site-directed mutagenesis with the ptsh gene of bacillus subtilis

T H E JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and MoleculaI ’ Biology, Inc

Val. 263, No. 32, Issue of November 15, pp. 17050-17054,1988 Printed in U.S.A.

Site-directed Mutagenesis with the ptsH Gene of Bacillus subtilis ISOLATION AND CHARACTERIZATION OF HEAT-STABLE PROTEINS ALTERED A T THE ATP-DEPENDENT REGULATORY PHOSPHORYLATION SITE*

(Received for publication, February 18, 1988)

Reinhard EisermannS, Josef Deutschert, Genevieve Gonzy-Treboulll, and Wolfgang HengstenbergS 11 From the Wbteilung Biologie der Ruhr-Uniuersitat Bochum, 0-463 Bochum, Postfach 102148, West Germany, the $Max-Planck Institut fur Systemphysiologie, 0 -460 Dortmund, Rkinlanddamm 201, West Germany, and the Wnstitut J q u e s Monod, Centre National de la Recherche Scientifique-Universite Paris VZZ, 75251 Paris Ceder 05, France

The codon for Ser-46 of the ptsH gene of Bacillus subtilis was modified by site-directed mutagenesis to the codons for Ala, Thr, Tyr, and Asp. The mutant genes were overexpressed, three of the corresponding proteins were purified to homogeneity with the excep- tion for the Asp derivative, which could not be detected, although the gene had the desired nucleotide sequence. The phosphotransferase activity of the altered proteins was determined to be 20-35% of wild type activity, which correlates well with the slow phosphorylation of heat-stable protein (HPr) by enzyme I and phosphoenolpyruvate. The ATP-dependent HPr kinase, which previously was shown to be involved in the regulation of carbohydrate uptake of Gram-positive bacteria by covalent phosphorylation of Ser-46 of HPr, is entirely inactive toward the OH group of Thr- 46 and Tyr-46 proteins.

In addition, we constructed a strain of B. subtilis, where the altered gene coding for the Ala-46 derivative of HPr was introduced into the bacterial chromosome. The physiological properties of this mutant are described.

The phosphocarrier protein HPr’ acts as the central dis- tributor of phosphogroups in the bacterial phosphotransferase system, which is involved in carbohydrate uptake of Gram- negative and Gram-positive bacteria. The following reaction sequence displays the protein components of the phosphotransferase system:

Me PEP + enzyme I - P-enzyme I + pyruvate

P-enzyme I + HPr P-HPr + enzyme I

P-HPr + enzyme 111 e P-enzyme 111 + HPr

Enzyme 11, Mg2i P-enzyme 111 + sugar,, > P-sugaa. + enzyme I11

Enzyme I and HPr are constitutively synthesized by the bacterial cell, whereas enzyme 111 and the membrane protein

* This work was supported by the Deutsche Forschungsgemein- schaft. The synthesis facility is supported by a grant from the “Min- ister fur Forschung und Technologie des Landes Nordrhein-West- falen.” The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence and reprint requests should be ad- dressed.

The abbreviations used are: HPr, heat-stable protein; PEP, P- enolpyruvate.

enzyme I1 are induced during growth on various carbohydrates. For further details, the reader is referred to the excellent review of Postma and Lengeler (1).

In addition to its well characterized function as a carrier of the phosphogroup from enzyme I to enzyme I11 proteins, which involves the phosphorylation of nitrogen 1 of the im- idazole ring of His-15, a second phosphorylation site at Ser- 46 was discovered (2) . In contrast to the phosphorylation at His-15, where PEP is the phosphoryl donor, Ser-46 is phosphorylated by an ATP-dependent protein kinase, which is stimulated by glycolytic intermediates and inhibited by inor- ganic phosphate (3). The occurrence of the corresponding HPr-Ser-P-phosphatase was also observed (4). The above observations together with the very low phosphotransfer activity of HPr-Ser-P led to a novel regulatory mechanism of carbohydrate uptake in Gram-positive bacteria (5). Originally, the ATP-dependent phosphorylation of HPr was discovered in Streptococcus pyogenes; it was further observed in Strep- tococcus lactis and faecalis (2, 6). HPr kinase activity is also present in Bacillus subtills (5), the HPr of which is a good substrate for the protein kinase of S. fuecalis (7). To perform site-directed mutagenesis, it is essential to have the isolated gene coding for the HPr protein (ptsH). Until now, among the ptsH genes of Gram-positive bacteria, only the gene coding for HPr of B. subtilis has been cloned and sequenced (a).*

In this paper, we shall describe the construction of site- directed mutations in the ptsH gene of B. subtilis resulting in altered HPr proteins, where Ser-46 is replaced by Ala, Thr, and Tyr. The mutant proteins were overexpressed and purified to homogeneity; the rate of the PEP-dependent phosphotransfer reaction and the rate of the ATP-dependent phosphorylation were estimated in vitro. In the case of the Ala-46 derivative, the corresponding gene was inserted into the bacterial chromosome, and the physiological properties of this mutant were investigated.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Growth Conditions-Escherichia coli TG1 was used for cloning experiments and phage growth in rich medium (9). Overexpression of HPr proteins was carried out either in E. coli K38 for the two vector system of Tabor and Richardson (10) or in JM107 for pUC vectors (11).

B. subtilis Strains and Plasmids-QB 151 (trpC2 metC3, Institut Jaques Monod) GM 329, QB 151 with deletion of ptsH and insertion of the erythromycin (erm) resistance gene from pHV1209; GM 2391, GM 329 with a deletion of erm resistance gene replaced with ptsHl from pTS223; GM 3292, relative isogenic strain of GM 3291, the erm resistence gene being replaced in GM 329 with ptsH (wild type) from pTS22.

The following B. subtilis plasmids were used pTS223, the HindIII-

G. Gonzy-Treboul, manuscript in preparation.

17050

Mutagenesis of the ptsH Gene 17051

Sac1 fragment (0.4 kilobase pair) of pTS22 (8) containing a wild type copy of ptsH was replaced with the same restriction fragment containing the mutant copy of ptsH (ptsHl), the codon for Ser-46 being changed to Ala by site-directed mutagenesis as described below; pTS 224, the EglII-Sac1 fragment (1.3 kilobase pairs) of pTS 22 was replaced by the BamHI-ClaI fragment (1.2 kilobase pairs) of pHV12903 harboring the erm resistance gene from pE194 (12).

Transformation Procedures and Media for B. subtilis-Erm resist- ant transformants of B. subtilis were selected on LB-agar plates supplemented with erm (0.8 pg/ml) and lincomycin (20 pg/ml). To prepare competent cells from phosphotransferase system-defective mutants, which do not use glucose, succinate, and glutamate, were added to the MGI and MGII transformation media as described previously (13). The phosphotransferase system phenotype of the transformants was tested by their ability to grow on C medium agar plates (14) supplemented with auxotrophic requirements (20 pg/ml) and mannitol or succinate plus glutamate (1 mg/ml each) as the carbon source. The growth of GM 3291 and GM 3292 was monitored in C liquid medium supplemented as previously described (14).

Construction of Recombinant DNAs-The phage Ml3mpl8ptsH was constructed by cloning a 468-base pair HindIIIISstI fragment containing the whole ptsH gene of B. subtilis' from the plasmid pTZ18RptsH into the polylinker region of M13mp18. For overexpression, the 486-base pair fragments were prepared from ds M13mpl8ptsH and altered derivatives and ligated into the polylinker region of pT76 or pUC19. All DNA manipulations were performed as described previously (15).

Site-directed Mutagenesis-Site-directed mutagenesis was carried out with excellent yields by the phosphorothioate method of Taylor et al. (9, 16). For hybridization with the Ml3mpl8ptsH template, the following oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer Model 381 A by the 0-cyanethyl phosphoramidite method (17) and purified as the trityl derivatives by high performance

dGTTAACCTTAAA GCT ATTATGGG for changing Ser-46 (TCT) liquid chromatography reversed phase chromatography:

into Ala-46 (GCT), dCCTTAAA TAT ATTATGGG for changing Ser-46 into Tyr-46 (TAT), dCCTTAAA ACT ATTATGGG for changing Ser-46 into Thr-46 (ACT), and m T T A A A GAT AT- TATGGG for replacement of Ser-46 with Asp-46 (GATmnal ly , mutations were confirmed by sequencing the single-stranded Ml3mplSptsH phage DNAs (18, 19).

Protein Purijication-All protein purification steps were performed at 4 "C. About 10 g of cells were suspended in 40 ml of 20 mM potassium phosphate, pH 6.8, containing 5 mg of DNase and disrupted by sonication. The extract was centrifuged at 35,000 X g for 60 min. Then the supernatant was applied to an anion exchange column (Q- Sepharose, 2.8 X 14 cm) equilibrated with 20 mM potassium phosphate, pH 6.8. HPr activity could be detected in the flow through after eluting with the above buffer. All HPr-containing fractions were collected and concentrated by pressure dialysis (Amicon) to a final volume of 40 ml. Finally, the protein solution (40 ml) was applied to a gel filtration column (Sephadex G-75, 5 X 90 cm) equilibrated with the volatile buffer 50 mM ammonium hydrogen carbonate, pH 8. After this step, the HPr was pure according to acrylamide gel electrophoresis in the native (20) and denatured (21) state. The lyophilized protein was stored at 4 "C.

Mutant Complementation Assay-The HPr mutant complementation assay to estimate the activity of HPr of Gram-positive bacteria was performed with crude extract of the Staphylococcus aureus strain S 797A defective for HPr as described earlier (22).

PEP-dependent Phosphorylation of HPr Proteins-Enzyme I was purified from S. jaecalis as described earlier (23). 40 pg of the HPr derivative of B. subtilis were incubated with 2 pg of enzyme I in 100 pl of 20 mM NH,HCOa buffer, pH 8.3, 2.5 mM PEP, and 2.5 mM MgClz at 37 "C. The reaction was stopped after 20 and 40 min by freezing the samples. The grade of phosphorylation was determined by electrophoresis of aliquots containing 10 pg of protein on native polyacrylamide tube gels (20).

ATP-dependent Phosphorylation of HPr Proteins-ATP-dependent HPr kinase was purified from S. jaecalis according to Ref. 3 until chromatography on DEAE-cellulose. For phosphorylation, 20 pg of the HPr derivative of E. subtilis was incubated together with 20 mM fructose 1,6-bisphosphate, 10 mM ATP, 10 mM MgCl', and 5 pl of HPr kinase (concentrate after the DEAE-cellulose run) in 20 p1 of Tris/HCl, pH 7.3, for 20 min at 37 "C. Subsequently, samples were loaded onto native polyacrylamide tube gels (20). This method al-

M. A. Petit and S. D. Ehrlich, unpublished results.

lowed separation of HPr and its phosphorylated derivatives after staining with Coomassie Brilliant Blue.

Materials-Restriction enzymes, Klenow fragment, T4 ligase, and polynucleotide kinase were purchased from Boehringer Mannheim. [ L U - ~ ~ S I ~ A T P was obtained from Amersham Buchler, Q-Sepharose, and Sephadex were from Pharmacia LKB Biotechnology Inc. [ L U - ~ ~ S ] dCTP was kindly provided by Dr. R. S. Goody (Max-Planck Institut fur Medizinische Forschung, Heidelberg). All other reagents were of the highest commercially available grade.

RESULTS

Oligonucleotide-induced Site-directed Mutagenesis-The possibility to introduce mutations by oligonucleotide mis- match is well known. However, methods, which gave the desired mutant clones in good yields, became available just recently. Among the various mutant enrichment methods (24), we applied the procedure developed in the laboratory of

H - 1 5 Y-37 F-6 Y-29

f ' , ' , . , . , . , , , ' , "

7 . 8 7 . 4 7 .O 6 . 6

PPM

FIG. 1. 500-MHz proton NMR spectrum of wild type HPr of B. subtilis and HPr Tyr-46. Upperpanel, spectrum of wild type HPr with the assignments of aromatic residues according to Kalbitzer et al. (27). The standard 1-letter code is used to mark the amino acids. Lower panel, spectrum of HPr Tyr-46, the arrows indicate the additional tyrosine signals in HPr Tyr-46. Protein concentration for both spectra: 10 mg/ml.

17052 Mutagenesis of the ptsH Gene

140% relative activity

120% - 100%

100%

80%

80%

40%

20%

ox

m Hh WT Ser48Ala Ser46Thr = S e r m

FIG. 2. Phosphotransfer activity of B. subtilis H P r and its derivatives. The activity was determined by the S. aureus mutant complementation assay (22). HPr concentrations were 20 PM each. Compared to S. aureus HPr, the activity of B. subtilis HPr was 74% at the above concentration in the assay system.

FIG. 3. PEP-dependent phosphorylation of HPr and its mutant derivative native polyacrylamide tube gels from left to right. 10 pg of HPr wild type, 10 pg of HPr wild type + enzyme I, 10 pg of HPr Ala-46, HPr Ala-46 + enzyme I, HPr Thr-46, HPr Thr- 46 + enzyme I, HPr Tyr-46, HPr Tyr-46 + enzyme I. Phosphorylation was run for 40 min before the sample was applied to the gel, the anode is at the bottom. The residual unphosphorylated band in the second lane is either unreacted HPr or hydrolyzed phospho-HPr. HPr of E. coli as an impurity can be excluded it migrates significantly slower in the native gel at pH 9.3.

Eckstein and co-workers (16), which allowed us to isolate the ptsH gene with the desired mutation in typical yields of about 90% as determined from DNA sequencing of isolated clones after mutagenesis.

Overexpression of the Mutated Genes-Among the established overexpression vectors, we have chosen the two-plasmid system of Tabor and Richardson (lo), where the selected gene is put under the control of the 410 promoter of the bacteriophage T7. The T7 RNA polymerase capable of tran- scribing the gene downstream of the T7 promoter is encoded by a second plasmid under control of the XpL promoter, which is inactivated by the CI repressor also encoded by the same plasmid. The polymerase is produced after a temperature shift to 42 "C to transcribe the desired gene from the 410 promoter. With this system, we obtained very high overexpression levels, which allowed us to isolate pure HPr proteins from 10 g of

a b c d e f g h i j "..".̂ ("I"" -

f

FIG. 4. ATP-dependent phosphorylation with wild type HPr and its mutant derivatives from B. subtilis. Phosphoryla- tion was performed as described under "Experimental Procedures." Lane a, 20 pg of HPr; lane j , 20 pg of HPr Ala-46. On all other lanes, 20 pg of HPr proteins incubated for either 20 or 40 min with the phosphorylation mix were applied lams b and c, HPr; lanes d and e, HPr Thr-46; lanes f and g, HPr Tyr-46; lanes h and i, HPr Ala-46. HPr Thr-46 and HPr Tyr-46 were found to migrate to the same position on native polyacrylamide gels as HPr Ala-46. The band pattern of the used kinase preparation is not shown on a separate lane. The impurities in the kinase at the position of P-Ser-HPr can be clearly derived from lanes h and i. The purity of the used HPr derivatives is convincingly demonstrated in Fig. 3.

cells (wet weight) in quantities of more than 100 mg after two chromatography steps.

Physiological Properties of the HPr Proteins Derived from the Mutated Genes-The amino acid exchange of the proteins after mutagenesis was generally deduced from the altered gene by DNA sequencing. In the case of the Ala-46 mutation, no phosphorylation occurred with ATP and the HPr kinase as one would expect. The Tyr-46 derivative was characterized by NMR spectroscopy, where one clearly sees the expected additional signals resulting from a 3rd Tyr residue (Fig. 1). The various derivatives at position 46 also possess very char- acteristic kinetic properties during the phosphotransferase reaction (Fig. 2) assayed by the mutant complementation assay with extracts of S. aureus (22).

Phosphorylation of the HPr proteins with PEP and enzyme I is shown in Fig. 3. Compared to wild type HPr, the rate of phosphorylation of mutant HPr is similarly lowered as the overall phosphotransferase reaction determined with the mutant complementation assay.

Since the evaluation of quantitative data for the kinetics of the ATP-dependent phosphorylation of HPr is rather tedious (7), we present qualitative data derived from a series of native gels to illustrate the different phosphorylation rates (Fig. 4).

It is very obvious that even the minor change in the Thr- 46 derivative results in the inability to become phosphorylated by the HPr kinase. For the more significant change in the Tyr-46 derivative, we expected no phosphorylation by the kinase.

Construction and Phenotype of the ptsHl Mutant-The ptsHl mutant (ptsH1 gene codes for the HPr Ala-46 protein) of B. subtilis was constructed in two steps. First, we constructed GM 329, in which ptsH and the beginning of ptsI (structural gene for enzyme I) were deleted and replaced with erm resistance gene (Fig. 5). Then, GM 329 was transformed by pTS223 or pTS22, and the class of transformants having integrated the plasmid pts fragment into the chromosome by homologous recombination through a double crossover (up- stream of the BgnI and downstream of the Sac1 site) were

Mutagenesis of the ptsH Gene 17053

PTS 22

98151

E Bq H T E

Ori ptsH ptsl

I - "

I" ermr cms

61329 "J E E

8 pTS223

1 pts+ cmS ermS

611132% E H E

ptsHl Ptsl "

FIG. 5. Construction of the B. subtilis strain GM 3291 containing the ptsHl gene (coding for HPr Ala-46) inserted into the bacterial chromosome. pTS22 was cut with BglII and SmI, the BumHI-ClaI fragment of pHV1209 containing an erythromycin resistance gene was inserted, giving pTS224; the insertion inactivates the BglII and SacI restriction sites. pTS224 was used to transform QB151 to erm resistance and chloramphenicol (cm) sensitivity, giving strain GM 329 by a double crossover. This strain is mannitol-negative (phosphotransfer system-negative phenotype). pTS22 was cut with HindIII and Sad, and the HindIII-Sac1 fragment of the pT76 derivative containing the ptsHl gene (coding for HPr Ala-46) was inserted, giving pTS223. Then, pTS223 and pTS22 as controls were used to transform GM 329 to a restored growth on mannitol, erm, and cm sensitivity, giving by a double crossover the mutant strain GM 3291 (ptsH1) and the wild type isogenic control strain GM 3292. The symbols B, Bg, C, E, H, and S represent the restriction sites BamHI, BglII, ChI, EcoRI, HindIII, and SacI, respectively. Ap represents ampicillin resistence gene, and ori the replication origin of pBR322.

TABLE I Comparative growth parameters of B subtilis GM 3291 (ptsH1) and

GM 3292 (wild type) GM 3291 GM 3292

Carbon source" Doubling ygb Doubling zy time time 14h

~

rnin rnin

Glucose (10 g/liter) 64' 5.6 66 9.7 Glucose (20 g/liter) 11.5 18.8 Mannitol (10 g/liter) 73 2.2 75 5.05 Glycerol (10 g/liter) 68 14.2 67 15.1 Succinate (10 g/liter) + 90 5.5 96 5.8

glutamate (10 g/liter)

The bacterial growth was performed in C liquid medium supple-

* Optical density measured in stationary phase after 14 h at 37 "C. mented as described under "Experimental Procedures."

All values were averaged from two identical experiments.

selected in both cases. These transformants grow on phosphotransferase system sugars and were chloramphenicol- and erythromycin-sensitive. The phenotype of both strains, GM 3291, carrying ptsH1, and its isogenic relative GM 3292, carrying the wild type gene, was examined. Although the mutant strain grew on phosphotransferase system sugars with rates similar to those of the wild type during exponential phase, a striking difference appeared at the level of the stationary phase, which is reached at optical densities lower for the mutant in the presence of phosphotransferase system sugars as the carbon source, but similar to those of the wild type strain in the presence of non-phosphotransferase system carbon sources like glycerol or succinate plus glutamate (Ta- ble I). In the case of glucose, two different initial concentrations were tested, and the remaining glucose was determined in the culture supernatants at the stationary phase. The results show that the level of the plateau is proportional to the initial concentration of glucose, which has been entirely consumed.

Therefore, it can be concluded that the absence of the regulatory phosphorylation site at Ser-46 of HPr leads to an energy waste resulting in a 2-fold lower bacterial mass on an average. The behavior of the mutant in presence of phosphotransferase system sugars could be explained at least in part by the activation of futile cycles. From the model of Reizer and Peterkofsky (5) concerning the dual control of the phosphotransferase system glycolysis cycle, it could be assumed that sugar uptake goes on despite the high level of fructose 1,6-bisphosphate in the mutant, which cannot regulate sugar uptake by the HPr kinase. This could induce back hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate, which is a useless energy expenditure. Another rather simple expla- nation would be the well known toxicity of the high concentration of phosphorylated carbohydrates in the cell.

DISCUSSION

Until recently, ATP-dependent phosphorylation of protein as a regulatory device was not well established in bacteria, whereas it is a common and extensively investigated mechanism of enzyme regulation in eucaryotic cells (25). The first example for regulation of an enzyme by covalent phosphorylation in bacteria was the isocitrate dehydrogenase, which essentially is inactivated by phosphorylation of Ser-113 (26). Site-directed mutagenesis of the gene to introduce other amino acids at this position clearly showed that substitution of Ser-113 by uncharged amino acids significantly lowered the activity. The results with the Asp-113 derivative were not clear cut; in the extract, where Asp-113 dehydrogenase was present, no in vivo protein phosphorylation was observed. It is assumed that this derivative is probably inactive in the dehydrogenase reaction. The second well characterized example of modification of a bacterial protein by phosphorylation is the HPr protein of Gram-positive bacteria, which is phosphorylated at Ser-46 (2). This prevents effective phosphorylation of His-15 by the PEP-dependent protein kinase enzyme 1. The phosphogroup can be removed from the serine residue by the specific HPr-Ser-P-phosphatase.

As shown in Figs. 2 and 3, the introduced amino acid exchanges at position 46 in HPr result in similar effects as observed by Thorsness et al. (26) for the isocitrate dehydrogenase.

The phosphotransfer activity of HPr Ala-46, Thr-46, and Tyr-46 is significantly lowered. In the case of the Asp-46 derivative, we were unable to detect the protein after overexpression with two different vector systems such as pUC and the Tabor-Richardson system (10). Even with polyclonal an-

17054 Mutagenesis of the ptsH Gene TABLE I1

Comparison of partial amino acid sequences of two HPr proteins of Gram-positive bacteria with HPr of E. coli . _

S . faecalis B . subtilis -Val-Asn-Leu-Lys E . coli

“

S . faecalis B . subtilis E . coli

Ser-Ile-Met-Gly-Val-Met-Ser-Leu-Glii

l I

tibody against HPr of S. faecalis, which cross-reacts well with wild type and mutant B. subtilis HPr, we failed to identify the protein.

There is, however, a very significant difference: in contrast to isocitrate dehydrogenase, HPr is not an enzyme; it is the substrate for enzyme I. Therefore, the low phosphoryl carrier activity of HPr is either caused by a significant conformational change of the native protein or Ser-46 is part of the recognition site of enzyme I, which is modified by the ATP- dependent phosphorylation to yield inactive HPr.

The qualitative data obtained by electrophoretic analysis of the PEP-dependent phosphorylation of the HPr proteins with enzyme I is substantial evidence that the rate of phosphorylation of HPr is the rate-limiting reaction in the overall phosphotransfer. We favor the second alternative, since NMR measurements, which were performed at HPr-Ser-P of S. faecalis, did not reveal a significant conformational change. As shown in Table 11, the sequence homology of HPr of B. subtilis and S. faecalis is very obvious around the phosphorylation site at Ser-46. HPr of E. coli also possesses a seryl residue at the same position, but the homology in this region is restricted to the positions 45 and 46. This is probably one of the reasons why HPr of E. coli is not phosphorylated by the HPr kinase of Gram-positive bacteria.

The mutant proteins isolated so far allow us to study the substrate specificity of the bacterial ATP-dependent protein kinase. Comparable to the phosphorylation behavior of the isocitrate dehydrogenase by its corresponding kinase, we also noticed that the Thr-46 and the Tyr-46 derivative could not be detectably phosphorylated under conditions where we observed almost quantitative phosphorylation of the wild type HPr (Fig. 4). This demonstrates that these well characterized bacterial protein kinases are very specific enzymes. As already mentioned under “Results,” we succeeded in constructing a strain of B. subtilis with the ptsHl gene (coding for HPr Ala- 46) inserted into the bacterial chromosome. The explanations given to interpret the abnormal growth behavior are still considered preliminary. So we did not exclude experimentally that the slower phosphorylation rate of the mutant HPr is partially due to an increased instability of the mutant phos- phoproteins. There is, however, evidence that the modification of Ser-46 by phosphorylation or mutagenesis does not have any effect on the protein conformation or the pK of the active site His residue. This could be demonstrated with P- Ser-HPr of S. faecalis and recently with the Tyr-46 derivative

of B. subtilis by NMR measurements: The further biochem- ical characterization of the mutant strain should give a deeper insight into the significance of the ATP-dependent regulatory phosphorylation site of the HPr proteins of Gram-positive bacteria.

Acknowledgments-We thank Dr. H. R. Kalbitzer, MPI Medizin- ische Forschung, Heidelberg, for recording the 500-MHz Fourier transform spectra. We are grateful to Dr. S. Tabor, Harvard Univer- sity, for permission to use the T7 promoter plasmid expression system. Automated oligonucleotide synthesis was performed by Dr. U. Kuck, Bochum.

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site-directed mutagenesis with the ptsh gene of bacillus subtilis

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