Unité de Biochimie Microbienne, CNRS URA 1300, Institut Pasteur,F-75724 Paris, France1
Institut de Biologie et Chimie des Protéines, CNRS UPR 412, F-69367 Lyon Cedex 07, France2
Laboratoire de Génétique des Microorganismes, INRA-CNRS URA 1925,F-78850 Thiverval-Grignon, France3
Laboratoire de Chimie Bactérienne, CNRS UPR 9043, F-13009 Marseille, France4
Laboratoire de Biophysique, URA 491, Université Louis Pasteur,F-67401 Illkirch Cedex, France5
Author for correspondence: Josef Deutscher. Tel: +33 1 30 81 54 47. Fax: +33 1 30 81 54 57. e-mail: jdeu{at}platon.grignon.inra.fr
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ABSTRACT |
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Keywords: PEP:sugar phosphotransferase system, HPr, Crh, catabolite repression
Abbreviations: FPr, fructose-specific HPr; HPr, histidine-containing protein; NPr, nitrogen-related HPr; PEP, phosphoenolpyruvate; PTS, PEP:sugar phosphotransferase system
a Present address: Unité de Régulation de lExpression Génétique, CNRS URA 1129, Institut Pasteur, F-75724 Paris, France.
b Present address: Dept of Biochemistry, The Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157-1016, USA.
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INTRODUCTION |
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Most bacteria possess only one ptsI and one ptsH gene encoding the general PTS components enzyme I and HPr, respectively. In Bacillus subtilis, the general PTS components participate in sugar transport and phosphorylation via the approximately 15 different sugar-specific enzymes II detected within the genome sequencing programme (Kunst et al., 1997 ). However, a few Gram-negative bacteria possess more than one enzyme I or HPr. Escherichia coli and Salmonella typhimurium, for example, contain a fructose-inducible diphosphoryl transfer protein which is composed of an N-terminal enzyme IIAFru and a C-terminal HPr-like domain (Waygood et al., 1984
; Geerse et al., 1989
) called FPr (fructose-specific HPr). The diphosphoryl transfer protein is phosphorylated by enzyme I at the HPr-like domain from where the phosphoryl group is subsequently transferred to the enzyme IIAFru domain. Interestingly, Haemophilus influenzae was found to possess a protein composed of an N-terminal enzyme IIAFru domain and two FPr domains fused in tandem to the C-terminus. This protein was also suggested to be active in fructose transport, but no specific function was attributed to the second FPr domain (Reizer et al., 1996
).
Evidence for PTS-mediated regulation of nitrogen assimilation and fixation was provided by the finding that in several Gram-negative bacteria the two genes ptsN and ptsO, which encode an enzyme IIAFru- and an HPr-like protein, respectively, were located in the vicinity of the rpoN gene (Reizer et al., 1992a ; Jones et al., 1994
). The rpoN gene encodes the alternate sigma factor
54, which is necessary for transcription of genes required for nitrogen assimilation and fixation, and
54 was thought to be regulated by PtsN and PtsO. These two proteins were therefore renamed enzyme IIANtr and NPr (for nitrogen-related HPr), respectively. Enzyme IIANtr and NPr were found to exchange phosphate with enzyme I, HPr and enzyme IIA of the PTS (Powell et al., 1995
). Loss of enzyme IIANtr activity diminished carbon-catabolite repression of
54-dependent transcription of the Pseudomonas putida xylS operon when expressed in E. coli (Du et al., 1996
).
Neither FPr nor NPr were detected in B. subtilis (Kunst et al., 1997 ). Nevertheless, B. subtilis was found to possess a protein exhibiting 45% sequence identity to HPr. It contained the phosphorylatable Ser-46, but the catalytic His-15 was replaced with a glutamine (Galinier et al., 1997
). As a consequence, no PEP-dependent, enzyme I-catalysed phosphorylation of this protein could be detected (Galinier et al., 1997
). However, the HPr-like protein can be phosphorylated by the ATP-dependent HPr kinase at Ser-46 (Galinier et al., 1997
). Similar to P-Ser-HPr, the seryl-phosphorylated HPr-like protein seems to interact with CcpA and to participate in catabolite activation of the ackA gene (Turinsky et al., 1998
) and in catabolite repression of the B. subtilis lev, xyn and iol operons (Galinier et al., 1997
, 1999
;Martin-Verstraete et al., 1999
) as well as the acsA gene (Zalieckas et al., 1998
). It was therefore called Crh (for catabolite repression HPr). By replacing Gln-15 of Crh with a histidine we wanted to study to what extent this mutation will enable Crh to carry out the catalytic and regulatory functions of P-His-HPr.
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METHODS |
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Standard procedures were used to transform E. coli and B. subtilis strains (Sambrook et al., 1989 ; Kunst & Rapoport, 1995
). Transformants were selected on agar plates containing ampicillin (100 µg ml-1), erythromycin (1 µg ml-1) plus lincomycin (25 µg ml-1) or chloramphenicol (5 µg ml-1). ß-Galactosidase activities were measured after growth of B. subtilis strains in CSK medium with or without 0·2% fructose using the method of Miller (1972)
. One unit of ß-galactosidase activity is defined as the amount of enzyme that produces 1 nmol of o-nitrophenol per min at 28 °C. Doubling times were determined by growing the bacteria in C minimal medium supplemented with glucose, fructose, mannitol, glycerol or glucitol (each at a concentration of 0·5%).
Plasmid constructions.
Plasmid pMTcrh1 was obtained by cloning a fragment of crh into the vector pMUTIN1 (Vagner et al., 1998 ) cut with HindIII and BamHI. The crh fragment was obtained by PCR using chromosomal DNA from B. subtilis strain 168 as template and the following two primers: CCCAAGCTTCGATTAAAGACAGGACTGCAAGCACG and CGCGGATCCGAACGTAAGCAGCAGCTTCTCC. The non-complementary HindIII and BamHI restriction sites are indicated in bold. A 1 kb DNA fragment of plasmid pRC12 containing the crh gene was cloned into M13mp19 to give M13mp19crh (Galinier et al., 1997
). The Muta-Gene M13 in vitro mutagenesis kit (Bio-Rad) was used to obtain the crhQ15H allele. A 434 bp PvuIIBstBI DNA fragment containing the promoterless crh gene or crhQ15H allele was inserted into pDR67 (Ireton et al., 1993
) cut with SmaI and ClaI to give plasmid pRC16 or pRC18, respectively. Plasmid pDR67 carries the pC194 chloramphenicol acetyltransferase gene cat and a spac promoter between two fragments of the B. subtilis amyE gene. The crh and crhQ15H alleles were integrated downstream of the spac promoter allowing induction of their expression with 1 mM IPTG after single-copy integration at the amyE locus of the B. subtilis genome. Appropriate oligonucleotides and pRC16 or pRC18 were used to amplify a DNA fragment carrying the crh or crhQ15H alleles containing the spac promoter and a BamHI site introduced 85 bp downstream of the crh stop codon. These fragments were integrated into plasmid pHT315 cut with EcoRI/BamHI giving plasmids pRC19 (wild-type crh) and pRC20 (crhQ15H), respectively. A 285 bp fragment containing the crhQ15H gene was amplified by PCR using RF M13mp19crhQ15H as template and two oligonucleotides creating an NdeI and a PstI restriction site at the 5' and 3' end, respectively. The NdeIPstI fragment was cloned into the expression vector pT7-7(6xHis) (Cortay et al., 1994
). The mutant protein CrhQ15H carrying a polyhistidine fused to the C-terminus was expressed from the resulting plasmid pAG6 after transformation in the E. coli strain BL21(DE3) (Novagen).
Plasmids pAG1, pAG2, pAG3, pQE30D and pQE30E, which were used for overproducing His-tagged Crh (His-tag attached to the C-terminus), HPr (His-tag added to the N-terminus), enzyme I, LevD and LevE, respectively, have been described previously (Galinier et al., 1997 ; Charrier et al., 1997a
). Transformants carrying one of the above plasmids were grown at 37 °C in 2xTY medium until the culture had reached an OD595 of 0·7. Expression of the genes encoding Crh, HPr, enzyme I, LevD or LevE was induced by adding IPTG to a final concentration of 1 mM, and incubation was continued for a further 4 h. Enzyme I(His)6, Crh(His)6 and HPr(His)6 (Galinier et al., 1997
) as well as LevD(His)6 and LevE(His)6 (Charrier et al., 1997a
) were purified on Ni-NTA-agarose columns.
Two-dimensional gel electrophoresis.
B. subtilis cells were disrupted by adding a solubilization solution containing 8 M urea, 4% CHAPS and 65 mM dithioerythritol to a cell suspension and by sonicating the cells three times for 20 s (Branson sonifier 450 equipped with a cuphorn). The volume of the added solubilization solution exceeded the volume of the cell suspension at least threefold. Aliquots of solubilized cells containing between 30 and 40 µg protein were used per silver-stained two-dimensional gel. Two-dimensional gel electrophoresis was performed using a commercially available immobilized pH gradient (IPG from Pharmacia Biotech) for the first dimension according to the method described on the web server (http://www.expasy.ch/ch2d/protocols.fm2.html#998745). The voltage was increased stepwise from 100 V to 3900 V over a period of 4 h and then maintained at 3900 V until a total of 150 kVh was reached. The temperature was maintained at 20 °C. The IPG strips were then equilibrated and transferred to a slab gel containing a 1220% polyacrylamide gradient, a 46% glycerol gradient and 0·1% SDS, and the electrophoresis was performed at 70 V overnight. Gels were silver stained using the Protein Silver Staining Kit from Pharmacia Biotech. The HPr migration position in the total cellular extract was first determined by comparison to the migration position of purified HPr (without His-tag). In the two-dimensional gels with the B. subtilis mutants GM1341 and CRH168, Crh and HPr were also identified by mass spectrometry of tryptic fragments generated by digestion of the proteins present in the corresponding spots cut out of the gel.
Protein phosphorylation assays.
Phosphorylation assays with [32P]PEP, enzyme I(His)6 and Crh(His)6, CrhQ15H(His)6 or HPr(His)6 were carried out in a total volume of 20 µl. One-and-a-half micrograms of Crh(His)6, CrhQ15H(His)6 or HPr(His)6 was incubated for 10 min at 37 °C with 2 µg enzyme I(His)6 in 50 mM Tris/HCl, pH 7·4, 15 mM MgCl2 and 10 µM [32P]PEP (0·5 µCi; 18·5 kBq). [32P]PEP was prepared from [-32P]ATP (Roossien et al., 1983
). LevR was phosphorylated with [32P]PEP, enzyme I(His)6 and HPr(His)6, Crh(His)6 or CrhQ15H(His)6 as described previously (Martin-Verstraete et al., 1998
).
Phosphorylation of LevD and LevE was carried out by incubating a reaction mixture containing 0·2 µg enzyme I(His)6, 0·5 µg LevD(His)6, 5 µg LevE(His)6, 12·5 mM MgCl2, 50 mM Tris/HCl (pH 7·4), 10 µM [32P]PEP (0·5 µCi) and 0·1 µg either HPr(His)6 or Crh(His)6 for 20 min at 37 °C in a total volume of 20 µl (Charrier et al., 1997a ). All phosphorylation reactions were stopped by adding an equal volume of sample buffer (Laemmli, 1970
) to the assay mixture. Proteins were separated by SDS-PAGE. Gels were dried for 2 h without prior fixation or coloration and exposed to X-ray film (Biomax MR, Kodak).
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RESULTS |
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Q15H mutant Crh can be phosphorylated by enzyme I and PEP
His-15 is the site of PEP-dependent enzyme I-catalysed phosphorylation of HPr. In Crh, His-15 was found to be replaced with a glutamine and no PEP-dependent, enzyme I-catalysed phosphorylation of wild-type Crh was detected (Galinier et al., 1997 ). We wanted to test whether replacement of Gln-15 of Crh with a histidyl residue would allow PEP-dependent enzyme I-catalysed phosphorylation of the mutant protein. A mutation exchanging Gln-15 for histidine was introduced into crh, the crhQ15H allele was inserted into a His-tag expression vector and His-tagged CrhQ15H was purified as described in Methods. PEP-dependent phosphorylation experiments were carried out with enzyme I and HPr, Crh or CrhQ15H. As previously reported (Galinier et al., 1997
), no PEP-dependent phosphorylation was observed for wild-type Crh (Fig. 3a
, lane 3). However, under the experimental conditions employed, the Q15H mutant Crh was found to be phosphorylated to a similar extent as HPr (Fig. 3a
, lanes 2 and 4). In contrast to wild-type HPr and Crh, the two His-tagged proteins were found to migrate to slightly different positions during SDS-PAGE. This is probably due to the different composition and location of the His-tag in HPr and Crh (see Methods section).
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Constitutive expression from the lev promoter in the ptsHDH strain becomes fructose-inducible when CrhQ15H is synthesized
In addition to phosphorylation of LevR at His-585 by enzyme I and HPr (Fig. 3b, lane 1), which stimulates its transcriptional activator activity, LevR was also found to be phosphorylated via enzyme I, HPr, LevD and LevE at His-869. This second phosphorylation inactivates LevR and occurs when fructose or mannose, the substrates of the lev-PTS, are absent from the growth medium. In the presence of one of its substrates, the components of the lev-PTS and consequently also His-869 of LevR are thought to become dephosphorylated leading to activation of LevR and to induction of the lev operon. In agreement with this concept, replacement of His-869 with alanine (Martin-Verstraete et al., 1998
) or mutations inactivating enzyme I, HPr, LevD or LevE (Martin-Verstraete et al., 1990
, 1995
; Stülke et al., 1995
) led to constitutive expression from the lev promoter. We wanted to test whether the synthesis of CrhQ15H would allow phosphorylation of LevR at His-869 and thus make the constitutive expression from the lev promoter in a ptsH deletion strain fructose-inducible. When grown in the absence of a sugar, the ptsH deletion strain QB7111 exhibited constitutive expression of a sacC'::lacZ fusion (Table 2
). The sacC gene encodes the enzyme levanase and is the fifth and last gene in the lev operon (Martin-Verstraete et al., 1990
). A similarly high ß-galactosidase activity was measured when the cells were grown in the presence of fructose. In contrast, the ptsH deletion strain QB7114 in which the crhQ15H mutant allele was integrated into amyE and expressed from the spac promoter exhibited very low ß-galactosidase activity in the absence of fructose. ß-Galactosidase activity was more than 150-fold higher when strain QB7114 was grown in fructose-containing medium (Table 2
), indicating that in the presence of CrhQ15H, LevR is inactivated by PEP-dependent phosphorylation catalysed by enzyme I, CrhQ15H, LevD and LevE. Similar to that observed for a ptsH+ strain (Martin-Verstraete et al., 1990
), expression from the lev promoter in QB7114 was fructose-inducible, as the uptake of fructose via the lev-PTS probably leads to dephosphorylation and activation of LevR.
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DISCUSSION |
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Nevertheless, P-His-CrhQ15H was capable of efficiently phosphorylating the transcriptional regulator LevR. LevR has been shown to be phosphorylated by HPr at His-585 and this phosphorylation was suggested to play a role in a CcpA-independent CCR mechanism operative for the lev operon (Martin-Verstraete et al., 1998 ). In addition, P-His-CrhQ15H could phosphorylate LevD, a fructose-specific enzyme IIA, and the phosphoryl group was subsequently transferred from phosphorylated LevD to LevE, a fructose-specific enzyme IIB. This phosphorylation cascade fulfils two functions. First, it is used for lev-PTS catalysed uptake of fructose (Charrier et al., 1997a
). However, fructose uptake via the lev-PTS is very slow (Gay & Delobbe, 1973
; Martin-Verstraete et al., 1990
). Although the lev-PTS is fully active in fruA or fruB mutants, which are defective in enzyme IIAFru or phosphofructokinase 1, respectively, two components of the main fructose metabolic pathway in B. subtilis, doubling times exceeding 360 min have been reported for these mutants when they were grown on fructose as the sole carbon source (Gay & Delobbe, 1973
). This low fructose-transport activity of the lev-PTS explains why the synthesis of CrhQ15H did not restore growth of a ptsH mutant on fructose (Table 1
), although the mutant Crh was found to phosphorylate LevD (Fig. 3
, lane 6). In addition, we cannot exclude that the mutant Crh phosphorylates LevD less effectively than HPr, which would further slow fructose uptake via the lev-PTS in the ptsH deletion mutant synthesizing CrhQ15H.
The second function of the enzyme IHPrLevDLevE phosphorylation cascade is phosphorylation of LevR at His-869 (Martin-Verstraete et al., 1998 ). Phosphorylation of LevR by phosphorylated LevE regulates induction of the lev operon. A ptsH mutant strain, in which the phosphorylation cascade leading to the formation of P-His869-LevR is interrupted, exhibited constitutive expression from the lev promoter (Martin-Verstraete et al., 1998
). Expression of the gene encoding CrhQ15H in a
ptsH mutant allowed reconstitution of the phosphorylation cascade from enzyme I to LevD to LevE and finally LevR, which prevented the constitutive expression from the lev promoter observed in the
ptsH mutant and restored its fructose-dependent induction.
Enterococcal glycerol kinases have been shown to be phosphorylated by PEP, enzyme I and HPr at a histidyl residue conserved in glycerol kinases of Gram-positive bacteria; phosphorylation increased glycerol kinase activity about 10-fold (Charrier et al., 1997b ). Enterococcus faecalis or B. subtilis mutants defective in enzyme I or HPr therefore have low glycerol kinase activity and cannot grow on media containing glycerol as the sole carbon source (Reizer et al., 1984
; Romano et al., 1990
; Gonzy-Tréboul et al., 1991
). Expression of crhQ15H was not able to restore growth of a B. subtilis ptsH deletion strain in the presence of glycerol. This is in agreement with the finding that although B. subtilis HPr could phosphorylate glycerol kinase from Ent. casseliflavus, no in vitro phosphorylation of this protein was observed in the presence of CrhQ15H. Again, the finding that Crh seems to contain certain characteristics of the PEP-dependent phosphorylation site of HPr from Gram-negative bacteria might explain that CrhQ15H is able to phosphorylate LevR, but not glycerol kinase. PEP-dependent phosphorylation of glycerol kinase has been observed only in Gram-positive bacteria (Charrier et al., 1997b
). In contrast, proteins containing the PTS regulation domain, which carries the site of P-His-HPr dependent phosphorylaton of LevR, have also been detected in Esc. coli and other Gram-negative bacteria (Stülke et al., 1998
).
In summary, the results presented here show that Crh in which Gln-15 has been replaced with a histidine is capable of carrying out some of the regulatory functions of HPr (phosphorylation of the transcriptional activator LevR at His-585 and His-869). The observed PEP-dependent enzyme I-catalysed phosphorylation of LevD/LevE in the presence of CrhQ15H suggests that CrhQ15H might be able to catalyse slow transport of fructose via the lev-PTS. However, the transport function for the most important PTS sugars glucose, fructose (via FruA) and mannitol as well as the P-His-HPr-dependent activation of glycerol catabolism could not be restored by the presence of CrhQ15H.
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ACKNOWLEDGEMENTS |
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Received 19 April 1999;
revised 2 July 1999;
accepted 9 July 1999.