7 Lysogeny and Transduction

7 Lysogeny and Transduction

John H Paul' and Sunny C Jiang 2 ~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 Screening marine bacteria for lysogeny Transduction assay Future directions

I N T R O D U C T I O N A N D B A C K G R O U N D

Lysogeny and transduction describe a type of phage/host interaction and a method of bacterial gene transfer (procaryotic sex), respectively. Although they are often reviewed together, these topics are linked only in 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, particularly 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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Figure 7.1. Cartoon depicting lytic, lysogenic, and pseudolysogenic phage/bacterial 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 abundance of both phages and host cells simultaneously.

originated in a bacterial host and are not a normal par t of the phage genome. Traits conferred to the host by conversion include immun i ty to superinfection, which means that the host becomes i m m u n e not only to infection by that part icular tempera te 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 botul inus toxins C and D; Ackermann and DuBow, 1987).

In a lysogenic symbiosis, there is a low rate of spontaneous reversion to virulence, in that, on average, 1 in 10 ~ produces a lyric event (Ackermann and DuBow, 1987). Thus, there is a constant product ion of tempera te phage in a lysogenic interaction. However , other interactions exist where there is a high-level product ion of both host cells and viral particles in a process termed ' p seudo lysogeny ' (Figure 7.1). Pseudolysogeny has been used synonymous ly with 'carrier state' and 'chronic infection', yet distinc- tions can be made between these conditions. By Ackermann and DuBow's (1987) definition, pseudo lysogeny 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 tempera te and virulent

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phage, that results in a constant supply of host and viral particles. This in many ways resembles the marine environment, where sensitive and resistant 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 adhesion-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

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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 and/or 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 mitomycin 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 mechanisms, 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 chromosome, 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 transducing 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 transductants 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

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during evolution has been inferred from nucleotide sequence compar- isons (Dr6ge et al., 1998). Among the bacterial gene exchange mechanisms, 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 environments (Morrison et al., 1978; Saye et al., 1987; 1990; Amin and Day, 1988; 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 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 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 transduction by bringing the host and phage into close contact with each other.

Compared with freshwater environments, less is known about transduction in marine waters even though a transducing marine bacteriophage 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 bacteriophages 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

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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 communi ty 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 gen t transfer between marine bacteria and enteric bacteria. He demonstra ted that five marine bacteria isolated from seawater were capable of spontaneous 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 chromosome 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 magni tude higher than those of t ransducing phages isolated from freshwaters (Saye et el., 1990; Ripp et el., 1994). Intergeneric transduction was also demonst ra ted in another startling report in which viral-like particles (VLP) produced by a hot-spring natural bacterial communi ty (predominated by hyper thermophilic chemolithotrophic sulfur bacteria) were capable of t ransducing loci

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to repair auxotrophic E. coli and Bacillus subtilis to prototrophy with an average efficiency of 10 ~' per VLP (Chiura et al., 1998). These results indicate 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 FOR L Y S O G E N Y

Isolates in cu l tu re

The protocol that follows has been used to screen marine bacterial isolates for inducible prophage and/or 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.

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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 environment 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 environment. Making a few assumptions, it is possible to estimate the proportion 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 investigated. 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.

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Equipment and supplies

• Membrex Rotary Biofiltration Device, equipped with 100 KD filter (note that this is necessary only for offshore samples and use withTEM enumeration

• 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 concentrat ion 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 concentrate (termed retentate) usually has a vo lume of 35-60 ml.

2. For concentrated samples, place 1 ml in a sterile 1.5 ml microcentrifuge tube or 5.0 ml of the retentate in a 15 ml conical centrifuge tube. Do this both for control (unamended) and treatment (mutagen amended) samples.

3. For unconcentra ted samples, add 25 ml each to a control or treatment, 50 ml sterile, conical centrifuge tubes. If several mutagens are to be investigated, increase the number of t reatment tubes accordingly.

4. Take an additional sample (1-5 ml for Membrex Retentate, 25 ml for unconcentrated) and fix with glutara ldehyde (2% final concentration) as a T = 0 control. If epifluorescence microscopy is to be used for enumerat ion, 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 mutagen (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% glutara ldehyde (for TEM) or 1°/, formalin (epifluorescence microscopy).

7. Samples for enumerat ion 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

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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 treatment, 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 vi ra l r e d u c e d m e t h o d

The method described above for detection of lysogeny in natural populations has the least amount of manipulat ion 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 communi ty through a 0.2 btm filter and washing the communi ty in viral-free water. In a seasonal s tudy of lysogeny currently unde rway in our laboratory, this procedure reduced viral direct counts by 62% while decreasing bacterial direct counts by 35%. In this s tudy over five samplings, prophage induction was detected only by the viral reduced method.


• All items listed in the section on Screening for lysogens in natural populations

• 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 estuarine waters because of the number of bacteria which are greater than 1 gm in size.

2. Prepare 0.02-btm-filtered water using one of the sterile polycarbonate filtration devices and the 47 mm 0.02 btm Anodisc filters.

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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 typically use 60 ml), turning off the vacuum when the volume is reduced to about 5 ml, making sure not to filter to dryness.

4. Add 40 ml of the virus-free sample water to the upper reservoir of the filtration device containing the 5 ml of filter-concentrated sample. Again filter until the volume 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 polymethylpentene flask. Using sterile forceps, remove the 0.02 btm filter and add it to the flask, along with 40 ml of addit ional 0.02-btm-filtered water.

6. Vortex for 30 s, then remove the filter with sterile forceps. 7. Bring the volume 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.

M a r i n e p r o p h a g e i n d u c t i o n assay

This protocol uses cultures of marine bacteria in an assay that can either be used to detect mutagenic activity of samples or compounds when using a known 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 may not be as great in the microtiter plate format because of limited aeration compared to rapidly shaking, well-aerated flasks.


• 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 number of bacteria to be assayed, designate four rows of the microtiter plate per strain, two for Mitomycin C, two for control.

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2. Inoculate a fresh flask (i.e. 10-25 ml) with the overnight culture. Monitor growth as A~,~,,, and when 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 unknown (treatment rows) add 55 gl of the appropriate unknown sample to the first well, and then 55 gl of a 1:5 dilution of the unknown 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 mult ichannel 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. Add 200 gl of the exponential ly growing cells to all wells. 8. Add 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 microcentrifuge tubes. Centrifuge the bacteria at 14 000 rpm in a microcentrifuge for 5 rain. Collect 200 gl of the supernatant and dilute appropriately for SYBR Gold counts. Positive induction is determined as a significant increase in viral counts over controls.

, , ~ , , t T R A N S D U C T I O N ASSAY

Transduction components and conditions

Three basic components are required for a transduction system: a donor, t ransducing phage and a recipient. In most transduction assays, both the donor and recipient should be sensitive to infection by the same bacteriophage.

Both cell-free phage lysates resulting from lyric phage product ion 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 (homoimmuni ty) from lytic attack (Miller et al., 1992).

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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 resistance 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 revertants from transduction (Levisohn et al., 1987). Cotransduction of closely linked loci will allow a more definitive identification of a unique transduced 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 plasmids 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

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Treatment

Control

HOPE -2 (1)DIB

Lysate

+

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) or

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 transduction be inferred.

reduce the infectivity (or virulence) of the phage, such that all putat ive 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 s imultaneously encounter ing 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 when the MOI was less than 0.05.

T r a n s d u c t i o n in c u l t u r e d isolates

To assay transduction in a cultured phage-hos t system, the following steps can be followed to establish a transduction system. However, the method presented here is highly generalized and can be adapted to various plasmids and phage-hos t systems.

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• 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 M Tris.HCI buffer pH 8.0 • N-methyI-N'-nitro-N-nitrosoguanidine (Sigma) • 0.2 lure membranes (Millipore) • Chloroform • E~Nase I (Sigma) • UV lamp • ~/ater bath

Methods

Construction of gen etically marked donor

The protocol below describes transferring an antibiotic resistant plasmid to a donor strain by triparental mating. Alternative methods of plasmid 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 sensitive 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.

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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

Protoco l

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 u s e .

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 production 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.

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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 bacteriophage and host systems

Protocol

1. Concentrate 20 to 100 1 of seawater from offshore environments using a Membrex vortex flow filtration system (Jiang et al., 1992) to 50 ml.

2. Mix 10 ml of this concentrate with various concentrations of transducing particles and allow phage to adsorb onto the bacteria at room temperature for 10 min.

3. Filter the mixture onto a 0.2 ~m filter and rinse with sterile artificial 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 antibiotics. 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 D I R E C T I O N S

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

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play a role in product ion of phage in the marine environment? And, do phytoplankton blooms crash because of induction of temperate algal viruses? Some of these questions are experimental ly difficult to answer with current technology, while others have yet to be investigated,

In comparison with freshwater environments, much 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 t ransduction in marine environments. Examples of these methods include: (1) transduction assays in flow through environmental chambers that are incubated at ambient temperature; (2) transduction with spontaneous induced phage without separa- tion and purification from donor bacteria (i.e. mixing the donor and recipient in an environmental chamber). In addition, several new approaches that may extend our current unders tanding of transduction in the marine environment are also wor thy 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 t ransduction 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 of0927 Tc 3.21s53 0 Td (be )T4n 0 Td Tc.0213 0 Tl6c7 0 Td95adea Td (i t )Tj 0.03333733 0 Td (199 Tc 976a8)Tj 03 0 Td (is70.09479 Tc 4.06to )Tj 0.0280 Tdd 0 Td (ar23.21s5 (auxot7d )Tj 0.0897n17.0854 Tc 2.04Td (and )l -1.275 Tec 3a Tj 0.0333373Hln0 Td (199 Tc 976e.0927 Tc 3.21s5 2Td (199 Tc 97 7iw0.05.t7d )Tj 0.r4 Tc 4.380499u.0280 Tdd 0 Td (ar23.2s0 Td (bacteria )Tj 0.068747.3671.3884 0 Td (are )Tj 0.(frequenj 0.2d (199 T59 0 Td (culturable )Tj Td (to )79 8957 Tc 1O(are )Tj 0.(ar23.210.06874 Tc 5968 0 Td ( 0.09999 s53 0.075 Tc 2 0 Td (less70.09479 0.351.06to )Tj 0.0280 Tdd 0 Tdequency )s5 4.0479 0 Ti8 0 Td (transfer )Tj 17xot7d )Tj 0.0897n1Td (marine0 Td (ar2uhic )Tj )Tj 0.08957 Tc 1d (to )Tj )Tj 0.010.8777xot7d )f luo2.8c5 0 Td (/F1 .09f Tdequency )8)Tj.07290 9.6 068.8 3)Tj mTc 4.380499 Td (as )9 Tc-34.7611 si tu Td (/F0 .09f Tdect )Tj 09 5307290 9.6 07)Tj 3b a c t e r i o p 0 . 0 9 T d ( a u x o t r o p ( i t ) T j 0 . 0 3 3 3 3 7 3 4 7 . 2 ( b e e n ) ] T u p t a k a r e ) T j 0 . ( a r 2 3 . 2 1 ) T j 0 . 0 7 5 T c 9 6 8 0 T d ( l y , ) T j 0 0 . 0 9 2 7 T c 9 0 t r a to / F 0 . 0 9 f T d b a c t e r i o p 9 5 3 0 7 2 9 0 9 . 6 2 8 7 . 0 6 j 3 0 . 0 6 j m T c P C R 8 0 T d ( l y , ) T j a l l

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List of suppliers Fisher Scientific 3970 Johns Ctvek Court Suwauee, GA 30024, USA 1-800-766-7000

Anodisc and Nuclepore filters, microtiter plates

Micron Separations Inc. 135 Flanders Road PO Box 1046 Westborou~h, M A 01581, USA 1-800-444-8212

Membrex /Osmonics Rotary Biofiltration Device

Sigma Chemical Corporation PO Box 14508 St. Louis, M O 63178, USA 1-800-325-3010

Electron microscopy grade glutaraldehyde Mitomycin C

Difco Laboratories P. 0 . Box 331058 Detroit, MI, 48232-7058, USA Phone: 800-521-0851 Fax: 313-462-8517

Microbiological media

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7 Lysogeny and Transduction