Gene, 55 (1987) 141-146

Elsevier 141

GEN 02021

Measurement of cut expression from growth-rate-regulated promoters employing fi-lactamase activity as an indicator of plasmid copy number

(Recombinant DNA; promoter fusion vectors; chloramphenicol acetyltransferase; nnB PI promoter; dot-blot

hybridization)

Robin-Ann Klotsky and Ira Schwartz

Department of Biochemistry, New York Medical College, Valhalla, NY 10595 (U.S.A.)

Received 12 January 1987

Accepted 16 March 1987

SUMMARY

Many promoter-fusion vectors contain an intact /I-lactamase (BLA) gene (blu) to allow measurement of BLA activity as an internal control for plasmid copy number. This approach rests on the assumption that blu is constitutively expressed. To use such vectors for comparison of promoter activity at different growth rates it was necessary to confirm that this is the case under all physiological conditions. The relationship between plasmid copy number and BLA activity at different steady-state growth rates in Escherichia coli HBlOl transformed with a ColEl-type plasmid (pBR325) was examined. Both BLA activity and plasmid copy number decreased in a parallel fashion as growth rate increased. This finding was tested further by measuring the growth-rate-regulated expression of the chloramphenicol acetyltransferase (CAT) gene under the control of the rrnB PI promoter in a plasmid pKK23 l- 1 fusion. The results indicate that BLA activity is a reliable indicator of copy number at a wide range of growth rates and that CAT/BLA ratios can be employed as a valid measure of promoter-specific activity in such plasmid fusions under these different physiological conditions.

INTRODUCTION

Promoter fusion vectors have been employed successfully in the isolation and identification of

Correspondence to: Dr. I. Schwartz, Department of Biochemistry,

New York Medical College, Valhalla, NY 10595 (U.S.A.)

Tel. (914)993-4064.

Abbreviations: Ap, ampicillin; BLA, j?-lactamase; blu, gene cod-

ing for BLA; bp, base pair(s); CAT, Cm acetyltransferase; cut,

gene coding for CAT; Cm, chloramphenicol; L broth, Luria

promoters and in quantitation of their activities. In general, these vectors consist of a promoterless gene which codes for a product whose activity can be easily measured (e.g., 1acZ or g&K). Insertion of a DNA fragment which contains a promoter into a cloning site situated immediately upstream from this reporter gene results in its expression (Casadaban and Cohen, 1980; McKenney et al., 1981). Brosius (1984) recently described such vectors (pKK23 l- 1 and pKK232-8) which contain a promoterless cat

broth; [ 1, designates plasmid-carrier state. gene preceded by a polylinker for fragment insertion.

0378-I 119/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

142

Vector pKK232-8 has been used in studies of the transcriptional regulation of the ~sU-d~~G-~o~ operon of E. coli and antitermination of transcription in rrnG (Lupski et al., 1984; Li et al., 1984). This vector has also been employed in our laboratory for the identification of multiple promoters in the thrS-infC-plT cluster in E. coli (Schwartz et al., 1986).

Vector pKK232-8 also contains an intact fl-lactamase gene. In the studies cited above (as well as in the earlier studies of Drahos et al., 1982) BLA activity was employed as an internal control for plasmid copy number since it can be readily deter- mined by simple and rapid assay procedures. This is based on the assumption that bia is a constitutively expressed gene under all physiological conditions and thus, BLA levels are directly related to the copy number of its gene.

In the course of our investigations with pKK232-8 it became clear that BLA activity varied greatly as a function of growth rate. To confirm the above assumption regarding BLA activity as an accurate indicator of copy number, a carefm analysis of plas- mid copy number and BLA activity at a variety of steady-state growth rates was undertaken. It was observed that both plasmid copy number and BLA levels decrease as the growth rate increases. In addi- tion, it is shown, employing a plasmid which contains the m?B PI promoter fused to cat in pKK231-1, that promoter activity responds to

growtb rate in a manner identical to that previous- ly demonstrated for PI by other techniques (S~entos and Cashel, 1983). These studies dem- onstrate that CAT/BLA ratios are a reliable measure of promoter-specific activity over a wide range of growth rates.

EXPERIMENTAL AND DISCUSSION

(a) Plasm&, DNA isolation aad ~~f~tion

Plasmid pKK275-4 was kindly provided by Jurgen Brosius. Plasmid DNA was prepared by the alkaline lysis method of Birnboim and Doly (1979). Transfor- mation was performed as previously described (Elseviers et al., 1982). The host E. coli strain for all the studies was HBlOl.

(b) Bacterial growth and lotion of cell extracts

To achieve steady-state growth at different rates, cells were grown in either L broth (1.0% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose) or M9 minimal salts (Miller, 1972) supplemented with either 0.4% glycerol, 0.4% glucose or 0.4% glucose plus 0.4% tryptone. All media were supplemented with 100 pg Ap/ml, Cells were grown overnight in L broth and inoculated, at a 50-fold dilution, into fresh media. Cells were harvested at midlog, washed once with 50 mM Tris * HCl (pH 7.8), 30 mM dithio- threitol and stored in the same buffer at -70°C. Cell extracts for enzyme assays were prepared by soni- cation and centrifugation to remove cell debris.

(c) Chloramphenicol acetyltransferase and /Mac- tamase assays

The activities of both CAT and BLA were deter- mined spe~~ophotome~c~y, essentially as de- scribed by Lupski et al. (1984) with minor mod& cations.

(d) Plasmid copy-number determination

Plasmid copy number was measured by a DNA- DNA hybridization method described by Adams and Hatfield (1984). The technique is rapid and can be peiformed directly on sonicates. Briefly, cell lysates prepared as above were dot-blotted onto nitrocellulose essentially as described by Maniatis et al. (1982). Fig. la is an autoradiogram of such a dot blot performed with lysates from pBR325-trans- formed HBlOl grown at several different growth rates. The amount of 32P-labeled pBR325 probe hybridized to each spot was quantitated by scintilla- tion counting. Fig. lb shows that there is a linear relationship between the radioactivity in each spot and the amount of lysate affiied to the filter, as previously shown by Adams and Hatfield (1984). If the lysates from cells grown at different rates are all prepared from a fured amount of cells then the slope of the curve in Fig, lb can be used to directly com- pare the plasmid concentrations (plasmid/&,,) under the different growth conditions.

143

a

5

IO

20

40

L MGT MG MGL

6i- / 2 x

E u

“0 10 20 30 40 50

VOLUME LYSATE BLOTTED (Uliter)

Fig. 1. Determination of plasmid copy number by blot hybrid-

ization. (Panel a) HBlOl[pBR325] were grown to midlog in

either L broth (L) or minimal medium supplemented with either

glucose plus tryptone (MGT), glucose (MG) or glycerol (MGL).

The growth rates were 1.64, 1.12, 0.91 and 0.61 doublings per

hour, respectively. Lysates were prepared and various amounts

were blotted to nitrocellulose as described in EXPERIMENTAL

AND DISCUSSION, section d. 32P-labeled pBR325 was pre-

pared by nick translation according to Rigby et al. (1977). The

blot was hybridized with s2P-labeled pBR325, washed and

processed for autoradiography as previously described

(Schwartz et al., 1983). Shown is an autoradiogram after 1 h

exposure at -70°C. Numbers along the left margin indicate the

volume of lysate blotted, in ~1. (Panel b) The dot blots shown in

panel a were cut into small squares corresponding to the areas

of radioactivity based on the autoradiogram. The radioactivity in

each spot was determined by Cerenkov counting in a Beckman

LS1800. The data shown are for lysates from growth in MG.

(e) Plasmid copy number and BLA activity at various

steady-state growth rates

In preliminary experiments with promoter fusions in pKK232-8, it was noted that BLA activity

decreased with increased growth rate. Since BLA activity was to be used as an internal standard for plasmid copy number, based on the assumption that bla is constitutively expressed, it was crucial that this assumption be re-examined. To investigate this question E. coli HB lOl[pBR325] was grown to midlog at four growth rates and the plasmid concen- trations (plasmid/&,) and BLA activities were determined. As growth rate increased there was a concomitant decrease in both plasmid concentration and BLA activity (Fig. 2). Moreover, the curves for both parameters parallel each other (except for a slight deviation for growth in minimal glucose- tryptone).

3

4

0 \

0.0 0.5 1.0 1.5 2.0

l \ ‘\

\

\ 0

JO \

\

l a. /I llOUBLINGS/HOUR)

Fig. 2. /I-Lactamase activity and plasmid copy number as a

function of growth rate. HBlOl[pBR325] was grown to steady

state in either L broth or minimal medium supplemented with

either 0.4% glucose + 0.4% tryptone, 0.4% glucose or 0.4%

glycerol. Growth was followed by monitoring the absorbance at

600 nm. Growth rates were determined from plots of log A,,, vs.

time. The slopes (k) of the linear portions of the growth curves

were used to measure g, the doubling time, using the relationship,

g = 0.693/2.303k. p, doublings per hour, is defined by, p = l/g.

The values for p for each growth condition were L, 1.64; minimal

glucose-tryptone, 1.12; minimal glucose, 0.91; minimal glycerol,

0.61. Cells were harvested at midlog and lysates were prepared

as described in EXPERIMENTAL AND DISCUSSION,

section b. BLA activity and plasmid copy number were deter-

mined on samples of the same lysate as detailed in EXPERI-

MENTAL AND DISCUSSION, sections c and d. Plasmid copy

number is shown as cpm hybridized to dot blots which is directly

proportional to copy number as discussed in EXPERIMENTAL

AND DISCUSSION, section d. Each data point represents the

average of three separate determinations. The values for BLA

activity and copy number at all growth rates are presented

relative to the values obtained from cells grown in L broth, which

were 19.5 nmol/min and 562 cpm hybridized per ~1 of lysate,

respectively, 0, /I-lactamase activity; 0, cpm hybridized.

144

These findings are consistent with the results of earlier studies on plasmid Rl which indicated that BLA activity was a linear function of copy number (NordstrUm et al., 1972) and that copy number decreased with increasing growth rate (Engberg and Nordstrom, 1975). In those studies, however, BLA activity appeared to increase linearly with growth rate. In a more recent report Lin-Chao and Bremer (1986) demonstrated that, for pBR322, piasmid copy number (expressed as plasmid/A,,,) decreased with increased growth rate. Indeed, their plot of copy

number vs. growth rate (doublings per hour) is essentially identical with that shown here in Fig. 2. The present investigation extends these studies further by demonstrating the relationship between BLA activity, copy number and growth rate for a ColE l-type plasmid.

The results presented here indicate that BLA activity can be used as an accurate indicator of copy number even for comparison of cells grown under different nut~tion~ conditions.

(f) Growth rate regutation of wnB PI

The ultimate purpose of this study was to ascertain whether growth-rate regulation of pro- moter utilization could be determined by CAT expression from a promoter fusion and represented as the CAT/BLA ratio for plasmid-transformed cells grown to steady state at different growth rates. The results presented in the preceding section suggested that this was the case since BLA activity and plasmid copy number varied in a parallel fashion at all the growth rates tested. To confm this, a fusion containing a well-characterized growth-rate-regu- lated promoter was tested.

Perhaps the best example of a growth-rate-regu- lated gene is the ribosomal RNA operon of E. coli.

These RNAs are transcribed from two tandem pro- moters, designated PI and P2 and a number of studies have shown that PI is the promoter responsi- ble for the growth-rate response (Glaser et al., 1983; S~ientos and Cashel, 1983). Therefore, plasmid pKK275-4 which contains the rrnB Pf promoter fused to cut in pKK231-1 and an intact bla was employed. E. coli HBlOl transformed with pKK275-4 was grown at four different growth rates and the CAT and BLA activities in crude lysates were determined. The results presented in Fig. 3

Fig. 3. The rrnB PI promoter activity as a function of growth rate. HBlOl[pKK275-41 was grown to midlog in either L broth, minimal glucose-tryptone, minimal glucose or minimal glycerol. The steady-state growth rates in these media were 1X3,1.18,0.78 and 0.64 doublings per hour, respectively. Lysates were prepared and the BLA and CAT activities were measured spectro- photometrically. pKK275-4 was constructed as follows: the PI

promoter was isolated on a 1032-bp DdeI fragment obtained from digestion of plasmid pKK3535, which extends from bp positions -984 to + 48 relative to the Pf transcription start point (Brosius et al., 1981a,b). The fragment was converted to blunt ends, modified by the addition of Hind111 linkers and inserted into the Hind111 site of pKK231-I immediately upstream from

gene cat (J. Brosius, personal communication).

show that at slow growth rates PI activity was low but increased dramatically as the growth rate was increased above one doubling per hour. This is the growth response which is characteristic for rRNA synthesis (Gausing, 1980).

Qu~titatively, similar findings for Rena PI have been reported by S~entos and Cashel (1983 ; Fig. 4) who measured actual transcript levels gener- ated from PI by gel electrophoresis. The present data indicate that the identical response is found for the rrnB PI promoter, as would be expected. More importantly, this experiment clearly demonstrates that promoter activity at different growth rates is accurately reflected by the CAT/BLA ratios obtained from promoter fusions to cat.

(g] Conclusions

The problems inherent in using fusion vectors for quantitation of promoter strength without correction for variation in copy number between different plas- mid constructs has been previously addressed (Adams and Hatfield, 1984). It was concluded that

145

copy number must be measured each time the indi- cator enzyme activity is assayed. The traditional methods for copy number determination, such as CsCl density gradient centrifugation and gel electro- phoresis (Shepard and Polisky, 1979; Projan et al., 1983), are rather tedious and therefore not the methods of choice for rapid estimation of copy num- ber on a large group of samples. Adams and Hatfield (1984) employed DNA blot hybridization for this purpose as described earlier. While this method has the advantage that it can be performed on small samples of the same lysate used for enzyme assays, it has several disadvantages. It requires the use of 32P-labeled probe which has a limited lifetime and must be prepared repeatedly. Second, the procedure involves autoradio~aphy which requires a minimum of several hours of exposure prior to the qu~titation of hybridized probe by scintillation counting. Rapid assay of a second enzyme activity which is expressed by the same plasmid is therefore preferred if it can be shown that the levels of this activity are directly proportional to plasmid copy number. The experi- ments presented here show that the assay of BLA activity meets these criteria and, furthermore, that CAT/BLA ratios accurately reflect the specific activity of the fused promoter. These studies suggest that plasmids such as pKK232-8 should be the vectors of choice for the analysis of promoter activity under different physiological conditions.

ACKNOWLEDGEMENTS

We thank JUrgen Brosius for providing pKK2754 and several helpful discussions. This work was sup- ported by NIH grant GM29265 to I.S.

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Communicated by G.N. Godson.