-
rescent protein (GFP) gene can be used, and transduction detected by
epifluorescence microscopy (Dahlberg et al., 1998).
In conclusion, marine transduction is still a y o u n g and growing field of
research. As new techniques are developed to study gene transfer in
natural populations, the overall importance of this process in the evolu-
tion of microbial populations in the environment will unfold.

References
Ackermann, H. W. and DuBow, M. S. (1987). Viruses qf Prokaryotes. Vol. 1. General
properties of bacteriophages. CRC Press, Boca Raton, FL.
Amin, M. K. and Day, M. J. (1988). Donor and recipient effects on transduction
frequency in situ. REGEM1 Program.
Baross, J. A., Liston, J. and Morita, R. Y. (1978). incidence of Vibrio parahaemolyticus
bacteriophages and other Vibrio bacteriophages in marine samples. Appl.
Environ. Microbiol. 36, 492-499.
Benzinger, R. and Hartman, P. E. (1962). Effect of ultraviolet light on transducing
phage P22. Virology 18, 614-626.
Borsheim, K. Y., Gratbak, G. and Heldal, M. (1990). Enumeration and biomass esti-
mation of planktonic bacteria and viruses by transmission electron microscopy.
Appl. Environ. Microbiol. 56, 352-356.

122
Buck, G. A. and Groman, N. B. (1981). Genetic elements novel for Corylwbacterium
diphtheriae: specialized transducing elements and transposons. I. Bacteriol. 148,
143-152.
Cavenagh, M. M. and Miller, R. V. (1985). Specialized transduction of PseudomoHas
aeruy,il~osa PAO by bacteriophage D3. J. Bacteriol. 165, 448-452.
Chiura, H. X. (1997). Generalized gene transfer by virus-like particles from marine
bacteria. Aquat. Microb. Ecol. 13, 75-83.
Chirua, H. X., Hato, K., Hiraishi, A. and Maki, Y. (1998). Gene transfer mediated by
virus of novel thermophilic bacteria in hot spring sulfur-turf microbial mats.
Eighth International Syrnposium on Microbial Ecology (1SME-8). Program and
Abstracts.
Cochran, P. K. and Paul, ]. H. (1998) Seasonal abundance of lysogenic bacteria in a
subtropical estuary. Appl. EH~qrolL Microbi~d. 64, 2308-2312.
Cochran, P. K., Kellogg, C. A. and Paul, J. H. (1998) Prophage induction of indige-
nous marine lysogenic bacteria by environmental pollutants. Mar. Ecol. Progr.
Set. 164, 125-133.
Dahlberg, C., Bergstrom, M. and Hermansson, M. (1998). hz situ detection of high
c-
levels of horizontal plasrnid transfer in marine bacterial communities. Appl. O
D~viron. Microbiol. 64, 2670-2675.
U
Denhardt, D. T., Dressier, H. H. and Ray, D. S. (eds). (1978). Sirrah' stra~ded DNA "O
Pha~es. Cold Spring Harbor Laboratory. e"
Dr6ge, M., Pi_ihler, A. and Selbitschka, W. (1998). Horizontal gene transfer as a
biosafety issue: a natural phenomenon of public concern. J. Biotech. 64, 75-90.
#
"D
Germida, J. J. and Khachatourian, G. G. (1988). Transduction of Escherichia colt in e,,

soil. CaJl. J. Microbiol. 34, 190-193. >,,

e-
Jain, R., Rivera, M. C. and Lake, J. A. (1999). Horizontal gene transfer among lla
genomes: the complexity hypothesis. Proc. Natl. Acad. Sci. USA 96, 3801-3806. O
Jarolmen, H., Bonke, A. and Crowell, R. I. (1965). Transduction of Staphylococcus
aim'us to tetracycline resistance iH vivo. J. Bacteriol. 89, 1286-1290.
Jensen, E. C., Schrader, H. S., Rieland, B., Thompson, T. L., Lee, K. W., Nickerson,
K. W. and Kokjohn, T. A. (1998). Prevalence of broad-host-range lyric bacterio-
phages of Sphaerotilus izatalls, Escherichi colt, and Pseudomonas aeruliJwsa. Appl.
DzviroH. Microbiol. 64, 575-580.
]tang, S. C. and Paul, J. H. (1994). Seasonal and diel abundance of viruses and
occurrence of lysogeny/bacteriocinogeny in the marine environment. Mar. Ecol.
Progr. Ser. 104, 163 172.
Jiang, S. C. and Paul, ]. H. (1996). Occurrence of lysogenic bacteria in marine
microbial communities as determined by prophage induction. Mar. Ec~d. Prog.
Set. 142, 27-38.
]tang, S. C. and Paul, J. H. (1998a). Significance of lysogeny in the marine environ-
ment: studies with isolates and a model for viral production. Microb. Ecol. 35,
235-243.
Jiang, S. C. and Paul, J. H. (1998b). Gene transfer by transduction in the marine
environment. Appl. E1zvirou. Microbiol. 64, 2780-2787.
]tang, S. C., Thurmond, J. M., Pichard, S. L. and Paul, ]. H. (1992). Concentration of
microbial populations from aquatic environments by vortex flow filtration. Mar.
Ecol. ProS. Set. 80, 101 107.
Keynan, A., Nealson, K., Sideropoulos, H. and Hastings, J. W. (1974). Marine
transducing bacteriophage attacking a luminous bacterium. J. Wr01. 14, 333-
340.
Kidambi, S. P., Ripp, S. and Miller, R. V. (1993). Evidence for phage-mediated
transfer among Pseluh~moHas aCI'HgiHosI7 o n the phylloplane. Appl. D~virotl.
Microbiol. 60, 496-500.

123
Kokjohn, T. A. (1989). Transduction: mechanism and potential for gene transfer in
the environment. In: Gene Transfer in the Environment (S. B. Levy and R. V. Miller,
Eds), pp. 73-97. McGraw-Hill, New York.
Levin, B. R. and Lenski, R. E. (1983). Coevolution in bacteria and their viruses and
plasmids. In: Coevolution (D. J. Futuyma and M. Slaktin, Eds), pp. 99-127.
Sinauer Associates, Inc., Sunderland, MA.
Levisohn, R., Moreland, J. and Nealson, K. H. (1987) Isolation and characterization
of a generalized transducing phage for the marine luminous bacterium Vibrio
fischeri MJ-I. 1. Gen. Microbiol. 13, 1577 1582.
Miller, R. V. (1992). Methods for evaluating transduction: An overview with envi-
ronmental considerations. In: Microbial Ecolo\~y: Principles, Methods, ~Tud
Applicatiopls (M. A. Levin, R. J. Seidler and M. Rogul, Eds), pp. 229-251.
McGraw-Hill, New York.
Miller, R. V., Ripp, S., Relicon, J., Ogunseitan, O. A. and Kokjohn, T. A. (1992).
Virus-mediated gene transfer in freshwater environment. In: Gene Transfers nnd
Etlviromnent (M. J. Gauthier, Ed.), pp. 51-62. Springer-Verlag, Berlin.
Morgan, A. E (1979). Transduction of Pseudomonas aeruginosa in a freshwater
environment. Appl. Environ. Microbiol. 36, 724-730.
Morrison, W. D., Miller, R. V. and Sayler, G. S. (1978). Frequency of Fl16 mediated
transduction of Pseudomonas aerugiuosa in a freshwater environment. Appl.
EHviron. Microbio[. 36, 724-730.
Novick, R. P. and Morse, S. I. (1967). hi vivo transmission of drug resistance factors
between strains of Staphyh~coccus aureus. J. Exp. Med. 125, 45-49.
Novick, R. P., Edelman, I. and Lofdahl, S. (1986). Small Staphylococcus aureus plas-
mids are transduced as linear multimers that are formed and resolved by
replicative process. J. Mol. Biol. 192, 209-220.
Paul, J. H. and Myers, B. (1982). The fluorometric determination of DNA in aquatic
microorganisms employing Hoechst 33258. Appl. Ewe,iron. Microbiol. 43,
1393-1399.
Ripp, S. and Miller, R. V. (1995) Effects of suspended particulates on the frequency
of transduction among Pseudomonas neruginosa in a freshwater environment.
Appl. D~viroH. Microbio[. 61, 1214-1219.
Ripp, S., Ogunseitan, O. A. and Miller, R. V. (1994). Transduction of a freshwater
microbial community by a new Pseudomonas aeruy,inosa generalized transducing
phage, UT1. Mol. Ecol. 3, 121-126.
Saye, D. J. and Miller, R. V. (1989). The aquatic environment: consideration of hori-
zontal gene transmission in a diversified habitat. In: Gene Transfer ii~ the
Ei1vironmel~t (S. B. Levy and R. V. Miller, Eds), pp. 223-259. McGraw-Hill, New
York.
Saye, D. J., Ogunseitan, O., Sayler, G. S. and Miller, R. V. (1987) Potential for trans-
duction of plasmids in a natural freshwater environment: effect of donor
concentration and a natural microbial community on transduction in
PseudomoJlas aeruginosa. Appl. Environ. Microbiol. 53, 987-995.
Saye, D. J., Ogunseitan, O. A., Sayler, G. S. and Miller, R. V. (1990). Transduction of
linked chromosomal genes between Pseudomonas aeruginosa during incubation
in situ in a freshwater habitat. Appl. Environ. Microbiol. 56, 140-145.
Scanlan, D. J. and Wilson, W. H. (1999). Application of molecular techniques to
addressing the role of P as a key effector in marine ecosystems. Hydrobiol. 401,
149-175.
Schichklmaier, P. and Schmieger, H. (1995). Frequency of generalized transducing
phages in natural isolates Salmonella typhimurium complex. Appl. Environ.
Microbiol. 61, 1637-1640.
Sternberg, N. L. and Maurer, R. (1991). Bacteriophage-mediated generalized trans-

124
 duction in Escherichia coli and Sahnonella typhimurium. Method Enzymol. 204,
19-43.
Stozky, G., (1989). Gene transfer among bacteria in soil. In: Ge~w Traus~er in the
ElzviromHent (S. B. Levy and R. V. Miller, Eds), pp. 165-222. McGraw-Hill, New
York.
Tapper, M. A. and Hicks, R. E. (1998) Morphology and abundance of free and
temperate viruses in Lake Superior. Limnol. Occauo?¢r. 43, 95-103.
Waterbury, J. B. and Valois, E W. (1993). Resistance to co-occurring phages enables
marine Synechococcus communities to coexist with cyanophages abundant in
seawater. Appl. EuviroJ~. Microbiol. 59, 3393-3399.
Weinbauer, M. G. and Suttle, C. A. (1996). Potential significance of lysogeny to
bacteriophage production and bacterial mortality in coastal waters of the Gulf
of Mexico. Appl. E1tviroJt. Microbiol. 62, 4374-4380.
Weinbauer, M. G. and Suttle, C. A. (1999). Lysogeny and prophage induction in
coastal and offshore bacterial communities. Aquatic Microbial Ecol. 18, 217-225.
Wichels, A., Biel, S. S., Gelderblam, H. R., Brinkhoff, T., Muyzer, G. and Sh(itt, C.
(1998). Bacteriophage diversity in the North Sea. Appl. E~viron. Microbiol. 64,
c-
4128-4133. O
, m

Wilcox, R. M. and Fuhrman, J. A. (1994). Bacterial viruses in coastal seawater: lytic

rather than lysogenic production. Mar. Ecol. Prog. Ser. 114, 35M5.
"O
Wilson, W. H., Turner, S. and Mann, N. H. (1998) Population dynamics of phyto- e-
plankton and viruses in phosphate-limited mesocosm and their effect on DMSP el

and DMS production. Estuar. Coastal Shelf Sci. 46, 49-59.

#
Wommack, K. E. and Colwell, R. R. (2000). Viroplankton: viruses in aquatic e-

ecosystems. Microbiol. Mol. Biol. Rev. 64, 69-114.

c-
Zeph, L. R. and Stotzky, G. (1989). Use of a biotinylated DNA probe to detect
bacteria transduced by bacteriophage P1 in soil. Appl. E1~viro11. Microbiol. 55, O
661-665.
Zeph, L. R., Onaga, M. A. and Stotzky, G. (1988). Transduction of Escherichia coil by
5"
bacteriophage P1 in soil. Appl. Environ. Microbiol. 54, 1731-1737.
Zinder, N. D. and Lederberg, J. (1952). Genetic exchange in Salmoi~ella. ]. Bactcriol.
64, 679-699.

List of suppliers
Fisher Scientific Sigma Chemical Corporation
3970 Johns Ctvek Court PO Box 14508
Suwauee, GA 30024, USA St. Louis, M O 63178, USA
1-800-766-7000 1-800-325-3010
Anodisc and Nuclepore filters, Electron microscopy grade
microtiter plates glutaraldehyde
Mitomycin C
Micron Separations Inc.
135 Flanders Road Difco Laboratories
PO Box 1046 P. 0. Box 331058
Westborou~h, M A 01581, USA Detroit, MI, 48232-7058, USA
1-800-444-8212 Phone: 800-521-0851
Fax: 313-462-8517
M e m b r e x / O s m o n i c s Rotary
Biofiltration Device Microbiological media

125