Communications to the Editor Ampicillin Effects on Five Recombinant Escherichia coli Strains: Implications for Selection Pressure Design

Kathryn Dennis, Friedrich Srienc. and James E. Bailey Department of Chemical Engineering, California Institute of Technology, Pasadena, California 9 1 125

Accepted for Publication January 7, 1985

INTRODUCTION

In genetics, selection refers to the use of growth environments in which only cells possessing certain genetic characteristics are able to grow. Selection for desired phenotypes is employed in several contexts in recombinant DNA technology. Selection is applied to identify plasmid-containing clones following trans- formation. Furthermore, imposition of selection pres- sure is a commonly used strategy for ameliorating negative effects of plasmid segregational instability on culture purity and recombinant fermentation process productivity.

As applied traditionally in genetics, selection is con- sidered as a binary, yes or no, growth or death situation. This perspective is not inappropriate in earlier contexts in which the phenotypes involved differ only in the presence or absence of a single gene copy yielding a functional product. The situation is more complicated and requires other conceptual and experimental ap- proaches when considering genes in plasmids. Cells containing these plasmids may possess hundreds or more plasmid, hence gene, copies. Furthermore, when the spectrum of number of gene copies is so much broader, the probability of substantial variability in single-cell plasmid gene content among the cells com- prising the population is great.

In this work two different experimental methods are employed to investigate the relationship between the selection pressure applied and the number of copies of the selection gene (the gene that when expressed endows host cells with the selected phenotype.) The system considered here is a recombinant strain of Escherichia coli in which p-lactam antibiotic resistance is provided by p-lactamase expressed from a plasmid gene. Five different recombinant strains containing plasmids propagated at different copy numbers are investigated to determine the effect of the number of selection gene copies. The selection pressure is de- termined by the ampicillin concentration.

The first experimental approach employed is deter- mination of the minimum inhibitory concentration (MIC)

for the antibiotic. While done here using suspension culture, the method is conceptually related to that proposed by Uhlin and Nordstrom.' The second ap- proach uses flow cytometry to characterize the alter- ation of single-cell morphology in the recombinant populations following exposure to ampicillin. When the drug concentration is sufficiently large that cells are susceptible, interference with cell wall synthesis results in growth of elongated cells, which can be readily identified and quantified on a single-cell basis using light-scattering measurements in a flow cytometer. This measurement has been presented previously as a means for determining the relative quantities of plas- mid-containing and plasmid-free E. coli cells in a mixed culture arising from plasmid segregational instability.*

Since the response of cells to different antibiotic concentrations is directly related to the amount of p- lactamase enzyme synthesized, the results here also provide new experimental insights into the relationship between the number of gene copies in a recombinant strain and the efficiency of cloned gene expression. Based on earlier theoretical model^,^ it has been pro- posed that the linearity reported previously' between number of gene copies and gene product activity should break down at high plasmid content. Cells containing very large numbers of plasmids may express plasmid genes less efficiently due to interference of plasmids and plasmid gene products with biosynthetic activities of the host ceII.

MATERIALS AND METHODS

Bacterial Strain

Host Escherichia coli strain HBlOl pro gal hsdR hsdM recAl transformed with pDM246, pDM247, pDM248, pFH 1 18, and RSF 1050 were kindly provided by David Moser. Plasmid RSFlO5O is a derivative of pMBl that has the same origin of replication and also contains the Amp' gene from Tn3. The RNA I wild- type fragment was removed from RSF1050 and an RNA I mutant was inserted giving the high copy number

Biotechnology and Bioengineering, Vol. XXVII, Pp. 1490-1494 (1985) 0 1985 John Wiley Lk Sons, Inc. CCC 0006-3592/85/101490-05$04.00

plasmid pFHl 18.4 The three other plasmids contain varying amounts of wild-type RNA I and mutant RNA I, or both, which result in variations of the plasmid copy number.'

Media and Cultivation

The growth medium was osmotically controlled L broth containing 10 g/L bactotryptone (Difco); 5 g/L yeast extract (Difco); 5 g/L glucose; 5 g/L NaCl; 3 g/L K,HP04, 1 g/L KH,P04 giving an osmolality of ca 325 mosmol/kg. Cultivation was in 250-mL Erlen- meyer flasks shaken at 250 rpm and 37°C in a tem- perature-controlled shaker (New Brunswick Scientific, Edison, NJ). Cell growth was monitored with a Klett- Summerson spectrometer with a green filter. Cells were harvested during early exponential growth and placed on ice for 15 min. Equivalent volumes of cells were centfiged at 10,OOO rpm, 10 min, 4°C; the supernatant decanted; and the centrifuge tube dried as much as possible. In this way any p-lactamase in the culture medium was removed. The pellets were resuspended with the appropriate amount of media at 37°C and incubated 15 min. Samples were then taken, following which ampicillin was added to the desired concentration. Cells were cultured for 90 min; samples were removed every 30 min. Samples were kept on ice until conclusion of the experiment when flow cytometric analysis was performed.

MIC Determination

The MIC of ampicillin (sodium salt; Sigma Chemical Co., No. A-9518) was determined for the five recom- binant strains in an osmotically controlled growth me- dium.* Cell growth was monitored with a spectropho- tometer (Spectronic 21, Bausch & Lomb, Rochester, NY) at 590 nm. Cells were harvested at an optical density of 0.1 and diluted 1 : 100. Tubes containing 3 mL media were inoculated with 50 p L of the diluted culture. MIC was defined for each strain as the lowest concentration of antibiotic necessary to suppress visible growth of the dilute culture after 18 h incubation at 37°C.

Flow Cytometric Analysis

The narrow-angle light-scattering properties of the cell populations were analyzed on a flow cytometer (Model 50H, Ortho Instruments, Westwood, MA) with an argon ion laser tuned at 514.5 nm and 0.25 W. Narrow-angle signals were processed in coincidence with right-angle signals at approximately 2000 cells/s. Data analysis was performed on a Cytomic 12 computer (Kratel GmbH, Stuttgart, FRG).

Copy Number Determinations

The plasmid copy number comparisons were made according to the method of Moser and CampbelL4

Cells were grown in L broth as described above and harvested at OD 0.5-0.6 for copy number analysis. These values will be termed reference copy numbers below since cells cultivated in ampicillin-containing medium and in possibly different growth states may have different copy numbers than cells grown under the reference conditions just described. However, be- cause the incubation time in different media is short, actual and reference copy numbers are not expected to differ significantly. Also, it is unlikely that the relative copy numbers from strain to strain will be substantially altered by brief exposure to nonreference conditions. Copy number measurements are not extremely reliable in an absolute sense. Since an internal standard is used, relative values are more accurate and more reproducible.

RESULTS

The MIC values for each recombinant strain are given in Table I. Also listed there is the fractional elongation for each strain expressed in terms of channel number, the standard measurement on the flow cy- tometer, which is directly proportional to the narrow- angle light-scattering intensity of single cells. The values listed are shifts in the mean value of the narrow-angle light-scattering distribution measured after 60 min in- cubation in medium containing 1500 pg ampicillin/mL. These shift values are normalized for each strain by the initial distribution mean.

The MIC values and fractional elongations are plotted versus corresponding reference copy numbers in Figures 1 and 2, respectively. For both parameters the rela- tionship has the expected overall trend. Increasing numbers of plasmid copies imply increasing single-cell content of Amp' genes coding for p-lactamase. Cells containing larger numbers of gene copies are expected to contain larger quantities of corresponding p-lactamase gene product and thus to be more resistant to ampicillin, implying higher MIC and smaller elongation values.

At copy numbers of less than 100 the relationship between copy number and elongation rate is approx- imately linear. However, MIC values estimated by linear extension of data for smaller copy numbers con- sistently overestimate the values measured at larger

Table I. combinant E. coli HBlOl strains.

Characteristic parameters for ampicillin effects on re-

~~~~~~~ ~ ~ ~

MIC pg ampicillin

Plasmid Fractional Plasmid copy number mL elongation"

pDM247 12 lo00 0.945 pDM246 24 1500 0.788 RSFIOSO 60 2500 0.284 pDM248 122 2900 0.227 pFH118 408 7700 -

a Measured at I500 pg ampicillin/mL, 60 rnin incubation.

DENNIS, SRIENC, AND BAILEY: AMPICILLIN EFFECTS ON €SCH€R/CH/A COLl 1491

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PLASMID COPY NUMBER

Figure 1. Relationship of MIC level with plasmid copy number.

copy numbers. Results for recombinant strains carrying pDM248 and pFHll8 at reference copy numbers of 122 and 408, respectively, deviate considerably from extrapolated lines based on the lower copy number measurement. Fractional elongation declines essentially linearly for lower copy number strains, but this trend is not followed for E. coli HBlOl[pDM248].

More detailed information on the relationship between copy number and response to ampicillin is indicated in Figure 3. The fractional elongation has been de- termined for four of the recombinant strains as a func- tion of medium ampicillin content. Solid symbols cor- respond approximately to 1 MIC values for each strain. So far as can be determined from these measurements, the fractional elongation is approximately linearly de- pendent on ampicillin concentration up to the 1 MIC value, after which a decrease in measured average elongation appears. Examination of the complete single- cell narrow-angle light-scattering distribution for these cases shows increasing quantities of small-magnitude scattering indicative of cellular debris (data not shown;

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Figure 2. Reference plasmid copy number correlation with mean fractional elongation as measured by flow cytometry.

INITIAL AMPICILLIN CCXENTRATIW (pghl )

Figure 3. Fractional elongation as function of ampicillin level for recombinant strains carrying plasmids pDM247 (0); pDM246 (0); RSFlOSO (A); pDM248 (0). Solid symbols represent (approximately) 1 MIC for each strain.

see, for example, measured light-scattering distributions in Dennis et a1.2).

Occurrence of complex interactions and single-cell heterogeneity are revealed by detailed examination of two-parameter flow cytometry measurements of re- combinant E. coli strains incubated in ampicillin. In Figure 4 several of these two-parameter distributions are shown for the recombinant strain carrying plasmid pDM246, incubated in medium containing at time zero 1500pg ampicillin/mL, equal to 1 MIC for this strain. Here the height of the mound at a particular x and y coordinate (single-cell narrow-angle and right-angle light-scattering intensity, respectively) represent the fraction of cells in the population giving those values of narrow-angle and right-angle single-cell light scat- tering. As this sequence of measurements clearly shows, the distribution of morphological forms in the culture shifts during the first 60 min of incubation toward larger cells as reflected by the increased values of single-cell light-scattering intensity. However, after 90 min incubation a shift in the reverse direction is ap- parent, although some cells in the culture have con- tinued to elongate and exhibit even greater light-scat- tering intensities than any seen in the 60-min sample. Thus, some cells in the population at 90 min continue to be inhibited in normal development while others appear, on the basis of these measurements, to possess essentially normal size and shape.

DISCUSSION

The data presented above show that the degree of selection pressure - the initial concentration of am- picillin in the cultivation medium in these experiments - has a continuous effect on the growth characteristics of recombinant organisms. The extent of growth in- hibition, and morphological change in the case of this

1492 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 27, OCTOBER 1985

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Figure 4. Two-parameter flow cytometry data on single-cell light-scattering statistics in a population of recombinant E. coli strain HB101[pDM246] after 0, 30, 60, and 90 min incubation in 1500 pg arnpicillin/mL (1 MIC). (X = narrow-angle scatter, Y = right-angle scatter, Z (vertical) axis = relative number of cells.)

selection agent, depends on the plasmid content of the cells. Therefore, on a single-cell basis, it is ap- propriate to consider the cell growth rate as a continuous function of plasmid content and environmental con- ditions, especially with respect to selection pressure.

A recombinant population carrying an unstable plas- mid will typically be heterogeneous with respect to single-cell plasmid content. This conclusion is supported by theoretical models based on realistic assumptions concerning plasmid propagation6” and is strongly sug- gested by the large degrees of heterogeneity revealed in experimental studies of recombinant microbial pop- ulation using flow cytometry.2’8 Indeed, the data shown in Figure 4, which indicates an increase of cells with relatively normal morphology late in the incubation while other cells continue to become larger due to drug effects, are strongly suggestive of significant het- erogeneity with respect to single-cell plasmid content in this culture. Therefore, it is an oversimplification to view recombinant populations simply as a mixture of two types of organisms, one devoid of plasmid and one containing plasmid. The plasmid-containing pop- ulation is actually comprised of many subpopulations characterized by their single-cell plasmid and selection gene product content.

Viewed from this perspective, the degree of selection pressure will alter the shape of the distribution of single-cell plasmid content in the population. As the selection pressure is increased, cells with lower copy numbers will become increasingly disadvantaged with respect to growth rate relative to those with higher

copy numbers, resulting in a shift toward higher copy numbers. Accordingly, the degree of selection pressure will determine to some extent the density in the pop- ulation of genes carried on plasmids and thereby the productivity of the process. However, in this connection it should be noted that the data presented above support earlier theoretical suggestions that production of a cloned gene product is not strictly proportional to cloned gene content, especially at high levels.

Another factor that may be important in the pop- ulation dynamics displayed in Figure 4 and that was also implicated in an earlier study of ampicillin effects on mixed cultures’ is commensal interaction. When the selective agent is an antagonist or inhibitor of growth that is decomposed, deactivated, or tightly bound by the product of the selection gene, commensal interaction between plasmid-carrying (or higher copy number) cells and plasmid-free (or lower copy number) cells is likely. Here, cells rich in selection genes reduce the concentration of the antagonistic selective agent, ameliorating the agent’s effects on the more sensitive cells. This is a general limitation and practical difficulty for all selection strategies of this type. For this reason, designing the host vector system to apply selection through complementation may offer advantages. Here, cells containing the cloned gene product have a complete metabolic and regulatory network while those missing the cloned gene lack a component (e.g., an enzyme, a repressor, or a tRNA species) essential for growth. In this case commensal interactions benefiting plasmid- free (or low-copy-number) cells are far less likely.

DENNIS, SRIENC, AND BAILEY: AMPICILLIN EFFECTS ON ESCHERICHIA COLl 1493

This work was supported by the National Science Foundation. 3. S. B. Lee and J. E. Bailey, Biotechnol. Bioeng., 26, 1383 (1984). 4. F. Heffron, M. So, and B. J. McCarthy, PNAS (US), 75, 6012

5 . D. R. Moser and J. L. Campbell, J . Bacteriol., 154, 809 (1983). 6. M. Hjortso and J. E. Bailey, Biotechnol. Bioeng., 26,528 (1984). 7. J.-H. Seo and J. E. Bailey, Biotechnol. Bioeng., 27, 156 (1985). 8. F. Srienc, J. L. Campbell, and J. E. Bailey, Biotechnol. Lett.,

(1978). References

1. B. Uhlin and K. Nordstrom, Plasmid, 1, 1 (1977). 2. K. Dennis, F. Srienc, and J. E. Bailey, Biotechnol. Bioeng., 25,

2485 (1983). 5, 43 (1983).

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