A method for plasmid copy number determination in recombinant Streptomyces

Journal of Microbiological Methods, 16 (1992) 69-80

69

© 1992 Elsevier Science Publishers B.V. All rights reserved 0167 - 7012/92/$05.00 MIMET 00515

A method for plasmid copy number determination in recombinant Streptomyces Carys A. Wrigley-Jones a, Hilary Richards b, Colin R. Thomas d and John M. Ward c aDepartment of Chemical and Biochemical Engineering, bDepart.ment of Biology, ¢Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, UK and dSchool of Chemical Engineering, University of Birmingham, Birmingham, UK (Received 3 February 1992; accepted 4 April 1992)

Summary A method for the measurement of plasmid copy number in Streptomyces mycelia is described. It is based on the preparation (on a mini-scale) of the total DNA present in the mycelium followed by gel electrophoresis with ethidium bromide, photography and densitometric scanning of photographic negatives. The method is suitable for plJ101-derived multi-copy plasmids and is shown to have comparable accuracy to similar methods developed for unicellular host organisms. A novel 'copy number' reconstitution approach was employed to define the sensitivity of the method. We recommend the optimisation of plasmid topoisomer separation since we have found evidence for transient changes in their relative proportions for certain Streptomyces plasmids~ Various plasmid topoisomers present in the ........................ y l . , , t , ~ i v e DNasel nicking t,~ p u r e asmid DNA. -.

Key words: Copy number; Plasmid; Recombinant; Streptomyces; Topoisomer

Introduction

Streptomyces are prokaryotic, Gram positive, filamentous micro-organisms which are mainly found in soil habitats. They can differentiate by forming spores on aerial hypae and this activity is thought to be linked with concomitant production of extracellular enzymes and secondary metabolites, some of which have antimicrobial activity. This property has made the genus very important to the pharmaceutical industry since Streptomycete are the source of many widely used antibiotics. Amongst antibiotics produced by bacteria, over 84% were derived from Actinomycetes [1]. Gene cloning can be applied to improve or diversify the secondary metabolite yield [2,3]. Streptomyces are also good producers of extracellular enzymes; they are therefore attractive candidates for heterologous gene expression. Correspondence to: J.M. Ward, Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WCIE 6BT, UK.

70 The genus is a fruitful source of extrachromosomal DNA elements which include cxamiAes of both linear and covalently closed circle (CCC) plasmid types [4]. Many cloning vectors, however, are derived from the S. lividans ISP5434 plasmid plJ101 (8.9 kb), and a series of constructs was made by Kieser et al. [5]. Such vectors are utilized in both academic and industrial laboratories during Streptomyces cloning experiments. So far, little is known regarding the stability and plasmid copy number of plJ101-derived vectors during laboratory (or large-scale) fermentations. Many genetically manipulated plasmids in Gram-negative and Gram-positive unicellular bacteria show segregational instability (i.e. the plasmid is lost during cell division). This eventually leads to loss of the plasmid-borne phenotype and poor yields of cloned gene product. Plasmid instability in such hosts is normally followed by plating samples on agar to reveal the proportion of colony forming units that exhibit a plasmid-borne phenotype, e.g., antibiotic resistance. Since Streptomyces are filamentous micro-organisms and almost always form microscopically sized clumps in most commonly used liquid media a simple plating method does not work because a colony on a plate is not derived from a single cell but from a clump in which only a small proportion of the mycelial filaments need to have a plasmid for a colony to be formed on selective agar. Another approach therefore must be found :.n order- to follow plasmid stability in liquid-grown Streptomyces cultures. Since segregational instability is often preceded by a decrease in plasmid copy number we have embarked on a study of vector stability in Streptomyces cultures by measuring plasmid copy number. In this paper we describe the development of our method which is fairly rapid and does not include autoradiography (which decreases speed and increases cost). The method relies on electrophoretic separation of total DNA, released from mycelia by neutral SDS lysis, and densitometric scanning of photographic negatives. Such features enable a number of samples to be processed simultaneously so as to reveal transient changes in plasmid copy number during the course of batch fermentations. Our plasmid copy number estimation method is based on those of [6] and [7] for recombinant unicellular bacteria, but involves DNA purification procedures adapted for Streptomyces mycelia. Isolation of good total DNA preparations from Streptomyces are rarely easy (compared to Escherichia coil) but, with care, the procedure described in this paper yields consistently good preparations from which plasmid copy number estimations can be made. The sensitivity of the method was tested by defining fluorescence linearity limits and by the application of a novel method of 'reconstitution' of separately purified chromosomal and plasmid DNA. The procedure is also suitable for following transient changes in the relative proportions of different plasmid topoisomers. Materials and Methods

Bacteria and plo~rnids Streptomyces lividans 66 strain TK24 [5] was used throughout. Plasmids plJ303, 10.7 kb [5] and plJ702, 5.6 kb [8] were obtained from Professor D.A. Hopwood, John Innes Institute, Norwich, UK. Piasmids pMT605 (12.6 kb) and pMT608 (6.4 kb) [8] were gifts from Dr. J. Cullum, UMIST, Manchester, UK.

71

Spore preporation and growth conditions Well sporulated cultures were obtair, cd by spreading liquid-grown mycelia onto modified R2YE agar (modified from [9]) containing 10 g-! -~ MgCI2, 10 g-! - i glucose, 22 g. 1- l . Bacteriological agar (Oxoid) made up in 100 mM Tris-HCl (pH 7.5) and after autoclaving the following sterile solutions were added; 5% w/v KH2PO4 at 1 ml.l -~ and 1 M CaCI2 at 20 ml-I -I. Agar-grown cultures were incubated at 30°C for several days. Recombinant S. lividans were grown on agar in the presence of 50 ttg" m l - ~ thiostrepton (a gift from S.J. Lucania, E.J. Squibb Inc., New Jersey, USA). Spores were harvested by gentle scraping with a sterile loop and suspended in a sterile solution of 20% v/v glycerol, 0.1% v/v Tween 80. Spore suspension aliquots were frozen at - 2 0 ° C until required. Liquid cultures were either grown in 250 ml Erlenmeyer flasks containing 50 ml of medium or 21 Erlenmeyer flasks containing 250 or 500 ml of medium. Either 0.2 ml or 1 ml, respectively, of thawed spore suspension was used to inoculate sterile medium. The media employed were modified MEP [10], containing 1% w/v malt extract broth, 1% w/v bacteriological peptone and 2% w/v glycerol; 10 mM potassium phosphatesupplemented modified MEP or YEME [9] (for protoplast preparation only). Liquid cultures of recombinant S. lividans were grown in the presence of 5 / t g - m l thiostrepton. Inoculated flasks were placed on an orbital shaker (2 inch throw, 200 rpm) and incubated at 28°C for at least 3 days.

Harvesting of mycelia S. lividans cultures were harvested by centrifugation in a bench centrifuge at maximum setting (1000 rpm) for 10 min. The mycelia were stored at - 2 0 ° C until required for DNA extractions.

Protoplast preparation and DNA transformation . . . . . . . r. ' - . ~ '.~ l t. -. ' * -.' ' ' ~. " . . . I-"'I-" . . . . u~.,o~,,u~u Ill [71. 1 lall~lOillli:ltlOll of D N A into protoplasts wa~ also performed as described by Hopwood et al. [9] using the rapid small-scale procedure.

DNA extraction methods Total DNA was prepared as described by Hopwood et al. [9] using the neutral lysis procedure 4. Approximately 100 mg wet weight mycelia were used and care was taken to remove all contaminating protein at the phenol/chloroform extraction steps. The propan-2-ol and spermine precipitation steps were performed on ice for a period > 30 min. Plasmid DNA was prepared using an alkaline lysis method as described in [11] and the propan-2-ol and spermine precipitation steps were conducted on ice for > 30 min. For larger quantities of plasmid DNA the volumes used were scaled up 10 times. The concentration of DNA in both plasmid and total DNA preparations was determined by measuring the absorbtion at 260 nm. It was assumed that 50/~g" m l double stranded DNA gives an absorbance reading of 1 at 260 nm [12]. The purity of DNA preparations was determined by measuring the ratio ODz60/OD28a. Restriction endonuclease digests. The restriction endonucleases Bglll and PstI were obtained from Ang!ian Biotechnology Ltd, U K , and digests were

72 performed at 37°C using a buffer containing 10 m M Tris-HCl (pH 7.5), 10 m M MgCI2 and 100 m M NaCI. All restriction digests were conducted to completion.

DNasel nicking reaction Bovine pancreatic DNasel was obtained from the Sigma Chemical Company and kept as a 1 mg. m l - ~stock solution in storage buffer containing 50 mM NaCI, 20 mM Hepes buffer (pH 7.9), 5 mM MgCI2, 5 mM CaCI2 and 20% v/v glycerol (modified from Maniatis et al. [12]) at -20°C. A dilution of this stock was made in the same buffer just prior to use and 0.5 pg of this DNaseI was added to approximately 25 pg of plasmid DNA. The reaction mixture was incubated at 37°C. Aliquots were removed from time zero onwards at 1-min intervals and p!aced in a solution containing 0.1 M sodium EDTA (ethylene-diamine~etra-acetic acid, pH 8.0) to stop the reaction. The reactk_,n's progress was followed by gel electrophoresis.

Gel electrophoresis 1% w/v or 0.7% w/v agarose gels were cast using 0.09 M Tris-base and 0.09 M boric acid buffer with 0.5/~g-ml-~ ethidium bromide. EDTA was not employed in either gel casting or electrophoresis running buffer since it causes the smearing of chromosomal DNA in the presence of impure EDTA. Such observations have been made in this laboratory and were also reported by Zhou et al. [13]. The same buffer was also used for submerged gel electrophoresis (again without EDTA). Electrophoresis was conducted at either 1.7 V . c m - l for 16 h ('slow') or 10 V - c m - ! for 2-3 h ('fast').

Photography and scanning densitometry Gels were visualized by illumination with UV light at 302 nm and photographed using Ilford HP5 film (llford, UK). The Polaroid land camera aperture was set at 4.5 and the exposure time was 15 s using an orange filter. The film was developed with Ilford Microphen developer and Ilford Microphen fixer according to manufacturer's instructions. Photographic negatives were scanned using a Joyce-Loebl Chromoscan 3 scanning densitometer fitted with a 0.1 mm diameter aperture and 530 nm filter. A built-in intergrator calculated the peak areas (in arbitrary units) which were re-calculated as necessary to eliminate background fluorescence. Peak integral values were then used to calculate plasmid copy number according to Projan et al.'s [6] equation: Plasmid copy number =

Plasmid Peak integral x Mw chromosome chromosome peak integral x Mw plasmid

All visible plasmid bands were used to calculate copy number. The size of the S. lividans TK24 chromosome was assumed to be 6.9 x l 0 9 Da [14].

73 15000

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12500

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oS°SS

~10000

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~7500.

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Ilg

p5000' 1.0

'

015

'

1.0

1~5

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2;0

2"5

0

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AMOUNTOF DNAPERBAND(pg) Fig. 1. Fluorescence intensity (peak integral, O) and peak height (A) plotted as a function of the amount of S. lividans TK24 chromosomal DNA per band.

Results

Linearity of DNA band intensity A wide range of chromosomal DNA and iinearized plasmid DNA levels were tested to establish the linearity of fluorescence (measured as darkness of bands on photographic negatives) as a function of the amount of DNA. The results of one such experiment are shown in Fig. 1. A linear relationship was found between Chromoscan peak integral and level of DNA up to 3/~g of DNA per band (a shallower slope was measured above 3/lg DNA per band). The lower limit of linearity was 0.15/~g of DNA (results not shown). These limits were observed for both chromosomal and linearized plasmid DNA. Also plotted in Fig. 1 is the Chromoscan peak height as a function of amount of DNA per band. A curve was observed which plateaus at levels of D N A above approximately 2-2.5 #g DNA. At such levels the band does not fluoresce with greater intensity but yields a broader band, hence the peak integral continues to increase while peak height does not.

Accuracy of plasmid copy number determination The accuracy of plasmid copy number estimation was tested by running known amounts of separately purified plasmid and chromosomal DNA in a single gel lane. A range of plasmid copy number values could thus be artificially generated through reconstitution of two sources of DNA. In all cases the plasmid DNA was linearized by restriction endonuclease digestion but the reaction was stopped before adding chromosomal DNA. Linearized plasmid DNA was used so that all plasmid DNA

74 appeared in a single band. A gel where decreasing quantities of linearized plJ303 plasmid were added to constant amounts of chromosomal DNA prior to loading resulted in the following, measured 'copy number' values; 566 (0.72 pg plJ303), 481 (0.6 pg plJ303) and 332 (0.48 pg plJ303). Since the levels of both plasmid and chromosomal DNA (1.2 pg) loaded into each lane was known then an expected 'copy number' could also be calculated. The expected 'copy number' values were 581,484 and 387, respectively. We therefore found that when known quantities of linearized plJ303 plasmid were loaded with constant levels of chromosomal DNA the resulting observed 'copy number' values agreed well with expected 'copy number' values. A similar agarose gel was also run using linearized pMT605 replication intermediates (Figs. 2,3). During our reconstitution experiment we have used both s ti ÷ and s t i - plasmids [15,16] and have found that the likely presence of a significant quantity of single stranded (ssDNA) molecules in the s t i - extracts probably leads to an overestimate of the amount of DNA that would appear on agarose gels. This is due to the quantitation of the total DNA sample being carried out by its absorption (which measures all forms of DNA), whilst the scanning of the agarose gels measures only DNA which has bound ethidium bromide, ssDNA molecules can only bind ethidium bromide LANES

1 2 3 4

CHROMOSOMAL DNA LINEAR PLASMID DNA

Fig. 2. Photograph of gel where aliquots of linearized Bglll digestion) pMT605 plasmid were added to constant levels of S. lividans TK24 chromosomal DNA prior to loading. Each lane was loaded with the following amounts of DNA from both sources: Lane 1 = 1.38/~g TK24 DNA + i.66/tg pMT605; Lane 2 = 1.38/~g TK24 DNA + !.23/tg pMT605; Lane 3 = i.38/tg TK24 DNA + 0.83/~g pMT605; Lane 4 = 1.38/~g TK24 DNA + 0.42/ug pMT605.

1.3

within those sequences which have base-paired. Care must therefore be taken when quantitating plasmid D N A extracts of sti- Streptomyces plasmids. The evidence from pIJ303 reconstitution gel experiments show that plasmid copy number estimations can therefore be made with reasonable accuracy provided the level of DNA in each band is within the linearity of fluorescence and/or film response. Determinations at very low (or very high) copy number are less accurate when the intensity of DNA bands approaches the limits of linearity.

Statbstical analysis of plasmid copy number values of parallel extracts of the same mycelium sample and of parallel lanes or a single extract The mean plasmid copy number of 15 parallel total DNA extracts of a single S.

lividans TK24 pi2303 mycelium sample was 497 and the standard error of the mean was 23, i.e., 4.6%. The results of plasmid copy number estimations of 9 parallel gel lanes of a single total D N A extract of S. lividans TK24 pIJ303 mycelium were 218,220, 236, 227, 164, 221, 180, 232 and 174. The mean value was 208 and the standard error of the mean was calculated as 9, i.e. 4.3%. We therefore assume that the accuracy of our plasmid copy number assessment method in total is approximately 9% (percentage standard error of the mean).

a

r'~t~

b

c

ZZ C~

d

<< Z7 t,-~ r'~

rn~

L) n

Br

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Fig. 3. Chromoscan traces of lanes !--4 of gel shown in Fizz. 2 (corresponding to a-d, respectively) where different aliquots of linearized pMT605 DNA were added to constant aliquots of S. lividans TK24 chromosomal DNA.

76

Plasmid topoisomer identification and separation It was found that open circle plasmid molecules of a certain size, e.g., plJ303 and pMT605, often co-migrate with chromosomal DNA during 'slow' electrophoresis in 1% w/v agarose gels. If such molecules remain hidden then plasmid copy number will be underestimated. Employing 0.7% w/v agarose gels helped to yield good band separatinn under 'slow' electrophoresis but adequate separation was, in addition, obtained with ~fast' electrophoresis, thereby increasing the speed of the procedure. Optimizing the separation of plasmid topoisomers is important and :his point has often been overlooked in previous plasmid copy number assessment ~rotocols e.g. [17]. It is sometimes difficult to positively assign a specific topoisome,ic form to a particular band in the gel. Results from our laboratory sugges~t that their relative proportions of the plasmid topoisomers can change during culth ation (manuscript in preparation). We therefore attempted to identify the different monomeric and dimeric plasmid topoisomers by progressive DNaseI nicking of pure plasmid DNA. The results of DNasel nicking of pIJ303 DNA is shown i~l Fig. 4. Both fast migrating bands disappear indicating that they are most likely to be supercoiled molecules (monomer and probably dimer, respectively). The t~.- slowest migrating bands increased in intensity and were consequently assigned as open circle (monomeric and dimeric) forms. A faint linear band was also seen in the central positions. The results of plJ702 nicking by DNasel are show,~ ,~ Fig. 5, dimeric forms were less evident here as was the open circle form at time zero. Actual total DNA extracts also contain chromosomal DNA which can complicate

LANES OPEN CIRCLE DIMER-~ u~'EN ~.,I~CLc. [vl~r~uIVlr-~t,~, LINEAR PLASMID-SUPERCOILED DIMER SUPERCOILED MONOMER r-"

1

2

3

4

5

6

7

8

9

10

11

23.13kb 9.416kb 6.56kb 4.361kb .~.322kb L027kb

Fig. 4. Photograph o f gel showing the progress of pIJ303 plasmid D N A nicking by DNaseI enzyme with increasing time; Lane 1 = 0 min; 2 = 1 min; 3 = 2 min; 4 = 3 rain; 5 = 4 min; 6 = 5 min; 7 = 6 rain; 8 = 7 min; 9 = 8 min; 1 0 = 2 0 min; 11 = Hindlll size marker. Slow electrophoresis conditions.

77 LANES

OPEN CIRCLE MONOMER-LINEAR PLASMIDSUPERCOILED MONOMER---

1

2

3 4. 5

6

7

8

9

10 11

l..~.

l ,..~INI,~

9.416kb 6.56kb "4.361kb 2.322kb 2.027kb

Fig. 5. Photograph of gel showing the progress of pIJ702 plasmid D N A nicking by D N A s e l enzyme with increasing time. Lane 1 = 0 min; 2 = 1 min; 3 = 2 min; 4 = 3 min; 5 = 4 min; 6 = 5 min; 7 = 6 min; 8 = 7 min; 9 = 8 min; 10 = 20 min; 11 = Hindlll size marker. Slow electrophoresis conditions.

the identification of bands. However, if 'slow' 0.7% agarose gel electrophoresis conditions are employed all plasmid forms seem to separate away from the chromosomal DNA as shown in Fig. 6. Therefore if precise measurements of plasmid copy number and the separation of bands are necessary, it is best to use 'slow' electrophoresis but this does decrease the speed of measurements. Discussion

The method described here is fairly rapid, inexpensive and suitable for the determination of plasmid copy number from many samples simultaneously. It was found to be accurate to within < 10% (standard error) of the mean. However this variation may also partly reflect heterogeneity of plasmid copy number distribution within the mycelium as well as errors inherent in its measurement. The accuracy of this method compares favourably with those quoted by Projan et al. [6] which was + 20% (standard deviation) of the mean and Lewington and Day [7] which was + 313% (it was not stated whether this was a percentage of the mean value). Another plasmid copy number assessment method specifically designed for Streptomyces mycelia was developed by Labes et al. [17]. No indication of the accuracy of this method was quoted and the large proportion of fluorescent material remaining in the wells shown in these authors' results would probably have led to erroneous estimates of plasmid copy number. The novel approach of using separately purified plasmid and chromosomal DNA proved very useful in defining the sensitivity of the method. However, it should be

78 LANES

CHROMOSOMAL DNA OPEN CIRCLE MONOMER LINEAR PLASMII~ SUPERCOILED DIME SUPERCOILED MONOM[

1

2

31 4

5

6

•CHROMOSOMAL DNA

OPEN CIRCLE MONOMER LINEAR PLASMID - SUPERCOILED MONOMER

Fig. 6. Photograph of gel comparing pure plasmid DNA (uncut), plasmid DNA (cut) and total DNA under slow electrophoresis conditions. Lane 1 = uncut PIJ303; 2 = Bglll cut plJ303; 3 TK24 plJ303 total DNA; 4 = uncut plJ303; 5 = Bglll cut plJ702; 6 TK24 plJ702 total DNA.

noted that when absorbance readings were used to quantify the concentration of DNA in s t i - plasmid extracts, wide discrepancies between the measured and expected plasmid copy number values were found. The reason for this was probably the presence of some ssDNA containing little or no base-pairing since it was not visualized via ethidium bromide binding. The error seemed to be consistent for each pMT60S and n]k/lT/~fl~ i-~/~IA preparation. In our experience plasmid dimers as well as monomers were often isGlated. The separation of all the resultant topoisomeric forms can be difficult, but necessary for accurate plasmid copy number estimations. It cannot be assumed that a particular plasmid topoisomer constitutes the same fraction of the total amount of plasmid DNA regardless of growth phase since we have found transient changes in the proportions of plasmid topoisomers occur as the culture ages. It is therefore necessary to separate as many plasmid topoisomers away from the chromosomal D N A as possible. This is best achieved through 'slow' electrophoresis using 0.7% w/v agarose. The final electrophoresis conditions will depend on the plasmid size and number of plasmid topoisomers present. Using this method we have observed an increase in plJ303 plasmid copy number from <200 t~ >400 between initial rapid growth phase and stationary phase (manuscript in preparation). Several multi-copy plasmids in the size range 5.6-12.6 kb have been studied using this protocol. The method should also be suitable for plasmid molecules outside this range. Low copy number vectors may need to be run at lower agarose gel concentrations since they are often larger molecules. Additionally, for total DNA extracts containing low (or very high) copy number plasmids it is

79 advisable to run concentrated and diluted samples in neighbouring lanes so as to obtain plasmid and chromosomal DNA bands which fall within the limits oflinearity. In general, however it is best to measure chromosomal and plasmid DNA bands from a single lane. The method has proved reproducible and suitable of the processing of many samples. In addition it should also be suitable for other Streptomyces host organisms provided good initial cell lysis, occurs.

Acknowledgements CW-J was supported by a SERC biotechnology Directorate ear-marked research studentship. HR and JMW acknowledge support from the Antibiotics and Recombinant D N A Club programme of the SERC Biotechnology Directorate, the companies participating in the Club programme were Glaxo Group Research, Beechams Pharmaceuticals, ICI and Celltech.

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80 15 Deng, Z., Kieser, T. and Hopwood, D.A. (1988) 'Strong incompatibility' between derivatives of tht Streptomyces multicopy plasmid plJl01. Mol. Gen. Genet. 214, 286--294. 16 Pigac, J., Vujaklija, D., Toman, Z., Gamulin, V. and Schrempf, H. (1988) Structural instability o f , bifunctional plasmid pZGI and single-stranded DNA formation in Streptomyces. Plasmid 19, 222230. 17 Labes, G., Simon, R. and Wohlleben, W. (1990) A rapid method for the analysis of plasmid conten and copy number in various Streptomycetes grown on agar plates. Nucleic Acids Res. 18, 2197.