Mierob Ecol (1989) 18:211-220

MICROBIAL ECOLOGY Springer-Verlag New York Inc. 1989

Related Plasmids Found in an English Lake District Stream R. W. Pickup Institute of Freshwater Ecology, Windermere Laboratory, Amblesidc, Cumbria, LA22 0LP UK

Abstract. An examination of the distribution ofplasmids carried by copper-tolerant bacteria from a freshwater stream revealed that > 60% carried at least one plasmid, and that large plasmids (> 100 kb) were predominant. A total of 10 copper-tolerant bacteria carrying the 54-kb plasmid, pFBA20, were detected at four sampling sites within the stream and, on consecutive occasions, at one site throughout a 1-year sampling period. The detection of this plasmid provides evidence that related plasmids can, under no apparent selective pressure, survive and disperse within the bacterial community. Two of the isolates that carried pFBA20 were phenotypically distinguishable. This would suggest that pFBA20 is transmissible.

Introduction

The ubiquity of plasmids in a large variety of bacteria from diverse environments is well documented [29], as is their ability to conjugally transfer under laboratory conditions [15]. Recently, and also as a result of the controversy surrounding the fate of recombinant bacteria released into the environment, much attention has been focused on the function, distribution, and transfer of plasmids in the natural environment [6, 11, 12]. Most environmental work has studied changes in bacterial communities and their plasmids in response to the introduction of xenobiotics [22], heavy metal pollution [10, 28], and the apparent transfer of antibiotic resistance at sewage or effluent polluted sites [25]. Some studies have compared the plasmid content of bacteria isolated from polluted and nonpolluted sites [6, 8, 11]. In each case, higher numbers of plasmid-containing strains were found at the polluted sites. Where a specific pollutant has been applied the increase in plasmid-containing bacteria is often complemented by an increase in the number of related plasmids within that population, each conferring a similar selective advantage on its host [16]. Most work has been conducted either in hospitals or heavily polluted sites. Little information exists on the characterization of plasmids not subjected to pollutant or extreme xenobiotic stress. As a consequence, Schutt [23] has identified areas of environmental plasmid research where information is lacking. These include plasmid distribution at various environmental sites, plasmid stability, genetic relatedness, physiological and regulatory functions, and DNA transfer in nature. This study was designed to examine plasmid distribution in copper-tolerant

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b a c t e r i a i s o l a t e d f r o m f r e s h w a t e r s t r e a m s i n a d i s u s e d m i n i n g area. T h e i n i t i a l a i m s were to d e t e r m i n e w h e t h e r t h e isolates h a r b o r e d c o p p e r - r e s i s t a n t p l a s m i d s a n d to e x a m i n e w h e t h e r r e l a t e d r e s i s t a n t p l a s r n i d s e x i s t e d a n d were a b l e to transfer in the aquatic e n v i r o n m e n t . Transferable plasmids conferring copper r e s i s t a n c e h a v e b e e n f o u n d i n b a c t e r i a i s o l a t e d f r o m piggery e m u e n t [26] a n d f r o m p l a n t s w h e r e c o p p e r s o l u t i o n s w e r e a p p l i e d to c o n t r o l p l a n t d i s e a s e s [3]. T h e g e n e t i c b a s i s o f c o p p e r r e s i s t a n c e is n o t u n d e r s t o o d , a l t h o u g h genes f r o m v a r i o u s b a c t e r i a l sources, w h i c h h a s b e e n s h o w n to c o n f e r c o p p e r r e s i s t a n c e , have b e e n cloned a n d sequenced. For copper resistance, D N A sequence analysis has p r e c e d e d b i o l o g i c a l u n d e r s t a n d i n g [24]. P l a s m i d s c o n f e r r i n g c o p p e r resist a n c e were n o t d e t e c t e d i n this i n v e s t i g a t i o n . H o w e v e r , r e l a t e d p l a s m i d s h a v i n g n o i n v o l v e m e n t i n c o p p e r r e s i s t a n c e were d e t e c t e d r a i s i n g q u e s t i o n s c o n c e r n i n g t h e i r f u n c t i o n a n d a b i l i t y to p e r s i s t i n t h e e n v i r o n m e n t . C o n s e q u e n t l y , t h e a i m s o f this i n v e s t i g a t i o n were r e f o c u s e d o n the d i s t r i b u t i o n o f t h e s e p l a s m i d s i n the s t r e a m .

Methods

Bacterial Strains and Plasmids The strains used are described in Table 1.

Sampling Site Church Beck is a freshwater stream in the Coniston Fells, Cumbria, UK. It is fed by Levers Water Beck, Low Water Beck, Red Dell Beck, and several unnamed tributaries as it flows ultimately into Coniston. Four areas were chosen for this study (Fig. 1): two independent sites were used in Deep Adit, a horizontal drainage shaft which flows into Red Dell Beck from the disused copper mine [sites 1 and 2: National Grid Reference (NGR) SD290987]; two central sites on Church Beck (sites 3 and 4: NGR SD293983 and SD304975); and a site at the inlet where Church Beck feeds into Coniston (site 5: NGR SD308970).

Isolation of Bacteria Sediment samples were taken from each site within the stream system using a sterile 400-ml widenecked bottle. All samples were returned to the laboratory within 45 min in an insulated container at 4*(2. Bacteria tolerant to I0 mM CuSO4 were isolated after sonicating each sample for 30 see at 20 Hz and then spreading 1 ml of sediment onto media comprising nutrient agar (Oxoid, Basingstoke, UK) supplemented with 10 mM CuSO4-6H20 and adjusted to pH 7.6 with 5 M NaOH before autoclaving. Single colonies which appeared after incubation at 20~ were purified on nutrient agar-copper sulfate selective media.

Isolation of Plasmid and Chromosomal DNA Plasmid DNA was prepared by the method of Wheatcrofi and Williams [30]. Chromosomal DNA was prepared from 10-ml nutrient broth (Oxoid) cultures grown to an ODrm of > 1. Cells were harvested by centrifugation at 7,500 x g for 2 rain at 20"C, washed once in 10 ml TES buffer (50

Plasmids in Aquatic Bacteria Table 1.

Bacterial strains used in this study

Strain FBA FBA FBA FBA FBA FBA FBA FBA FBA FBA

213

1230 1338 1324 1317 1316 1009 1051 1045 1014 1039

Phenotype" CUt CUt Cu' Cu t Cut Cut Cu t Cut Cu' CUt

PIasmid

Origin

pFBA20 pFBA20 pFBA20 pFBA20 pFBA20 pFBA20 pFBA20-1 pFBA20 pFBA20 pFBA20

This This This This This This This This This This

study study study study study study study study study study

CusSmrtrp -

--

C.J. Duggleby

CusSmrpro leu-thi-

--

(5)

Pseudomonasputida PaW 340

Escherichia coli HB 101

a t and s denote isolates tolerant or sensitive to 10 m M CuSOa in nutrient agar, respectively; Sm T denotes resistance to streptomycin up to 1 mg ml -t for PAW340 and 50 ug ml -t for HBI01; trp-, pro-, and thi- denote the requirement for tryptophan, proline, or thiamine, respectively

m M Tris:HC1 pH 8.0, 10 m M EDTA, 50 m M NaCI) followed by centrifugation at 7,500 x g for 2 rain at 20~ The cell pellet was resuspended in 0.5 ml of solution A [2 mg ml -t lysozyme, 100 mg ml -~ sucrose, 0.5 mg ml -~ heat-treated RNase (Sigma, Poole, UK) in TES buffer]. After 20 rain at room temperature, 0.25 ml of solution B (24 mg ml -j n-lauroylsarcosine in TES buffer) and 0.5 ml TES buffer were added. The lysis mixture was then vortexed for 3 rain followed by the addition of 1.25 ml TES buffer, 100 ul ethidinm bromide (20 mg m l - 0 , and 5.0 ml cesium chloride (1.12 g ml -a in TES buffer). The cesium chloride gradients were centrifuged at 100,000 x g for 18 hours at 20*(2. The D N A baud was removed and purified by standard procedures [18].

DNA Restriction and Electrophores& In each case, 40 t~l of D N A was digested by restriction endonucleases according to the suppliers' instructions (Gibco-BRL, UK). Electrophoresis was carried out as previously described [19].

Sizing of the Plasmid Restriction endonuclease digests of plasmid D N A were sized as previously described [ 19], except that instead of pWWO (9), HindlII-dJgested ~ D N A (Gibeo-BRL, Paisley, UK) or EcoRI SPPI D N A (P&S Biochemicals, Liverpool, UK) were used as a size standard.

Comparison of Plasmid Structure by Restriction Endonuclease Digestion All plasmid fragment profiles generated by HindlII digestions were compared. Those plasmids which appeared to be similar (i.e, those which shared multiple c o m m o n fragments) were reisolated, digested with HindIII, and subjected to electrophoresis on the same gel. Further digestions with

214

R.W. Pickup

other restriction enzymes, both singly and in combinations, were used for additional confirmation of structural similarities.

Bacterial Conjugation Overnight nutrient broth cultures of donor and recipient bacteria were mixed, and 200/~1 aliquots were spread immediately, at varying dilutions, onto appropriate selective agar plates. Both donor and recipient bacteria were spread separately onto the selective agar to act as negative controls. When the transfer frequency of liquid matings was low, 0.5 ml donor and 0.5 ml recipient cultures were filtered through a 0.45-#m miUiporefilterwhich was laid on a nutrient agar plate and incubated at 25~ for 16-24 hours. The bacterial growth was resuspended in 0. l M phosphate buffer (pH 7.5) and spread, at varying dilutions, onto selective agar.

Mutagenesis Plasmid curing was carried out using acridine orange [7] except that growth at the maximum concentration occurred after 48 hours at 25"C. Screening for plasmid-free derivatives was carried out using a crude cell lysis method [30].

Results

Plasmid Content and Size Distribution The analysis o f the plasmid content o f 370 unidentified aquatic bacteria, tolerant to 10 m M copper sulfate in nutrient agar, is presented. W h o l e p l a s m i d extraction [30] revealed that 60% o f the isolates contained one plasmid a n d 21% contained two or more. Conversely, < 2 0 % o f the isolates contained no detectable plasmids. T h e size distribution o f 303 plasmids isolated in this study was e x a m i n e d by restriction endonuclease digestion with HindIII. The m a j o r i t y o f plasmids ( > 8 5 % ) fell in the size range o f 8 0 - 2 0 0 kb with some as large as 240 kb. The p o p u l a t i o n o f copper-tolerant bacteria was therefore d o m i n a t e d by large plasmids with > 5 0 % larger than 100 kb. Small plasmids were infrequently encountered in this population.

Detection and Distribution of Common Plasmids O f the 303 plasmids analyzed in this investigation, only four distinct plasmid types (denoted as A, B, C, a n d D), which h a d related restriction profiles, were detected. T h e y were distributed a m o n g the five sampling sites (Fig. 1). In each case, for plasmid types B, C, a n d D (plasmid profiles n o t presented), only two isolates per plasmid type were detected. N o two identical plasmids were f o u n d at the same site. The 54 kb type A plasmids, however, were detected at four o f five sites in 10 i n d e p e n d e n t isolates (Fig. 1). P l a s m i d profiles (Fig. 2) s h o w e d that all type A plasmids were similar. Digestion with HindIII showed that nine plasmids shared all 10 restriction fragments; these plasmids were n a m e d pFBA20. The remaining plasmid p F B A 2 0 - 1 , f o u n d in strain F B A 1045, s h o w e d

Plasmids in Aquatic Bacteria

215 Low Water

,~k Lever.sWater

~nt~

Cht~h Beck

/~1

~1" 3:-

~k

1: A/D

A/A"/BIC

4: A/C

5: A/BID

Fig. 1. Distribution ofplasmid types A, B, C, and D at five sites on Church Beck in the English Lake District. 1 and 2 represent independent sites within a horizontal mine shaft. A' represents a plasmid type (pFBA20-1) with a similar restriction profile to that o f the type A plasmids (pFBA20).

an obvious difference. The HB fragment was lost and replaced by three novel fragments with an overall gain of 6 kb. The plasmids were confirmed as highly related by DNA hybridization ([ 18]; data not presented). The presence of small HindIII fragments of pFBA20 (difficult to visualize in Fig. 3) was confirmed by end-labeling with 32P-dCTP by standard methods ([18]; data not presented). Type A plasmids were detected in the stream system at sites 1, 2, 4, and 5 (see Table 2) throughout the sampling period of 1 year, except for one occasion. At site 1, six isolates were found to carry a pFBA20-1ike plasmid, yet four of them were isolated on different occasions, each separated by a 6- to 8-week interval. This indicated that the pFBA20-1ike plasmids are stably maintained in the bacterial population with respect to time within the stream system.

Properties of Type A Plasmids There was no detectable transfer of copper tolerance from any of the isolates to Pseudomonas putida PaW 340 or Escherichia coli HB 101 when selected on nutrient agar supplemented with Sm (15 #g ml) and 10 m M copper sulfate (pH 7.6). Plasmid curing using acridine orange at 100 #g m1-1 produced plasmidfree strains of FBA 1230 and FBA 1045. No detectable difference in growth on nutrient agar (10 m M CuSO4) was observed between the plasmid § and plasmid- strains. No differences in ability to grow in the presence of several

216

R.W. Pickup

Fig. 2. HindIII digestion of type A plasmids found in independently isolated copper-tolerant bacteria. The major bands are indicated at the left. Lanes: 1, X DNA (HindIII); 2, FBA1338; 3, FBA1324; 4, FBAI230; 5, FBAI316; 6, FBA1317; 7, FBAI009; 8, FBA1045; 9, FBA1051; 10, FBA1014; 11, FBA1039; 12, ), DNA (HindIII). Note that fragment B (lowerarrow)is missing in FBA1045 (lane 8) and is replaced by three unique fragments (upper arrows).

antibiotics used in previous tests on aquatic bacteria [ 14] were f o u n d between the plasmid + and plasmid- strains. This was also the case when challenged with several h y d r o c a r b o n and aromatic c o m p o u n d s as sole carbon source and toxic heavy metals (data not presented). Similarly, the plasmid was found not to confer U V resistance [13]. On the basis o f these results pFBA20-1ike plasmids were considered to be cryptic in that they carried no detectable function.

Evidence for Plasmid Transfer in the Stream System All except two o f the isolates, which carried type A plasmids, were p h e n o t y p ically and morphologically indistinguishable as judged by API 2 O N E and A P I Z Y M test strips (API Systems, Basingstoke, UK), growth characteristics, cell morphology, motility, and G r a m stain. T h e y were tentatively identified as P. fluorescens. T h e two exceptions were FBA1051 and FBA1039 which were identified as Pseudomonas sp. and were most likely P. putida. These differences were further confirmed by restriction endonuclease digestion o f their chrom o s o m a l D N A . This revealed that several o f the host strains were indeed identical. Only FBA 1051 and FBA 1039 p r o d u c e d distinct c h r o m o s o m a l D N A digest patterns (Fig. 3). Restriction e n z y m e fingerprinting o f c h r o m o s o m a l D N A has been used to distinguish unique but morphologically indistinguishable isolates, including biovars within a species [ 1]. These differences indicate that as

Plasmids in Aquatic Bacteria

2t 7

Fig. 3. HindlII digestion of genomic DNA from the l0 isolates carrying pFBA20 and oFBA20-1. Lanes: 1, strain FBA1338; 2, FBAI324; 3, FBAI230; 4, FBAI316; 5, FBAI317; 6, FBA1009; 7, FBAI051; 8, FBA1045; 9, FBAI014; 10, FBA1039; 11, SPPI DNA (EcoRI digested). Note that only FBA1051 (lane 7) and FBA1039 (lane 10) show distinct differences in restriction profile.

the basic c h r o m o s o m a l u n i t is n o n h o m o l o g o u s (as j u d g e d b y restriction digest only), the h o s t strain is t h e n distinct f r o m the o t h e r isolates w h i c h all h a v e v e r y s i m i l a r c h r o m o s o m a l digest patterns.

Discussion

T h e analysis o f p l a s m i d c o n t e n t a n d p l a s m i d size d i s t r i b u t i o n w i t h i n the s t r e a m s y s t e m s h o w s the c o n j u g a l t r a n s f e r p o t e n t i a l p o s s e s s e d b y a q u a t i c bacteria to be high. A l t h o u g h small p l a s m i d s w e r e detected, p l a s m i d s w i t h a size > 100 k b were p r e d o m i n a n t in the p o p u l a t i o n o f c o p p e r - t o l e r a n t bacteria. T h i s c o n trasts with p l a s m i d p o p u l a t i o n s f o u n d in the C h e s a p e a k e Bay, U S A [11] a n d

Table 2. Location and time of isolation of Type A

plasmids Isolate FBA 1338 FBA 1324 FBAI230 FBA1317 FBAI316 FBA 1009 FBAI051 FBA1045 FBAI014 FBA1039

Site~

Time ~

1 1 1 1 1 1 2 2 4 5

1 1 1 2 3 5 1 1 2 1

See Fig. 1 b Represents consecutive sampling times at intervals o f 6--8 weeks. "1" represents time first sample was taken; "'2" represents second sample taken, 6-8 weeks later; and "5" represents the final sample

218

R.W. Pickup

River Ely, U K (P. A. Rochelle, M. J. Day, and J. C. Fry, personal communication), where small plasmids were frequently encountered in bacterial populations. Large plasmids (100-200 kb) were also encountered in the River Ely in high numbers. A m i n i m u m plasmid size of 30 kb for the F plasmid and 1520 kb for RP4/RK2 [31] is required for conjugal transfer. If this is reflected in the plasmids of the aquatic community, then >90% of the plasmids tested, which represents > 60% of the total isolates, are large enough to encode transfer functions. Even the smaller plasmids can be transferred under certain circumstances by mobilization or co-integrate formation with a co-resident plasmid [27]. Failure to detect plasmids in about 20% of the isolates does not confirm that they are plasmid-free. It is possible that their plasmids may be refractory to purification by the method employed in this study. Bacterial populations respond to selective environments by shifts in taxonomic diversity and by changing their plasmid content [10]. Polluted sites show a consistent increase in both plasmid size and in plasmid number within the population [6, 11]. Specific pollutants can select for uniformity in plasmid structure and function (e.g., mercury [16]). Lundquist and Levin [ 17] suggest that plasmid populations can be established and maintained if their replication rate is sufficient to prevent segregational loss, by positive selection for plasmiddetermined characters or by infectious transmission. However, cryptic plasmids under no apparent selection should be lost from the population due to lack o f selection and/or due to the additional metabolic burden imposed on their host [21 ]. No such metabolic burden seems to be imposed by pFBA20 on its hosts. This is reflected in the ability of the host to survive over a period of time at a particular site. Furthermore, its detection at other sites implies dispersal and recolonization has occurred. Transfer of pFBA20 can also be inferred through its presence in at least two distinct hosts. The plasmid, therefore, demonstrates both vertical and horizontal transfer in an apparently nonselective aquatic environment. Similar studies have shown the dispersal and maintenance of plasmids in microbial populations where environmental pollutants provide a strong selective pressure [2, 20]. Therefore, the question arises as to whether pFBA20 is really a cryptic plasmid. This study has shown that pFBA20, as yet, does not confer any adaptive functions on the host, although the tests carried out cannot be considered exhaustive. Its undoubted ability to survive within the stream system would indicate that selective pressures ensure its maintenance. Alternatively, the plasmid may be efficiently and actively partitioned to the cell progeny so that the possibility of loss at cell division is minimized. The role of this plasmid is unknown and to define it as cryptic may be premature a s i t has. the capacity to encode several functions. If it is the case that pFBA20 does not confer any adaptive functions on the host, then its persistence may not be due to direct selective pressures. Lundquist and Levin [17] showed that plasmids could be maintained and transferred under no selective pressure in a chemostat. Furtherrnore, Bouma and Lenski [4] have shown that the presence of a plasmid can affect the fitness of the host. The antibiotic-resistant plasmid they studied was initially antagonistic to the fitness of the host in the absence of selection but was mutualistic under antibiotic selection. However, after 500 generations of selective growth it became mutualistic in both the presence or absence of

Plasmids in Aquatic Bacteria

219

t h e s e l e c t i v e p r e s s u r e . E x p e r i m e n t s h a v e s h o w n t h a t t h e g e n e t i c c h a n g e res p o n s i b l e f o r t h e n e w m u t u a l i s t i c effect a r o s e i n t h e h o s t c h r o m o s o m e . I t is p o s s i b l e t h a t t h e i s o l a t e s r e p o r t e d in t h i s s t u d y m a y h a v e u n d e r g o n e t h i s c h a n g e in a period when pFBA20 conferred some selective advantage. Once the unknown selective pressure was removed the plasmid persisted because of a m u t u a l i s t i c effect w h i c h i n c r e a s e d t h e fitness o f t h e h o s t . T h i s s t u d y h a s i m p l i c a t i o n s r e g a r d i n g t h e fate o f r e c o m b i n a n t D N A r e l e a s e d into the environment. It has shown that in the absence of an apparent selective p r e s s u r e , p l a s m i d s m a y s u r v i v e a n d d i s p e r s e in t h e a q u a t i c e n v i r o n m e n t . T h e f i n d i n g o f a p F B A 2 0 - 1 i k e p l a s m i d in a m o d i f i e d f o r m i n a n o t h e r h o s t suggests t h a t t h e s e a r e t r a n s f e r a b l e p l a s m i d s i n a c o n s t a n t s t a t e o f s t r u c t u r a l flux. I t d o e s indicate that recombinant plasmids, which rely on selective pressures for their maintenance, may not be lost so easily once released into the environment. In a d d i t i o n , t h e i r fate m a y b e difficult t o p r e d i c t . T h e s p r e a d o f p F B A 2 0 i n t h e c o p p e r - s e n s i t i v e b a c t e r i a l p o p u l a t i o n in t h e f r e s h w a t e r s t r e a m is b e i n g i n v e s t i g a t e d u s i n g D N A p r o b e s specific t o t y p e A p l a s m i d s . Acknowledgments. This research was funded by the Natural Environment Research Council, UK. I wish to thank Prof. J. G. Jones and Dr. J. R. Saunders for their helpful comments.

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