Effect of introducing genetically engineered microorganisms on soil microbial community diversity

FEMS MicrobiologyEcolo~ 86 (1991) 109-176 © 1991 Federation of European Micmbiclog;.calSoc~ie!ies0168-6496/9t/$03.50 Published by Elsevier

169

FEMSEC 00361

Effect of introducing genetically engineered microorganisms on soil microbial community diversity Asim K. Bej, Michael Perlin and Ronald M. Atlas LMlmrZmem of Biotob~. University of Louisville, Loulsvi?le, ICY, U.S.A. Received 28 May 1991 Revision received and accepted 15 August 1991

1. SUMMARY Introducing the genetically engineered microorganism Pseudomonas cepacia ACll00 into soil microcosms resulted in elevated taxonomic diversity determined by phenotypic analyses of culturable isolates and genetic diversity determined by analysis of the heterogeneity of total microbial community DNA reannealing kinetics. The greatest impact occurred when P. cepacia ACll00 was introduced along with the herbicide 2,4,5-T, which P. cepacia ACll00 can degrade. The data suggests that both changes in the balance of populations and genetic recombination contributed to the increased diversity. 2. INTRODUCTION The potential ecological impacts of deliberately releasing geneticafiy engineered microorganisms into the environment has been an area of concern to both the scientific community and the public [1-3]. Studies have addressed the re-

CorreSpondence to: R.M. Atlas, Department of Biology, University of Louisville, Loui~Ue, KN 40292, U.S.A.

sponses of microbial communities to introduction of genetically engineered microorganisms; these studies have examined survival and persistence of the introduced genetically engineered microorganisms, gene exchanges between the genetically engineered microorganism and the indigenous microbial populations, and alterations in microbially-mediated biogeochemical cycling reactions [4-9]. Taxonomic diversities have been measured iry characterizing representative randomly selected organisms, characterizing those organisms and subjecting them to numerical taxonomic analyses and cluster analysis to ~stablish taxonomic groupings [10]. The Shannon diversity index has been widely used [11-16], but other diversity indices have also been applied [17-19]. The Shannon diversity index measures both the occurrences of individual species and the evenness of distn'bution within the community [20,21]. Additionally, the genetic diversity of microbial communities has recently been measured by Torsvik and colleagues [15,16]. DNA reassoclation kinetics is a measure of the diversity of the total pool of DNA within the biological community. Torsvik et aL [16] reported that there is a high degree of correlation between taxonomic diversity measurements based upon phenotypic

t70 characterization of isolates and genetic diversity based upon DNA reassociation kinetics; the greater the diversity of the DNA the slower the reassociation and the lower the genetic diversity the faster the DNA reanncals. In this study we examined the response of a soil microbial community to the introduction of Pseudomonas cepacia ACll00. Pseudomonas cepacia ACII00 is a 'genetically engineered', 2A,5-trichlorophenoxyacetic acid (2,4,5-T) degrader [22-27]; the specific strain of P. cepacia ACI100 used in this study also is resistant to nalidixic acid [28]. P. cepacia ACII00 was formed through molecular breeding which favors recombinational processes and hence the evolution of new strains by bringing together diverse genes and mobilizable genetic elements. We measured both taxonomic diversity and genetic diversity following introduction of P. cepacia ACll00 into soil microcosms alone or in combination with the herbicide 2.4,5-T.

3. MATERIALS AND METHODS

3.1. Soil microcosms 2-kg replicate samples of an Oregon silt loam soil were placed into polyethylene beakers to establish test soil microcosms. Soil properties, determined by standard methods for soil analysis, were as follows: organic content, 6%; sand, 28%; silt, 44%; and clay. 28%. Soil moisture was adjusted to 60% water-holding capacity by addition of sterile water. Replicate microcosms (4 each) received no other treatment (control microcosms), were treated with 2,4,5-T to achieve a final concentration of 100 ppm, were inoculated with P. cepacia ACI100 at a concentration of 10~/g soil, or were treated with l0 s P. cepacia/g soil + 100 ppm 2,4,5-'1". For inoculation, P. cepacia ACll00 was grown in basal salts medium (BSM) [23] containing 1 mg 2,4,5-T/ml to early log phase. The cells were sedimented by centrifufation and washed and resuspended in 0.1 M sodium phosphate buffer (pH 6.8). Cell numbers were estimated by spectrophotometric absorbance (A540) based on a standard curve determined by viable plate counts;

P. cepacia ACll00 was added to the microcosms to give a final concentration of approximately 105 cells per g soil which was approximately 0.01% of the viable heterotrophic bacterial population (determined as colony forming units on trypticase soy agar) in the soil. Ten germinating radish seeds were planted into each soil microcosm at a depth of 1 cm. The soil microcosms were incubated at 25°C with alternating 12-h light-dark cycles. Periodically replicate 2.5-¢m cores were recovered. The sites where samples were collected were replaced with glass tubes. 3.2. Enumeration of Pseudomonas cepacia ACl lO0 Selective plate counts for P. cepacia ACll00 were performed by plating serial dilutions from 10-g soil samples onto trypticase soy agar (BBL) supplemented with naladixic acid (200 p~g/ml, Sigma). All plates were incubated at 25°C for 7 days. P. cepacia ACll00 detection was confirmed by colony hybridization performed using a high stringency procedure [29] to minimize nonspecific hybridization. Autoradiography was performed by exposing the filters to x-ray film (X-Omat AR, Kodak) for 4 h at - 70°C. The gene probe used to detect P. cepacia ACll00 consisted of a 1.3-kb restriction fragment that was complimentary *o a repeat sequence (RS-1100) which is present in 15-20 copies on this organisin's chromosome and plasmids [30]. 3.3. Taxonomic diversity Bacterial colonies from each sample were randomly selected for isolation from the trypticase soy afar plates. These isolates were considered as representative of the major populations of the bacterial community since they were obtained from countable plates of greatest dilution. The primary isolates were subcultured on trypticase soy afar to ensure purity and viability. Approximately 40 phenotypic characteristics were determined for each isolate. Phenotypic characteristics examined included those of the API 20E test supplemented by cellular and colonial morphologies, temperature growth ranges, and abilities to utilize additional substrates. Data were subjected to cluster analysis to determine taxonomic groupings (phenotypic clus-

171

ters) using the simple matching coefficient (S m) and unweighted average linkage clustering. Taxonomic groups were defined at approximately the 90% similarity level. The number of taxonomic groups and the number of individuals within each group were used to calculate the Shannon diversity index, H ' as previously described [12]. An H' value near 0 represents a community with low diversity; H ' values of approximately 4 or higher represent rather high diversity.

3.4. Genetic diversity Total DNA from soil samples (100 g) was extracted by following the direct lysis extraction procedure described by Ogram et al. [31] as modified by Steffan et al. [32]. DNA was purified by cesium chloride centrifugation and hydroxyap. atite chromatography [32]. The purity of the DNA was determined by spectrophotometric readings at 230, 260 and 280 nm wavelengths, in TE buffer (10 mM Tris-HCI, pH 7.0, 1 mM Na2EDTA). The samples were stored in the freezer (-20°C) until analyzed. Each DNA solution was diluted in TE buffer and subjected to shearing by using a Cell Sonic Disrupture (Heat Systems, NY). Sonication was performed in a 20-mI volume with a total sonication time of 15 min. The tubes were maintained at < 5 ° C during sonication. After sonication the DNA was precipited by 2.5 volumes ethanol overnight at -2ff'C. The DNA pellet was collected by centrifugation at 12000 × g for 15 rain at 4°C in a Sorvall high speed centrifuge. The DNA pellet was washed once in 70% alcohol, centrifuged and dried in a vacuum. The DNA was resuspended in 1 × SSC solution (0.18 M NaCI, 0.01 M Na2HPO4-7H20 and 0.001 M

Na2EDTA, pH 7.0) and stored at 4°C for future use. An aliquot of each of the sonicated DNA samples was used for DNA size analysis by electrophoresis as described by Ausubel et al. [33]. These analyses indicated the mean size of the sheared DNA was 0-5 kb and that > 99.5% of the DNA was double stranded. Thermal denaturation and reassociation was measured using a Lambda III spectrophotometer with an automatic temperature controller (Perkin-Elmer Inc., Norwalk, CT). Thermal melting was carried out with a total of 280-300 ~g of DNA per ml of 1 × SSC for each sample for 10 rain to melt the DNA completely. The reassociation of DNA was initiated by lowering the temperature rapidly ( > 5 ° C rain) to 60~C. A26o measurements were made, initially every minute and subsequently as the absorbance became constant every day or week. Percent reassociation was calculated at time t (in s e c o n d s ) ~ A o - A t / A × 100, where At ffi absorbance at 260 nm at time t, A,~ ffi absorbance at 260 run when DNA was completely melted, and A ffi absorbance at 260 nm for 100% melted DNA - absorbance at 260 nm for 0% DNA melted. DNA reassociation values (Cot) were calculated as tool x l -t × s [15,16]. Cotl/2 and Cots~4 values were calculated corresponding to the Cot value at 50% and 75% DNA reassociation, respectively.

RESULTS

4. L Persistence of P. cepacia ACl lO0 'ze concentrations of P. cepacia initially increased following introduction into microcosms and subsequently decreased (Table 1). This ge-

Table 1 Concentration of Pseudomonas cepacia ACII00 (Io8 number per g soil) after treatment with the herbicide 2,4,5-'1" and the 2,4,J-11" degrading bacterium Pseudomonas cepacia AC1100 alone and in combination Time (days)

Control

2,4,5-T

P. cepacia ACII00

2,4,5-T+ P. cepacia ACII00

0 7 21 42

<0

<0 <0 <0 <0

5.0 6.0 7.2 2.7

5.0 7,1 8.0 4.3

<0

<0 <0

172 Table 2 Taxonomic diversitymeasured as the Shannon index (l-I') after treatment with the herbicide 2,4,5-T and the 2,4,5-T degrading ~ c t e H a m Pseudomon~ ce~c/a ACII00 alone and in combination Time (days) 0 7 21 42

Control 3.S 3.8 3.7 3.6

2,4,S-T 3.8 3.7 3,3 3.1

P. cepada ACII00 3.8 4.0 3,9 3.7

2,4,q-T+ P. ceoacia ACII00 3.8 4.0 4.3 3.8

0/s minimalvalue for H', 4.6 is maximalvalue for experimental ¢rolocol.

netically engineered microorganism persisted in microcosms with and without the addition of 2A,5-T for the full 6 weeks of the experimental period. Concentrations of P. cepacia after 6 weeks were somewhat higher in the microcosms where 2,4,5-T was present than in those where it was absent. P. cepacia A C l l 0 0 was not detected in microcosms that had not been inoculated. Screen. ing o f several hundred colonies from control samples showed no cross-hybridization to the probe and no hybridization occurred with D N A extracted from the total microbial communities of the control and other microcosms not inoculated with P. cepacia AC1100, demonstrating that P. cepac/a ACI100 does not occur naturally in the soil used in this study [34].

4.2. Taxonomic diversity The addition of 2,4,5-T alone caused a decrease in taxonomic diversity, a typical response

to chemical pollutants that disturb the microbial community that result in the elimination of some bacterial populations a n d / o r the proliferation of others (Table 2). Only those populations that are culturable under the in vitro growth conditions are analysed for taxonomic diversity. Surprisingly, the addition of Pseudomonas cepacia resulted in a slight increase in taxonomic diversity of c u r e r able populations, particularly when 2,4,5-T was added along with this herbicide degrading microorganism.

4.3. Genetic diversily The Cot plots that measured D N A reassociation kinetics (genetic diversities) were consistent with the patterns of changes observed for taxo. heroic diversities (Table 3). These analyses ot total extracted D N A measure the genetic heterogeneity of both culturable and nonculturable microorganisms in the s~il, The addition of 2,4,5-'I

Table 3 Genetlc'dlversiWme~ured as 50% DNA rcassociation(C0tl/2) and 75% DNA reassociation (C0ra/4) after treatment with the herbicide 2,4,5-T and the 2,4,5.'1"degrading baelerium Pseudomonaseepae[a ACII00 alone and in combination Control

2,4,5-T

P. cepacia ACI104]

2,4,5-T~ P. ¢elaTciaACI100

0 "/

3.1 × 104 3.1 X 10 4

3A x 10 4 3 . 0 X 10 4

3.1 x 10 4

3.1 × 10 4

3.9 x 104

3.9 x 104

14 21

3.0x I0' 2Ax 104

L9× I0' 1.4× 104

2.4x I0* i.IX 104

4.9x 104 2.9× 104

9 . 6 x 10 4

9.6X 104 9.6x 104 9.0X 104 4.2× 104

9.6~<104 3.2x l0s 7.2 X 104 6.0x 104

9.6× 10' 1.2× 106 7.7 X 10 7 4.1 x 10 4

Time (days)

C0¢112

Cet~/4 0 7 14 21

9.6x 104 6,9x 104 3.6X 104

Cot values="molxl-I xs.

173 alone resulted in lowered Cot values indicative of decreased genetic diversity. In contrast the addition of P. cepacia ACllO0 resulted in increased Cot values, reflective of an increase in diversity within the gene pool of the soil microbial community. The increased genetic diversity was particularly noticeable in measurements of Cot3/4 va]ues which have a greater sensitivity than Cot1~2 values at detecting the presence of rarer gene sequences. AS indicated by the DNA reassociation analyses as well as by the taxonomic diversity analyses the initial impact on the community was transient and by the end of the 6 week experiment the community was returning to reference soil community levels of both taxonomic and genetic diversity.

5. DISCUSSION The addition of the genetically engineered 2,4,5-T-degrading microorganism P. cepacia appears to have caused an imbalance within the microbial community, such that despite the addition of a plasmid-bearing microorganism the evenness of distribution of populations within the community was enhanced, as is reflected in the slight increase of the Shannon diversity. The initial taxonomic diversity of the microbial community in the soil tested was characteristic of an unstressed community; that is a Shannon index approximately equal to 4. The highest diversity occurred when the maximal number of P. cepacia ACll00 was fnund. The increase in numbers of P. cepac/a following inoculation into the soil microcosms could have been due to the ability of this organism to utilize diverse substrates in the soil or to continue metabolism of substances in the medium used to grow the inoculum. The increase in diversity may have resulted from changes within the balance of microorganisms within the community. This hypothesis is supported by the increased taxonomic diversity which reflected greater evenness of populations within the community. The metabolic activities of P. cepacia ACll00 may have co~tr~b,Jted substratus for other populations leading to a transient imbalance within the community. Changes

in population interactions typically result in decreased diversities because only a few populations generally axe able to outcompete the others, but in this case the opposite occurred. Unlike Torsvik et al. [15,16], we did not separate bacterial cells from the soil matrix and eukaryotic members of the soil microbial community prior to DNA recovery. Our direct recovery of DNA should include ¢ukaryotie and prokaryotic DNA. As in the study of Torsvik et al. [15], the reassociation kinetics in our study did not follow second-order kinetics and is not a direct measure of species richnm,s. The DNA reasscsziation kinetics in our study had a biphasic character indicati,,e of the presence of significantly differ~ ing populations of DNA. Eukaryotic DNA has a higher number of repeat sequences than bacterial DNA and therefore should show more rapPt reassociation of DNA. The 1.3-kb repeat sequence of P. cepacia also would contribute to rapid DNA reassociation. The Cotl/2 values represent a hybrid measure of the relatively rapidly reassociating and the more diverse DNAs. The Cot3/+ values are largely reflective of the slower reassoelation of diverse DNAs, probably due to the bacterial populations including the rarer species. The fact that the C0tt/2 values decreased but that the Cot3/+ values were relatively unchanged when 2,4,5-T atone was added suggests that 2,4,5T may have affected primarily euka~otic algae. The magnitude of the genetic diversity increase suggests that in addition to population shifts, the introduction of P. cepacia ACI100 may have resulted in genetic transfer and/or recombination. P. cepacia ACII00 has plasmids and a multicopy 1.3-kb sequence that has been shown to be a transposon [30]. Mobilizable genetic elements were used in the molecular breeding (evolution) of P. cepac/a ACII00 and could well contribute to new genetic combinations that would increase the genetic diversity of the microbial cemmunity. There is potential for enhanced evolution when mobilizabIe and novel DNA is added to a microbial community, particularly when chem.;cal disturbance enhances the adaptiveJtess of recombiaants. The increased diversity was transient indicating that if genetic recombination was contributing to the increased genetic

174 diversity m o s t or all o f t h e r e c o m b i n a n t s nonfunctional or noncompetitive.

wcrc

ACKNOWLEDGEMENTS This research was supported by a cooperative research a~reement with the U.S.E.P.A. Corvallis Research Laboratory.

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