FEMS Microbiology Letters Published by Elsevier
231
34 (1986) 231-235
FEM 02401
Covalent modification of proteins in Escherichia coli growing anaerobically with nitrate as electron acceptor (Covalent
modification;
protein
phosphorylation;
2-dimensional
gel electrophoresis;
Escherichia co/i)
Armin Quentmeier and Garabed Antranikian Institut ftir Mikrobiologie der Georg-August Universiiiii Gijttingen, Grisebachstrasse 8, 3400 Giitingen, F. R.G.
Received 16 December 1985 Revision received 30 December 1985 Accepted 31 December 1985
1. SUMMARY
Escherichia coli cells were labeled with [ 32P]orthophosphate under anaerobic conditions using nitrate as electron acceptor. By 2-dimensional gel electrophoresis, 20 protein species were found to be radioactively labeled; their isoelectric points and molecular masses were determined. Treating the labeled extract with alkaline phosphatase revealed evidence for the presence of 9 proteins modified by phosphorylation. Treatment with snake venom phosphodiesterase also indicated that 2 protein species in E. coli were modified by phosphate-containing compounds which were bound to the protein by a phosphodiester bond.
[4]. Some protein species in E. coli, Salmonella typhimurium, Rhodocyclus gelatinosus and Clostridium sphenoides were reported to be phosphorylated [5-lo]. Evidence has also been presented for the covalent modification of nitrogenase in Chromatium oinosum [ll]. So far the only known bacterial enzymes regulated by phosphorylation/dephosphorylation are the NADP+dependent isocitrate dehydrogenase from E. coli [12,13] and the citrate lyase ligase from C. sphenoides [14]. All these results prompted us to conduct further radioactive experiments with E. coli in order to detect new protein species which are modified by covalent modification.
3. MATERIALS
AND
METHODS
2. INTRODUCTION Many proteins in eukaryotic cells are subjected to covalent modification such as phosphorylation, ADP-ribosylation or nucleotidylylation [l-3]. In contrast, few proteins have been detected in prokaryotes which undergo covalent modification. The regulation of the glutamine synthetase system in enteric bacteria is an example for adenylylation/ deadenylylation and uridylylation/deuridylylation 0378-1097/86/$03.50
0 1986 Federation
of European
Microbiological
3.1. In vivo labeling of proteins E. coli strain K-12 (DSM498)
was used in all experiments. Cells were grown in a medium with low phosphate concentration (1 mM) containing in addition (w/v): 1.19% 4-(2-hydroxyethyl-lpiperazineethanesulfonic acid (Hepes); 0.09% (NH,),SO,; 0.02, MgSO, - 7 H,O; 0.01% L-cysteine - HCl. H,O, and 1 ml/l of tenfold concentrated trace element solution SL 4, according to Societies
232 [15]. Glucose was added as carbon and energy source to a final concentration of 20 mM. The final pH was 7.0. Cells were grown anaerobically at 37°C to an absorbance of 0.34 at 578 nm, then harvested by centrifugation at 11 000 × g and resuspended anaerobically under a nitrogen stream in 10 ml fresh medium containing 20 mM glucose and 0.66% (w/v) KNO 3. 700 ~Ci [32p]orthophosphate was then added to the cell suspension, and incubation was conducted at 37°C for 1 h. Glucose concentration was then 9.2 mM.
3. 2. Preparation of cell extracts After incubation of E. coli cells for 1 h at 37°C in the presence of [32p]orthophosphate, 5 ml of cell suspension was taken and cells were centrifuged and washed twice with 10 ml of fresh medium without glucose or KNO 3. Cells were resuspended in 0.5 ml of 10 mM 2-amino-2-hydroxymethylpropane-l,3-diol (Tris) buffer, pH 7.4, containing 10 mM MgC12, and cell extracts were prepared and treated with DNase and RNase as described previously [9,10]. Aliquots of these extracts were incubated for 2.5 h with either alkaline phosphatase (0.1 mg/ml) at 30°C or snake venom phosphodiesterase (0.1 m g / m l ) at 37°C. 3.3. Electrophoretic techniques One-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described earlier [16]. 2-Dimensional gel electrophoresis was performed according to O'Farrell [17] with some modifications as described by Averhoff et al. [9]. For detection of DNA molecules, gels were soaked in ethidium bromide solution (1 mg/1) for 25 min. After washing the gels in distilled water for 20 min, DNA fragments were visualized with ultraviolet (UV) light at 254 nm. Photographs were taken using a Polaroid positive/negative film (type 665). 3.4. Enzymes and chemicals The chemicals for gel electrophoresis were purchased from Serva (Heidelberg, F.R.G.). Calf intestine alkaline phosphatase (1500 U / m g protein), snake venom phosphodiesterase (52 U/mg), pancreatic DNase (1300 U/mg), pancreatic RNase (50 U/mg), proteinase K (20 U / m g ) and phenyl-
methylsulfonylfluoride were obtained from Boehringer (Mannheim, F.R.G.). [32p]Orthophosphate was purchased from Amersham (Braunschweig, F.R.G.). All other chemicals were obtained from E. Merck (Darmstadt, F.R.G.).
4. RESULTS AND DISCUSSION
4.1. One-dimensional gel electrophoresis of 32p_ labeled extracts For the detection of protein species covalently modified by phosphate or phosphate-containing groups, 32p-labeled cell extracts were applied to 1-dimensional SDS-PAGE. Autoradiography of these gels showed numerous 32p-labeled bands after exposure times of approx. 1 day. Treatment of gels with ethidium bromide (Fig. 1A), however, revealed that these bands represented not only 32P-labeled proteins, but to some extent also DNA, which was highly labeled during growth of bacteria with [3Zp]orthophosphate. Even incubation with DNase (and RNase) at a concentration of 2/~g/ml, as described by Dadssi and Cozzone [18], did not completely eliminate all DNA molecules (Fig. 1A, lane 3). At a 5-fold higher concentration of DNase/RNase, some DNA bands could still be detected (Fig. 1A, lane 4). When the concentration of DNase/RNase was raised to 100 /tg/ml, almost all DNA molecules had disappeared (Fig. 1A, lane 5). Autoradiography of gels after separation of radioactively labeled extracts showed that untreated extracts yielded numerous 32p-labeled band~ (Fig. 1B, lane 1). Only small changes were observed when the extract was treated with proteinase K, indicating that these bands were not caused by 32p-labeled proteins (Fig. 1B, lanes 3 and 4). Incubation with DNase/RNase at a concentration of 100 ~ g / m l eliminated most of the 32p-labeled bands completely (Fig. 1B, lane 2). Two labeled bands were affected by neither D N A s e / R N A s e nor proteinase K. When bacteria were grown in the presence of [32p]orthophosphate, DNA- and RNA-species were highly labeled, caused strong bands during autoradiography, and spread over proteins modified with 32P-containing compounds carrying only one label.
233
Fig. 1. One-dimensional separation of 32P-labeled cell extracts from E. coil (A) DNA fragments from E. coli cell extracts separated by SDS-PAGE. Samples containing 200 btg of protein were treated as follows: lane 1, crude extract; lane 2, crude extract treated with proteinase K (1 m g / m l ) at 37°C for 1 h; lane 3, crude extract treated as in (2) and then with 2 #g D N a s e / R N a s e per ml at room temperature for 1 h; lane 4, crude extract treated as in (2) and then incubated with 10 # g / m l D N a s e / R N a s e at room temperature for 1 h; lane 5, crude extract treated as in (2) and then incubated with 100 # g / m l D N a s e / R N a s e at room temperature. After electrophoresis the gel was soaked for 25 rnin in ethidium bromide (1 m g / l ) and rinsed with distilled water for 20 min. DNA bands were visualized with UV light at 254 nm. (B) 32p-labeled bands after 1-dimensional gel electrophoresis. 32P-labeled samples containing 150/~g of protein were applied. Exposure time 18 h. Lane 1, crude extract; lane 2, crude extract after 1 h incubation with D N a s e / R N a s e (100 #g/ml); lane 3, crude extract after 1 h incubation with proteinase K (1 m g / m l ) at 37°C; lane 4, crude extract after 1 h incubation with proteinase K (2 mg/ml) at 37°C.
Based on these results, 1-dimensional gel electrophoresis does not seem to be a reliable method for the detection of in vivo 32p-labeled proteins. Further experiments were therefore conducted using 2-dimensional gel electrophoresis after sample treatment with high concentrations of DNase/RNase (100 #g/ml).
and M r. Isoelectric focusing was carried out using 2% ampholytes of pH 3-10, resulting in a broad pH range. For staining, a recently developed Coomassie blue-based staining technique was used [19]. The vast majority of proteins was found to have an isoelectric point between 5.5 and 7.5 (Fig. 2). Therefore, the radioactively labeled extracts were separated by isoelectric focusing using 1.6% ampholines of pH 5-7 and 0.4% of pH 3-10, yielding improved resolution in the range pH 5.2-7.1. Autoradiography of these gels showed the presence of 20 32P-labeled proteins (Fig. 3a). The isoelectric points and M r are shown in Table 1. To reveal the nature of the 32p-containing compounds, we incubated aliquots of labeled cell extracts with either alkaline phosphatase or phosphodiesterase. The samples were subjected to 2-dimensional gel electrophoresis and autoradiography. Incubation with alkaline phosphatase caused the loss of the 32p label from 10 protein species (Fig. 3b). As shown in Fig. 3c, treatment with phosphodiesterase instead of alkaline phosphatase affected only 3 spots: the radioactivity of spots o and r was completely released, and spot p became significantly fainter. It is quite likely that these proteins are modified by a nucleotide or its derivative. The 32p-labeling of 9 spots was removed only by incubation with alkaline phosphatase, indicat-
a ..e
W ¢I
E
"5 td _¢ O
4.2. In vivo labeling of proteins by phosphate-containing groups By 2-dimensional electrophoretic separation of cell extracts from E. coli incubated under anaerobic conditions in the presence of KNO 3 as electron acceptor, more than 650 protein species were resolved according to their isoelectric point
4.5
5.5
6.5
7.5
8.5
pH
Fig. 2. 2-dimensional electrophoresis of E. coil cell extract by the O'FarreU technique. For isoelectric focusing, 2% ampholytes pH 3-10 were used. Cell extracts containing 150 /~g of protein were applied to electrophoresis. The gels were subjected to a highly sensitive Coomassie blue-based staining procedure.
234
68.0 45.0
2 5.0
12.5
I
I
I
I
I
I
I
I
ing that these proteins are really covalently modified with orthophosphate residues. 8 protein species were affected by neither alkaline phosphatase nor phosphodiesterase. These results did not rule out covalent modification by phosphorylation or nucleotide binding; it is possible that these enzymes could not access the 32P-containing groups because of steric hindrance or protein folding. The patterns of proteins labeled with 32p-containing compounds in E. coli, reported by Enami and Ishihama [7], Desmarques et al. [6] and our results, are quite different. The first group has grown E. coli in a mineral medium with glucose, the second with acetate under aerobic conditions and we used glucose in the presence of KNO 3 under anaerobic conditions. It is therefore likely that the number and species of covalently modified proteins is dependent on the growth conditions, indicating that phosphorylation or binding of other phosphate-containing compounds plays a major role in controlling metabolic activities in E. coli, as has been established for the regulation of
z
I 68.C
Table 1
C
Molecular mass and isoelectric points of 32p-labeled proteins from E. coli
45.C n
q 25.0 -
~
Protein
Mr
Isoelectric point
Alkaline phosphatase a
a b c d e f g h i j k 1 m n o p q r s t
52000 52000 52000 52000 45000 41000 45 000 38000 45 000 46000 44000 43 000 35000 37000 32000 32000 31000 29000 26000 21000
5.6 5.7 5.8 6.0 5.6 5.6 5.8 5.8 6.0 6.2 6.4 6.4 6.3 6.4 6.4 6.6 6.2 6.2 6.3 5.6
+
p
$
_
5.5
I
I
I
I
5.9
6.2
6.5
6.9
pH
Fig. 3. Covalent modification of proteins with 32p-containing compounds after incubation of E. coli cells with [32p]orthophosphate under anaerobic conditions in the presence of K N O 3. Cell extracts without treatment (a) and cell extracts treated with alkaline phosphatase (b) or phosphodiesterase (c) were subjected to 2-dimensional gel electrophoresis. Isoelectric focusing was carried out using 1.6% ampholytes pH 5-7 and 0.4% ampholytes pH 3-10. After drying, gels were exposed to X-ray films for 7 weeks at - 7 0 ° C .
Phosphodiesterase
+
+ + +
+ + (+) + + + +
+
" + , Release of radioactivity by incubation with alkaline phosphatase or phosphodiesterase; ( + ), incomplete release.
235 N A D P + - d e p e n d e n t isocitrate d e h y d r o g e n a s e reversible p h o s p h o r y l a t i o n .
by
ACKNOWLEDGEMENTS
W e express o u r a p p r e c i a t i o n to Prof. G . G o t t s c h a l k for advice a n d his critical review of the m a n u s c r i p t . S u p p o r t of this w o r k b y the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t is g r a t e f u l l y a c k n o w l edged.
REFERENCES [1] Rubin, C.S. and Rosen, O.M. (1975) Ann. Rev. Biochem. 44, 831-887. [2] Krebs, E.G. and Beavo, J.A. (1979) Ann. Rev. Biochem. 48, 923-959. [3] Chock, P.B., Rhee, S.G. and Stadtman, E.R. (1980) Ann. Rev. Biochem. 49, 813-843. [4] Magasanik, B. (1977) Trends Biochem. Sci. 2, 9-12.
[5] Wang, J.Y.3. and Koshland, D.E. (1978) J. Biol. Chem. 253, 7605-7608. [6] Desmarquets, G., Cortay, J.C. and Cozzone, A.J. (1984) FEBS Lett. 173, 337-341. [7] Enami, M. and Ishihama, A. (1984) J. Biol. Chem. 259, 526-533. [8] Ferro-Luzzi, Ames, G. and Nikaido, K. (1981) Eur. J. Biochem. 115, 525-531. [9] Averhoff, B., AntranikJan, G. and Oottschalk, G. (1986) FEMS Microbiol. Lett., 33, 299-304. [10] Antranikian, G., Herzberg, C. and Gottschaik, G. (1985) FEMS Microbioi. Lett. 27, 135-138. [11] Gotto, J.W. and Yoch, D.C. (1985) Arch. Microbiol. 141, 40-43. [12] Garnak, M. and Reeves, H.C. (1979) J. Biol. Chem. 254, 7915-7920. [13] Borthwick, A.C., Holms, W.H. and Nimmo, H.G. (1984) Biochem. J. 222, 797-804. [14] Antranikian, G:, Herzberg, C. and Gottschalk, G. (1985) Eur. J. Biochem. 153, 413-420. [15] Pfennig, N. and Lippert, D. (1966) Arch. Microbioi. 55, 245-256. [16] Laemmli, U.K. (1970) Nature 227, 680-685. [17] O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007-4021. [18] Dadssi, M. and Cozzone, A.J. (1985) FEBS I_,¢tt. 186, 187-190. [19] Neuhoff, V., Stamm, R. and Eibl, H. (1985) Elcctrophoresis 6, 427-448.