Persistence and stability of genetically manipulated derivatives of Enterobacter agglomerans in soil microcosms
Elena Evguenieva-Hackenberg *, Sonja Selenska-Pobell, Walter Klingmiiller
Department of Genetics, University of Bayreuth, Universitiitsstr. 30, D-95440 Bayreuth, FRG
(Received 7 January 1994; revision received 6 June 1994; accepted 10 June 1994)
Abstract: Molecular methods and conventional plating were applied to monitor Enterobacter agglomerans 339 derivatives carrying a Tn5-Mob or an nptI-cassette in unsterile soil microcosms. The plate counts of the introduced bacteria decreased continuously in time until undetectable on selective media. In contrast, hybridization of the total DNA directly isolated from inoculated soil samples showed that the target sequences detected corresponded to a much higher number of bacteria than indicated by plating. By PCR-amplification and hybridization of the soil DNA we could show that a significant number of target sequences still persisted in the soil microcosms, even when the inoculated bacteria were not able to make colonies on selective agar plates. The Tn5 marker caused instabilities in the genome of the bacteria studied. Some of the clones that grew in the soil samples had rearrangements in their genome. The detection of E. agglomerans 339 derivatives carrying the immobile nptI-cassette was also dependent on its location in the bacterial genome.
The fate of either deliberately or accidentally released genetically engineered microorganisms and their recombinant DNA could be at least partly predicted if enough data are collected by experiments in appropriately designed micro- cosms. Direct monitoring by molecular methods [1-13] is quite important because in many cases the microorganisms of interest could perhaps be- come unculturable on laboratory media after a
long exposure to natural conditions [14,15]. Tn5 is one of the most frequently used markers in laboratory [16-22] and field studies (for a review, see [23]). However this transposable element can generate rearrangements in the genome of host cells [24] and so alter their characteristics. On the other hand, since all kinds of genetic exchange have been shown to occur in the environment [18,25-30], one cannot exclude the possibility of transfer of Tn5 to some representatives of the natural communities. There is little information available about the possible consequences of the introduction of large amounts of bacteria labeled with transposable elements into soil [23].
This work aimed to study the persistence and
SSDI 0168-6496(94)00052-2
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stability of a Tn5-Mob labelled Enterobacter ag- glomerans 339 strain introduced into unsterile soil microcosms. Perhaps a more stable and safer way to label bacteria is the insertion of antibiotic resistance markers in the form of cassettes [31,32]. Hence, in the present work we monitored three derivatives of E. agglomerans 339, carrying an nptI-cassette from pUC4K [33], in soil micro- cosms. In one of the strains the cassette was located on a high copy number plasmid, in the second on a low copy number plasmid, and in the third on the chromosome.
Materials and Methods
Bacterial strains and plasrnids The strains used in this study are listed in
Table 1. The plasmids harboured in the Es- cherichia coli strains were used for the isolation of DNA fragments carrying the sequences used for the hybridization experiments. For inocula- tions of the soil microcosms and for plate mating experiments, derivatives of the E. agglomerans 339 strain from our own collection [34] were used. E. agglomerans 339 contained a large (200 kb), indigenous, self-transmissible low copy number nif-plasmid pEA9 [35-37]. E. agglomerans 19-1-1 was labeled with Tn5-Mob [16] by random muta- genesis [37] and contained three copies of the transposon in its genome [5] (see Table 1). E. agglomerans 339- was cured of pEA9 [35]. E. agglomerans 339CR1 was a 339- derivative, car- rying an nptI-sacB-sacR cassette on the chromo- some [38]. E. agglomerans 339-12 contained, in
addition to pEA9, a recombinant high copy num- ber pBR322-derivative carrying a 3 kb fragment of the pEA9 nil-cluster including the nifN and nifX genes with the nptI-cassette inserted in front of the nifX gene (pBR322::nptI) [39]. E. agglomerans 339-25 carried the same nptI-cas- sette inserted in front of the nil X gene of pEA9 (pEA9::nptI). This strain was obtained by curing of the above mentioned E. agglomerans 339-12 of the pBR322::nptI. In this case a site-directed double replacement of the wild-type nil sequence of pEA9 by the recombinant nif::nptI sequence occurred [Liu, C., unpublished].
Microcosms A sandy loam soil was used from the experi-
mental field of the University of Bayreuth (1.48% organic C; C / N ratio 13.2; pH 5.6). The soil was air-dried to 7.6% 50 g of unsterile soil were distributed in Erlenmeyer flasks and then inocu- lated with 2 x 107 bacteria per g of soil, resus- pended in 4 ml Luria broth or saline. The result- ing water content of the soil was 14.4%. The cells were evenly distributed with a spatula. The flasks were stoppered with cotton, weighed and incu- bated at 22°C in darkness [40]. Every week the humidity was adjusted to the initial value by adding sterile water and the soil was mixed thor- oughly with a spatula. One day later 2 g of soil were withdrawn for isolation of DNA by direct lysis and another 2 g were used for enumeration of the labeled bacteria by plating. To establish the detection limits, soil samples were inoculated with decimal dilutions of an overnight culture, starting from 1 × 107 and decreasing to about one
E. coli S17-1 pSUP5011 16 E. coli K-12 JM83 pUC4K 33 E. agglomerans 19-1-1 pEA9: :Tn5-Mob Rif r, Nal r, K r n r / N m r. The K m r / N m r was 5, 37
due to two copies of Tn5-Mob Rif r, Str r Rif r, Km r, SaC. The Km r and Sac r were due to an nptI-sacB-sacR cassette Rifr Rifr
E. agglomerans 339- E. agglomerans 339CR1
E. agglomerans 339-12 E. agglomerans 339-25
pEA9, pBR322::npt I pEA9:: npt I
35 38
39 Liu, personal communicat ion
cell per gram soil. Samples were incubated at room temperature for 15 min. Afterwards, the DNA content and the colony forming units (cfu) of the target bacteria were analysed. The experi- ments were performed in duplicate and repeated at least once. All values were calculated per g of wet soil. The cells used for the inoculation of the soil microcosms were grown in liquid LB medium without antibiotics.
Enumeration of the labelled bacteria in soil by plating
Soil samples were shaken in an equal volume of saline on a rotary shaker at 180 rpm for 30 min and allowed to settle for 15-20 min. From the supernatant, aliquots were taken, dilution series were done if necessary, and platings were made on LB plates supplemented with antibiotics. The npt II gene of Tn5 confers resistance to kanamycin (Km) and neomycin (Nm). Therefore, the Tn5- Mob containing E. agglomerans 19-1-1 cells were recovered from soil on plates supplemented with Km at 20/zg m1-1, Nm at 100, and cycloheximide and nystatin (antifungal agents) at 50 ~g m1-1. The nptI-labelled bacteria (this marker confers resistance to Km) were recovered on plates with Kin, cycloheximide and nystatin in the concentra- tions mentioned above. The plates were incu- bated up to seven days at 30°C. To distinguish between the target strain and indigenous Kmr/ Nm r bacteria, replica plating on LB-agar supple- mented with 100 /zg m1-1 rifampicin (Rif) were done [40]. Plating was performed in duplicate.
Plate matings Donor (E. agglomerans 19-1-1 or its derivatives
recovered from soil) and recipient (E. agglomer- ans 339-) cells were grown as overnight cultures in LB medium with appropriate antibiotics: Km and Nm (concentrations as above) for the donors, and streptomycin (Sm) (200 /xg m1-1) for the recipients. They were washed, resuspended in saline and mixed 1:1. From this mixture, 0.4 ml samples plus 1.6 ml LB were spread onto LB plates and incubated for 16 h at 30°C. The cell suspension was washed, appropriately diluted and plated on LB plates supplemented with antibi- otics to identify donors, recipients and exconju-
181
gants. The transconjugant-selective plates con- tained Km, Nm and Sm at the concentrations already mentioned above. The donor plates con- tained Km and Nm and the recipient-selective plates contained Sm. The experiments were per- formed twice in duplicate.
DNA isolation and manipulation Soil DNA was isolated by direct lysis as de-
scribed by Selenska and Klingmiiller [5]. Total bacterial DNA from overnight culture was iso- lated by the method of Masterson et al. [41]. The plasmid content was analysed by a mini-scale procedure of Casse et al. [42]. Preparative isola- tions of small plasmids were done by the alkaline lysis method [43], and large plasmids were iso- lated according to Kao et al. [44].
All other DNA manipulations were performed by standard methods as described by Sambrook et al. [43].
Two different probes were applied for detec- tion of the Tn5-Mob sequences. The first one consisted of the 4.3 kb BgllI fragment of the core part of the Tn5 derivative. For the second one, the 1.3 kb HindlII-HpaI fragments of the two IS50 elements of the same transposon were used. All Tn5-Mob specific sequences were isolated from pSUP5011 [16]. A 1.2 kb PstI fragment, containing the nptI gene was isolated from pUC4K [33]. The fragments were biotinylated and used for Southern blot and slot blot hy- bridizations by the non-radioactive PhotoGene detection procedure according to the protocol provided by the manufacturer (BRL, Life Tech- nologies).
Enumeration of the target genomes present in the soil samples by DNA-DNA hybridization
The soil DNA samples were concentrated by a HYBRISLOT 24-Well Filtration Manifold (BRL) onto a PhotoGene nylon membrane (BRL). To avoid false positives, not more than 5/zg of DNA per slot were loaded.As a negative control, DNA isolated from uninoculated soil was used. Two- to four-fold dilutions containing between 1 and 100 ng of the total DNA of the strain used for inocu- lation of the microcosms were taken as a stand- ard. The intensity of the signals obtained was
182
compared both visually and by using the 2D- densitometer of the computer program Cy- berTech CAM version 2 (Cybertech, Berlin). The amount of target genomes calculated by the in- tensity of the hybridization signals was expressed as genome equivalent. In the above calculations, an amount of 6 fg of DNA per bacterial cell was assumed [2].
Amplification of the nptI and nptlI fragments in soil DNA by PCR
The primers used for the amplification of a 400 bp fragment of the nptI gene and a 550 bp fragment of the nptII gene of Tn5 were the same as described by Selenska et al. [39]. Taq poly- merase and the PCR incubation buffers were supplied by Amersham. The PCR reaction mix- ture was 200/~M of each dNTP, 0.4/~M of each primer, 1 ~1 (about 0.1-0.2 ~g) of soil DNA and 2 U of the enzyme in a final volume of 25 ~1. The MgC12 concentration was adjusted to 3 mM and the reaction mixture was supplemented with 0.1% BSA [11]. The conditions for DNA amplification were as follows: 3 min denaturation at 95°C and then 35 cycles using 1 rain at 95°C, 1 min at 55°C and 1.5 rain at 72°C. The reaction was extended 7 rain at 72°C before 8 ~1 of the reaction mixture was analysed in a 1.2% agarose gel.
Results and Discussion
Efficiency of recovery, sensitivity and specifity of the methods used
The recovery of freshly introduced bacteria (15 min after inoculation), carrying either Tn5-Mob or npt I-cassette by shaking the unsterile soil sam- ples with saline and plating on selective media, was in the range of 10-30%. The limit of detec- tion was 10 inoculated cells per g of soil (Fig. 1). Hence, more than 70% of the introduced bacteria could not be recovered by this method, most probably due to their adsorption to soil particles. This result is comparable with that achieved by Holben et al. [1], where 33-35% of the bacteria could be released from soil. About 105 indige- nous cells per g of soil were able to form colonies on kanamycin and neomycin containing plates,
I00
8o
60
40
~ 20
7 6
I"1 by plating • by hybridization
5 4 3 2 1 0
log no. inoculated cfu/g soil
Fig. 1. Detection of E. agglomerans 19-1-1 in unsterile soil by plating and by hybridization of soil DNA. The number of the introduced cells is set to 100%. Results are means of two independent experiments and both were performed in dupli- cate, The values obtained by plating did not deviate by more than 15% and those obtained by hybridization did not deviate
by more than 22%.
comparable with the results of others [21,32]. None of them were rifampicin resistant. Total DNA of 100 K m r / N m r indigenous bacteria was isolated and slot blot hybridization with the Tn5 specific probes was performed. No hybridization signals were obtained, which confirms that nptII can be used as reliable marker for the introduced bacteria in the soil samples analysed in this work. This result is in agreement with the results of other studies [1,21].
Direct DNA isolation from the soil samples and subsequent slot blot hybridization with Tn5 specific probes resulted in detection of 70-80% of the introduced target genomes (Fig. 1), which is consistent with the results of Tebbe and Vah- jen [13]. As a negative control 5/~g DNA isolated from uninoculated soil was used where no Tn5 specific signals were obtained. When more than 5 /xg DNA were used, very weak hybridization sig- nals were detected. The unspecifity of these sig- nals was confirmed using 1 -1 0 / zg of E. agglom- erans 339- total DNA as a control. Using dilu- tions of total bacterial DNA from pure cultures, the limit of detection was 0.5 pg of the target sequence, which represents about 104 target
183
genomes. Because we did not load more than 5 ~g soil DNA per slot, specific hybridization sig- nals were obtained from DNA samples where at least 105 E. agglomerans 19-1-1 cells per g of soil were present. This detection limit is comparable with that achieved by others. For example, Tebbe and Vahjen [13] detected 104 target cells per g soil (eight targets per cell) and Smalla et al. [12] detected about 106 target cells per g soil (three target sequences per cell) using slot blot hy- bridization of soil DNA. The direct DNA isola- tion method gave a higher percentage of recov- ery, but the detection limit of 10 cells per g of soil of the plating technique was much lower. Thus, for detection of culturable bacteria able to ex- press the selective marker, conventional plating was more sensitive in comparison with hybridiza- tion of non-unamplified DNA.
The limit of detection of PCR amplification of the nptlI fragment was 6 X 103 E. agglomerans 19-1-1 cells per g of soil (about 20 target se- quences in a reaction mixture at three copies of the target per cell). The limit of detection of the nptI-gene by PCR was in the same order of magnitude. Control reactions using soil DNA iso- lated from uninoculated samples always gave neg- ative results. The sensitivity obtained is consistent with those achieved in other studies. For exam- ple, Smalla et al. [12] detected 103 P. fluorescens cells per g soil (about 20 target sequences per reaction and three targets per cell) and Picard et al. [6] detected 104 Agrobacterium tumefaciens cells per g of soil (one copy of the target was sufficient for successful amplification and one target per cell was present), while Tsai and Olson [9] detected 5 X 102 E. coli cells per g soil (about 20 targets per reaction and seven targets per cell). However, lower detection limits in the range of 10 Hansenula polymorpha cells per g of soil (8 targets per cell) [13] or one Pseudomonas cepacia cell per g of sediment (15-20 targets per cell) [3] were also reported, which shows that further im- provement of the sensitivity is possible.
Monitoring of E. agglomerans 19-1-1 in long-term experiments
As shown in Fig. 2, during the first ten days of incubation number of the E. agglomerans 19-1-1
1010
~. 106
"~ - -X-- plating 104
.~ ~ ----O-- hybridiz
102 t~
100 ~ t " ~ _ _ X ~ : _ ~ 0 10 20 30 40 50
days of incubation
Fig. 2. Detection of E. agglomerans 19-1-1 in unsterile soil by plating and by hybridization (hybridiz.). The bacterial inocu- lum was resuspended in Luria broth and added to the soil. Results are means of two independent experiments and both were performed in duplicate. The replicate values obtained by both plating and by hybridization did not deviate by more
than 60%.
cells detected by plating was comparable with the number estimated by slot blot hybridization with the Tn5 specific probes. After this period, how- ever, the number of target bacteria detected on selective plates decreased rapidly in time. In con- trast, the number of target genome equivalents determined by hybridization remained almost constant - in the range of 107 per g of soil. After 5 weeks of incubation, less than 10 inoculated bacteria per g of soil were able to grow on the selective plates, and by the sixth week no cfu were obtained. At that time the number of target genome equivalents estimated by DNA-DNA hy- bridization was still higher than 106 per g of soil, and amplification of the npt II sequences by PCR was successful. Hence, the decrease in the num- ber of the introduced bacteria determined on plates was not parallelled by a decrease in the number of target molecules detected by molecu- lar methods.
In a previous study, where E. agglomerans 339 was introduced into unsterile soil microcosms and monitored by plating on selective media, it was shown that the viable counts decreased rapidly from 1 X 10 7 to about 1 × 104 cfu per g of dry soil in 3 weeks [45], which is in agreement with our plating results (Fig. 2). From this [45] and also from related experiments [40] it was assumed that E. agglomerans 339 survives poorly in soil. Later it was shown that Tn5 sequences of the E. ag-
184
glomerans 19-1-1 strain could be detected by hy- br id izat ion of the D N A isolated directly from soil samples up to 70 days after their inoculat ion. At that t ime the in t roduced bacter ia were no longer able to grow on agar plates conta in ing antibiotics [5]. The results p resen ted here are in ag reemen t with the above. In addit ion, we quant i f ied the hybridizat ion signals (see Methods) and com- pared the n u m b e r of the so detec ted genome equivalents with the n u m b e r of E. agglomerans 19-1-1 cells able to grow on selective agar plates.
Since the long pers is tence of target D N A in na tu- ral condi t ions was already demons t ra t ed [10,11], it is impor tan t to est imate what fract ion of the
detected specific sequences represen ted bacter ial cells or D N A released from dead cells.
Here a quest ion arises about the source of the ob ta ined hybridizat ion signals and PCR products, since one can expect de tec t ion of D N A from the inocula ted bacter ia as well as of naked D N A released from them. Slot blot hybridizat ions with marker -homologous probes and P C R amplifica- t ions of soil D N A can not answer this quest ion. By these methods, relatively small D N A frag- ments are targeted, thus specific sequences can be detected even from strongly sheared [6] or degraded D N A [7]. Sou the rn blot hybridizat ion
Fig. 3. Detection of Tn5-Mob containing EcoRI fragments in soil DNA. The results presented under (A), (B), and (C) and those presented under (D) are from two independent experi- ments. In the first experiment (A, B, and C) the bacterial inoculum was resuspended in saline and in the second (D) - in Luria broth. (.4,) Electrophoresis of EcoRI digested soil DNA recovered at different times after inoculation with E. agglomerans 19-1-1. The number of the introduced bacteria per g soil, able to make colonies on selective plates, is given. 1:0 time - 2;<107, 2:3 days - 1×107; 3:7 days - 1×106; 4: 28 days - 1 x 103; 5:35 days - 3 ;< 102; 6:56 days - 1 × 102; 7: 70 days - no introduced bacteria grew on selective plates; 8: DNA recovered from non-inoculated soil; 9: total DNA of E. agglomerans 19-1-1 digested with EcoRI; 10: kilobase ladder. (B) Southern blot of (A) and PhotoGene detection of Tn5-Mob core sequences. (C) Lane 5 of (B) - shorter film exposure. (1)) Southern blot of an EcoRI digested soil DNA sample and PhotoGene detection of Tn5-Mob core sequences. The DNA sample was recovered from soil inoculated with E. agglomer- ans 19-1-1 after one week of incubation when 1 × 10 7 target bacteria per g of soil were detected by plating. --* indicates a
new band in the hybridization pattern.
can help to be t te r analyse the fate of the target D N A in the env i ronmen t [7] and to moni to r the pers is tence of microorganisms and their D N A in soil samples [5]. If a specific hybridizat ion pa t t e rn of the in t roduced bacter ia can be ob ta ined in total soil DNA, then by compar ison of the pat- terns ob ta ined in different samples, conclusions about the survival of the bacter ia in these, rear- r angemen t s in their genome and probably also about horizontal gene t ransfer could be made [1]. For these purposes, gent le procedures should be applied, like the one developed by Selenska and Klingmiil ler [5] for direct isolation of D N A from
A) 1 2 3 4 5 6 7 8 9 10
? : 2
- 12kb - 6kb
B) 1 2 3 4 5 6 7 8 9 10
J
c)
- 12kb 6 kb
- 12 kb - 6kb
D)
- 12kb
D 6 kb
185
soil, which yields high molecular weigth DNA digestable with restriction enzymes.
We analysed the Tn5 hybridization patterns of EcoRI digested soil DNA recovered from our inoculated microcosms after different periods of incubation. In almost all analysed DNA samples we obtained the same EcoRI fragments which were about 34 kb, 21 kb and 9.6 kb in size and hybridized with the Tn5 core probe. In soil DNA isolated from uninoculated samples no specific sequences were detected. The limit of detection was comparable with that of the slot blot hy- bridization. A representative example of the hy- bridization patterns is shown in Fig. 3B. The intensity of the hybridization signals decreased in time (see Fig. 3B, lanes 2, 3 and 4, with exception of the sample in lane 5 (isolated after 35 days of incubation), where strong propagation of Tn5 containing rearranged bacteria was detected. This exception will be discussed below. In samples withdrawn from this microcosm at days 28 (lane 4), 56 (lane 6) and 70 (lane 7), specific hybridiza- tion signals at the limit of detection were ob- tained. Because of the presence of nucleases in the environment, naked DNA is degraded until adsorbed to particulates [29,46,47]. It was shown that free DNA is subjected to significant frag- mentation in an unsterile soil [7]. Further, shorter DNA fragments are adsorbed to soil to a greater extent than longer fragments, and it was assumed that fragments larger than 30 kb are unlikely to persist in the environment [48]. The distinct hy- bridization patterns obtained in our experiments could be explained by persistence of intact bacte- rial genomes in the microcosms. It is possible that some of these intact genomes were harboured in dead cells still protecting the DNA from nucle- ases. However, we suggest that the majority of the detected specific DNA-sequences in our in- vestigation were isolated from surviving E. ag- glomerans 19-1-1 cells, since otherwise the natural microbial activity should lead (even with delay) to degradation of most target DNA in the micro- cosms. The long persistence of the introduced bacteria in the unsterile microcosms could be explained by the fact that E. agglomerans 339, which is the parent strain of E. agglomerans 19-1-1, is a soil bacterium [34] well adapted to
such an environment. If the introduced cells sur- vived in soil but were unable to grow on agar plates containing antibiotics, they could be in a dormant or non-culturable state or they could be unable to express the selective marker. It was shown in several studies that the expression of the nptlI gene decreases in time when bacteria containing this marker were exposed to natural environments [4,8,19]. Other studies showed that Tn5-1abelled Pseudomonas fluorescens cells rapidly lost their culturability in soil [22].
Stability of genetically manipulated E. agglomerans 19-1-1 in soil
The stability of E. agglomerans 19-1-1 in the soil microcosms was studied by analysis of clones recovered after different incubation times. A to- tal of 140 colonies isolated from five consecutive experiments, where cells resuspended in LB medium were added to the microcosms (the re- suits of two of these experiments are summarized in Fig. 2), was investigated. A total of 160 colonies, obtained by plating dilutions of the overnight cultures used for inoculation of the microcosms, was analysed as a control.
The plasmid content of the 140 clones recov- ered from soil was assayed. All except five con- tained the indigenous plasmid pEA9. Four clones had smaller pEA9 derivatives (data not shown). The rearranged derivatives of the plasmid pEA9 were isolated and their restriction analysis was performed. Two were found to be identical. Their size was reduced by approximately 30% (data not shown). The clones harbouring these deletion derivatives were used as donors in plate matings. Their conjugation rate was increased (5 × 10 -6 transconjugants per donor) in comparison with the conjugation rate with E. agglomerans 19-1-1 (4.8 x 10 -7 transconjugants per donor). This ex- ample shows how some rearrangements could increase the probability of spread of genetic in- formation in the environment. The other two pEA9 derivatives did not have such extensive deletions and the clones carrying them had conju- gation rates comparable with those of the parent strain. One of the isolated clones was cured of pEA9. The loss of pEA9 without further genomic rearrangements in this clone and the rearrange-
186
ments on the pEA9 derivatives mentioned above were also confirmed by pulsed-field gel elec- trophoresis analysis. In these cases, insertion of pEA9 in the chromosome was excluded (unpub- lished results). In the 160 control clones obtained from overnight cultures, one case of a small dele- tion on pEA9 was detected.
Total genomic D N A was isolated from each of the 140 clones recovered from soil, digested with E c o R I and analysed by Southern blot hybridiza- tion. The Tn5 hybridization patterns of the clones were compared with the hybridization pat tern of the strain used for inoculation. Tn5-Mob does not contain a recognition site for E c o R I , and that is why the number of hybridization signals corre-
A)
1 2 3 a
sponds to the number of the Tn5-sequences which exist in the genome of the bacteria studied. Changes in the hybridization pat tern of ten of the clones indicated rearrangements in their genomes (Fig. 4). Transpositions of the full-length Tn5- Mob and of the IS50 element and other rear- rangements were found in addition to the already described deletions on pEA9 and spontaneous curing of this plasmid. The Tn5-Mob transposi- tions were detected as new hybridization bands appearing in the patterns of certain clones after using both the IS50 and the Tn5-Mob core probe (Fig. 4, lines 9 and 10a). The IS50 transpositions were detected only by using the IS50 specific probe (compare Fig. 4A, lanes 7 and 8 with B,
4 5 6 7 8 9 10a
-12 kb
-6 kb
13)
1 3a 3b 4 5 6 7 8 9 lOa lOb
~,~mwr ~ ~ n a ~ , I i / - 12 kb
" " -6 kb
Fig. 4. Detection of genomic rearrangements in Tn5-Mob containing E. agglomerans 19-1-1 clones recovered after exposure to unsterile soil. (A) Southern blot hybridization using Tn5-Mob core probe with EcoRI digested DNA of E. agglomerans 19-1-1 derivatives. (B) Southern blot hybridization using IS50 probe with EcoRI digested DNA of E. agglomerans 19-1-1 derivatives. 1. Total DNA of E. agglomerans 19-1-1, containing three copies of the transposon; 2. DNA of the indigenous plasmid pEA9,
containing one of the transposon copies; 3-10 b total DNA of E. agglomerans 19-1-1 derivatives, isolated after incubation in soil.
187
lanes 7 and 8). Deletions and other rearrange- ments appeared as a change in the position of certain bands (Fig. 4, lanes 3a,b, 4 and 6). In the D N A of the clone, cured of pEA9, the plasmid- specific hybridization band was missing (Fig. 4, lane 5). Clones 3a, 3b and clones 10a, 10b carried identical rearrangements, but the rearrangements of clones 3a,b were different from those of the 10a, b. Lanes 3b and 10b are missing on Fig. 4A. In the 160 control clones obtained from overnight cultures, two different cases of Tn5-Mob transpo- sition were detected (not shown).
The rearranged clones were often isolated from soil in clusters. For example, in one case when 16 clones were recovered from the same soil sample, four were altered: one of them carried a new inserted IS50 copy (Fig. 4, lane 8), another one a new inserted Tn5 copy (Fig. 4, lane 9) and the other two clones had identical hybridization pat- terns with two additional Tn5 copies (Fig. 4B, lanes 10a and 10b). In another case, three of six analysed clones isolated from the same soil sam- ple had deletions on pEA9. Two of them were identical, as already mentioned (Fig. 4B, lanes 3a and 3b). In the hybridization pat tern of the third one, the upper band was a doublet (Fig. 4, lane 4).
The number of rearrangements observed in clones isolated after incubation in soil was higher than that in clones isolated from LB-medium. Only about 2% of the E. agglomerans 19-1-1 cells from ovemight cultures (without antibiotics) were rearranged (Table 2). The same strain when re- covered after exposure to soil conditions (1-7 weeks of incubation) had 7% of cells with rear- rangements. However, the percentage of rear- ranged bacteria was 18% in samples drawn after 5 to 7 weeks of incubation. Hence, most rear- rangements were found in clones recovered after 5 - 7 weeks of incubation in the microcosms (see Table 2). This is the time when less than 10 labelled bacteria per g of soil were able to grow on selective plates (Fig. 2). Clones with identical rearrangements were isolated from the same soil sample, but never from different microcosms.
Perhaps the clones with the additional Tn5 copies, integrated in some particular places in the genome, could have selection advantages on K m /
Table 2 Stability of Tn5-Mob containing E. agglomerans 19-1-1 in Luria broth and in soil conditions
Clones isolated from Luria Soil: Soil: broth 1-7 5-7
weeks weeks ofincu- of incu- bation bation a
Number of analysed clones 160 140 50 Number of rearranged clones 3 10 9 Number of rearrangements 3 13 11
Results are the summary of five consecutive experiments. In only three of them were rearrangements detected. The bacte- rial inoculum was resuspended in LB. The rearrangements found in clones isolated from overnight cultures were not identical with those detected in clones recovered from soil after different incubation periods. a The clones analysed after 5-7 weeks of incubation are also included in the previous column where the analysis of the clones isolated after 1-7 weeks of incubation is presented.
Nm containing plates, when isolated at a time when the most of the cells probably enter a dormant or non-culturable state. However, this selective pressure cannot explain the preferential isolation of bacteria with deletions or IS50 trans- positions. These events are consequences of transpositions and could happen during the growth on the plates, but the fact that colonies with identical rearrangements were always iso- lated from the same soil sample argues against this explanation. We suggest that at least some of the rearranged clones propagated in our soil mi- crocosms. Whether these rearrangements oc- curred in soil or in the overnight cultures with which the microcosms were inoculated is unclear. In general, by inoculation of soil with Tn5-Mob labelled E. agglomerans 19-1-1, about 2% of the introduced population was already altered.
In two of about one hundred soil D N A sam- ples analysed the Tn5 hybridization EcoRI re- striction pat tern was changed (see Fig. 3, C and D). In these two cases the number of rearranged bacteria present in the samples was above our limit of detection by hybridization. These two samples were from independent experiments. In both experiments, the control hybridization pat- terns of the total D N A of the culture used for inoculation were not altered. The result pre-
188
sented in Fig 3A-C, lane 5 is interesting because the observed change in the hybridization pattern was detected after a relatively long exposure of bacteria to the soil conditions. As seen in Fig. 3A, the amount of DNA recovered from this soil sample was larger than in the other samples, where no changes were observed. In addition, in this sample the number of Tn5 carrying bacteria was much higher than in samples taken one or four weeks earlier (Fig. 3, B and C). It is impossi- ble to say at which time the rearranged bacteria grew in soil, but they likely formed a clone in a particular site in the microcosm. This is the rea- son why in the other samples taken from the same microcosm no rearrangements were de- tected. Such differences between samples with- drawn from the same microcosm could be ex- pected, because soil is not a homogenous envi- ronment. In this specific case (Fig. 3, A, B and C) soil was inoculated with bacteria resuspended in saline and no LB was added. The rearranged bacteria grew despite the presence of an indige- nous population and without any selection pres- sure applied. The second case of growth of rear- ranged bacteria in the unsterile microcosm (Fig. 3D) was detected in a sample withdrawn 1 week after inoculation of soil with bacteria resus- pended in LB. It was already shown that one day after inoculation of E. agglomerans 339 cells re- suspended in LB, the number of the inoculated bacteria and that of the culturable indigenous bacteria increased [40]. It is possible that the growth of the rearranged bacteria was enabled by the LB amendment, but no selection pressure was applied. The origin of the new hybridizing bands is not clear. These bands could represent rearrangements in the genome of Enterobacter agglomerans 19-1-1, or they could come from a Tn 5 recipient in the natural bacterial community.
Particular cases when Tn5 and IS50 confer growth advantages to their E. coli host cells in comparison to the parent bacteria without special selective pressure were shown by Biel and Hartl [49] and by Hartl et al. [50]. These observations could explain growth of some rearranged clones in our microcosms. The increased fitness of such clones could also explain their preferential isola- tion by plating in the last stages of our experi-
ments. These results contrast with those obtained by van Elsas et al. [20,51] that Tn5 and nptII decrease the fitness of a P. fluorescens popula- tion as a whole in soil. Obviously the place of insertion and the nature of the induced mutations could be responsible for these different effects.
Transposons and IS-elements may cause rear- rangements in the genome of their host cells and because of that are very interesting from an evo- lutionary point of view [24,52,53]. The transposi- tion rate of Tn5 in E. coli was shown to be 10-3_10 2 [24,53]. Therefore, it is not surprising that Tn5 caused instabilities in the genome of E. agglomerans 19-1-1, which is also a representative of the family Enterobacteriaceae (the transposi- tion rate in overnight culture without selection pressure was 2 × 10-2). The high proportion of rearranged clones isolated from soil and their propagation in the microcosms is somewhat sur- prising. Several authors have reported that Tn5 can be used as a relatively stable marker for monitoring of some bacteria in soil [17,21]. Its reliability for detection based on phenotypic ex- pression of the kanamycin resistance however was doubted by others [8,19]. In addition, rearrange- ment events in bacteria carrying Tn5 sequences occurring in the soil environment have also been discussed [54,55]. Jansson et al. [54] detected an increased proportion of rearranged bacteria in soil samples by restriction fragment length poly- morphism analysis of cellular DNA of bacteria extracted from soil. In the present study the detection of genomic rearrangements was done in DNA recovered from soil by direct lysis without a previous extraction of bacteria.
The results of our study show that (i) Tn5-Mob caused rearrangements in the genome of a genet- ically engineered soil bacterium exposed for a long time to unsterile soil, (ii) some of the rear- rangements could increase the probability of spread of genetic information in the environment and (iii) rearranged Tn5-Mob-containing bacteria possibly grew in unsterile soil to numbers de- tectable by hybridization (e.g., more than 105 per g of soil) without applied selective pressure and, in one case without energy source supplementa- tion.
It is necessary to notice that the number of the
10 8 - - X - - on pBR
~'~.---e ---43-- on chrom -- 106 ~ ~ o n pEA9 "5
104
102
100 i i ~ i ~ t 0 10 20 30 40 50 60
days of incubation
Fig. 5. Detection of the nptI-cassette labelled E. agglomerans 339 derivatives in unsterile soil by plating. The inoculum was resuspended in saline and added to the soil. In E. agglomer- ans 339-12 the cassette was on pBR322, a high copy number plasmid (*), in E. agglomerans 339-25 the cassette was on the low copy number plasmid pEA9 ( • ) and in E. agglomerans 339CR1 the cassette was on the chromosome ([]) . Results are means of three independent experiments and the values did
not deviate by more than 60%.
field tests with Tn5 labelled recombinant mi- croorganisms performed from 1986 to 1991 was relatively high [23], most probably due to the simplicity with which this marker can be intro- duced into bacteria. On the other hand, acciden- tal releases of Tn5 labelled bacteria cannot be excluded because of the widespread use of this transposon as a genetic engineering tool, and probably such releases occur continually. Hence, in some environments Tn5 sequences could be already abundant [56, 57]. The consequences of such releases are difficult to predict but we sug- gest to avoid this marker in future deliberate releases.
Monitoring of nptI-cassette labelled bacteria in soil samples
The detection of the nptI-labelled bacteria after different periods of incubation in soil was dependent on the location of the marker in the genome. The results obtained by plating are shown in Fig. 5. Bacteria labelled with the nptI- cassette in the chromosome were not detect- able by plating after 4 weeks of incubation. How- ever, 3 weeks later detection of nptI sequences was still possible by PCR (data not shown).
189
After 7 weeks of incubation, bacteria containing pBR322::nptI (high-copy number) were not de- tectable by plating, whereas their detection by PCR was possible. Bacteria with the npt I-cassette on the low-copy number pEA9 were not de- tectable by PCR when they did not form colonies on selective media. The fact that a case of spon- taneous curing of pEA9 in E. agglomerans 19-1-1 in soil conditions occurred (see above), shows that labelling of the strain with the npt I-cassette on pEA9 is not very reliable.
When the npt I marker was located on the high copy number pBR322::nptI, it was detectable for a long time. It was already shown by Byrd and Colwell [15], that pBR plasmids can be main- tained in E. coli for a long time even when the target bacteria enter a nonculturable state. How- ever, such plasmids could be released from the bacteria into the environment [58] and moreover they could be accepted by some indigenous recip- ients by natural transformation [28,29] or mobi- lization [27]. On the other hand, it was shown by Stotzky [59] that the frequency of conjugal trans- fer of chromosomal genes (10 -5 to 10 -4) in the soil environment was generally higher than that of plasmid borne genes (10 -7 to 10-5). Neverthe- less, it is preferable to introduce the markers into the chromosome because by using appropriate endonucleases a unique restriction pattern of the fragments carrying the marker could be gener- ated. This can be applied for the monitoring of the target bacteria as well as the marker. In contrast, when the marker is plasmid located, independently of its transfer to other soil recipi- ents, its restriction pattern will not be changed. In addition, as mentioned before, plasmids could be lost in environmental conditions, while the chromosomal DNA represents a more stable part of the bacterial genome. In this respect, introduc- tion of markers into the bacterial chromosome could be proposed to be more appropriate for monitoring purposes.
Acknowledgements
We are grateful to Armin Lechler for the help with the graphics and to Ilona Seebauer for the
190
technical assistance in some of the experiments. We thank Dr. J.D. van Elsas for his constructive critical remarks and suggestions as well as the two anonymous reviewers who helped to improve our manuscript. The English corrections made by Molly Daniel are gratefully acknowledged. The strain E. agglomerans 339-25 was obtained from Liu Chengjun.
References
1 Holben, W.E., Jansson, J.K., Chelm, B.K. and Tiedje, J.M. (1988) DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl. Environ. Microbiol. 54, 703-711.
2 Steffan, R.J., Goksoyr, J., Bej, A.K. and Atlas, R.M. (1988) Recovery of DNA from soils and sediments. Appl. Environ. Microbiol. 54, 2908-2915.
3 Steffan, R.J. and Atlas, R.M. (1988) DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Appl. Environ. Microbiol. 54, 2185-2191.
4 Pichard, S.L. and Paul, H.J. (1991) Detection of gene expression in genetically engineered microorganisms and natural phytoplaneton populations in the marine environ- ment by mRNA analysis. Appl. Environ. Microbiol. 57, 1721=1727.
5 Selenska, S. and Klingmiiller, W. (1991) DNA recovery and direct detection of Tn5 sequences from soil. Lett. Appl. Microbiol. 13, 21-24.
6 Picard, C., Ponsonnet, C., Paget, E., Nesme, X. and Si- monet, P. (1992) Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reac- tion. Appl. Environ. Microbiol. 58, 2717-2722.
7 Romanowski, G., Lorenz, M.G., Sayler, G. and Wacker- nagel, W. (1992) Persistence of free plasmid DNA in soil monitored by various methods, including a transformation assay. Appl. Environ. Microbiol. 58, 3012-3019.
8 Selenska, S. and Klingmiiller, W. (1992) Direct recovery and molecular analysis of DNA and RNA from soil. Mi- crob. Rel. 1, 41-46.
9 Tsai, Y.-L. and Olson, B.H. (1992) Detection of low num- bers of bacterial cells in soils and sediments by polymerase chain reaction. Appl. Environ. Microbiol. 58, 754-757.
10 Recorbet, G., Picard, C., Normand, P. and Simonet, P. (1993) Kinetics of the persistence of chromosomal DNA from genetically engineered Escherichia coli introduced into soil. Appl. Environ. Microbiol. 59, 4289-4294.
11 Romanowski, G., Lorenz, M.G. and Wackernagel, W. (1993) Use of polymerase chain reaction and electropora- tion of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils. Appl. Environ. Microbiol. 59, 3438-3446.
12 Smalla, K., Cresswell, N., Mendonca-Hagler, L.C., Wolters,
A. and van Elsas, J.D. (1993) Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated amplification. J. Appl. Bacteriol. 74, 78-85.
13 Tebbe, C.C. and Vahjen, W. (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and yeast. Appl. Environ. Microbiol. 59, 2657-2665.
14 Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, S.A. and Palmer, L.M. (1985) Viable but non- culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engi- neered microorganisms. Bio/Technology 3, 817-820.
15 Byrd, J.J. and Colwell, R.R. (1990) Maintenance of plas- mids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment. Appl. Environ. Microbiol. 56, 2104-2107.
16 Simon, R. (1984) High frequency mobilization of Gram- negative bacterial replicons by the in vitro constructed Tn5-Mob transposon. Mol. Gen. Genet. 196, 413-420.
17 Frederickson, J.K., Bezdicek, D.F., Brockman, F.J. and Li, S.W. (1988) Enumeration of Tn5 mutant bacteria in soil by using a most-probable-number-DNA hybridization pro- cedure and antibiotic resistance. Appl. Environ. Microbiol. 54, 446-453.
18 Riebaume, A., Angle, J.C. and Sadowsky, M.J. (1989) Influence of soil variables on in situ plasmid transfer from Escherichia coli to Rhizobium fredii. Appl. Environ. Micro- biol. 55, 1730-1734.
19 Pillai, S.D. and Pepper, I.L. (199l) Transposon Tn5 as an identifiable marker in Rhizobia: Survival and genetic sta- bility of Tn5 mutant bean Rhizobia under temperature stressed conditions in desert soils. Microb. Ecol. 21, 21-33.
20 Van Elsas, J.D., van Overbreek, L.S., Feldmann, A.M., Dullemans, A.M. and Leeuw, O. (1991) Survival of geneti- cally engineered Pseudomonas fluorescens in soil in com- petition with the parent strain. FEMS Microbiol. Ecol. 85, 53-64.
21 Recorbet, G., Givaudan, A., Steinberg, C., Bally, R., Nor- mand, P. and Faurie, G. (1992) Tn5 to assess soil fate of genetically marked bacteria: screening for aminoglycoside- resistance advantage and labelling specifity. FEMS Micro- biol. Ecol. 86, 187-194.
22 Binnerup, S.J., Jensen, D.F., Christensen, H.T. and Sorensen, J. (1993) Detection of viable, but non-culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique. FEMS Microbiol. Ecol. 12, 97- 105.
23 Wilson, M. and Lindow, S.E. (1993) Release of recombi- nant microorganisms. Annu. Rev. Microbiol. 4, 913-944.
24 Berg, D.E. (1989) Transposon Tn5 In: Mobile DNA (Berg, D.E. and Howe, M.M., Eds.), pp. 185-210. American Society for Microbiology, Washington, DC.
25 Schilf, W. and Klingmiiller, W. (1983) Experiments with E. coli on the dispersal of plasmids in environmental samples. Rec. DNA Tech. Bull. 6, 101-102.
26 Smit, E., van Elsas, J.D., van Veen, J.A. and de Vos, W.M. (1991) Detection of plasmid transfer from Pseudomonas
fluorescens to indigenous bacteria in soil using phage R2f for donor counterseleetion. Appl. Environ. Microbiol. 57, 3482-3488.
27 Henschke, R.B. and Schmidt, R.J. (1990) Plasmid mobi- lization from genetically engineered bacteria to members of the indigenous soil microflora in situ. Curr. Microbiol. 20, 105-110.
28 Lorenz, M,G. and Waekernagel, W. (1990) Natural genetic transformation of Pseudomonas stutzeri by sand-adsorbed DNA. Arch. Microbiol. 154, 380-385.
29 Khanna, M. and Stotzky, G. (1992) Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA. Appl. Environ. Microbiol. 58, 1930-1939.
30 Zeph, R.L., Onaga, M.A. and Stotzky, G. (1988) Trans- duction of Escherichia coli by bacteriophage PI in soil. Appl. Environ. Microbiol. 54, 1731-1737.
31 Kokotek, W. and Lotz, W. (1989) Construction of a LacZ- kanamycine-resistance cassette, useful for site-directed mutagenesis and as promotor probe. Gene 84, 467-471.
32 Smit, E. and van Elsas, J.D. (1992) Conjugal transfer in the soil environment: New approaches and developments. In: Gene Transfers and Environment (Gauthier, M.J., Ed.), pp. 79-94. Springer Verlag, Heidelberg-Berlin.
33 Vieira, J. and Messing, J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and se- quencing with synthetic universal primers. Gene 19, 259- 268.
34 Kleeberger, A., Castorph, H. and Klingmiiller, W. (1983) The rhizosphere microflora of wheat and barley with spe- cial reference to Gram-negative bacteria. Arch. Microbiol. 136, 306-311.
35 Singh, M., Kleeberger, A. and Klingmiiller, W. (1983) Location of nitrogen fixation (nil) genes on indigenous plasmids of Enterobacter agglomerans. Mol. Gen. Genet. 190, 373-378.
36 Kreutzer, R., Steibl, H.-D., Dayananda, S., Dippe, R., Halda, L., Buck, M. and Klingmiiller, W. (1990) Genetic characterization of nitrogen fixation in Enterobacter strains from the rhizosphere of cereals. In: Nitrogen Fixation (Polsinelli, M. et al., Eds.), pp. 25-36. Kluwer Academic Publishers, Dordrecht.
37 Klingmiiller, W., Herterich, S. and Min, B.W. (1989) Self- transmissible nif-plasmids in Enterobacter. In: Nitrogen Fixation with Non-legumes (Skinner, F.A. et al., Eds.), pp. 173-178. Kluwer Academic Publishers, Dordrecht.
38 Rappold, C. (1992) Klonierung, Sequenzierung und Muta- genese des recA-Gens yon Enterobacter agglomerans 339. Dissertation, Universit[it Bayreuth, Bayreuth, F.R.G.
39 Selenska, S., Schentzinger, S. and Klingmiiller, W. (1992) Direct detection of particular DNA-sequences in soil. In: Gene Transfers and Environment (Gauthier, M.J., Ed.), pp. 3-7. Springer Verlag, Heidelberg-Berlin.
40 Klingmiiller, W. (1991) Plasmid transfer in natural soil: a case by case study with nitrogen-fixing Enterobacter. FEMS Microbiol. Ecol. 85, 107-116.
41 Masterson, R.V., Prakash, R.K. and Atherly, A.G. (1985)
191
Conservation of symbiotic nitrogen fixation gene se- quences in Rhizobium japonicum and Bradyrhizobiurn japonicum. J. Bact. 163, 21-26.
42 Casse, F., Boucher, C., Julliot, M.M. and Denari6, J. (1979) Identification and characterization of large plas- raids in Rhizobium meliloti using agarose gel electrophore- sis. J. Gen. Microbiol. 113, 229-242.
43 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molec- ular Cloning. A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory, NY.
44 Kao, J.C., Perry, K.L. and Kado, C.I. (1982) Indoleacetic acid complementation and its relation to host range speci- fying genes on the Ti plasmid of Agrobacterium tumefa- c/ens. Mol. Gen. Genet. 188, 425-432.
45 D6hler, K. and Klingmiiller, W. (1988) Genetic interaction of Rhizobium leguminosarum biovar viciae with Gram- negative bacteria. In: Risk Assessment for Deliberate Re- leases (KlingmiJller, W., Ed.), pp. 18-28. Springer Verlag, Heidelberg.
46 Aardema, B.W., Lorenz, M.G. and Krumbein, W.E. (1983) Protection of sediment-adsorbed transforming DNA against enzymatic inactivation. Appl. Environ. Microbiol. 46, 417-420.
47 Romanowski, G., Lorenz, M.G. and Wackernagel, W. (1991) Adsorption of plasmid DNA to mineral surfaces and protection against DNase I. Appl. Environ. Microbiol. 57, 1057-1061.
48 Ogram, A.V., Mathot, M.L., Harsch, J.B., Boyle, J. and Pettigrew, C.A.J.R. (1994) Effects of DNA polymer length on its adsorption to soil. Appl. Environ. Microbiol. 60, 393-396.
49 Biel, S.W., and Hartl, D.L. (1983) Evolution of trans- posons: natural selection for Tn5 in Escherichia coli K-12. Genetics 103, 581-592.
50 Hartl, D.L., Dykhuizen, R.D., Miller, L.G. and de Fra- mond, J. (1983) Transposable element IS50 improves growth rate of E. coli ceils without transposition. Cell 35, 503-510.
51 Van Elsas, J.D., Wolters, A.C., Clegg, C.D., Lappin-Scott, H.M. and Anderson, J.M. (1994) Fitness of genetically modified Pseudomonas fluorescens in competition for soil and root colonization. FEMS Microbiol. Ecol. 13, 259-272.
52 Shapiro, J.A, Adhya, S.L. and Bukhari A.I. (1977) New pathways in the evolution of chromosome structure. In: DNA Insertion Elements, Plasmids, and Episomes. (Buk- hari, A.I. et al., Eds.), pp. 3-11. Cold Spring Harbor Laboratory, NY.
53 Berg, D.E. (1977) Insertion and excision of the transpos- able kanamycin resistance determinant Tn5. In: DNA Insertion Elements, Plasmids, and Episomes (Bukhari, A.I. et al., Eds.), pp. 205-212. Cold Spring Harbor Laboratory, NY.
54 Jansson, J.K., Holben, W.E. and Tiedje, J.M. (1989) De- tection in soil of a deletion in an engineered DNA se- quence by using DNA probes. Appl. Environ. Microbiol. 55, 3022-3025.
55 Sayler, G.S., Burns, R., Cooper, J,E., Gray, T., Pederson,
192
J., Prosser, J. and Selenska, S. (1992) Detection methods for modified organisms in the environment. In: The Re- lease of Genetically Modified Microorganisms - REGEM 2. (Stewart-Tull, D.E.S. and Sussman, M., Eds.), pp. 119- 122.
56 Left, L.G., Dana, J.R., McArthur, J.V. and Shimkets, L.J. (1993) Detection of Tn5-1ike sequences in kanamycin-re- sistant stream bacteria and environmental DNA. Appl. Environ. Microbiol. 59, 417-421.
57 Smalla, K., van Overbreek, L.S., Pukall, R. and van Elsas, J.D. (1993) Prevalence of nptlI and Tn5 in kanamycin-re- sistant bacteria from different environments. FEMS Mi- crobiol. Ecol. 13, 47-58.
58
59
Lorenz, M.G., Gerjets, D. and Wackernagel, W. (1991) Release of transforming plasmid and chromosomal DNA from two cultured soil bacteria. Arch. Microbiol. 156, 319-326. Stotzky, G. (1992) Gene transfer among and ecological effects of genetically modified bacteria in soil. In: Proceed- ings of the 2nd International Symposium on the Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms (Casper, R. and Landsmann, J., Eds.), pp. 122-134. Biologische Bundesanstalt fiir Land- und Forstwirtschaft, Braunschweig, Germany.
