relaxed Escherichia coli K12 strain pair

Microbiol. Res. (1996) 151, 99 -103

Microbiological Research © Gustav Fischer Verlag Jena

Influence of relA locus on electrophoretic mobility of a stringentI relaxed Escherichia coli K 12 strain pair Burkhard Gitter1, Alexander Hummel 2 , Franz Glombitza 2 1 Hans-Kn6ll-Institut fUr Naturstoff-Forschung e. v., BeutenbergstraBe 11, D-07745 Jena, Federal Republic of Germany 2 Consulting and Engineering GmbH, Fachbereich Biotechnologie, Chemnitzer StraBe 13, D-09224 Griina, Federal Republic of Germany

Accepted: October 15, 1995

Abstract Stringent (relA + ) and relaxed (rei A - ) controlled Escherichia coli cells differ in their regulation of many bio-

chemical pathways such as phospholipid and lipopolysaccharide metabolism (LPS) after amino acid limitation. Because such differences could result in various cell envelopes, cells of stringent controlled E. coli strain CP78 (relA +) and relaxed controlled E. coli strain CP79 (reIA) were studied regarding their electrophoretic mobility. The graphs of the mobility distributions of both strains were different: cells of strain CP79 caused secondary peaks in addition to the main peaks whereas the mobility distributions of cells of strain CP78 showed only one maximum. In the pH range from 6.0 to 8.0 the location of the main peaks of cells of strain CP79 were changed to less negative values after induction of relaxed response. In contrast to this the stringent response in strain CP78 caused no change of the mobility distributions.

Key words: Electrophoretic mobility relaxed response - Escherichia coli

stringent and

Introduction Wild type Escherichia coli cells synthesize guanosine5'-diphosphate-3'-diphosphate (ppGpp) in response to amino acid starvation (stringent response). The actions of ppGpp adapt the stringent controlled cells

Corresponding author: B. Gitter

to new environmental conditions. ppGpp is synthesized in a ribosome dependent manner by (p )ppGpp synthetase I which is the product of the relA gene. Cells bearing a mutation in this gene are defective in synthesizing ppGpp after the onset of amino acid limitation, they are defined as relaxed controlled cells (reIA) (for review see Cashel & Rudd, 1987). The pleiotropic low molecular weight effector ppGpp influences among many other metabolic reactions the biosynthetic pathways offatty acids and phospholipids (Cashel & Rudd, 1987). ppGpp controls also the biosynthesis and release oflipopolysaccharides (LPS) (Ishiguro et al., 1986). Comparative studies with stringent and relaxed controlled Escherichia coli cells showed that increased levels of recombinant proteins could be found in supernatants of relaxed cell cultures after amino acid starvation. For instance significant amounts of Pglucanase (Riethdorf et al., 1990), p-Iactamase, interferon exl (Gitter et al., 1995) and miniantibodies (B. Gitter, unpublished observations) were excreted by these cells. Besides the different excretion efficiency, stringent controlled cells reply to an amino acid limitation with a change of the fatty acid pattern of their membranes whereas the relaxed controlled counterpart do not (Gitter et al., 1995). Moreover, first results indicate a different shift of the ratio of the head groups in the lipid fraction of both strains after addition of valine (Gitter, unpublished results). All these findings are indications for different characteristics of the cell envelopes of both strains Microbiol. Res. 151 (1996) 1

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which could also affect the cell surfaces and the electrophoretic mobilities (EPM). Bayer & Sloyer (1990) compared some E. coli strains with significant differences in their cell envelope such as capsules or LPS type. Here, we measured the electrophoretic mobility distribution of stringent and relaxed controlled Escherichia coli K12 cells in order to test whether these isogenic strains except their relA locus are different regarding their cell surfaces.

Materials and methods Bacterial strains. For our studies the Escherichia coli strains CP78 (thr, his, arg, leu, thi, relA + ) and CP79 (thr, his, arg, leu, thi, reIA-) (Fiil & Friesen, 1968) were used. Medium, culture conditions. The glucose mineral salt medium contained per litre: 2.7 g KH 2P0 4, 2.8 g Na2P04, 0.54 g NH 4Cl, 5.1 g NaCl, 1.1 g Na2S04, 20 mg MgCI 2, 5 mg FeCI 3 , 4 mg MnCI 2, 50 mg L-threonine, 50 mg L-histidine, 50 mg L-arginine, 50 mg L-leucine, 10 mg thiamine, 50 mg ampicillin and 2 g glucose. Cells were grown in 50 ml shaking flask cultures at 28 DC and pH 7.0. The addition of valine (final concentration 0.24 g 1-1) induced partial isoleucine limitation and led to stringent respectively relaxed response. Cells taken from the log phase and 2 hours after addition of valine were harvested by centrifugation. Sample preparation. Cell pellets from centrifugation were resuspended in 0.25 mM NaCl to ODsso = 0.1 and the samples were adjusted to pH 6.0, 7.0, 8.0 and 2.0, respectively with NaOH or HCI. The measurement of electrophoretic mobility distribution was carried out three times at 25 DC. Measurement of mobility distribution. The measurements of electrophoretic mobility (EPM, defined as the ratio of particle velocity in /lm s - 1 to field strength in Y cm - 1) were carried out with Delsa 440 electrophoresis instrument (Coulter Scientific Instruments, Inc., Hialeah, FI, U.S.A.). The Delsa 440 is a He - N e-Iaser based multi angle particle electrophoresis analyzer that measures the electrophoretic mobility of particles or cells (0.02 - 30 /lm) in liquid suspensions by using the electrophoretic light scattering technique (ELS). To increase the resolution and to facilitate the separation of multimodal mobility distributions the Doppler frequency spectra are obtained at four different scattering angles simultaneously. The four measurements were converted automatically into four 256 channel histograms of the EPM distribution and values for the corresponding electrophoretic mobilities were obtained by

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using operating and analysis software provided by Coulter Electronics. The rectangular chamber of DELSA 440 was filled with 0.8 ml of cell suspension. The electric field applied was set according to the recommendations of the manufacturer in dependence on the conductivity of the electrolyte medium.

Results EPM in the physiological range of pH 6.0 to pH 8.0

In order to characterize the functional properties of cell surface of stringent controlled E. coli CP78 and relaxed controlled E. coli CP79 the electrophoretic mobility (EPM) of these strains were determined as described in materials and methods. For clarity, the mobility spectra in figures 1,2 and 3 were limited to those derived from 26.0 0 scattering angle of DEL SA 440. In the physiological range of pH 6.0 to pH 8.0 the graphs of mobility distribution of strain CP79 showed secondary peaks in addition to the main peaks, whereas cells of strain CP78 caused only one maximum at each pH. The location of the main peaks of strain CP79 and the maxima in strain CP78 were approximately the same at a mobility of about - 4.0 /lm cm Y -IS -1. The secondary peaks of CP79 cells were less negative in comparison to the main peaks at a mobility of about -3.0 /lm cm y- 1 s- 1 (Fig. 1, lA, IIA, IlIA). After induction of stringent respectively relaxed response the location of the main peaks of both strains were clearly different. While the main peaks of strain CP79 shifted to more positive mobilities, the mobility distributions of strain CP78 remained nearly unchanged (Fig. 1, IB, lIB, IIIB). Moreover the graphs of mobility distribution of strain CP79 showed an additional third peak (Fig. 1, IB) and an additional shoulder (Fig. 1, IIIB). EP M at the extreme pH of 2.0

At the extreme pH of 2.0 the appearance of the mobility distribution without addition of valine was the same as at pH of 6.0 to 8.0: cells of CP79 produced a double peak whereas the mobility distribution of strain CP78 was approximately a Gaussian distribution (Fig. 2, A). After addition of valine we could observe a big shift of the curve of strain CP78 to a more positive electrophoretic mobility whereas the curve of strain CP79 showed no significant change of EPM in comparison to the values before addition of valine (Fig. 2, B). The main peaks for CP78 and CP79 cells were located at about 2.0 /lm cm Y -IS -1 and 0.0 /lm cm y- 1 2- 1 respectively.

Relative intensity

IA

--~~------------,

IB

IIA

III A

liB

IIIB

~~~~~'-~~~==~~I~I~~~I~~==~~~~I~~I~ r --~~~~===r==~~I~~1 -5

-1

-3

-5

-3

-1

-5

-3

-1

EPM in IJm c!'1 V " s ., EPM in IJm em V" s"

Fig. 1. Electrophoretic mobility (EPM) of cultures of CP78 (_) and CP79 (::::) after adjustment of pH 6.0 (I), pH 7.0 (II) and pH 8.0 (III), A = without valine, B = 2 hours after addition of valine. Relative Intensity

A

IB

-1

3

EPM In IJm em V ·'S·1

Fig. 3. Electrophoretic mobility (EPM) of a mixture (1: 1) of cultures of CP 78 and CP 79 2 hours after addition of valine and adjustment of pH 2.0, 5 min (::: :), 10 min (::::) and 15 min (_) after mixing. -1

3

EPM in IJm em V"s "

Fig. 2. Electrophoretic mobility (EPM) of cultures of CP78 (_) and CP79 (::::) after adjustment of pH 2.0, A = without valine, B = 2 hours after addition of valine.

EP M of mixtures In order to check our measurements regarding the location of the maxima we mixed samples of strain CP78 and CP79 after addition of valine and adjustment to pH 2.0, because we found at these conditions clearly separated main peaks for cells of strain CP78 and strain CP79 (see Fig. 2, B). Figure 3 shows the kinetic of the EPM distributions of this 1: 1 mixture. As results of the measurements double peaks with one maximum around O.O!lm cm y-1s-l and another maximum at about 1.5!lm em y-1s-l were obtained.

Discussion The measurement of the electrophoretic mobility (EPM) of cell suspensions of Escherichia coli CP78 and CP79 were carried out three times. Because approximately the same distribution graphs were produced, the used method is in our opinion a reproducible tool for determination the electrophoretic mobility distributions of E. coli cell suspensions. The overall charge carried by the surface of cells is determined by the chemical structure and the number of ionogenic surface groups, which are controlled by the surface morphology, and the composition of the suspension medium (Mozes et al., 1989). The outer membrane of E. coli is an asymmetric bilayer consisting of phospholipids and proteins in the inner monolayer and mainly lipopolysaccharides (LPS) Microbiol. Res. 151 (1996) 1

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and proteins in the outer leaflet. Above all, the core regions of the LPS molecules are rich in charged groups, especially phosphate, carboxyl and amino groups (Nikaido and Vaara, 1987). Because the phosphate groups and the carboxyl groups of the 2keto-3-deoxyoctonic acid (KDO) are the dominant charged groups, the cell surface of E. coli is negatively charged under physiological conditions. This was confirmed by our results (as shown in Fig. 1) and by a study of Bayer and Sloyer (1990) in which some E. coli strains with different cell envelopes were tested. However, they found the highest EPMs in capsulated cells of E. coli K1 strain with a maximum at about -1.8 while our uncapsulated E. coli K12 cells produced maxima at about -4.0 Moreover, in this previous study very different E. coli strains concerning their cell envelope (LPS type, capsules) were investigated (Bayer and Sloyer, 1990). We obtained clearly distinguishable EPMs "merely" after addition of valine to cultures of the stringent and relaxed controlled E. coli cells. Before induction of stringent respectively relaxed response the cells of both strains did not differ in their walls. The addition of valine to the culture medium induced a partial isoleucine limitation in the cells, what led to different responses in strain CP78 (stringent response) and in strain CP79 (relaxed response) by action of different concentrations of guanosine-5 '-diphosphate3' -diphosphate (ppGpp). There are findings showing, that ppGpp influences among other processes the metabolism of components of the cell envelope such as phospholipid and fatty acid synthesis (Cashel and Rudd, 1987) and the LPS biosynthesis and release (lshiguro et al., 1986). Because of the different genotypical background regarding the allelic state of the relA gene, one could expect, that the cell envelope of stringent and relaxed controlled cells are distinguishable after the onset of amino acid limitation. Moreover, amino acid starved cells of CP79 excreted significant amounts of ~-glucanase (Riethdorf et al., 1990), ~-lactamase, interferon cd (Gitter et al., 1995) and miniantibodies (B. Gitter, unpublished observations) into the medium in contrast to stringent controlled cells of CP78, and both strains differ in their fatty acid patterns after amino acid starvation (Gitter et al., 1995). The supposition, that these different characteristics of the cell envelopes of both strains probably affect the electrophoretic mobilities were confirmed by our results represented in Figure 1: amino acid starved cells of strain CP78 and CP79 showed clearly distinguishable distribution graphs for the electrophoretic mobility (Fig. 1, IB, lIB, IIIB). After induction of partial isoleucine starvation the mobility distributions of the stringent controlled strain CP78 remained nearly unchanged, which could be an 102

Microbiol. Res. 151 (1996) 1

indication for a functional regulatory system via ppGpp and the ability of these cells to keep the composition of their cell surface unchanged even after changing environmental conditions. That is in contrast to the reaction of isoleucine starved cells of strain CP79. The main peaks shifted to less negative values in comparison to the mobility distributions before induction of partial isoleucine limitation. Altogether it was surprising, that cells of relaxed controlled strain CP79 produced in all measurements mobility graphs consisting of a main peak and additional peaks or shoulders (Fig. 1, 2, 3). The mobility spectra represent an average value of all cells in the suspension. Therefore, the deviation from a Gaussian distribution could point to a splitting of the strain CP79 into subpopulations. However, lightmicroscopical studies did not yield indications for such a reaction (micrographs not shown here). It is possible however, that subpopulations were only produced by application of an electrical field, such as during measurements with the DELSA 440. At pH 2.0 all charged groups of the cell surface should be protonated resulting in positive EPM values (James, 1991). That could be confirmed by us both for the stringent controlled strain CP78 and the relaxed controlled strain CP79 (Fig. 2). The measurements of cell suspensions at this extreme pH may result in irreversible surface disorganization (James, 1991). It is possibly the reason, that under these conditions in contrast to conditions of physiological pH (Fig. 1) cells of both strains were not able to keep the nature of their cell surface constant after the onset of leucine starvation. The comparable location of the maxima in Fig. 3 and in Fig. 2, B is in our opinion a further confirmation for the reproducibility of the used method, even if, however, the intensity of the second peak was lower than that of the first one. In summary, it can be said that the divergent response to the addition of valine and the different electrophoretic mobility distributions derived from cells of the stringent controlled strain E. coli CP78 and the relaxed controlled strain E. coli CP79 refer probably to various cell surfaces of these cells. Which structural differences in the cell envelopes cause these unequal EPM distributions and whether they are correlated with the observed different excretion efficiency for recombinant proteins should be the subject of further studies. Acknowledgement B. G. was supported by grant of the Deutsche Forschungsgemeinschaft (A7, SFB 197).

References Bayer, M. E., Sloyer, J. L. Jr. (1990): The electrophoretic mobility of Gram-negative and Gram-positive bacteria: an electrokinetic analysis. J. Gen. Microbiol. 136, 867-874. Cashel, M., Rudd, K. E. (1987): The stringent response. In: Escherichia coli and Salmonella typhimurium - cellular and molecular biology (Editor in chief: Neidhardt, F. C.). American Society for Microbiology, Washington, D. C., 1410 -1438. Fiil, N., Friesen, J. D. (1968): Isolation of "relaxed" mutants of Escherichia coli. J. Bacteriol. 95, 729-731. Gitter, B., Diefenbach, R., Keweloh, H., Riesenberg, D. (1995): Influence of stringent and relaxed response on excretion of recombinant proteins and fatty acid composition in Escherichia coli. Appl. Microbiol. Biotechnol. 43,89-92. Ishiguro, E. E., Vanderwel, D., Kusser, W. (1986): Control of lipopolysaccharide biosynthesis and release by Esche-

richia coli and Salmonella typhimurium. J. Bacteriol. 168, 328-333. James, A. M. (1991): Charge properties of microbial cell surfaces. In: Microbial cell surface analysis - structural and physicochemical methods (Eds.: by Mozes, N., Handley, P. S., Busscher, H. J., Rouxhet, P. G.) VCH Publishers, Inc., New York, 221- 261. Mozes, N., Amory, D. E., Leonard, A. J., Rouxhet, P. G. (1989): Surface properties of microbial cells and their role in adhesion and flocculation. Colloids and Surfaces 42, 313 - 329. Nikaido, H., Vaara, M. (1987): Outer membrane. In: Escherichia coli and Salmonella typhimurium - cellular and molecular biology (Editor in chief: Neidhardt, F. C.). American Society for Microbiology, Washington, D.C., 7-22. Riethdorf, S., Ulrich, A., Volker, U., Hecker, M. (1990): Excretion into the culture medium of a Bacillus ~­ glucanase after overproduction in Escherichia coli. Z. Naturforsch. 45c, 240-244.

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