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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1991, p.

2873-2879

Vol. 57, No. 10

0099-2240/91/102873-07$02.00/0 Copyright C 1991, American Society for Microbiology

Effect of 2-Hydroxybenzoate on the Maintenance of NaphthaleneDegrading Pseudomonads in Seeded and Unseeded Soil
0. A. OGUNSEITAN, I. L. DELGADO, Y.-L. TSAI, AND B. H. OLSON* Environmental Analysis and Design, Program in Social Ecology, University of California, Irvine, California 92717
Received 20 February 1991/Accepted 12 July 1991

The addition of specific nontoxic inducers of catabolic operons to contaminated sites is an approach that may enhance the efficiency of in situ biodegradation. We determined the genetic response of six pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of available carbon, and their DNA sequences show significant homology to the nahAB genes of the degradative plasmid NAH7. Duplicate nonsterile soil cultures were incubated for up to 30 days. Experimental soil cultures were seeded with naphthalene-degrading strains (108 CFU g-1) originally isolated from the soil and amended with salicylate (16 or 160 ,ug g-). Soil samples were analyzed periodically for the population density of heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype in the bacterial community. At 160 ,ug g1, salicylate sustained the density of naphthalene degraders at the introduced density for 30 days in addition to producing a two- to sixfold increase in the occurrence in the bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 ,ug of salicylate g-1, but this concentration of salicylate induced a significant increase in the level of nah-related genes in the population.

Biodegradative genotypes are abundant in microbial communities that colonize polynuclear aromatic hydrocarbon (PAH)-contaminated environments, and bacterial degradation of both substituted and unsubstituted PAHs has been studied in detail (2, 5). However, the complete removal of these compounds from contaminated sites in situ has yet to be demonstrated. Many reasons have been proposed for the inefficiency of bioremediation in situ (3, 4). Two approaches to improve environmental bioremediation have been well demonstrated: (i) the deliberate release of degradative bacteria into contaminated environments, and (ii) the nonspecific stimulation of autochthonous bacterial populations with nitrate, phosphate, and oxygen. Our laboratory has proposed a third approach that uses specific inducers of genetic operons to enhance the degradation of toxic compounds (7). This approach offers two direct benefits to bioremediation: increased specificity of the target population to be stimulated, and predictability of intermediates and end products because known degradative pathways are stimulated. Salicylate (also known as 2-hydroxybenzoate) has been identified as an intermediate in the naphthalene degradation pathway and as an inducer of both the upper (nah) and lower (sat) operons carried on the NAH7 plasmid for the degradation of naphthalene and salicylate, respectively (8). This study examines the effect of salicylate, as an inducer of genetic expression, on the maintenance of degradative phenotypes and genotypes in a soil bacterial community. Nucleic acid hybridization methods developed for direct and indirect determinations of genotype distribution in natural environmental samples were used to characterize the maintenance of reintroduced natural bacteria in soil.
MATERIALS AND METHODS Soil source. The test soil was obtained from a manufactured-gas plant site (active between 1903 and 1916), located
*

Corresponding author.
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0.6 km from the ocean, in Venice, Calif. The soil samples came from the lamp black sump of the defunct plant and therefore contain a variety of PAHs including phenanthrene, pyrene (benzo[deA]phenanthrene), methylnaphthalene, and naphthalene. The total concentrations of PAHs measured in the dry soil and in the oil-water slurry were 7.07 ,ug g-1 and 9.4 jig ml-', respectively. The concentrations of naphthalene in the soil and in the oil-water scum were 0.043 ug g-1 and 0.247 ,ug ml-', respectively (gas chromatography-mass spectrometry analyses performed by Global Geochemistry Corp., Canoga Park, Calif.). The water content of the sandy soil was 10% at the time of sampling, and the cation exchange capacity was 0.09 meq g-1. Soil samples were aseptically collected in stainless steel vessels equipped with Teflon caps and kept at 4C until needed. Media and growth conditions. Bacteria were isolated by serial dilution of the soil in phosphate-buffered saline (PBS). Heterotrophic bacterial population density was determined on half-strength plate count agar (Difco, Detroit, Mich.). The population density of bacteria able to utilize naphthalene as the sole carbon source was determined on minimal salts medium (MMN) containing (wt/vol) 0.4% NaNO3, 0.015% KH2PO4, 0.05% Na2HPO4, 0.02% MgSO4 7H20, 0.00005% FeCl3 6H20, and 0.001% CaC12 .2H20. The medium was solidified With purified agar (Beckton Dickinson, Cockeysville, Md.) and exposed to naphthalene vapor from pure crystals (Sigma Chemical Co., St. Louis, Mo.). To further confirm that bacteria that formed colonies on minimal medium with naphthalene vapor actually degraded naphthalene, we inoculated the colonies into MMN broth cultures containing 2-hydroxybenzoate or naphthalene as the sole source of carbon. After the inoculated broths became turbid, cells were removed by centrifugation and the supernatant was scanned at 220 to 420 nm. Degradation of naphthalene was confirmed by observing the production of a metabolite with maximum absorbance at 310 nm (2). Broth cultures of selected bacteria were grown in minimal medium supplemented with 2.0 mM sodium succinate (MMS). Bacterial

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(A)

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1-.g

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fluorescens

(lanes

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putida (lanes i to s),strains isolated from PAH-contaminated soil used in this study. Lanes a and t represent plasmid size markers. (B) Autoradiograph of hybridization of DNA molecules shown in panel A to a radiolabeled nahAB probe. Lane f contains the positive control strain PpG7(NAH7).

colonies

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incubation at 23C. Identification of bacteria. Bacterial colonies that grew on MMN were purified and identified with the Analytical Profile Index rapid NFT system (Analytab, Plainview, N.Y.). The selected bacteria were screened for the presence of plasmids (1), and genetic homology to the first two genes of the naphthalene degradation operon, nahAB [cloned from Pseudomonas putida PpG7(NAH7), a gift from B. Ensley, Amgen Corp., Thousand Oaks, Calif.], was determined through colony hybridization to 32P-labeled nahAB genes. Among the strains isolated from the contaminated soil, six pseudomonads were chosen for further study because of the high intensity of DNA hybridization to the nahAB genes. The strains were identified as P. putida VNM5, VNM11, VNM43, and VNM45 and P. fluorescens V01 and V22. These strains contain plasmids that bear molecular resemblance through size and hybridization relationships to the NAH7 of P. putida PpG7(NAH7), ATCC 17485 (Fig. 1). Soil culture experimental design. Soil cultures contained 50 g of subsurface soil from the contaminated site in loosely capped cylindrical polyethylene 250-ml culture flasks (Nalgene model; internal diameter, 6 cm; height, 10 cm). Duplicate sets of flasks were inoculated with individual strains to a final cell density of approximately 108 CFU of mid-logphase cultures (grown in MMS and washed in PBS) per g of nonsterile soil. All experimental (seeded) and blank (unseeded) soil cultures were allowed to equilibrate at 23C for

1 h with intermittent mixing, for even distribution of bacteria in the soil. For each strain, a solution of 2-hydroxybenzoate (sodium salt; Fisher Scientific, Fairview, N.J.) was added to a duplicate set of soil cultures to obtain final concentrations of 16 or 160 ,ug per g of soil. A third set of soil cultures was inoculated with individual bacterial strains without 2-hydroxybenzoate. An uninoculated set of nonsterile soil cultures was also studied that contained 0, 16, or 160 ,ug salicylate per g of soil. Salicylate solutions or sterile water was added to the soil cultures in a final liquid volume of 1 ml. The soil cultures were incubated at 23C in a humiditycontrolled chamber (Lunaire Environmental Inc., Williamsport, Pa.). Soil samples (2 g) were collected after 0, 1, 3, 5, 7, 15, and 30 days of incubation for determination of bacterial densities and at 1 and 30 days for nucleic acid extractions. Direct extraction of bacterial DNA from soil. A simplified freeze-thaw method was used for extracting bacterial DNA from 1.0-g subsamples of soil (10). The advantage of this method over traditional direct-extraction techniques is that only a small amount of soil (1 g) is needed. Briefly, bacteria were dislodged from soil particles by thorough washing of 1.0 g of soil three times in 0.12 M phosphate buffer (pH 8.0). The soil slurry was then incubated in a 1.5% lysozyme solution containing 0.15 M NaCl and 0.1 M EDTA (pH 8.0) at 37C for 3 h with occasional agitation. The soil samples were then transferred onto ice, and a 10% sodium dodecyl sulfate (SDS) solution (pH 8.0) was added; this was followed by three freeze-thaw cycles in dry ice-ethanol (5 min) and 65C water (10 min) baths. The soil was then extracted with Tris-buffered phenol. An aliquot of the aqueous phase was extracted once with a 1:1 solution of phenol and (chloroform-isoamyl alcohol [24:1]) and once with chloroformisoamyl alcohol (24:1). Nucleic acids were pelleted from the aqueous phase with an equal volume of cold isopropanol at -200C. Direct extraction of bacterial mRNA from soil. The acid guanidine thiocyanate extraction method (11) was used to isolate bacterial transcripts directly from 10.0 g of soil. The method consists of washing 10-g soil samples twice with 0.12 M phosphate buffer (pH 5.2) containing 1.0% diethyl pyrocarbonate. The soil was then incubated, for 1 min with constant agitation, with a 4.0 M solution of guanidine thiocyanate containing 0.025 M sodium citrate, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. The slurry was extracted once with a 1:1 mixture of phenol and chloroform-isoamyl alcohol (24:1) and once with chloroform-isoamyl alcohol (24:1). The RNA was precipitated with isopropanol at -20C. The pellets were dissolved in 0.5% SDS and treated with DNase before gel electrophoresis or hybridization. Nucleic acid hybridizations. Nylon membrane discs (Biotrans; ICN, Irvine, Calif.) were used for colony lifts, and nitrocellulose membranes (Schleicher & Schuell, Keene, N.H.) were used for hybridizations involving nucleic acids bound directly to membranes by using a microfiltration manifold (Schleicher & Schuell). Hybridization membranes were processed as recommended by the manufacturer. The DNA fragment containing nahAB was excised from its vector by restriction with EcoRI and HindlIl. Agarose gel-purified nahAB fragments were labeled with 32P by random priming (BRL, Bethesda, Md.). The specific activity of radiolabeled DNA was greater than or equal to 108 dpm ,g in all cases. Hybridization of 1.0 pLg of extracted DNA to radiolabeled nahAB was carried out at 42C in 50% formamide solution, followed by high-stringency washes. For colony hybridizations, X-ray autoradiography was used

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EFFECT OF SALICYLATE ON NAPHTHALENE-DEGRADING BACTERIA

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Time (d) Time (d) FIG. 2. Bacterial population densities in soil microcosms. Heterotrophic (A) and naphthalene-degrading (B) bacterial population densities respectively, in uninoculated soil. Heterotrophic (C) and naphthalene-degrading (D) bacteria population densities, respectively, in soil inoculated with 108 CFU of P. putida PpG7(NAH7) per g. Salicylate was added to the soil samples at 16 (0) or 160 (A) ,ug/g. Soil samples were also incubated without salicylate (O).

to detect signals from colonies that contained nahAB sequences. For quantifying the occurrence of nahAB sequences in purified DNA extracted from the soils, the radioanalytic imaging system (AMBIS, San Diego, Calif.) was used. The counts per minute retained on DNA hybrids correlated to the abundance of nah homologous DNA (r = 0.91).

duced bacteria. Changes that occurred in bacterial densities of representative soil cultures during incubation are illustrated in Fig. 2 to 4. In most cases, the addition of 160 ,ug of salicylate per g to soil sustained the bacterial density at 108 CFU g-' for at least 3 days. The majority of the sustained

RESULTS Biodegradative potential of test soil. The total heterotrophic bacterial population of the test soil, determined at the beginning of the study, was (4.47 1.9) x 106 CFU g-1. Approximately 63% of the isolated colonies were able to utilize naphthalene as the sole carbon source. Of the colonies that degraded naphthalene, 29% + 4.8% contained DNA sequences that hybridized with nahAB, suggesting a certain degree of genetic diversity in the population of naphthalene-degrading bacteria. Effect of salicylate on the survival of indigenous and intro-

bacteria represented naphthalene-degrading subpopulations as indicated by colony counts on MMN (Fig. 2B, 3B, and 4B). The specific stimulation of naphthalene degraders began to decline relative to the total bacterial population by 15 days. In the absence of salicylate, the population density of naphthalene-degrading bacteria declined to less than 105 CFU g-1. The density of P. putida PpG7(NAH7) declined from the initial 2 x 108 CFU g-1 to between 105 and 106 CFU g-1 within 30 days, except in the presence of 160 ,ug of salicylate per g of soil (Fig. 2C and D). The addition of 16 ,g of salicylate per g of soil did not produce a consistent stimulatory effect on the maintenance of naphthalene-degrading strains (Fig. 2B) or on total cell density (Fig. 3C). The fraction of the total bacteria that were

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Time (d) Time (d) FIG. 3. Bacterial population densities in soil microcosms that were inoculated with P. putida VNM11 (A and B) and VNM43 (C and D). Panels A and C represent bacterial population densities enumeratvd on plate count agar. Panels B and D represent population densities enumerated on MMN. Salicylate was added to the soil samples at 16 (0) or 160 (A) pg/g. Soil samples were also incubated without salicylate (O).

able to degrade naphthalene (calculated as the number of colonies enumerated on MMN plates divided by the number of colonies enumerated on plate count agar plates) exhibited strain- and incubation period-dependent variation. The addition of salicylate to soil at either concentration did not significantly affect the proportion of bacteria that expressed the degradative phenotype. Effect of salicylate on the maintenance of the nah genotype in test soil. The proportion of total colonies which degraded naphthalene and contained DNA sequences homologous to nahAB was determined by colony hybridization (approximately 250 colonies were probed for each experiment). The proportion of the heterotrophic population isolated on nonselective media that hybridized to nahAB was lower than the fraction that exhibited phenotypic naphthalene degradation. Although the latter represented between 80 and 100% of the population during the 5- to 7-day period for all soil cultures that were amended with salicylate, the proportion of total colonies that hybridized to nahAB fluctuated between 5 and 80% (data not shown). This may be due to molecular

divergence of the nah operon in the environmental strains, but further investigation of DNA sequence similarities under different hybridization stringencies will be necessary to fully characterize the level of divergence of the naphthalenedegradative operons in the strains found in the soil. Effect of salicylate on the density of nahAB genes in DNA extracted from soil. The electrophoresed nucleic acid molecules extracted from soil culture samples are represented in Fig. 5A. Extensive shearing of the DNA did not occur, and the linear molecules were about 23 kb. The DNA extracted from the soils after a 1-day incubation contained a significant number of RNA molecules (Fig. 5A, panel 1). These RNA molecules were absent in the extractions performed after a 30-day incubation, suggesting a decline in overall metabolic activity of the soil organisms through the 30-day incubation period (Fig. 5A, panel 2). The intensity of autoradiographic signals from DNA-DNA hybrids obtained after probing with nahAB is shown in Fig. SB. The density of nah genes per unit quantity of soil increased in response to salicylate concentration (Table 1). There were three major types of response.

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Time (d) Time (d) FIG. 4. Bacterial population densities in soil microcosms that were inoculated with P. fluorescens V22 (A and B) and V01 (C and D). Panels A and C represent bacterial densities enumerated on plate count agar, whereas panels B and D represent densities enumerated on MMN. Salicylate was added to the soil samples at 16 (0) or 160 (A) ,ug/g. Soil samples were also incubated without salicylate (O).

(i) The uninoculated soil showed no detectable response to the addition of either concentration of salicylate after 1 and 30 days of incubation. (ii) The P. putida strains typified by PpG7(NAH7) and VNM43 (Table 1) showed higher levels of nahAB density per gram of soil in response to 160 ,ug of salicylate per g of soil after a 30-day incubation. (iii) The response of the P. fluorescens strains (typified by the response of strain V22 [Table 1]) showed an increased intensity of DNA-DNA hybridization after a 1-day incubation in response to the concentration of salicylate. However, at the end of a 30-day incubation, the response to salicylate was not as pronounced as in the soil cultures inoculated with the P. putida strains (Table 1). Effect of salicylate on the accumulation of nah transcripts in bacteria inoculated into soil. The presence of genetic transcripts derived from nah genes was detected 30 days after three strains (VNM11, VNM43, and VNM45) were inoculated into soil. The radioactive signals from the mRNA-DNA hybrids were too low in all cases to be quantified by the radioanalytic image system. Therefore, detection was done by X-ray autoradiography (Fig. SC). In the absence of

salicylate amendment, no transcripts were detectable by hybridization. In the soils amended with 16 ,ug of salicylate per g, the occurrence of nahAB transcripts was detected only in soil inoculated with strains V22, VNM5, VNM11,
and VNM43 (Fig. SC, lane 1). In soils amended with 160 ,ug of salicylate per g of soil, nahAB transcripts were detected in

soil samples inoculated with strains V22, VNM5, VNM11, VNM43, and VNM45. The absence of detectable transcripts in soil inoculated with P. putida PpG7(NAH7) is probably due to the low cell density of this laboratory strain after a 30-day incubation in soil (Fig. 2C and D).

DISCUSSION
The data generated by this study demonstrate two points. First, naphthalene-degrading bacteria respond positively to salicylate, a known inducer of the nah operon. Hence, salicylate at certain concentrations can be used directly in contaminated soil to stimulate the maintenance of genetic activity in bacteria that have the potential to degrade naphthalene in situ. Second, methods now available to study the

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FIG. 5. (A) Electrophoresis of nucleic acids extracted directly from 1.0 g of soil samples from the microcosms after 1 day (panel 1) and 30 days (panel 2). The absence of RNA molecules from the extractions after a 30-day incubation suggests that there were lower metabolic activities at the end of the incubation period. Lane a contains DNA size markers (phage lambda restricted with HindIII). Sample loading wells are not shown. The largest DNA fragments extracted were approximately 23 kb. (B) Autoradiograph of signals from nahAB hybridization of 1.0-,ug aliquots of DNA extracted directly from soil microcosms inoculated with the following strains in order from columns a to h, or i to p: PpG7(NAH7), V22, VNM45, VNM11, VNM43, VNM45, V01, and uninoculated soil. DNA was extracted from samples taken after 1 day (row 3, columns a to h, no salicylate added; row 3, columns i to p, 16 ,ug of salicylate added per g of soil; row 2, columns a to h, 160 ,ug of salicylate added per g of soil and DNA extracted after a 30-day incubation; row 2, columns i to p, no salicylate added to soil; row 1, columns a to h, 16 ,ug of salicylate added per g of soil; row 1, columns i to p, 160 ,ug of salicylate added per g of soil). (C) Autoradiograph of signals from mRNA-nahAB hybrids from 10-,ug aliquots of total RNA extracted directly from soil after a 30-day incubation. Lanes 1 and 2 represent samples from soil that was amended with 60 and 160 ,ug of salicylate per g of soil, respectively. Bacterial inocula: row a, V22; row b, VNM11; row c, VNM43; row d, VNM45; row e, V01. Signals were detected after a 6-day autoradiographic exposure to X-ray film.

ecological impact of environmentally introduced bacteria


can also be used for comparative analyses of the performance of strains that are considered significant in environmental bioremediation or other field applications. Salicylate is a utilizable carbon source for a variety of soil microorganisms and is now shown to be able to stimulate a genetic response in a subpopulation of biodegradative bacteria. By comparing the survival and genetic response of strains that were isolated and reintroduced into a contaminated soil with those of the prototype NAH7-containing strain, it can be concluded that native strains possess certain characteristics that enable them to survive and express better in their natural habitat than exogenous strains can. The test soil had been exposed to a variety of PAHs for a protracted period; consequently, bacteria with the ability to degrade PAHs, at least naphthalene, were abundant (greater than 60% of the recoverable heterotrophic population) in the soil. Without the addition of salicylate, the bacterial density of the soil stabilized at approximately 106 CFU g-1 under the conditions of the 30-day incubation. Addition of salicylate at 16 ,ug g-1 did not have a notable effect on this stable cell

density. However, when salicylate was added to soil samples at 160 ,ug g-1, the bacterial densities in both the uninoculated soil and soil that was inoculated with naphthalene-degrading bacteria (108 CFU g-1) increased and was maintained at between 107 and 108 CFU g-' through the 30-day incubation period. The effect of salicylate on the total heterotrophic bacterial population was not distinguishable from the effect on the naphthalene degraders, probably because a high proportion of the natural soil population was able to degrade naphthalene and presumably responded to salicylate induction (Fig. 2 to 4). The effect of salicylate on the maintenance of naphthalene degraders was apparent within the first 5 days of incubation during which 80 to 100% of the heterotrophs exhibited the ability to utilize naphthalene. In the absence of sufficient salicylate per gram of soil, the naphthalene degradation phenotype declined from near 100% after 7 days to approximately 20% after 30 days in soil that was inoculated with strain PpG7. Thus, a periodic dose of salicylate could be used to maintain an active degradative phenotype in the population. The addition of a single high dose of salicylate,

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TABLE 1. Effect of salicylate on the occurrence and maintenance of nahAB genes in soil
Bacterium and salicylate concn (p.g/g of soil)

CPM/g of soila on:


day 1 day 30

Soil only 0 16 160 P. putida NAH7 0 16 160 P. putida VNM43 0 16 160 P. fluorescens V22 0 16 160

68.1 5.6 41.2 4.4 67.2 5.6 1,050.9 15.2 4,340.5 44.8 2,377.6 33.2
344.8 12.6 849.4 19.8 1,392.9 25.4

37.5 4.2 55.6 5.1 54.7 + 5.0 499.5 15.2 176.1 9.0 677.5 + 17.5
102.9 6.9 242.8 10.6 510.6 15.4

73.7 5.8 246.5 10.7 544.9 15.9

74.1 5.9 53.3 5.0 91.7 6.5

a Bacterial DNA was extracted directly from 1.0-g soil samples. Then 1.0 ,ug of the extracted DNA was probed with radiolabeled nahAB. Radioactive signals from DNA-DNA hybrids were quantified with the AMBIS system.

been designed to characterize the microbial diversity with respect to the relative abundance of specific gene sequences which might be expected to be amplified as a consequence of population invasion (9). In an attempt to quantify community response at the genetic level, DNA probes were applied to nucleic acid molecules extracted directly from the soil cultures (Fig. 5). The addition of salicylate to soil samples that were inoculated with naphthalene-degrading bacteria clearly enhanced the occurrence of the genetic determinants of naphthalene degradation in the bacterial community. Experiments to characterize the contribution of the enhanced genetic potential (abundance of genetic determinants in the population irrespective of genetic expression) to the rate of naphthalene degradation in soil have been conducted (6). The direct application of salicylate or other nontoxic inducers of genetic pathways of biodegradation into contaminated environments may be an efficient means of achieving environmental bioremediation, particularly where the degradative bacteria have evolved naturally. Furthermore, the high-density reinoculation of native bacteria which respond to specific inducers of biodegradation may prove better than the introduction of engineered or exogenous strains into contaminated environments.
ACKNOWLEDGMENTS This study was supported by research grant no. 8000-25 from the environment division of the Electric Power Research Institute (EPRI), Palo Alto, Calif.
REFERENCES 1. Anderson, D. G., and L. L. McKay. 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46:549-552. 2. Cerniglia, C. E. 1984. Microbial metabolism of polycyclic aromatic hydrocarbons. Adv. Appl. Microbiol. 30:31-71. 3. Goldstein, R. M., L. M. Mallory, and M. Alexander. 1985. Reasons for possible failure of inoculation to enhance biodegradation. Appl. Environ. Microbiol. 50:977-983. 4. Jain, R. K., and G. S. Sayler. 1987. Problems and potential for in situ treatment of environmental pollutants by engineered microorganisms. Microbiol. Sci. 4:59-63. 5. Mueller, J. G., P. J. Chapman, and P. H. Pritchard. 1989. Action of fluoranthene-utilizing bacterial community on polycyclic aromatic hydrocarbon components of creosote. Appl. Environ. Microbiol. 55:3085-3090. 6. Ogunseitan, 0. A., and B. H. Olson. Unpublished data. 7. Olson, B. H., and R. A. Goldstein. 1988. Applying genetic ecology to environmental management. Environ. Sci. Technol. 22:370-372. 8. Scheli, M. A. 1985. Transcriptional control of the nah and sal hydrocarbon degradation operons by the nahR gene product. Gene 36:301-309. 9. Torsvik, V., K. Salte, R. Sorheim, and J. Goksoyr. 1990. Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria. Appl. Environ. Microbiol. 56:776781. 10. Tsai, Y.-L., and B. H. Olson. 1991. Rapid method for the direct extraction of DNA from soils and sediments. Appl. Environ. Microbiol. 57:1070-1074. 11. Tsai, Y.-L., M. J. Park, and B. H. Olson. 1991. Rapid method for direct extraction of mRNA from seeded soils. Appl. Environ. Microbiol. 57:765-768.

however, maintained the degradative phenotype around the inoculum density throughout the 30-day incubation period. The native P. putida strains showed a similar but less drastic response to salicylate compared with that of strain PpG7. In the unseeded soil, the lower concentration of salicylate produced a higher proportion of naphthalene degraders. This may be because the higher concentration of salicylate also provided a general nutrient source from breakdown products for nondegraders. The effect of salicylate on the fraction of the naphthalenedegrading population that contain DNA sequences which hybridize to nahAB was more dependent on the concentration of salicylate added to the soil than on the type of strain. The population of the nah-containing colonies fluctuated considerably in the absence of sufficient salicylate. When salicylate was added at 160 jig per g of soil, a distinct increase in the proportion of nah-containing clones became apparent after a 15 day incubation, reaching near 100% for all the strains and for the uninoculated soil. The colony hybridization data suggest a certain degree of evolutionary divergence of the naphthalene-degradative genes present in the natural test soil. This is supported by the evidence presented in Fig. 1, in which only 7 of 18 naphthalene degraders from the soil exhibited significant homology to nahAB (at the hybridization stringency of 95%). The limitation of the colony hybridization technique for assessing genotype maintenance, however, is that the colonies that were examined must be abundant enough in the soil to be detected by the plating technique and must be capable of forming independent colonies. For this and other reasons, several investigators have pursued alternative methods for direct assessment of bacterial genotypes in environmental samples. The studies that have been conducted on the direct extraction of nucleic acids from environmental samples have

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