AG Physiologie der Mikroorganismen, Ruhr-Universität Bochum, ND 06/744, Universitätsstr. 150, D-44780 Bochum, Germany1
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany2
Institut für Immunologie, Abteilung Proteinstrukturlabor, MA 2/143, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany3
Author for correspondence: Wolfgang Hengstenberg. Tel: +49 234 3224247. Fax: +49 234 3214620. e-mail: wolfgang.hengstenberg{at}ruhr-uni-bochum.de
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ABSTRACT |
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Keywords: antitermination, glucose-specific phosphotransferase system, regulation, Staphylococcus carnosus, protein phosphorylation
Abbreviations: DIG, digoxigenin; ESI, electrospray ionization; PTS, phosphotransferase system; PRD, PTS regulatory domain; RAT, ribonucleic antiterminator; RBD, RNA-binding domain
The GenBank accession number for the complete ORF of the gene encoding GlcT is Y14029.
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INTRODUCTION |
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The second type is so-called non-processive antitermination. In this case antitermination is mediated by antiterminator proteins interacting with nascent mRNA sequences. These ribonucleic antiterminator (RAT) sequences overlap -independent transcription terminators. A complex of the RAT with the antiterminator protein stabilizes the secondary structure of the RAT and thus prevents an alternative formation of the terminator loop (Aymerich & Steinmetz, 1992
; Arnaud et al., 1992
).
Various genes and operons are controlled by antitermination, including rRNA synthesis and production of proteins involved in carbohydrate transport and utilization (Landick et al., 1990 ; Yanofsky et al., 1987
; Rutberg, 1997
).
Antitermination of catabolic operons appears to be substrate-induced via reversible phosphorylation of the antiterminator proteins. The transmitters are components of the phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS), responsible for uptake of carbohydrate under concomitant phosphorylation. Reversible phosphorylation of the antiterminator proteins is thought to be mediated by the general components (HPr, enzyme I) as well as by the carbohydrate-specific components (enzyme II) of the PTS (Schnetz & Rak, 1988 , 1990
; Arnaud et al., 1996
).
The antitermination systems of several catabolic operons involved in the metabolism of sugars like glucose, sucrose and ß-glucosides are assigned to the bgl-sac family based on sequence similarities with regard to antiterminator proteins and RAT sequences (Rutberg, 1997 ; Stülke et al., 1998
). The antiterminator proteins of this family are composed of three structural domains. The N-terminal residues are involved in binding the RAT sequence (Manival et al., 1997
). The two other domains, called PRD-I and PRD-II (PTS regulatory domain), contain two similar regions which are phosphorylated at conserved histidine residues by components of the PTS (Stülke et al., 1998
). Genetic and biochemical evidence points to a modulation of the binding of the antiterminator to its corresponding RAT sequence by PTS components (Amster-Choder & Wright, 1992
, 1997
; Arnaud et al., 1992
).
In this study we show in vitro and in vivo studies which provide evidence for regulation of the ptsG operon of Staphylococcus carnosus via antitermination. glcT was cloned and the antiterminator protein GlcT was purified. A PTS-mediated phosphorylation and its influence on quaternary structure was observed. Because of the high similarities to the ptsG antitermination system of Bacillus subtilis, the complementation between these two systems was investigated by in vivo studies in B. subtilis and analysis of RNA-binding activity through surface plasmon resonance.
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METHODS |
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Bacterial strains and growth conditions.
Escherichia coli DH5 and XL-1 Blue (Sambrook et al., 1989
), and S. carnosus TM300 were used for cloning experiments. E. coli BL21(DE3) (Sambrook et al., 1989
) was used for expression of S. carnosus GlcT. B. subtilis GlcT was expressed in E. coli FT1. E. coli was grown in TBY medium (10 g tryptone l-1, 5 g yeast extract l-1, 5 g NaCl l-1, 50 µg ampicillin l-1).
B. subtilis strains QB5448 [trpC2 amyE(LA ptsG''lacZ aphA3); Stülke et al., 1997
] and GP109 [trpC2
glcT8 amyE(
LA ptsG''lacZ aphA3); Bachem & Stülke, 1998
] were grown in SP or CSE medium (Faires et al., 1999
). Media were supplemented with carbon sources as indicated.
DNA manipulations.
All manipulations with recombinant DNA were carried out by standard procedures (Sambrook et al., 1989 ). Restriction enzymes and T4 ligase used in recombinant DNA experiments were used according to the specifications of the supplier. DNA fragments were purified from agarose gels with the Nucleo Spin Extract kit from Macherey & Nagel. Genomic DNA from S. carnosus TM300 was purified using the Genomic DNA kit from Qiagen. DNA sequences were determined with the ALF DNA Sequencer (Pharmacia; Kristensen et al., 1988
).
Cloning of the glcT gene.
To generate a digoxigenin (DIG)-labelled fragment, oligonucleotide primers were designed according to the partial ORF upstream of the gene glcA. Primers glcT1 (5'-CCAGAAGATGAGATTGG-3') and glcT2 (5'-GTTGTGTGAAGTGATTG-3') correspond to amino acids 15 and 132138 of the truncated ORF localized on pUC18-7k (see below), respectively. These primers were used for PCR with construct pUC18-7k which bears the partial ORF of the putative glcT as template (Christiansen & Hengstenberg, 1996 , 1999
). The PCR mixture (50 µl) contained 200 ng pUC18-7k, 50 pmol each primer, DIG-dNTP mix (Boehringer Mannheim) and 2·5 U Taq polymerase (Life Technologies) in the recommended buffer.
Genomic DNA was prepared from S. carnosus TM300 by using the genomic isolation kit from Qiagen. Southern blots of digested genomic DNA were probed with DIG-labelled DNA (see above) using the Genius kit from Boehringer Mannheim. A subgenomic S. carnosus library was constructed using a size-fractionated (23 kb) pool of BstYI-digested chromosomal DNA in pUC20 (Boehringer Mannheim), transformed into competent E. coli XL-1 Blue cells. Colonies were screened by hybridization with the DIG-labelled probe.
One positive clone was detected, the cloned genomic fragment was digested with EcoRI and the subfragments were cloned into the EcoRI site of pUC20 for determination of the complete nucleotide sequence. During a three-step procedure the complete ORF of the putative glcT, including the ShineDalgarno box, was cloned into the vector pT7-6 (Tabor & Richardson, 1985 ). The gene could be expressed under the control of the T7 promoter in E. coli BL21(DE3) (Stratagene). This construct was named pT7-6-glcT.
Expression and purification of GlcT.
E. coli strain BL21(DE3) was transformed with pT7-6-glcT. The antiterminator protein GlcT was expressed by growing cells in TBY medium containing 50 µg ampicillin ml-1 at 37 °C until they had reached an A578 of about 1. IPTG was subsequently added to a final concentration of 0·33 mM and cells were incubated for an additional 3 h at 37 °C.
Cells (4 g) were resuspended in 10 ml buffer A (Tris/HCl, pH 7·5, 1 mM EDTA, 1 mM NaN3, 1 mM PMSF, 1 mM DTT) and disrupted by sonication. Cell debris was removed by centrifugation for 50 min at 50000 g and the supernatant was loaded on a Q-Sepharose column (3x10 ml; Pharmacia) equilibrated with buffer A. The proteins were eluted with a 600 ml linear gradient of 00·6 M NaCl in buffer A. GlcT-containing fractions (0·330·39 M NaCl) were identified. The pool was adjusted to 25% ammonium sulfate and applied on a Butyl TSK column (5x22 cm; Tosohaas) pre-equilibrated with 25% ammonium sulfate in buffer A. The proteins were eluted with a 2 l linear gradient (250% ammonium sulfate in buffer A). GlcT eluted at 20% ammonium sulfate; the pool was concentrated by ammonium sulfate precipitation (85%), the pellet was resuspended in 20 ml buffer A and loaded onto a Sephadex G75 gel filtration column (5x90 cm; Pharmacia) which was eluted with 2 l buffer A. The GlcT pool was stored as an ammonium sulfate precipitate (in 85% ammonium sulfate). All purification steps were checked by SDS-PAGE (12%; Laemmli, 1970 ).
Phosphorylation of GlcT and separation of phosphorylated GlcT.
Phosphorylation was achieved by incubation of GlcT with enzyme I and HPr in a sixfold excess of GlcT. The reaction was performed in 25 mM Tris/HCl buffer, pH 7·5, 2 mM MgCl2 and 25 mM phosphoenolpyruvate. Phosphorylation was detected by using 1012% native gels (Hjerten et al., 1965 ). For separation of phosphorylated GlcT, 3 nmol protein was used and after 30 min incubation at 37 °C with 0·5 nmol enzyme I/HPr the phosphorylation mixture was loaded for FPLC gel filtration (Superose 12; Pharmacia) at a flow rate of 0·5 ml min-1.
In vivo assay of GlcT activity.
In vivo assays were performed in E. coli DH5 (Sambrook et al., 1989
) and B. subtilis QB5448 and GP109 (Stülke et al., 1997
; Bachem & Stülke, 1998
).
For studies in E. coli, the promoter region of glcA was translationally fused with gfp, encoding the green fluorescent protein. The promoter region was subcloned into the HincII site of pUC20 through HaeIII restriction of pUC18-N1B (Christiansen & Hengstenberg, 1996 , 1999
). Restriction with PstI led to cloning into the PstI site of pGFP (Clontech). The resulting construct was named pRAT/To-gfp. After cloning in pUC20 via EcoRI restriction, the insert was amplified by PCR using reverse and universal primers (Boehringer Mannheim). The fragment containing RAT/To-gfp was cloned into the SmaI site located downstream of the glcT gene of pUC21-glcT (all amplified fragments were sequenced). For in vivo studies, the construct was used with RAT/To-gfp in the opposite orientation glcT (named pGG1). As a negative control a construct was used with the amplified fragment RAT/To-gfp cloned into the SmaI site of pUC21 (named pGG0).
Cells of E. coli DH5 were transformed with pGG0 and pGG1. Fluorescence was detected after growth in TBY medium at 30 °C starting from an A578 of 0·2. IPTG was added to a final concentration of 0·3 mM and growth was stopped at an A578 of 1·0. The cells were centrifuged and resuspended in 0·8 ml 10 mM Tris/HCl, pH 7·5, 50 mM NaCl. Cells were disrupted by sonication, centrifuged at 10000 g for 1 h and the supernatant was used for fluorescence measurement with a spectrofluorometer (Jasco) at 395 nm excitation and 509 nm emission. As a measure of relative fluorescence, the fluorescence measurement was divided by the cell density.
For studies with B. subtilis, two constructs were made. (i) The glcT gene was cloned by KpnI/BamHI restriction into the shuttle vector pHT304 (Arantes & Lereclus, 1991 ) to give pH- T304-glcT. (ii) To clone the N-terminal RNA-binding domain (RBD), two primers were designed according to the amino acids 18 (pRBD1; 5'-AAACCCGGGACAAAGGAGCTGATTAGCATGAGCAACTACGTCATAGAG-3') and 5260 (pRBD2; 5'-ATATCTAGATTAATCTTTCTTTTGCTCTAATTTATAAAC-3'). Moreover, primer pRBD1 contains a B. subtilis ShineDalgarno box and a SmaI site.
For PCR, 200 ng pUC21-glcT was used and the reaction was performed using standard procedures. The amplified fragment was cloned into the HincII site of pUC21, sequenced and cloned into the vector pBQ200 through SmaI restriction to give pBQ200-rbd.
B. subtilis was transformed with plasmids pHT304-glcT and pBQ200-rbd according to the two-step protocol described by Kunst & Rapoport (1995) . Transformants were selected on SP plates containing chloramphenicol (5 µg ml-1), kanamycin (5 µg ml-1) or erythromycin plus lincomycin (1 and 25 µg ml-1, respectively).
Quantitative studies of lacZ expression in B. subtilis in liquid medium were performed as follows. Cells were grown in CSE medium supplemented with the carbon sources indicated. Cells were harvested at an OD600 of 0·60·8 for cultures in CSE medium and 0·81 for cultures in CSE medium with sugar. Cell extracts were obtained by treatment with lysozyme and DNase. ß-Galactosidase activities were determined as previously described using ONPG as substrate (Miller, 1972 ). One unit is defined as the amount of enzyme which produces 1 nmol o-nitrophenol min-1 at 28 °C.
Liquid chromatography (LC)-MS analysis of non-phosphorylated and phosphorylated GlcT.
LC-MS/MS spectra were recorded on a Finnigan TSQ 7000 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source. GlcT was dissolved in ammonium hydrogen carbonate buffer (pH 8·0), additionally purified through HPLC (C4 column; flow rate, 80 µl min-1) and introduced on-line into the ESI source. Phosphorylated GlcT was rebuffered through G25 high trap columns (Pharmacia) in ammonium hydrogen carbonate buffer (pH 8·0) and prepared as described for non-phosphorylated GlcT.
For identification of the phosphorylation site, 2 nmol phosphorylated protein was digested with LysC protease (Promega) at 37 °C overnight. HPLC separation of the peptides was performed on a 300 µmx25 cm C18 column at a flow rate of 4 µl min-1. The peptides were eluted by a linear gradient (550%) of solvent B over 90 min (solvent A, 0·025% trifluoroacetic acid; solvent B, 0·02% trifluoroacetic acid, 80% acetonitrile).
Limited LysC digestion.
For the digestion experiments, LysC protease (Promega) was used at a ratio of 1:60 (w/w, protease/substrate) as recommended by the supplier. For isolation of proteolytic fragments, the digestion reaction contained 4 nmol GlcT and 4 µg LysC protease and was incubated for 20 min at 37 °C. Purification was performed by FPLC gel filtration (Superose 12; Pharmacia). Fragments were identified by N-terminal sequencing with an Applied Biosystems Sequencer 473A.
Assay of proteinRNA interaction by surface plasmon resonance analysis.
Sequence-specific proteinRNA interactions were detected by surface plasmon resonance analysis using the BIAcore X optical biosensor (Pharmacia Biosensor). 5'-Biotinylated synthetic RNAs were immobilized onto a streptavidin-containing chip. The RNA, dissolved in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0·005% polysorbate 20), was coupled to the streptavidin surface of the sensor chip at a flow rate of 10 µl min-1. Since 1000 resonance units (RU) might correspond to a surface concentration of 1 ng mm-2, the different RNA molecules were immobilized to surface concentrations between 1·4 and 1·8 ng mm-2. The standard running buffer was HBS-EP buffer and all reagents were introduced at a flow rate of 10 µl min-1. GlcT from S. carnosus and B. subtilis was used at concentrations of about 16 µM. The sensor surface was regenerated between assays by injecting 20 µl 50 mM NaOH (in 1 M NaCl) to remove bound analyte. For processing the data, the program BIAevaluation 3.0 was used.
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RESULTS |
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The corresponding nucleotide sequence was used to construct a DNA probe to screen a genomic library of S. carnosus. The complete ORF of the gene encoding GlcT was isolated as a fragment of 2258 bp. The fragment was subcloned into the vector pT7-6 and expressed in E. coli strain BL21(DE3).
Overexpression was analysed by SDS-PAGE (major band at 33 kDa). GlcT partially formed inclusion bodies. However, sufficient amounts of GlcT were detected in the supernatant. Purification was performed as described above and each step was analysed by SDS-PAGE. The mass of the purified protein was determined via ESI-MS (see Fig. 2). The measured mass of 33504 Da confirmed the theoretical mass of 33500 Da with the expected cleavage of the first amino acid, methionine, during expression in E. coli. From about 1 l culture we obtained a yield of about 11 mg purified protein. Interestingly, the purified protein showed a pattern of two bands after analysis on native PAGE (Fig. 1
), suggesting a monomerdimer distribution. On SDS-PAGE the pure protein was present as a single band around 30000 Da with a trace of dimer at 60000 Da.
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Phosphorylation of GlcT by PTS components and its effect on the structure of GlcT
The enzyme I/HPr-dependent phosphorylation of B. subtilis GlcT at residue His104 has been suggested to cause inactivation of the protein (Bachem & Stülke, 1998 ). According to the proposed active dimeric state, phosphorylation was expected to lead to monomerization of GlcT. Phosphorylation of S. carnosus GlcT by the general components of the PTS was analysed by native PAGE and ESI-MS (see below). Two changes could be observed. First, on native gels the pattern changed upon phosphorylation. The band representing the monomeric protein disappeared. Second, the band of the dimer shifted. In general a band shift indicates an additional negative charge through a phosphoryl group. Consequently, this shift suggested a phosphorylation via the general components of the PTS (Fig. 1a
). Surprisingly, in contrast to GlcT of B. subtilis, enzyme I/HPr-dependent phosphorylation of GlcT of S. carnosus clearly facilitated dimerization. The monomerdimer distribution shifted almost quantitatively to the dimeric state. Separation via gel filtration confirmed this observation since two peaks were observed. Retention time of these two fractions suggests masses of 30 kDa (monomer) and 60 kDa (dimer). Phosphorylation caused partial disappearance of the monomer fraction (Fig. 1b
).
According to genetic studies, the formation of the antiterminator/RAT complex should be carbohydrate-specific and dependent on complete enzyme IICBA1 as the sensor for glucose. Therefore, the influence of the glucose-specific IIBA domain on the monomerdimer distribution was studied: neither the phosphorylated form of IIBAGlc1 nor the non-phosphorylated domains altered the oligomeric state of GlcT of S. carnosus. In addition, the interaction of spin-labelled IIBA in various phosphorylation states with GlcT was also investigated via electron paramagnetic resonance (EPR) spectrometry, but no interaction between GlcT and IIBAGlc could be detected. However, these data do rule out that intact glucose-specific enzyme IICBA of S. carnosus is still involved in regulation of GlcT under in vivo conditions.
Determination of the phosphorylation site
For several antiterminator proteins, multiple phosphorylation sites at conserved histidyl residues were observed (Stülke et al., 1998 ). Enzyme I/HPr-dependent phosphorylation of LicT leads to three phosphorylated residues, whereas in SacY two histidyl residues were phosphorylated (Lindner et al., 1999
; Tortosa et al., 1997
).
Since PTS-dependent phosphorylation of GlcT of S. carnosus has not been investigated via ESI-MS yet, the number of phosphoryl groups per molecule GlcT was determined. In previous studies, phosphorylation was visualized by labelling with radioactive phosphate. This technique does not allow detection of the number of phosphates per molecule. A mass increase of 80 Da suggested a single phosphorylated residue (80 Da corresponds to one phosphoryl group; Fig. 2a.) Since four highly conserved histidyl residues exist in the PRDs of antiterminator proteins, one of these four histidines might be the candidate for enzyme I/HPr-dependent phosphorylation. To localize the phosphorylated residue, phosphorylated GlcT was digested with the endoproteinase LysC and the masses of the generated fragments were measured by ESI-MS.
All observed fragments corresponded to the predicted masses except a single peptide with a mass of 1829·2 Da. This mass shows good agreement to the mass of the fragment containing His105 with an additional phosphoryl group (Fig. 2b). Three lines of evidence suggest that His105 is phosphorylated. (i) Acid-lability of the phosphorylated GlcT pointed to a phosphorylated histidine residue (data not shown). (ii) Residue His104 was described as the target for phosphorylation of the closely related GlcT of B. subtilis (Bachem & Stülke, 1998
). (iii) Residue His105 is the only histidine present in the putatively phosphorylated fragment.
Considering the in vivo experiments, phosphorylation studies with HPr of E. coli and B. subtilis were performed. As expected because of the high similarities, GlcT was efficiently phosphorylated by B. subtilis HPr and this phosphorylation led to dimerization. In contrast, HPr from E. coli is not able to phosphorylate GlcT to a detectable extent.
In vivo assays of GlcT of S. carnosus in B. subtilis
As already mentioned, the two ptsG-RAT sequences of B. subtilis and S. carnosus are highly similar (Langbein et al., 1999 ). Since the possible complementation of these two systems was expected, B. subtilis strains were transformed first with plasmids containing the whole glcT gene (pHT304-glcT) and with the sequence encoding the first 60 aa of GlcT (pBQ200-rbd). Two strains (wild-type QB5448 and
glcT mutant GP109) with an insertion in the amyE locus, including the lacZ gene fused with the ptsG-promoter, were used. This fusion allows convenient GlcT activity tests via the expression of ß-galactosidase (Stülke et al., 1997
; Bachem & Stülke, 1998
). As shown in Table 1(a)
, GlcT of S. carnosus recognizes the ptsG-RAT sequence of B. subtilis and causes antitermination, as visualized by ß-galactosidase activity in both strains. GlcT of S. carnosus was active with or without glucose added to the growth medium. The first 60 aa forming the RBD also showed this constitutive antitermination activity (Table 1b
).
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Unexpectedly, phosphorylation by the general components of the PTS is still possible as is dimerization (Fig. 5.)
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DISCUSSION |
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Since His105 is a conserved histidine residue in PRD-I of GlcT, this result seems to be contradictory to the conception of PRD-I as a target of negative regulation of antiterminator proteins (Stülke et al., 1998 ).
We suggest that HPr-dependent phosphorylation of GlcT followed by dimer formation may be a control device to ensure the proper function of the general PTS components enzyme I and HPr. Induction of glucose-specific enzyme II only is reasonable if intact general PTS components are available for enzyme II phosphorylation.
On the other hand HPr is involved in regulation of carbohydrate metabolism in Gram-positive bacteria through ATP-dependent phosphorylation at Ser46 (Kravanja et al., 1999 ). This regulatory phosphorylation of HPr inhibits phosphoenolpyruvate-dependent phosphoryl transfer to HPr and thus would also affect GlcT activity. This might be a control mechanism to inhibit excessive induction of glucose-specific enzyme II.
Dimerization potential of structural domains of S. carnosus GlcT
As shown in B. subtilis glucose metabolism, the N-terminal domain of GlcT interacts as a dimer with the corresponding RAT sequence. This leads to constitutive synthesis of glucose-specific PTS (Bachem & Stülke, 1998 ). Structural studies on the similar RBD of the SacY antiterminator protein suggested a dimer as the RNA binding form (van Tilbeurgh et al., 1997
; Manival et al., 1997
).
To further estimate whether isolated PRDs without the RBD possess dimerization potential, PRD-I and PRD-II were produced by treatment with the endoproteinase LysC. This fragment could still be phosphorylated by the general PTS components, confirming the definition of the functional independence of domains. Moreover, this fragment still dimerizes. The in vivo assays in B. subtilis with the RBD from GlcT of S. carnosus suggested dimerization because of the observed antitermination. These results led to the conclusion that two dimerization motifs exist in a single molecule. One motif is localized in the RBD causing spontaneous dimerization. This process may be responsible for the monomerdimer pattern which was observed for the overexpressed, non-phosphorylated GlcT. The second motif is located in the PRDs, as shown for the E. coli antiterminator protein BglG (Boss et al., 1999 ). Dimerization is under control of the phosphorylation via the general PTS components.
Possible mechanism for glucose-specific antitermination
In the absence of glucose the phosphodimer must be inactivated somehow or be unavailable for antitermination. Upon the addition of glucose as an inducer, the phosphorylated GlcT should become accessible and interact with the RAT sequence. So far we have not been able to detect any effect on the monomerdimer distribution of phosphorylated GlcT by the isolated domains IIBA or IIA of the glucose-specific enzyme IICBA. No additional phosphorylation in stoichiometric amounts of the potential histidyl residues requiring enzymes IIA, IIB or IIAB could be detected in vitro with mass spectrometric methods. In vivo studies with GlcT of B. subtilis showed that a mutant of IIBA (H620A) which could no longer be phosphorylated but which contained an intact IIC domain still showed glucose-inducible antitermination (Bachem & Stülke, 1998 ). Thus an additional direct phosphorylation or dephosphorylation of phosphorylated GlcT by enzyme IIBA alone could not be observed in vitro and may be not involved in glucose induction. However, there is still the possibility that the IIC domain may be required for an additional phosphorylation/dephosphorylation of phosphorylated GlcT.
During in vivo studies with B. subtilis GlcT, S. carnosus GlcT also led to antitermination, as monitored by the lacZ fusion, but induction of antitermination could not be initiated by adding glucose. GlcT of S. carnosus behaved constitutively, similar to the N-terminal fragment of B. subtilis GlcT which is dimeric, as observed for the homologous fragment of the SacY antiterminator but the specific interaction with glucose-specific enzyme II probably mediated by the PRDs is lacking. The studies described above confirm that the antiterminator proteins of the bgl-sac family perform antitermination in the dimeric state. For S. carnosus GlcT phosphorylation of His105 clearly enhances dimer formation. The basal level dimeric enzyme IICBA probably in its phosphoform during starvation must somehow complex the dimeric GlcT which prevents binding to RAT. The binding of glucose to the phosphorylated enzyme IICBA should then release the GlcT dimer now capable of performing antitermination. In the in vivo studies with S. carnosus GlcT, the complexing and releasing procedure inside the B. subtilis cell may not function properly due to imperfect protein interaction between B. subtilis enzyme IICBA and S. carnosus GlcT. An alternative explanation would be an excessive expression of the S. carnosus GlcT protein in B. subtilis. The amount of free unbound GlcT may then also lead to constitutive antitermination.
Preliminary support for this tentative mechanism of antiterminator release may be the observation that membrane fragments of S. carnosus containing induced enzyme IICBA appeared to specifically remove dimeric GlcT from the monomerdimer mixture. Such a mechanism would require a strong conformational change in the enzyme IICBA structure upon binding and phosphorylation of the carbohydrate. Such an effect has been observed with the mannitol-specific enzyme II during micro-calorimetric experiments (Meijberg et al., 1998 ). According to these studies on the mannitol-specific enzyme II (also of the IICBA type) this protein was shown to be active as a dimer (Pas et al., 1988
). In the case of the mannitol-specific enzyme II a very strong, unexpected conformational change of the IIBA domains upon binding of the substrate or its analogue was observed. Such a massive conformational change could lead to the release of the dimeric antiterminator complexed by enzyme IICBA. Further biochemical and genetic experiments will be necessary to support or abandon the proposed glucose-dependent induction mechanism.
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ACKNOWLEDGEMENTS |
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Received 8 March 2000;
revised 24 May 2000;
accepted 23 June 2000.