varied resonses in gene expression of culturable heterotrophic bacteria isolated from the...

Appl Microbiol Biotechnol (1992) 37:818-824 @pried

Microb/o bgy Biotechnology © Springer-Verlag 1992

Varied responses in gene expression of culturable heterotrophic bacteria isolated from the environment

Christoph C. Tebbe*, Oladele A. Ogunseitan, Paul A. Rochelle, Yu-Li Tsai, and Betty H. Olson

Environmental Analysis and Design Division, School of Social Ecology, University of California, Irvine, CA 92717, USA

Received 19 September 1991/Accepted 27 May 1992

Summary. Gene expression in heterotrophic bacteria isolated from environmental samples was studied using a combination of non-selective and selective plating techniques and gene probe methodology. The gene probes and their respective phenotypes were nahAB, for naphthalene degradation, merA, for narrow-spectrum mercury resistance, and merB, for broad-spectrum mercury resistance. Gene-probe-positive organisms could be placed into one of three categories: (1) organisms that could express their genetic information immediately upon isolation from the environment; (2) organisms that expressed their genotype only after cultivation before selection for the genotype; and (3) organisms that did not express their genotype at all in our hands. For all three probes it was found that most organisms fell into cate- gory 2. This phenomenon was also observed with bacteria isolated from lake water that probed positive with the nitrogenase (nifHDK) gene probe. The data suggest that the numbers of isolates identified by gene probes merely reflect the genetic potential of a community whereas various expression data suggest that differences in the actual activity of those genotypes exist in the natural environment.

Introduction

Assessing the occurrence of specific capabilities in environmental microbial communities is often restricted to evaluating phenotypic traits of isolated microorganisms under laboratory conditions, e.g. on selective media (Mahler et al. 1986) or through measurement of catabolic products in reaction vessels (Nakamura et al. 1990; Garcia-Valdez et al. 1988). Anticipating that such an ap- proach might be insufficient in judging the actual capabilities of microbial consortia in situ, numerous studies

* Present address: Department of Soil Biology, FAL, Bundesallee 50, 3300 Braunschweig, Federal Republic of Germany

Correspondence to: B. H. Olson

have assessed the density of genetic determinants using gene probe technology (Sayler and Layton 1990). Speci- fically, genotype occurrences involved in adaptation to pollutants have been investigated. These studies have included heavy metal resistance in bacteria (Barkay et al. 1985, 1989; Diels and Mergeay 1990; Rochelle et al. 1991), and bacterial degradation of organic compounds such as toluene (Sayler et al. 1985; Jain et al. 1987), naphthalene (Sayler et al. 1985; Blackburn et al. 1987) and chlorinated biphenyls (Pettigrew and Sayler 1986; Furukawa et al. 1989; Walia et al. 1990).

Sayler et al. (1985) reported on a positive correlation between the proportion of bacteria carrying DNA homologous to toluene degradative genes in microcosm sediments and the concentration of aromatic pollutants in those sediments. This indicated bioavailability of toluene and suggested expression of degradative genes under in situ conditions. Barkay and Olson (1986) found that narrow-spectrum mercury-resistance genes were present in lake sediments that had most of the mercury contam- ination removed by dredging 15 years earlier. Walia et al. (1990) assayed the capability of non-selectively isolated bacteria with genes homologous to those-that encoded for chlorobiphenyl degradation to express their genotype under laboratory conditions. They found that six out of ten isolates revealed positive gene expression. In a similar investigation on 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria it was found that five out of 12 colonies expressed their assumed genotype (Amy et al. 1990). Another report demonstrated that gene expression among non-selectively isolated organisms with zinc-resistance genes was 100% (Diels and Mergeay 1990). Therefore the question of whether environmental bacteria isolated on media, and characterized by colony hybridizations with specific gene probes, are actually capable of expressing their genotype needs to be addressed more thoroughly.

To gain more insight into the relationship between gene occurrence and gene expression, four genetic determinants were selected for this study. We used DNA probes capable of detecting four different cistrons, which represent independent phenotypic properties of

environmental bacteria: naphthalene biodegradation (nahAB genes), narrow and broad spectrum mercury resistance (merA and merB), and nitrogen fixation (nifHDK genes). Expression was determined by using selective conditions and in one case (nif genes) by enzyme assays with whole cells. In three assessment studies gene expression upon immediate isolation from the environment was compared to gene expression obtained upon non-selective isolation. Thus, we were able to distin- guish between genotypes that (1) expressed their potential trait immediately, (2) only expressed upon non-selective recovery from the environment (soil or sediments) or (3) could not express at all.

Materials and methods

Isolation o f bacteria. Organisms were isolated from environmental samples by inoculating agar plates with suspensions of soil, sediment, or lake water in 50 mM potassium phosphate buffer, pH 7.2. For studies of gene expression in mercury-resistant and naphthalene-degrading bacteria, non-selective isolation was performed on PCA or R2A media (Difco, Detroit, Mich., USA). Additional- ly, prototrophic bacteria were isolated non-selectively on a mineral based agar (Sayler et al. 1985) with glucose (5 mM) and succinate (10 mM) as organic carbon sources. Selective isolation of naphthalene-utilizing organisms was performed on the same medium with no organic carbon except naphthalene vapor. In this case plates were stored in air-tight containers in which an open beaker containing naphthalene crystals was placed. Organic carbon and phosphate solutions were filter sterilized and added to the other autoclaved minimal media components after cooling to 50 ° C.

Narrow-spectrum mercury-resistant bacteria were isolated selectively on PCA containing 5 or 25 mg Hg 2+ as HgClz/1, and for broad-spectrum mercury-resistant bacteria, PCA was amended with 0.6mg or 3.0rag methylmercury (CH3Hg + [as (CH3Hg)OH)/1]). All mercury solutions were added at the stated concentrations after autoclaving and cooling of the base media to 50 ° C. Stock solutions of mercury were stored at 4 ° C for a maxi- mum of 2 weeks. Nitrogen-fixing bacteria were isolated non-selectively by inoculating R2A agar plates (Reasoner and Geldreich 1985; Difco) with dilutions of lake water. All plates were incu- bated at 25°C ( - 2 ° C) for 3-5 days except for naphthalene-utilizing bacteria (7-10 days).

Gene probes. The characteristics of the gene probes and their respective cloning vectors are shown in Table 1. The cloned DNA fragments were separated from their cloning vector, using endonuclease restriction (Boehringer Mannheim, Indianapolis, Ind., USA) and 0.8°70 agarose gel electrophoresis resolution. The frag-

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ments were electroeluted from the agarose at 100 V with gel slices in dialysis tubes and purified by passage through Elutip-D co- lumns (Schleicher and Schuell, Keene, N. H., USA). All proce- dures were carried out as described by Sambrook et al. (1989). DNA was radiolabeled [c~-32p]dCTP (ICN Biomedicals, Irvine, Calif., USA) by random priming (Feinberg and Vogelstein 1983; Random primers labeling kit, Bethesda Research Laboratories, Gaithersburg, Md., USA). The sizes of the cloned DNA templates used for our gene probes ranged from 460 to 6400 bp. They cov- ered 8.5-25°70 of the complete operons from which they were derived (Table 1).

Hybridization assays. Bacterial colonies were transferred onto nylon membranes (Biotrans; ICN) and cells were lysed in 1.5 M NaC1 solution containing 0.5 M NaOH. The membranes were then neutralized in 3 M sodium acetate, pH 5.5, air-dried, and baked at 80 ° C under vacuum for 1 h. The occurrence of nif genes in bacterial cells was determined by using a microfiltration manifold (Schleicher and Schuell) to apply cell lysates to nitrocellulose membranes. Bacteria were subcultured in R2A medium (2 ml) at room temperature until visible growth was achieved. Cell density was determined by turbidity measurements (560 nm) which were calibrated to colony-forming units (cfu). Cells grown on R2A broth were pelleted in a microcentrifuge (4 ° C) and resuspended in 1 ml lysis solution [lysozyme (3 rag/l) in 25 mM TRIS-HC1, pH 8.0, 25 mM EDTA, and 10070 (w/v) sucrose]. The lysates were fil- tered through the nitrocellulose membrane, which was neutralized and phenol-extracted as described by Landers (1987). All hybridizations were performed for 8-24 h at 42°C in a shaking water bath (100 rpm). Membranes were washed under conditions of high stringency for nitrocellulose-bound DNA, as described by Sam- brook et al. (1989), and for nylon membranes as described by the manufacturer (50070 formamide, two 30-min washes at 65°C in 15 mM sodium chloride, 1.5 mM sodium citrate, 0.5% sodium dodecyl sulfate).

Experimental controls. In all hybridization assays, positive and negative controls were included to prevent misrepresentation of data due to variable signal-to-background ratio. Positive controls were Pseudomonas putida G7 harboring plasmid NAH7 (Dunn and Gunsalus 1973; for nahAB), P. aeruginosa PU21 with plasmid RIP64 (kindly provided by G. Jacoby; for merA), Pseudo- monas sp. OR3 (for merB), Frankia sp. CpII and Thiobacillusfer- rooxidans TFI-35 (for n/fHDK). Negative controls were P. aeruginosa strains, lacking plasmid NAH7, or RIP64, Escherichia coli DHS-a, P. aeruginosa PU21 RIP64 (for merB). In the n/f-occurrence studies negative controls consisted of P. aeruginosa. In the latter study it was found that when using the microfiltration de- vice, non-specific hybridization signals occurred when the cell concentration exceeded 6 x 10 7 cells per well. Therefore, when investi- gating environmental strains the cell concentration was adjusted to 2 x 106-2x 107 cells per well.

Table 1. Characterization and origin of gene-probes used

Name Origin Cloning Size a Coverage References Source vector (kb) (%)b

nahAB Pseudomonas putida pUC9 c 6.4 17.3 Kurkela et al. (1988) AMGEN nifHDK Klebsiellapneumoniae pSA30 a 6.0 25.0 Cannon et al. (1979) J. Ligon merA Serratia mareescens pT7/T3-18 e 1.0 18.6 Nucifora et al. (1989); S. Silver

Griffin et al. (1987) merB S. marcescens pT7/T3-18 e 0.46 8.5 Griffin et al. (1987) S. Silver

Gene probes: nahAB, naphthalene degradation; merA, narrow- spectrum mercury resistance; merB, broad-spectrum mercury resistance; n/fHDK, nitrogen fixation a Size of the DNA template for gene probes

b Indicates the percentage length (base pairs) of the cloned DNA relative to the whole operon c Cloning was performed in Escherichia coli GM119 d Cloning was performed in E. coli C600 e Cloning was performed in E. coli DH5-a

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Cell lysis on membranes was detected after colony hybridization and autoradiography by staining membranes in 0.25 070 methylene green for 2 min and rinsing in deionized water for 5 min.

Determination of gene occurrence and gene expression. For the merA, merB, and nahAB gene assessment studies, the population density of the respective genotype in a given environmental sample was calculated by determining the percentage of colonies detected by gene probes among total colonies recovered upon isolation on media that did not select specifically for the phenotype. Imme- diate gene expression was determined by scoring growth of buffer- diluted environmental samples under selective conditions, i.e. in the presence of H g 2+ , CH3Hg +, or naphthalene as a sole source of organic-carbon-amended plates. The expression of genes in bacteria isolated on media containing no mercury was determined by transferring PCA-grown colonies with sterile toothpicks to selective media and on PCA as a replica control. To obtain a random selection, all isolated colonies from certain plates (with up to 40 colonies) were subculture& Utilization of naphthalene by several nahAB-positive colonies was confirmed by subculturing strains in mineral-broth cultures containing salicylate or naphthalene as a sole carbon source (22°C, 120rpm). When inoculated broth cultures reached visible turbidity, cells were removed by cen- trifugation and the supernatant was photometrically scanned (220-420 nm). Degradation of naphthalene or salicylate was confirmed by observing the production of a metabolite at 310 nm. This metabolite was likely to be catechol (Barnsley 1975).

Fig. 1A-C. Autoradiography of representative membranes from colony lifts obtained from PCA plates (see Materials and methods), inoculated with soil or sediment dilutions; hybridized with A nahAB, B merA, and C merB

Results

Detection o f genotypes and efficiency o f colony lysis

Colonies tha t car r ied h o m o l o g o u s D N A sequences to our gene p robes could always be detected when environ- menta l samples were p la ted on to m e d i u m no t selective for the p h e n o t y p e (Fig. 1). No t all o f the colonies isola ted f rom soil or sediments and t r ans fe r red on to membranes could be lysed. F o r example , o f 715 colonies isola ted f rom one sediment 45.1 07o underwen t lysis (Fig. 2). The lysis eff iciency o f the subcul tu red s trains was similar (57°7o) to tha t ob ta ined u p o n immed ia t e i so la t ion f rom the sediment , ind ica t ing tha t a representa t ive p ro- po r t i on o f bac te r ia had been subcul tured .

Naphthalene-degrading bacteria and expression of nahAB

Naph tha l ene -deg rad ing bac te r ia were i so la ted f rom sub- surface soil (2.2 m depth) o f a fo rmer manu fac tu r i ng p lan t (Los Angeles , Cal i f . , USA) under three d i f fe ren t

Fig. 2A, B. Colony hybridization of PCA subcultured environmental strains with merA A and detection of lysis after methylene green staining B. Arrows indicate example for positive (1) and negative (2) detection of merA in lysed colonies

condi t ions : non-select ively on R 2 A agar for he tero- t roph ic bac te r ia , on a minera l m e d i u m (MM) a m e n d e d with glucose and succinate for p r o t o t r o p h i c bac te r ia , and selectively on M M with naph tha l ene vapor as a sole source o f organic ca rbon . The d a t a are summar ized in Tab le 2.

Gene hybr id i za t ion revealed tha t 3 . 7 7 x 10 3 Cfll or 35°70 o f all colonies on R2A conta ined D N A sequences h o m o l o g o u s to nahAB. Of all cfu i sola ted on M M with glucose and succinate , 1.78 x 103 c f u / g or 6707o o f the to ta l con ta ined nahAB-homologous genes, indicat ive o f the p o p u l a t i o n densi ty o f po ten t ia l p r o t o t r o p h i c naph-

Table 2. Occurrence of naphthalene-utilizing and nahAB-positive bacteria isolated from Venice soil on selective and non-selective media, and expression of nahAB-positive colonies

Media for immediate Total bacteria in soil Percentage of total nahAB-positive colo- Percentage of nahAB colo- isolation (cfu/g) colonies able to de- nies in cfu/g nies able to degrade naph-

grade naphthalene in (o70 of total) thalene broth

R2A 1.07 x 105 MM + glucose + succinate 2.80 x 103 MM + naphthalene 2.67 x 103

25070 3.77 x 10 3 (35070) 76.5070 (68 tested) 5107o 1.78 × 103 (67070) 85.807o (113 tested)

100070 9.17 x 10 2 (34070) 100070 (56 tested)

Single X 2 = 14.7, df= 2, p < 0.005: cfu, colony-forming units. For R2A and MM media, see Materials and methods

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Table 3. Occurrence of bacteria with mercury (Hg)-resistant phenotypes and genotypes upon selective and non-selective isolation from Oakridge sediment A

Media for immediate isolation

Total bacteria in sediment (cfu/g)

Percentage of total colonies that express following transfer to Hg ~+ media

mer-Positive colonies in sediment in cfu/g (% of total)

Percentage of mer-positive colonies that express mercury resistance

PCA 1.77 × 105 N.A. 4.04 × 104 (22.8%) 28.5% (43 tested) PCA, 5 mg Hg2+/1 6.87 x 104 84.1% (151 tested) 2.73 x 104 (39.7%) 100% (43 tested) PCA, 25 mg HgZ+/1 2.57 x 104 41.1% (151 tested) 1.37 X 104 (53.3) 100% (43 tested)

B

PCA 1.77 × 105 N.A. 2.12 × 104 (11.9%) 16.5% (28 tested) PCA, 0.6 mg CH3Hg+/1 5.26× 102 64.1% (170 tested) 6.49× 101 (12.3%) 89.3% (25 tested) PCA, 3.0 mg CH3Hg+/1 <3.3 × 101 16.5% (170 tested) Not detectable 53.6% (15 tested)

A Narrow-spectrum resistance and merA (single X 2= 32.7; df=2; p<0.005). B Broad-spectrum resistance and merB (single X2= 19.3; df=2; p <0.005). For PCA medium see Materials and methods: N.A. = not applicable

thalene degraders. Although the number of cfu isolated on MM with naphthalene as a sole organic carbon source was as high as the cfu isolated on glucose and succinate, only 34% were found to hybridize with nahAB. Thus, isolation on media not selective for naphthalene utilizatioia led to a higher recovery of nahAB- positive organisms than selective isolation. Twenty-five percent of the culturable bacteria on R2A and 51% of the prototrophic (MM, glucose and succinate), were able to express their genotype immediately under the selective conditions. All of several nahAB-positive colonies oc- curring on MM-naphthalene plates and tested for naphthalene degradation in broth cultures metabolized the compound. To determine whether the bacteria that contained nahAB but could not utilize naphthalene as sole carbon source contained non-functioning operons, colonies were transferred from both non-selective media onto MM with naphthalene. It was found that 76.5070 of colonies isolated on R2A that contained nahAB were also able to grow on mm-naphthalene. For colonies isolated on MM with glucose and succinate the number was slightly higher (85.5%). This demonstrated that a significant proportion of the bacteria (X2=14.7, dr=2, p < 0.001) needed non-selective isolation to express their nah genes and some strains did not express even after non-selective recovery.

Mercury-resistant bacteria and expression of merA and merB

The source of organisms was sediment (Oakridge, Tenn., USA) known to be contaminated with both Hg 2+ and CH3Hg + (approximately 80 mg total Hg/kg sediment). To study immediate gene expression the environmental samples were cultivated non-selectively (PCA) and selectively (PCA with HgZ+). In this investigation, we included two concentrations of mercury, Hg 2+ (5 and 25 mg/1) and CH3Hg + (0.6 and 3 rag/l), to evaluate the effect of a selective gradient. Preliminary experiments indicated that a significant proportion of

the mercury-resistant bacteria were inhibited by high concentrations of the heavy metal in the growth medium, whereas too low concentrations resulted in scoring mercury-sensitive colonies as resistant (false posi- tives).

To determine the effect of initial nutrient enrichment on the isolation of Hg2+-resistant bacteria, colonies isolated on PCA were transferred to Hg2+-amended PCA. Different colony morphologies indicated that a variety of bacterial species was selected. Of 151 isolates, 84.1% grew on PCA containing 5 mg Hg2+/1, and 41.1% on PCA containing 25 mg HgZ+/1.

Amendment of PCA with the low Hg 2+ concentration (5 mg/1) reduced the number of cfu upon immediate isolation from 1.77 × 105 on PCA to 6.87 × 10 4 (or 36O7o), and with high Hg 2+ (25 rag/l), to 2.57 × 104 (or 12.8%) (Table 3). The number of organisms carrying genes homologous to merA was 4.04 × 104 cfu/g sediment (22.8% of the total cfu on PCA). However, on media with 5mg Hg2+/1 this fraction was only 2.73 × 104 (39.7%), and with 25 rag/1 it was 1.37 × 10 4 (53.3%). This demonstrated that a significant proportion of the merA-positive and PCA-culturable bacteria could not fully express the met genes upon selective isolation from the environment. Inability to express immediately was in this case correlated to the concentration of the selective agent. However, approximately one- third of the merA-positive isolates failed to express even in the presence of 5 mg Hg2+/1 (Table 3A).

Isolates that contained merA represented 28.5°7o of the subcultured strains. This was similar to the merA in- cidence found upon immediate isolation on PCA (22.8%), which indicated that a representative fraction of the heterotrophic bacteria was selected for subcultivation. All 43 merA-positive colonies from this subcultured group could express their genotype on media amended with both concentrations of Hg 2+. This indicated that all detected genotypes had the potential to express their trait even at a concentration of 25 mg Hg 2 +/1 (Table 3A).

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Methylmercury exhibited a higher toxic effect on the bacterial population than Hg 2÷ . Bacterial survival on PCA containing 0.6 mg CH3Hg ÷/1 was less than 1% of the total whereas on PCA containing 3.0 mg/1 only single colonies were detectable when media were inoculated with 1 ml of 10-1 sediment dilution (Table 3B). The potential for CH3Hg ÷ resistance in the sediments was higher, as determined by subsequent subcultivation of non-selectively isolated colonies: out of 170 colonies, 64.1% resisted 0.6mg CH~Hg+/1 and 16.5% resisted 3.0 mg CH3Hg ÷/1 (Table 3B). Culturable merB-positive isolates were present at a relatively high concentration (12% of the total population), as determined by colony hybridization of organisms grown on PCA. At the lower concentration of CH3Hg + only 5.26 x 10 a cfu, or 0.3% of these merB-positive isolates were detectable, indicat- ing lack of full expression of merB immediately upon isolation. At 3.0 mg CH3Hg+/1, no merB-positive isolates could be found. Out of the 170 isolates, that were subcultured selectively, 28 (or 16.5%) were merB-positive. Thus, occurrence of merB in the randomly selected colonies was similar to that upon non-selective isolation (11.9%). The expression of broad-spectrum resistance depended on the concentration of the heavy metal: 89.3% on low- and 53.6% on high-level CH3Hg +- amended media. Thus, a significant proportion of isolates (10.7-46.4%) did not express merB under our laboratory conditions.

Expression of nifHDK genes

Genes for nitrogen fixation were detected in bacteria isolated non-selectively from oligotrophic lake water (Lake Waramaug, Conn., USA). Out of 52 colonies, which showed a considerable variety in morphology, 12 showed positive nifHDK hybridization signals. Two of these strains, designated A-13 and A-26, exhibited a higher signal-to-cell ratio in slot-blot hybridization assays that had been normalized for cell density. Gene expression was tested by subculturing all 12 strains in a nitrogen-deficient mineral medium with multiple carbon sources. Only A-13 and A-26, the two strongly hybridizing organisms grew well. Expression of nif-genes was confirmed in these two strains by a positive acetylene reduction assay (Burris 1972).

Discussion

In this study we determined the expression of four independent genotypic properties of bacteria recovered from soil, sediment, or lake water. All of the chosen genotypes commonly occurred in the given environments as indicated by positive hybridizations. For example, 33% of colonies isolated on mineral medium with naphthalene as a sole source of organic carbon, hybridized with the nahAB probe. Fifty-three percent of the bacteria scored as narrow-spectrum mercury-resistant hybridized to merA. All genotypes were also detectable upon non- selective isolation: 22.8°7o of all cfu recovered from mer-

cury-polluted sediment hybridized with merA and 12% with merB. Nif-genes could be detected in 23°70 of 52 isolates from lake water and nahAB genes in 35.2% of all non-selectively recovered colonies from soft contaminated with polyaromatic hydrocarbons. Mercury-resistance genes similar to our probe have been found to ac- count for a large proportion of the mercury-resistant bacteria isolated from contaminated environments (Bar- kay and Olson 1986; Rochelle et al. 1991). The nifHDK gene probe, originally isolated from Klebsiella pneumoniae, is known to have homologies to other nitrogen- fixing Gram-negative and Gram-positive microorganisms (Mazur et al. 1980; Rufkun and Ausubel 1980; Normand and Bousquet 1989; Kennedy 1989). Thus, most nitrogen-fixing environmental bacteria were likely to hybridize with our gene probe. The genes for naphthalene dioxygenase (nahAB) of P. putida are known to have homologies to other bacterial dioxygenases (Kurke- la et al. 1988). The homologies, however, are below 70% and thus did not result in the detection of false- positive hybridizations in our studies since the applied stringency condition was above 80%.

Three different categories of operon expression could be differentiated in this investigation: 1. Some organisms could express their genotype immediately upon selective isolation. 2. A large number of organisms only expressed their genotype after initial non-selective isolation. 3. Complete inability to express a genetic trait was observed for all genes, except merA. A similar phenomenon was observed when bacteria were recovered after long-term starvation from well water (Caldwell et al. 1989).

Differences between the number of genotypes detected upon non-selective isolation and the number that is able to express the phenotype upon selective isolation has also been described by other authors. In the Diels and Mergeay (1990) study, various results were obtained between non-selective and selective isolation. In at least one instance, zinc-resistant bacteria under selective conditions represented only 30% of the genotype-positive colonies obtained under non-selective conditions. This result is similar to ours on narrow-spectrum mercury resistance, where Hg 2+ selection (25 mg/1) reduced the number of colonies containing merA genes to 33.9% compared to the total number of merA-hybridizing colonies on unamended PCA. Although organisms carried the genetic potential for mercury resistance, many failed to express this trait immediately. With CHaHg + as a selective agent, immediate expression was reduced by more than one order of magnitude, probably due to the higher specific toxicity of methylmercury than inorganic mercury. Inability to recover strains from an environmental sample on selective media has been investigated with introduced strains of known genotypic and phenotypic characters. For example, a chlorobenzoate-degrading Alcaligenes sp. lost the capability to form colonies on selective media but remained catabolically active under in-situ conditions, as judged by correlation between genotype occurrence and chlorobenzoate concentration in water-sediment microcosms (Fulthorpe and Wyndham

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1989). E. coli strains harboring plasmid-encoded Hg 2+- resistance and introduced into soil or aquatic microcosms, did not express mercury resistance upon immediate selective isolation, but the potential for mercury resistance was still detectable (Devanas et al. 1986; Chaudry et al. 1989).

Divergence between genotype occurrence and immediate expression has also been described for catabolic genes, such as those encoding for toluene and naphthalene degradation (Sayler et al. 1985). In microcosm studies, the reduction in bacterial population densities isolated on mineral medium with naphthalene as a sole source of carbon was above 99.9%, which indicated to the authors the presence of co-metabolizing organisms. In this study, we demonstrated that auxotrophy was not the sole reason for non-recovery on selective medium. On a mineral medium with glucose and succinate, the number of nah-positive colonies was twice as high as on the same medium with naphthalene as a sole source of organic carbon (Table 2). Subsequent sub-cultivation showed expression of nahAB genotypes to be above 75°70 and indicated that the majority of operons recovered non-selectively had the potential to express, and thus enable the organism to utilize naphthalene as a sole carbon source.

It is probable that the inadequacy of selective recovery in the assessment studies with merA, merB and nahAB, was associated with the nutritional status of the cells coming directly from the environment. Sediment organisms that are adapted to low concentrations of nutrients or dormant organisms that have ceased to meta- bolize because of low nutrient concentrations have to adapt to eutrophic levels of nutrients upon isolation from the environment. The proportion of organisms that expressed immediately depended on the in-situ selective pressure imposed on the cells as well as on their ability to adapt to different nutrient conditions. Thus the data suggest that it may be a very small proportion of the community. Inability of immediate gene expression for part of the bacterial community was thus associated with the inability to induce detoxifying genes, as evidenced by the higher number of bacteria that could express detoxification genes after non-selective isolation. An explanation for non-induction of potentially functioning operons might be that the organisms are not exposed to sufficient concentration of inducer compounds in situ, and thus the genotypes were inactive. It is likely that a specific genotype with already induced resistance genes will grow better on an artificial growth substrate with a selective agent than the same genotype that is not induced. Consequently, gene probe methodology by itself is likely to merely characterize the genotypic potential of microorganisms in their environment and should not be interpreted as an indicator of microbial activity in situ.

The expression of merA genes in bacteria isolated non-selectively was found to be 100%, as measured by growth on selective medium (25 rag/l). The same level of expression has been described for non-selectively-isolated zinc-resistant genotypes, when colonies were transferred to ZnZ+-selective plates (Diels and Mergeay

1990). In all other studies, except for those discussing merA, a variable proportion of colonies failed to express even upon non-selective recovery from the environment (depending on the genetic system (10-85%)). The highest rate of non-expression was found with colonies detected with the nif probe. In the current study, slot-blot assays were used instead of colony hybridization. Only the two strains that showed strong hybridization signals were able to express their genotype. Non- expression in the weakly hybridized group could also be attributed to the abundance of incomplete operons or related DNA sequences. The DNA templates from which our probes were derived represented 8-25% of the complete operons (see Table 1) and thus hybridization did not necessarily mean the detection of a complete functioning operon.

Very likely, inducer and nutrient concentrations, as well as physical factors such as humidity and temperature have a significant effect on gene expression under artificial (laboratory) conditions. The different responses in gene expression as measured in this investigation may also be the result of the in-situ history of the microbial community, e.g. presence and availability of inducers, nutrients or growth-inhibiting factors. Thus, any report on gene occurrence related to population or community factors must be accompanied by several as- sessments of gene expression. Further, this report indicates that studies linking gene occurrence and function in the environment have to be carefully interpreted. In light of this study, it is important to qualify these types of findings by using the term genetic potential when re- ferring to functions in situ. Finally, our observations indicate the value of direct extraction of mRNA from environmental samples and point to the need to develop a quantitative measurement of mRNA in environmental samples.

Acknowledgements. The study was supported by the Electric Pow- er Research Institute, grant 8000-25. C.C.T. was supported by DAAD, Sonderstipendium Genetechnologie, Bonn, Federal Re- public of Germany.

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varied resonses in gene expression of culturable heterotrophic bacteria isolated from the...

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