interferon-a1 gene in Escherichiu coli TG1 was studied in chemostat cultures under non-selective. (medium without antibiotics), selective (medium with p-lactam ...
Acta Biotechnol. 15 (1995) 4, 375-380
Akademie Verlag
Short Commun'ication Stability of the pBR322 Plasmid Derivative pBB210 in Escherichiu coli TG1 under Non-Selective and Selective Conditions LOSER. c.
UFZ - Umweltforschungszentm Leipzig- Halle GmbH Sektion Sanierungsforschung Permoserstr. 15 04318 Leipzig, Germany
Summary The stability behaviour of the pBR322 plasmid derivative pBB210 withp-lactamase gene and human interferon-a1 gene in Escherichiu coli TG1 was studied in chemostat cultures under non-selective (medium without antibiotics), selective (medium with p-lactam antibiotic ampicillin) and modified selective (medium with ampicillin and thep-lactamase inhibitor sulbactam) conditions. Under non-selective conditions, a behaviour typical of unstable systems was found. Under selective conditions, the behaviour predicted by the models was obtained - the fraction of plasmid-bearing cells in the population approached a constant value which was dependent on the ampicillin concentration in the feeding and on the cell concentration in the chemostat. Under modified selective conditions, the higher the concentration of sulbactam in the medium was, the higher the fraction of plasmid-bearing cells was in steady state conditions.
Introduction
The Escherichiu coli host strain TGl transformed with the plasmid pBB210 was used in the pilot-scale human interferon-a1 production by means of fed-batch high-celldensity cultivation. The amount of interferon obtained in this process was smaller than expected, caused by a large fraction of unproductive plasmid-free segregants at the end of cultivation. Since plasmid pBB2lO bears aJ-lactamase gene which mediates ampicillin resistance [l], ampicillin was used to suppress the rising plasmid-free cells. But contrary to expectations, despite the ampicillin application, the accumulation of unproductive plasmidfree segregants could not be prevented. Hypothetical mathematical models (without any experimental background) describing the plasmid stability behaviour in chemostat cultures were derived in [2, 3.43. The models of CHANG [2] and OLLIS [3] describing the behaviour of mixed plasmid-bearing antibiotic resistant and plasmid-free antibiotic sensitive cell populations in chemostat cultures with antibiotic in the feeding predict coexistence between plasmid-bearing and plasmid-free cells in steady state condi-
376
Acta Biotechnol. 15 (1995) 4
tions.These model predictions have not yet been thoroughly checked by experimental studies. But the experihental investigation by CAULCO'IT [5] dealing with the stability of plasmid pCI70 encoded forJ-lactamase in Escherichia coli in a chemostat culture with carbenicillin-containing medium refers to a coexistence between plasmid-bearing and plasmid-free cells. In this report, the plasmid stability behaviour of plasmid pBB2lO in Escherichiu coli TG1 is studied under non-selective and selective conditions in chemostat cultures to understand the cause of the inefficiency of antibiotic application and to confirm the model predictions. If a commensal-interaction-like process between plasmid-bearing and plasmid-free cells is responsible for their coexistence, a higher stability may be expected by the reduction of plasmid-mediated antibiotic degradation caused by the use of thefl-lactamase inhibitor sulbactam. Material and Methods Microorganisms The Escherichiu coli host strain TGl is a K-12 strain with the following phenotype: A (lac-pro), supE, thi, hsdD5 / FtraD36, proA B +,lac1 q.lacZdM15 [6]. Neither intact host cells nor their lysates showed any#-lactamase activity. Escherichiu coli TGl(pBB210) was obtained by the transformation of the plasmid pBB2lO into the host [l]. The 3.783 kb plasmid pBB210 is a derivative of plasmid pBR322 with the blu gene, encoded for ampicillin resistance @lactamase is expressed constitutively), and a heterologous expression unit of the staphylokinase gene of Stuphylococcur aureus phage 042D and of the human interferon-a1 gene [I]. +
Medium The medium used was a mineral salt medium with the following composition: 20 mM KHzP04, 20 mM NazHPO,, 10 mM NH4Cl. 7.5 mM NazS04. 0.2 mM MgC12, 0.02 mM FeCl3, 0.02 mM MnC12,S g / 1 NaCI and 0.005 g / 1 thiamine x 2 HCl (the pH was adjusted to 7.0 with 1 N NaOH). 0.2 to 0.8 g / 1 glucose, which acts as the growth-limiting factor, was used as the carbon source. When required, the following p-lactam antibiotics were added as sterile-filtered stock solutions: ampicillin-Na (PHARMAKHIM, Bulgaria) and/or sulbact;lm-Na (PFIZER Inc.). Cultivation Conditions Cultivation took place in a chemostat (180 ml working volume, aeration with 30 litres of air per hour, at a temperature of 29 O C and a dilution rate of 0.4 h -1). The fed-in medium was replaced at 5-day intervals and was stored at 2 "C to diminish the spontaneous hydrolysis of ampicillin in the medium. Shake flask cultures were used as the inoculum (0.05 g / l of ampicillin-Na was added to the precultures).
Analysis For determining the fraction of plasmid-containing cells (in the following designated by the symbol F), a new method wils developed to obtain exact results with little effort. SinceJ-lactamase negative segregants never contained plasmids (as proved with gel electrophoresis) theJ-lactamase is used as an indicator for plasmid content. For this purpose, the cells of a culture were individualized by cultivation on an antibiotic-free submerged medium containing 2 g/l of glucose solidified with 20 g / l
377
L M R , C., pBR322 Plasmid Derivative pBB2lO in Escherichiu coli TGI
of agar-agar in PETRI dishes at 35 "C. When the colonies had reached a size of about 1 mm in diameter, the surface bf the emerged medium was dried by storing the PETRI dishes without cover in an incubator at 35 "C for about 2 hours. After drying, the emerged medium in the dishes was coated with a special mixture. For this purpose, 1.5 ml of starch solution (20 g / l in 50 mM potassium phosphate buffer of pH 7.0). 1 ml of ampicillin-Na solution (1.5 g /1 in water), 0.1 ml of iodinepotassium-iodide solution (90 mM I? and 3.600 mM KI in water), and 2.5ml of agarose solution (10g/l agarose molten in 50 mM potassium phosphate buffer at a pH of 7.0 at 100 "C) were mixed, and the plate was immediately coated with this hot blue-coloured mixture. Colonies which have been grown from plasmid-bearing cells produce a decolourization in their surroundings within a few minutes. The plasmid-encoded J-lactamase hydrolyzes the ampicillin of the coating gel, and the hydrolysis product ampicilloic acid binds the iodine, which leads to the decolourization of the blue iodine-starch complex. By contrast, colonies consisting only of plasmid-free cells retain their dark colouring. The fraction F of the sample is the quotient of the number of decolourized colonies and the total number of colonies tested (for each value about 800 colonies).
Results and Discussion Plasmid Stubility under Non-Selective Conditions
The plasmid stability under non-selective conditions was investigated in a chemostat culture with 0.8 g / l glucose and without antibiotics in the feeding. The fraction of plasmid-bearing cells (F) decreases only very slowly at first and then more and more rapidly (Fig. 1). When F < 0.5, the decrease slows down again and, as expected, it then approaches the value F = 0 asymptotically. The decrease of F i n time showed the typical kinetics described in other literature (e.g., [7, 81). The slow initial decrease is to be attributed to the small plasmid loss rate of plasmid pBB210, caused by a relatively large average plasmid copy number of about
ki o-6
0.4
0.2
0
t
1
0
50
100
150
ZOO
250
300
350
400
450
Time [h]
Fig. 1. Fraction of plasmid-bearing cells (F)in chemostat culture under non-selective conditions (CSO = 0.8 g/l, CAO = 0 g/1, c 1 0 = 0 g 11)
378
Acta Biotechnol. 15 (1995) 4
20 copies per cell. The slowly progressing decrease of F is due to the small difference between the specific kowth rates of E. coli TG1 and E. coli TGl(pBB210) (in the conditions under examination j~ = 0.4906 h-* and 0.4733 h -1, respectively). Plasmid Stability under Selective Conditions
Seven chemostat experiments with different ampicillin and glucose concentrations in the feeding were carried out (Fig. 2 and Fig. 3). In contrast to the results obtained under non-selective conditions, the steady state value of F was greater than zero. This behaviour under selective conditions is to be explained as follows: When the fraction of plasmid-bearing cells in the chemostat culture is high, the ampicillin is intensively degraded by the plasmid-bearing cells, and the ampicillin concentration in the medium of the chemostat is correspondingly low. Therefore, the ampicillin has little influence on the growth of plasmid-free cells and their growth-rate advantage leads to an increase of their proportion in the population and to a decrease in F (because of the constant total cell concentration). As a result of the decrease in F, the ampicillin degradation is reduced, the concentration of ampicillin in the medium increases, and the growth rate of the plasmid-free cells decreases under antibiotic influence until the steady state is reached. The steady state value of F is directly proportional to the ampicillin concentration in the nutrient medium of the chemostat, because a higher ampicillin feeding requires a greater effort in ampicillin degradation and hence a correspondingly higher concentration of plasmid-bearing cells. The steady state value of F is inversely proportional to the glucose concentration in the feeding, because a certain ampicillin feeding requires a
\\ o*2
t\
0 0
v
I
1
50
100
-
150
C , = 0 3 ~
..-
c, = 0.8 gn
I
I
I
I
I
1
200
250
300
350
400
450
Time (h]
Fig. 2 Fraction of plasmid-bearing cells (F)in chemostat cultures under selective conditions with thw different glucose concentrationsin the feeding (C*o = 0.02 g/s CIO = 0 g/l)
379
L ~ RC.,, pBR322 Plasmid Derivative pBB210 in Escherichia coli TG1
c, = 0.10
0.6
g/l
-
a 0.4
-
0.2 -
I
I
I
I
I
I
I
I
Fig. 3. Frwtion of plasmid-baring cells (F)in chemostat cultures under selective conditions with three different ampicillin concentrationsin the feeding (CSO = 0.4 g /I, C,O= Og 11)
CIO= 0.005 g/l
c,o = 0.002
g/l
0.6 L
0.4 -
0.2 rn
01 0
I
c,o = o.oO0
g/l
I
I
I
I
I
I
I
I
1
50
100
150
200
250
300
350
400
450
Time [hl
Fig.4. Fraction of plasmid-hearing cells ( F ) in chemostat cultures under modified selective conditions with four different sulbactam concentrations in the feeding (C,,= 0.4 g1L ,c, = 0.02 g/l)
certain cell concentration of plasmid-bearing cells, and an increase in the overall cell concentration in the chemostat leads therefore inevitably to a decrease in F.
380
A a a Biotechnol. 15 (1995) 4
Plasmid Stability under Modified Selective Conditions
Modified selective conditions were created by the combined use of thep-lactam antibiotic ampicillin and the J-lactamase inhibitor sulbacm. Three chemostat experiments with 0.4 g l 1 of glucose, 0.02 g / 1 of ampicillin-Na and different sulbactam concentrations in the nutrient medium were carried out. As under selective conditions, the fraction of plasmid-bearing cells (F) also approaches a steady state value under modified selective conditions (Fig. 4). The higher the s u l b a c m concentration in the nutrient medium, the higher F i n steady state conditions Was.
The increase in Fat a steady state in the presence of sulbactam is caused by the inhibition of theJ-lactamase in the plasmid-bearing cells, which results in a reduced ampicillin degradation, which in turn causes a shift of the cell concentration of plasmidbearing cells at an equilibrium towards higher values. Nomenclature CAO
-
G o
- giucose concentration in the nutrient medium of the chemostat culture
ampicillin-Na concentration in the nutrient medium of the chemostat culture
CIO - sulbactam-Na concentration in the nutrient medium of the chemostat culture
F
-
t
- cultivation time
fraction of plasmid-bearing cells in the cell population of the chemostat culture
Received 29 August 1994 Received in revised form 30 January 1995 References BREITLING, R.: Konstitutive und induzierbare Expression heterologer Gene in Escherichiu coli und Bacillus subrilis. Jena: Zentralinstitut fur Mikrobiologie und Experimentelle Therapie. doctoral thesis, 1988. CHANG, Y. K.,LIM, H. C.: Static Characteristics of a Continuous Flow Bioreactor Containing Antibiotic Resistant Recombinant Cells. Biotechnol. Bioeng. 29 (1987). 950-961. OLLIS,D. F.: Competition between Two Species when Only One Has Antibiotic Resistance: Chemostat Analysis. AIChE Meeting in San Francisco, 1984. COOPER, N. S., BROWN,M. E., CAULCOT". C. A.: A Mathematical Model for Analysing Plasmid Stability in Micro-Organisms. 3. Gen. Microbiol. 133 (1987). 1871-1880. CAULCOTT, C. A.: Competition between Plasmid-Positive and Plasmid-Negative Cells. Biochern. Soc. Trans. 12 (1984). 1140-1 142. CARTER. P.. BEDOUELLE, H.. WINTER, G.: Improved Oligonucleotide Site-Directed Mutagenesis Using M13 Vectors. Nucl. Ac. Res. 13 (1985). 4 4 3 1 4 3 . WEBER, A. E.. SAN.K.-Y.:Persistence and Expression of the Plasmid pBR322 in Escherichiu coli K12 Cultured in Complex Medium. Biotechnol. Lett 9 (1987). 757-760. CHEW, L.C. K., TACON, W. C. A., COLE, J. A.: Effect of Growth Conditions on the Rate of Loss of Plasmid pAT153 from Continuous Cultures of Escherichiu coli HB101. FEMS Microbiol. Lett. 56 (1988), 101-104.
