Dec 9, 1988 - Laboratory of Microbial Ecology, Department ofBiology, New York ... transducing phage P1 and E. coli, were confirmed to be lysogenic for ...
APPLIED
AND
ENVIRONMENTAL MICROBIOLOGY, Mar. 1989, p. 661-665
Vol. 55, No. 3
0099-2240/89/030661-05$02.00/0 Copyright © 1989, American Society for Microbiology
Use of a Biotinylated DNA Probe To Detect Bacteria Transduced by Bacteriophage P1 in Soil L. R. ZEPH AND G. STOTZKY* Laboratory of Microbial Ecology, Department of Biology, New York University, New York, New York 10003
Received 20 October 1988/Accepted 9 December 1988
Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E. coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIll 3 fragment of bacteriophage P1. No Pl lysogens of indigenous soil bacteria were detected with the DNA probe. The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively. In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared. These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.
There is limited knowledge concerning the fate of genetically engineered microorganisms released to the environment and the transfer of their novel genes, via conjugation, transformation, or transduction, to indigenous microorganisms (20; G. Stotzky, in S. B. Levy and R. V. Miller, ed., Gene Transfer in the Environment, in press). Studies on the transfer in soil of novel genes from genetically engineered microorganisms to either indigenous or added bacteria have focused primarily on bacterial conjugation. Transduction by bacteriophage has been reported in marine and freshwater aquatic habitats (2, 12, 17) and in soil (5, 24). Understanding and predicting the frequency of gene transfer in situ by any mechanism from genetically engineered microorganisms planned for release would be helpful in addressing questions concerning the long-term persistence and potential ecological effects of the novel DNA in the environment. We have demonstrated the transduction of bacterial resistance genes for chloramphenicol (CAM) and mercury into Escherichia coli when the transducing coliphage P1 was added to nonsterile soil as either lysates or E. coli lysogens (24). One objective of the present study was to confirm that these E. coli transductants were lysogenic for phage P1 by employing DNA-DNA hybridization techniques with a biotinylated DNA probe constructed with DNA from phage P1. The second objective was to determine whether phage P1 transduced any indigenous soil bacteria. The third objective was to determine the specificity and sensitivity of the probe for target DNA. Nonradioactive biotin-labeled DNA probes are an attractive alternative to radiolabeled DNA probes in DNA-DNA hybridizations, as they are stable for long periods of storage and are relatively safe to handle. DNA labeled with biotin by nick translation has been successfully employed in DNADNA hybridizations with Southern blots, dot blots, and colony DNA (7, 14, 22, 25). The highly specific biotinlabeled phage P1 DNA probe was used to assess the utility of this technique for screening for phage P1 transductants in soil.
tryptone, 0.5% yeast extract, 0.5% NaCl). Lysates of bacteriophages P1 Cm cts and P1 Cm cts::TnSOl were prepared by heat induction at 42°C (15) of cultures of E. coli AB1157 lysogenic for either of the phages grown in Luria broth containing 0.1% glucose, 10 mM MgSO4 7H20, and 2 mM anhydrous CaCl2 (LCB) and filtered (0.45-p.m membrane filter; Millipore Corp., Bedford, Mass.). Titers of the lysates were determined on E. coli J53(RP4) grown in the same preparation plus 1.5% Bacto-Agar (Difco Laboratories, Detroit, Mich.) for the bottom agar and 0.75% agar for the top agar. Plaque assay plates were incubated at the permissive temperature of 42°C. Lysates of phage F116L of Pseudomonas aeruginosa were prepared, and titers of the lysates were determined as described by Miller and Ku (11). Lysates of bacteriophages 4ilda and 4i13, the T bacteriophages (including phage BF23), and P7 were kindly supplied as titered phage stocks by Jean Poindexter, Barbara Stitt, and June Scott, respectively. Isolation of phage P1 DNA. Phage P1 Cm cts::TnSOJ was purified by equilibrium centrifugation in CsCl (CsCl density, 1.47 g/ml) by the method of Mural et al. (13). The phage suspension was dialyzed overnight at 4°C against 2 liters of TM buffer (6 mM Tris, 10 mM MgSO4 7H20 [pH 7.2]) to remove the CsCl. The phage particles were lysed at 37°C for 10 min in 15 mM EDTA (pH 8.0) containing 0.1% (vol/vol) sodium dodecyl sulfate. Proteins were removed with phenol and chloroform-isoamyl alcohol and precipitated with ethanol-sodium acetate (10). The concentration and purity of the DNA were determined from the A260 and A280 (10) (model 552A spectrophotometer; The Perkin-Elmer Corp., Norwalk, Conn.). Restriction endonuclease digestion of phage P1 DNA. The DNA from phage P1 Cm cts::Tn5Ol was digested with 10 U of HindlIl per ,ug of DNA in React 2 buffer (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) at 37°C for 1 h, and then HindtIl was heat inactivated at 60°C for 5 min. The anticipated four fragments, designated HindIII-1, -2, -2', and -3, were obtained (1). The HindIll 3 fragment, which contained the region coding for the repA gene (1.2 kilobase pairs [kbp]) of phage P1, was used for construction of the DNA probe. Agarose gel electrophoresis of DNA. Preparative gel electrophoresis was conducted on 1% agarose gels at 2 V/cm (3 mA/cm) for 17 h in TAE buffer (40 mM Tris-acetate, 2 mM
MATERIALS AND METHODS Bacteria, plasmids, and bacteriophages. Bacteria were grown overnight at 30°C, with shaking, in Luria broth (1.0% *
Corresponding author. 661
662
ZEPH AND STOTZKY
EDTA). DNA from phage lambda digested with HindIII and EcoRI (International Biotechnologies, Inc., New Haven, Conn.) was used for DNA size standards. The gels were photographed with Polaroid 667 film and a 302-nm UV light source (model TS-15; UVP Inc., San Gabriel, Calif.). Individual bands of DNA were recovered from the gels by electroelution (100 V) into dialysis bags (10). Construction of the DNA probe from phage P1 DNA. The HindIll 3 fragment was labeled by nick translation with biotinylated dATP, according to the specifications of the manufacturer for the nick translation system (Bluegene Nonradioactive Nucleic Acid Detection System, Bethesda Research Laboratories). The biotin-labeled DNA was recovered by precipitation in ethanol-sodium acetate as described above. Isolation of bacterial colonies from soil. Phage P1 Cm cts or P1 Cm cts::TnSOJ and E. coli W3110(R702) or J53(RP4) were added to nonsterile soils, the characteristics and preparation of which have been described elsewhere (24), in LCB at 106 or 108 PFU and 105 to 106 CFU/g of equivalent oven-dried soil, respectively. Presumptive P1 transductants of E. coli were isolated by plating the 1:1,000 dilution of the inoculated soils on MacConkey agar (MAC) containing 75 pLg of CAM per ml. This medium is selective for CAM-resistant gramnegative bacteria and differential for lactose-utilizing bacteria (essentially for the added E. coli, as most soil bacteria are lactose negative). Presumptive transductants of indigenous bacteria were isolated by plating the 1:100 dilution of soils inoculated with phage P1 Cm cts::Tn5OJ on MAC containing 30 FM HgCl2 or on MAC with CAM. Phage P1 was also heat induced to confirm presumptive E. coli and indigenous bacterial transductants isolated from soil (24). Lysis of bacteria and phages on nitrocellulose filters. Colonies of presumptive P1 transductants of E. coli W3110(R702) and J53(RP4) were purified by streak plating on MAC with HgCl2 or CAM. After purification, dot blots of 18-h cultures of E. coli and other bacteria (108 CFU/ml) and of bacteriophages (108 to 109 PFU/ml) used in testing the specificity of the DNA probe were prepared by spotting two 5-,u samples of the bacteria and four 5-rl samples of the bacteriophages onto a nitrocellulose filter (HATF 08225; Millipore) and allowing the samples to air dry between applications. Colonies of gram-negative bacteria isolated from soil were transferred directly to nitrocellulose filters by placing the filters over the colonies on the surface of the appropriate dilution plates. Positive control dot blots of E. coli J53(RP4)(P1 Cm cts), prepared as described above, were placed on areas of the nitrocellulose filters that contained no colonies of soil bacteria. The procedure of Haas and Fleming (7) was used for the lysis of both colony and dot blot preparations. The filter was immersed in 0.5 M NaOH at room temperature for 15 min, placed for 15 min on Whatman no. 2 paper (Whatman, Inc., Clifton, N.J.) saturated with 0.5 M Tris (pH 8.0), and washed three times (1 min each wash) in 30 ml of ice-cold 50 mM Tris (pH 8.0). Any intact cells remaining on the filter were lysed with 30 ml of ice-cold lysing solution (1.5 mg of lysozyme per ml and 25% sucrose [wt/vol] in 50 mM Tris [pH 8.0]) at 4°C for 10 min. The filter was warmed for 2 min at 37°C in two changes (30 ml each) of SSC (lx SSC is 150 mM NaCl and 15 mM sodium citrate, pH 7.2) and then treated for 1 h at 37°C with 30 ml of SSC containing 200 ,ug of proteinase K (International Biotechnologies, Inc.) per ml. The filter was then washed twice in 30 ml of SSC for 1 min each time and twice in 90% ethanol for 2 min each time. After 30 min of air drying, the filter was gently swirled in 50 ml of 25:24:1
APPL. ENVIRON. MICROBIOL.
phenol-chloroform-isoamyl alcohol (vol/vol/vol; phenol was equilibrated with deionized H20) and then washed twice in 25 ml of chloroform for 20 min each time. After being air dried for 30 min, the filter was baked at 80°C for 1 h under vacuum and stored under vacuum at room temperature. An alternative procedure for the deproteinization of colony DNA from soil bacteria was also evaluated (F. Rafii and D. Crawford, personal communication) in order to reduce the amount of nonspecific color development often associated with the use of biotin-labeled DNA probes. After treatment with NaOH and lysozyme as described above, the filter was placed in 200 ml of 3x SSC containing 0.1% sodium dodecyl sulfate, and colony debris was removed by gently rubbing the filter while wearing gloves. This was repeated with fresh 3x SSC-0.1% sodium dodecyl sulfate, and the filter was incubated for 1 h at 37°C in 25 ml of 6x SSC containing 0.5 mg of proteinase K per ml and 0.1% sodium dodecyl sulfate, washed twice in 100 ml of 5 x SSC, and baked as described above. Binding of purified DNA to nitrocellulose. DNAs from phage P1 Cm cts::Tn5OI and phage lambda (International Biotechnologies, Inc.) were diluted separately in Tris-SSC, and 0, 20, 50, 100, and 200 pg of DNA were individually applied in 5-,u volumes to a nitrocellulose filter. The DNA denaturation and baking procedure of Maniatis et al. (10) was used to bind the purified DNA to the nitrocellulose filter. Hybridization conditions. Prehybridization, hybridization, and visualization of the biotin-labeled DNA probe were conducted according to the specifications of the manufacturer. A concentration of 50 or 100 ng of biotinylated HindIII-3 DNA per ml was used for colony and dot blot hybridization. The purified lambda and P1 DNA samples were hybridized with 100 ng of biotinylated-lambda DNA (HindIll-digested lambda DNA; Bethesda Research Laboratories) and biotinylated HindIII-3 DNA per ml, respectively. Colorimetric visualization of the streptavidin-alkaline phosphatase conjugate bound to the biotin-labeled DNA normally occurred in less than 30 min. RESULTS DNA analysis and restriction endonuclease digestion of P1 DNA. Phage P1 DNA of high purity was isolated, as evident from an A26JA280 ratio of 1.9. The HindIll digestion of P1 DNA yielded a restriction pattern of four fragments of 59.8, 29.8, 9.2, and 1.2 kbp (the latter was designated HindIII-3), identical to that reported by Bachi and Arber (1). At the agarose concentration used for Fig. 1, the 59.8- and 29.8kilobase fragments could not be distinguished. However, the HindIll 3 fragment clearly separated from the larger fragments on preparative gels, from which it was isolated and used to prepare the biotin-labeled DNA probe. Probe sensitivity and cross-hybridization. Known amounts of DNA (0 to 200 pg) isolated from phage P1 Cm cts::Tn5OJ were hybridized with the biotinylated HindIII-3 DNA probe (100 ng/ml) to assess probe sensitivity (Fig. 2). As little as 50 pg of target DNA from P1 was detected, whereas with phage lambda, the hybridization signal was visible with 20 pg of lambda target DNA. No color development was detected in controls containing buffer only. The cross-reactivity of the biotinylated HindIII-3 probe prepared from P1 DNA was assessed with DNA from several bacteria and bacteriophages prepared as dot blots. No cross-reactivity was evident with P1 DNA on dot blots from 21 of 22 bacteria of unknown DNA homology to the HindIII-3 probe, as only Klebsiella pneumoniae DNA gave a
VOL. 55, 1989
USE OF DNA PROBE TO DETECT BACTERIA
663
12 3 4 5 A
B Y~
C,
t
E F
FIG. 1. Agarose gel electrophoresis of HindIll-digested DNA from phage P1 Cm cts::TnSOl (right lane). The left lane contains DNA size standards, in kilobase pairs (kbp), from EcoRI- and HindIII-digested DNA from phage lambda (21.0, 5.1, 4.9, 4.2, 3.5, 2.0, 1.9, 1.5, 1.3, 1.0, and 0.8 kbp). The 1.2-kbp HindIII-3 band migrated near the 1.3-kbp phage lambda band.
slight hybridization signal (light pink rather than the characteristic blue; Fig. 3A). From nine bacteriophages other than P1, only DNA from bacteriophage F116L of P. aeruginosa hybridized weakly with the probe (Fig. 3B). No crossreaction of the probe with purified phage lambda DNA was observed (data not shown). Hybridization of the biotinylated HindIII-3 DNA probe with presumptive phage P1 transductants. Dot blots prepared from presumptive P1 Cm cts::Tn5OJ transductants of E. coli J53(RP4) and from P1 Cm cts transductants of E. coli W3110(R702) isolated from soil hybridized with the HindIll3 DNA probe, whereas no hybridization was observed with nearly 300 indigenous soil bacteria presumed to be transductants on the basis of phenotype (i.e., gram negative, lactose negative, and resistant to 30 ,uM HgCl2 or 75 ,ug of CAM per ml) (Table 1). These isolates also did not produce phage P1 after induction at 42°C (24). Dot blots of presumptive P1 transductants of E. coli W3110(R702) hybridized with as little as 50 ng/ml of HindIII-3 probe DNA (Fig. 2). Colony DNA from the indigenous gram-negative soil bacteria that was deproteinized by the method of Rafii and Crawford pi
:
LAMIDA DNA
:
FIG. 3. Specificity of the biotinylated HindIII-3 DNA probe as assessed by dot blot hybridization with DNA from various bacteria and bacteriophages. Three presumptive P1 transductants of E. coli W3110(R702) were individually isolated from soil (D5, El, and E2). The probe concentration during hybridization was 50 ng/ml. The bacteria and bacteriophages were obtained from our culture collection except where noted in Materials and Methods. Boxes contained the following bacteria, bacteriophages, and lysogens: Alcaligenes faecalis (Al), Salmonella pylorum (A2), Staphylococcus saprophyticus (A3), Bacillus cereus (A4), E. coli B (A5), Micrococcus agilis (Bl), Agrobacterium radiobacter (B2), Bacillus megaterium (B3), Bacillus subtilis (B4), Staphylococcus aureus (B5), Acinetobacter anitratum (Cl), Proteus mirabilis (C2), K. pneumoniae (C3), Pseudomonas fluorescens (C4), Shigella boydii (CS), Staphylococcus epidermidis (D1), Arthrobacter globiformis (D2), Enterobacter aerogenes (D3), Enterobacter cloacae (D4), E. coli W3110(R702)(Pl Cm cts) transductant (D5), E. coli W3110(R702)(Pl Cm cts) transductant (El), E. coli W3110(R702)(Pl Cm cts) transductant (E2), E. coli HB101(pBR322) (E3), P. aeruginosa PAT2 (E4), E. coli J53 (E5), no sample (Fl), P7 (F2), F116L (F3), (lda (F4), 413 (F5), no sample (Gl through G5), BF23 (Hl), T4 (H2), Tl (H3), T5 (H4), T6 (H5), no sample (I1), E. coli AB1157(Pl Cm cts) (12), no sample (I3 through I5).
(personal communication) did not exhibit hybridization with the probe; however, there was virtually no nonspecific color development as a result of cellular residue on the filter, as frequently occurred with the procedure of Haas and Fleming
(7). E
100
TABLE 1. Verification by heat induction of lysis and with the biotinylated DNA probe of presumptive transductants of E. coli and indigenous soil bacteriaa
so
No. positive/no. tested
z
Presumed transductant
a 20
Soil
Heat induction of phage plb
Nonsterile Sterile Nonsterile Sterile Nonsterile
0/297 15/15 8/8 13/13 25/25
Hybridization
with P1 DNAc
0
Indigenous
0
E. coli W3110(R702)
2. DNA hybridization of a biotinylated lambda DNA probe purified phage lambda DNA (lane Lambda DNA) and a biotinylated Hindlll-3 DNA probe with purified phage P1 Cm FIG.
with
cts::Tn5OJ probes
was
DNA (lane P1
100
ng/ml.
DNA). The concentration of both DNA
E. coli J53(RP4)
HgCl2
on MAC containing either (30 aIsolated b For methods, see reference 24. c Colonies on dot blots. ND, Not determined.
0/297 ND 8/8 ND 7/7
p.M) or CAM (75 FLg/ml).
664
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ZEPH AND STOTZKY
DISCUSSION Successful DNA-DNA hybridization depends on the use of a DNA probe that has a unique nucleotide sequence in order to prevent nonspecific binding of the probe to nontarget DNA. The genes located in the immunity (or incompatibility) region of the genome of phage P1 include repA, which functions in the replication of the independent prophage
replicon and, therefore, should contain DNA that does not share homology with replication genes found in other plasmid incompatibility groups. Phage P1 is a member of incompatibility group Y, which includes plasmid pIP231 (3), a cryptic plasmid from E. coli 15 (9), and phage P7 (18). The closely related phage P7 is not thought, however, to share homology with P1 in the HindIII 3 fragment (23), as was also shown in this study. The biotinylated HindIII-3 DNA probe was sensitive enough at a concentration of 100 ng/ml to give detectable color development with as low as 50 pg of purified P1 DNA. Zwadyk et al. (25) reported a similar minimum sensitivity with a 1-kbp biotinylated DNA probe for a beta-lactamase gene, using the same commercial kit for DNA labeling and probe visualization. The lambda DNA probe used as a control was more sensitive, probably as a result of its higher molecular mass. Dot blots with target DNA from E. coli lysogenic for P1 confirmed the observation of Yang (22) that biotinylated DNA probes are capable of hybridization with as few as
105
to 106 bacterial cells on the filter.
Nonspecific binding of the streptavidin-alkaline phosphatase conjugate to cellular components other than DNA is a frequent problem in the utilization of biotinylated DNA probes with target DNA obtained from crude bacterial preparations. This was evident in the present study on hybridization filters that had not undergone sufficient deproteinization after cell lysis. Deproteinization of lysed bacteria and bacteriophages on dot blot preparations was conducted with the method of Haas and Fleming (7). However, for colony DNA hybridization, which was used in the screening of some indigenous bacteria for potential P1 lysogens, the procedure of Rafii and Crawford was more consistently successful in limiting the amount of nonspecific color development (personal communication). Nevertheless, a weak hybridization signal with nontarget DNA is not necessarily undesirable, as it facilitates matching the pattern of colonies on the filter with the dilution plate from which the colonies were obtained. Dot blot hybridizations with DNA from pure cultures of several bacteria and bacteriophages demonstrated that the specificity of this probe was satisfactory for detecting P1 DNA. The lack of hybridization of the HindIll3 probe with colony DNA from numerous soil bacteria indicated that cross-hybridization with this probe was not a problem with bacteria isolated from the soils used in this study. No phage P1 transductants of indigenous bacteria were detected with the P1-specific probe or by heat induction of phage lysis. Although the genera Enterobacter and Klebsiella are potential non-E. coli hosts for transduction by phage P1 in soil, only P1-sensitive mutants of these genera are infected by this phage (4, 6), and the probability of transduction is dependent on the numbers of these phage-sensitive strains present in soil. Therefore, if P1-transduced indigenous bacteria were present in these soils, they were below the detectable limit of 102 transductants per g of soil (oven dried), as the 1:100 soil dilution plates were used as the source of colonies for hybridization. No indigenous E. coli was evident at these dilutions, as there were no lactose-
positive colonies on MAC. As the biotic and physicochemical characteristics of soil affect phage survival (19, 21), more soil types and environmental conditions should be tested for their effects on transduction of indigenous soil bacteria, as it is important to determine whether novel genes can be transferred to indigenous bacteria. The biotin-labeled probes prepared from P1 DNA were employed successfully to confirm that strains of E. coli added to soil with transducing phage P1, either as lysates or in lysogens, were lysogenized by this phage (24). The sensitivity and long storage life of the biotinylated DNA probe and its low background hybridization signal made it a convenient alternative to radiolabeled DNA probes. The detection of any P1-transduced indigenous soil bacteria with this DNA probe would require a selective medium that would allow plates prepared from lower soil dilutions to be used for colony hybridization, an enrichment technique that would allow the number of P1-transduced bacteria to increase selectively, preparation of purified DNA from soil bacteria for use in Southern blot analysis (8) for the detection of the phage P1 repA gene sequence, or amplification of phage P1 target DNA sequences through the polymerase chain reaction technique (16). ACKNOWLEDGMENTS These studies were supported, in part, by cooperative agreements CR812484, CR813431, and CR813650 between the U.S. Environmental Protection Agency Corvallis Environmental Research Laboratory and New York University. The able assistance of Xiaoyan Lin is appreciated. LITERATURE CITED 1. Bachi, B., and W. Arber. 1977. Physical mapping of
BglIl,
BamHI, EcoRI, HindIII, and PstI restriction fragments of bacteriophage P1 DNA. Mol. Gen. Genet. 153:311-324. 2. Baross, J. H., J. Liston, and R. Y. Morita. 1974. Some implications of genetic exchange among marine vibrios, including Vibrio parahaemolyticus, naturally occurring in the Pacific oyster, p. 129-137. In T. Fujino, G. Sakaguchi, R. Sakazaki, and Y. Takeda (ed.), International symposium on Vibrio parahaemolyticus. Saikon Publishing Co., Ltd., Tokyo. 3. Briaux, S., G. Gerbaud, and A. Jaffe-Biachet. 1979. Studies of a plasmid coding for tetracycline resistance and hydrogen sulfide production incompatible with the prophage P1. Mol. Gen. Genet. 170:319-325. 4. Gerhardt, P., R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Philips (ed.). 1981. Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. 5. Germida, J. J., and G. Khachatourians. 1988. Transduction of Escherichia coli in soil. Can. J. Microbiol. 34:190-193. 6. Goldberg, R. B., R. A. Bender, and S. L. Streicher. 1974. Direct selection for P1-sensitive mutants of enteric bacteria. J. Bacteriol. 118:810-814. 7. Haas, M. J., and D. Fleming. 1986. Use of biotinylated DNA probes in colony hybridization. Nucleic Acids Res. 14:3976. 8. Holben, W. E., J. K. Jansson, B. K. Chelm, and J. M. Tiedje. 1988. DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl. Environ. Microbiol. 54:703-711. 9. Ikeda, H., M. Inuzuka, and J. Tomizawa. 1970. P1-like plasmid in E. coli 15. J. Mol. Biol. 50:457-470. 10. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 11. Miller, R. V., and C.-M. C. Ku. 1978. Characterization of Pseudomonas aeruginosa mutants deficient in the establishment of lysogeny. J. Bacteriol. 134:875-883. 12. Morrison, W. D., R. V. Miller, and G. S. Sayler. 1978. Frequency of F116-mediated transduction of Pseudomonas aerug-
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18. 19.
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