BglF, the sensor of the E.coli bgl system, uses the same site to phosphorylate both a sugar and a regulatory protein
1997; Springer Nature; Volume: 16; Issue: 15 Linguagem: Inglês
10.1093/emboj/16.15.4617
ISSN1460-2075
Autores Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle1 August 1997free access BglF, the sensor of the E.coli bgl system, uses the same site to phosphorylate both a sugar and a regulatory protein Qing Chen Qing Chen Department of Molecular Biology, The Hebrew University-Hadassah Medical School, POB 12272, Jerusalem, 91120 Israel Search for more papers by this author Jos C. Arents Jos C. Arents E.C. Slater Institute, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, 1018 TV, Amsterdam, The Netherlands Search for more papers by this author Rechien Bader Rechien Bader E.C. Slater Institute, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, 1018 TV, Amsterdam, The Netherlands Search for more papers by this author Pieter W. Postma Pieter W. Postma E.C. Slater Institute, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, 1018 TV, Amsterdam, The Netherlands Search for more papers by this author Orna Amster-Choder Corresponding Author Orna Amster-Choder Department of Molecular Biology, The Hebrew University-Hadassah Medical School, POB 12272, Jerusalem, 91120 Israel Search for more papers by this author Qing Chen Qing Chen Department of Molecular Biology, The Hebrew University-Hadassah Medical School, POB 12272, Jerusalem, 91120 Israel Search for more papers by this author Jos C. Arents Jos C. Arents E.C. Slater Institute, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, 1018 TV, Amsterdam, The Netherlands Search for more papers by this author Rechien Bader Rechien Bader E.C. Slater Institute, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, 1018 TV, Amsterdam, The Netherlands Search for more papers by this author Pieter W. Postma Pieter W. Postma E.C. Slater Institute, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, 1018 TV, Amsterdam, The Netherlands Search for more papers by this author Orna Amster-Choder Corresponding Author Orna Amster-Choder Department of Molecular Biology, The Hebrew University-Hadassah Medical School, POB 12272, Jerusalem, 91120 Israel Search for more papers by this author Author Information Qing Chen1, Jos C. Arents2, Rechien Bader2, Pieter W. Postma2 and Orna Amster-Choder 1 1Department of Molecular Biology, The Hebrew University-Hadassah Medical School, POB 12272, Jerusalem, 91120 Israel 2E.C. Slater Institute, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, 1018 TV, Amsterdam, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:4617-4627https://doi.org/10.1093/emboj/16.15.4617 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Escherichia coli BglF protein is a sugar permease that is a member of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). It catalyses transport and phosphorylation of β-glucosides. In addition to its ability to phosphorylate its sugar substrate, BglF has the unusual ability to phosphorylate and dephosphorylate the transcriptional regulator BglG according to β-glucoside availability. By controlling the phosphorylation state of BglG, BglF controls the dimeric state of BglG and thus its ability to bind RNA and antiterminate transcription of the bgl operon. BglF has two phosphorylation sites. The first site accepts a phosphoryl group from the PTS protein HPr; the phosphoryl group is then transferred to the second phosphorylation site, which can deliver it to the sugar. We provide both in vitro and in vivo evidence that the same phosphorylation site on BglF, the second one, is in charge not only of sugar phosphorylation but also of BglG phosphorylation. Possible mechanisms that ensure correct phosphoryl delivery to the right entity, sugar or protein, depending on environmental conditions, are discussed. Introduction The Escherichia coli BglF protein, which is a member of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), catalyses concomitant transport and phosphorylation of β-glucosides (Fox and Wilson, 1968). In addition to its role in sugar transport, BglF functions as a negative regulator of bgl operon expression (Mahadevan et al., 1987). This property of the enzyme is due to its unprecedented ability to phosphorylate not only its sugar substrate but also a protein essential for operon expression, BglG, a property not yet demonstrated for any other protein (Amster-Choder et al., 1989). BglG is an RNA-binding protein which controls bgl operon expression by transcriptional antitermination (Mahadevan and Wright, 1987; Schnetz and Rak, 1988; Houman et al., 1990). The ability of BglG to override termination of transcription depends on its phosphorylation state; BglF phosphorylates and dephosphorylates it depending on β-glucoside availability (Amster-Choder et al., 1989; Amster-Choder and Wright, 1990; Schnetz and Rak, 1990). It was further shown that the reversible phosphorylation of BglG regulates its activity by modulating its dimeric state (Amster-Choder and Wright, 1992). Thus, in the absence of β-glucosides, BglF phosphorylates BglG; BglG∼P is a monomer that cannot bind to its target RNA site and is inactive as a transcriptional antiterminator. In the presence of β-glucosides, BglF dephosphorylates BglG∼P, allowing it to dimerize and function as a positive regulator of operon expression. The phosphoryl flux in PTS starts with a phosphoryl group donated by phosphoenolpyruvate (PEP) which is passed down a phosphoryl transfer chain which consists of Enzyme I (EI) and HPr, which are not sugar specific, and Enzyme IIs (EIIs), which are specific for different sugars. The EIIs are composed of at least three well-recognized functional domains whose order is not conserved: IIA is a hydrophilic domain that possesses the first phosphorylation site, a conserved histidine which can be phosphorylated by P-HPr; IIB is a second hydrophilic domain that possesses the second phosphorylation site, usually a conserved cysteine that can be phosphorylated by P-IIA; and IIC is a hydrophobic domain which includes 6-8 transmembrane helices that presumably form the sugar translocation channel and at least part of its binding site (reviewed in Saier and Reizer, 1992; Postma et al., 1993). BglF is the PTS EII for β-glucosides and is also designated IIbgl. Based on sequence comparisons with other PTS EIIs, several conserved residues in BglF were suggested to play a role in the transfer of the phosphoryl group from HPr to BglF and from BglF to the sugar. These residues were subjected to site-directed mutagenesis and the phosphorylation performance of the respective mutants was studied (Schnetz et al., 1990). His547 (H547), located in the IIA domain, was identified as the first phosphorylation site (site 1), which is phosphorylated by HPr and transfers the phosphoryl group to the second phosphorylation site (site 2). Two residues were candidates for the second phosphorylation site, Cys24 (C24) and His306 (H306), both shown to be essential for the transfer of the phosphoryl group to the sugar. Based on the vast amount of information available on phosphorylation sites in related permeases (Postma et al., 1993, and references therein), it was logical to assume that C24, which is located in the IIB domain, and not H306, located in the IIC domain, is the residue which accepts the phosphoryl group from H547 and transfers it on to the sugar, while H306, located in the IIC domain, is involved in transporting the sugar. In vitro phosphorylation experiments with distinct domains have shown unambiguously that the second phosphorylation site resides in the IIB domain of BglF and not in the IIC domain (Q.Chen and O.Amster-Choder, unpublished data). Which site on BglF phosphorylates the transcriptional regulator BglG? The basis for the ability of a protein to phosphorylate such different entities as a carbohydrate and a protein is unknown. Knowledge of whether a single phosphorylation site performs both transfer reactions or whether two different sites are involved, one for each reaction, is crucial for elucidating the relationship between recognition and phosphorylation. It was suggested previously that each of the two phosphorylation sites on BglF is in charge of a different phosphorylation function (Schnetz and Rak 1990), i.e. the site on IIAbgl phosphorylates BglG and the site on IIBbgl phosphorylates the sugar. These authors also suggested that IIAglc, which is homologous to the IIAbgl domain (Bramley and Kornberg, 1987) and was shown to complement BglF mutated in site 1 (Schnetz et al., 1990), can transfer phosphoryl groups not only to site 2 of BglF but also to BglG. However, the observation that no [32P]BglG was detected when non-phosphorylated BglG was incubated with [32P]PEP and a soluble fraction of a Salmonella typhimurium strain overproducing EI, HPr and IIAglc (Amster-Choder et al., 1989) did not support transfer from IIAglc to BglG. Here we provide both in vivo and in vitro evidence that the site on BglF which transfers a phosphoryl group to β-glucosides, site 2, is the same one that is used for transfer of a phosphoryl group to BglG. Thus, the phosphoryl group is transferred from site 1 to site 2 and then to either the sugar or to BglG. Therefore, not only is BglF unique in its ability to phosphorylate both a sugar and a regulatory protein, but, more interestingly, the phosphoryl group is donated to these totally different entities by the same site. Possible mechanisms that ensure correct phosphoryl delivery to the right entity, depending on environmental conditions, are discussed. Results To test which site(s) on BglF are involved in transfer of a phosphoryl group to β-glucosides and BglG, we mutated each of the two phosphorylation sites on BglF. His547 was mutated to an arginine (H547R), and Cys24 was mutated to a serine (C24S) (see Materials and methods). We then followed the ability of the mutant proteins to be phosphorylated and to donate the phosphoryl group to β-glucosides and to BglG in vitro on one hand, and to mediate β-glucoside utilization and to modulate BglG activity in vivo on the other hand. Phosphorylation of wild-type and mutant BglF proteins Membranes containing wild-type BglF, or BglF mutated in either one of its phosphorylation sites (C24S or H547R), were incubated in the in vitro phosphorylation system described previously (Amster-Choder et al., 1989). The system, which will be referred to as system A, is crude and contains [32P]PEP, a cytoplasmic extract prepared from the mutant strain of S.typhimurium LJ144 which expresses increased amounts of EI, HPr and IIAglc (Saier and Feucht, 1975), and membranes prepared from E.coli K38 cells expressing the bglF alleles under the control of phage T7 promoter. All three BglF derivatives were detected by autoradiography following SDS-PAGE (Figure 1A, lanes 1-3). This polypeptide could not be detected when membranes of cells containing a similar plasmid which lacks the bglF gene were included in this in vitro system (Figure 1A, lane 4). Figure 1.Phosphorylation of BglF mutated in either one of its phosphorylation sites. (A) Membranes of cells that overproduce the various BglF derivatives were incubated with [32P]PEP and a soluble protein extract prepared from the Salmonella typhimurium LJ144, which is enriched for EI, HPr and IIAglc for 10 min (phosphorylation system A). (B) The various BglF derivatives were overproduced in LM1, a crr and nagE E.coli strain. Membranes were incubated with [32P]PEP and purified EI and HPr (phosphorylation system B) for 10 min without (lane 1-4) or with (lanes 5-8) IIAglc. H547R and C24S: mutations in the first and second phosphorylation sites of BglF (‘site 1’ and ‘site 2’) respectively. No BglF: membranes from cells which do not overproduce BglF, but are otherwise identical to the other membrane preparations used in each experiment, were included in the phosphorylation systems described above. Samples were analysed by SDS-PAGE followed by autoradiography. Molecular masses of protein standards are given in kilodaltons. Arrowheads indicate the positions of BglF, EI and IIAglc. Download figure Download PowerPoint Subsequently, we have expressed the three bglF alleles (wild-type and the two mutants) in E.coli LM1, a strain deleted for the crr and nagE genes (and thus not expressing the IIAglc and IInag proteins which can substitute for IIAbgl). The overproduction of the three BglF derivatives in this strain was demonstrated by metabolic labelling with [35S]methionine (data not shown). Membranes prepared from LM1 producing the different BglF derivatives were incubated with [32P]PEP and purified EI and HPr (referred to as system B). Phosphorylated proteins were detected by autoradiography following SDS-PAGE, and the results, presented in Figure 1B (lanes 1-3), demonstrate that the site 2 mutant (C24S) behaved like wild-type BglF, while a mutation in site 1 (H547R) abolished the ability of BglF to be phosphorylated by HPr. The control reaction contained membranes of LM1 bearing a similar plasmid lacking the bglF gene (Figure 1B, lane 4). Addition of purified IIAglc restored site 1 mutant phosphorylation (compare lanes 3 and 7 in Figure 1B) while it did not affect phosphorylation of wild-type BglF and the site 2 mutant (Figure 1B, lanes 5 and 6 versus lanes 1 and 2). Thus BglF mutants are behaving as expected in the two in vitro systems utilized by us; BglF mutated in site 1 cannot accept a phosphoryl group from HPr, but this mutation can be complemented by IIAglc, while a mutation in site 2 does not interfere with BglF phosphorylation by HPr. This is in agreement with the previously suggested heterologous phosphoryl transfer from IIAglc to site 2 of BglF (Schnetz et al., 1990). Dephosphorylation of wild-type and mutant BglF proteins by β-glucosides All of the published evidence to date suggests that the second phosphorylation site on BglF is the one involved in transferring the phosphoryl group to the sugar substrate. We have tested the ability of our mutant BglF proteins, pre-labelled by incubation with [32P]PEP, and purified EI and HPr to donate a phosphoryl group to the β-glucoside salicin. As seen in Figure 2, the site 1 mutant protein (H547R), once phosphorylated by IIAglc, behaves like wild-type BglF and is completely dephosphorylated upon addition of salicin (compare lane 3 with lane 4 and lane 5 with lane 6, for the wild-type and site 1 mutant, respectively). The phosphorylated site 2 mutant protein (C24S), on the other hand, is not chased by salicin (Figure 2, lanes 7 and 8). Figure 2.BglF mutated in site 1, but not in site 2, is dephosphorylated by β-glucosides. The various BglF derivatives (wild-type, H547R and C24S) were overproduced in ZSC112ΔG, a pstG strain. Membranes were incubated with [32P]PEP, purified EI, HPr and IIAglc. The mixtures were incubated further with (+) or without (−) 0.2% salicin for 5 min. Samples were analysed by SDS-PAGE followed by autoradiography. Molecular masses of protein standards are given in kilodaltons. Arrowheads indicate the position of BglF, EI and IIAglc. Download figure Download PowerPoint IICBglc, which is present in our membrane preparations in a significant amount (as demonstrated by Western blot analysis using monoclonal antibodies raised against this protein, data not shown), was reported to be phosphorylated by the IIA domain of BglF (Vogler et al., 1988; Schnetz et al., 1990). It can therefore lead to some dephosphorylation of BglF, which is independent of β-glucosides, due to the the presence of residual glucose contamination, detected occasionally in commercial salicin. To avoid this complication, the membranes containing the various BglF derivatives were prepared from strain ZSC112ΔG, which is mutated in the ptsG gene encoding IICBglc. This strain expresses the crr gene at a relatively lower level (P.W.Postma, unpublished data). Therefore, to ensure phosphorylation of site 2 of the H547R mutant, IIAglc was included in the phosphorylation reaction. Our conclusion from the results presented in this section is that our mutants behave as expected with regard to sugar phosphorylation (i.e. only the one that contains an intact second phosphorylation site can transfer the phosphoryl group to the sugar) and should thus serve as a reliable tool to study phosphorylation reactions catalysed by BglF. Phosphorylation of BglG by wild-type and mutant BglF proteins We have shown before that BglF, phosphorylated in vitro, can transfer a phosphoryl group to BglG (Amster-Choder et al., 1989). The physiological significance of this result was demonstrated by the correlation between the behaviour of BglG mutants in vivo and their phosphorylation behaviour in vitro. BglF-dependent phosphorylation of BglG was also demonstrated in vivo (Amster-Choder and Wright, 1990). Thus the ability of BglG to be phosphorylated in vitro is an excellent indication for the in vivo situation. We have tested the effect of the mutations in the phosphorylation sites of BglF on its ability to phosphorylate BglG. An extract of cells overproducing BglG (see Materials and methods) was added to mixtures containing the different BglF variants that had been pre-labelled for 10 min in system A. As was shown in Figure 1A, all three BglF variants examined (wild-type and mutants) are phosphorylated in this system (the site 1 mutant is labelled due to the presence of IIAglc in this phosphorylation system, see above) and we could thus test their ability to transfer the phosphoryl group to BglG. The results, presented in Figure 3, demonstrate that a mutation in the first phosphorylation site of BglF does not prevent this protein from phosphorylating BglG. Some phosphorylation of BglG was detected 1 min after the addition of the BglG-containing extract, and the amount of phosphorylated BglG increased with time of incubation (Figure 3, lanes 9-12). The slight difference from the phosphorylation pattern of BglG by wild-type BglF (Figure 3, lanes 1-4) can be explained by the fact that H547R is labelled by IIAglc and the phosphoryl flow is expected to be less efficient in this heterologous system than in the wild-type situation which involves intramolecular transfer of the phosphoryl group. In contrast to the behaviour of the site 1 mutant, no phosphorylation of BglG occurred with BglF mutated in site 2, even after incubating the labelled C24S with the BglG-containing extract for 15 min (Figure 3, lanes 5-8). Longer periods of incubation gave the same result (data not shown). Figure 3.BglF mutated in site 1, but not in site 2, phosphorylates BglG. Membranes containing the various BglF derivatives (wild-type, C24S and H547R) were labelled in phosphorylation system A, as described in Figure 1A. Extract of cells that overproduce BglG was added, and incubation was continued for the times indicated. Samples were analysed by SDS-PAGE followed by autoradiography. Lane 13 contains a 35S-labelled sample of BglG. Molecular masses of protein standards are given in kilodaltons. Arrowheads indicate the positions of BglF and BglG. Download figure Download PowerPoint To assay for BglG phosphorylation by BglF in a purified system, and in light of the difficulty in purifying BglG due to its irreversible precipitation in inclusion bodies upon overproduction (A.Wright, unpublished data), we decided to measure phosphorylation of the fusion protein MBP-BglG (BglG fused to maltose-binding protein) which is soluble and can be purified on an amylose column (see Materials and methods). We first demonstrated that this fusion protein can be phosphorylated by wild-type BglF in vitro (Figure 4A, lane 2). To ensure that it is the BglG, and not the MBP moiety, that is phosphorylated by BglF, we incubated purified MBP with pre-labelled BglF and demonstrated that MBP, though present in the reaction in an amount which is equimolar to that of MBP-BglG (see Figure 4B for Western blot analysis), is not phosphorylated by BglF (Figure 4A, lane 3). We subsequently added purified MBP-BglG to the BglF variants that had been pre-labelled in system B. The results, presented in Figure 5, demonstrate that MBP-BglG can be phosphorylated by the site 1 mutant which was labelled in a reaction supplemented with IIAglc (lane 7), but not by the site 2 mutant (lanes 3 and 6). No phosphorylation of MBP-BglG could be detected when it was added to membranes of cells that do not produce BglF, which were pre-labelled in phosphorylation system B (Figure 5, lane 1). Thus, phosphorylated EI and HPr cannot phosphorylate BglG. Figure 4.BglF recognizes and phosphorylates BglG fused to MBP. BglF was labelled in phosphorylation system B (lanes 1), then further incubated for 15 min in the presence of either MBP-BglG (lanes 2) or MBP (lanes 3). Proteins were fractionated on a 5-12.5% SDS-polyacrylamide gradient gel and then blotted onto a nitrocellulose filter. The blot was probed with anti-MBP antibodies and analysed by autoradiography. (A) Autoradiography. (B) Western blot analysis. Molecular masses of protein standards are given in kilodaltons. Arrowheads indicate the positions of BglF, MBP-BglG and MBP. EI co-migrates with BglF in this gel system (see Figure 5). Download figure Download PowerPoint Figure 5.BglF mutated in site 1, but not in site 2, phosphorylates MBP-BglG. The various BglF derivatives (wild-type, C24S and H547R) were labelled in phosphorylation system B in the absence (lanes 2-4) or presence (lanes 5-7) of IIAglc. The mixtures were incubated further in the presence of MBP-BglG for 15 min. Proteins were fractionated on a 5-12.5% SDS-polyacrylamide gradient gel followed by autoradiography. Lane 1 contains a control with membranes from cells that do not overproduce BglF that were labelled in phosphorylation system B; it demonstrates that phosphorylated EI co-migrates with BglF in this gel system. Molecular masses of protein standards are given in kilodaltons. Arrowheads indicate the positions of MBP-BglG, BglF, EI and IIAglc. Download figure Download PowerPoint Taken together, these results show conclusively that the second phosphorylation site in BglF (C24), and not the first, is in charge of delivering the phosphoryl group to BglG. These results also rule out the possibility raised before (Schnetz and Rak, 1990) that phosphorylated IIAglc can deliver the phosphoryl group to BglG (see Figure 5, lane 6). β-glucoside phosphotransfer mediated by wild-type and mutant BglF proteins Next we decided to substantiate our in vitro results regarding BglF-dependent BglG phosphorylation by in vivo studies. We first verified that our mutants behave as expected with regard to β-glucoside utilization. To analyse the ability of the various BglF derivatives to transfer β-glucosides into the cell while phosphorylating them, we used strains defective in the bglF gene, and carried out complementation analyses with a series of plasmids encoding BglF derivatives: pMN5 and pCQ-F encode wild-type BglF; pCQ-F1 and pCQ-F2 encode BglF mutated in the first and second phosphorylation sites (H547R and C24S), respectively. Positive complementation of the chromosomal mutation in the bglF gene by the plasmid-encoded alleles was indicated both by growth on minimal medium containing arbutin as the sole carbon source and by the formation of red colonies on MacConkey arbutin plates. Utilization of the β-glucoside arbutin depends on the ability of the plasmid-encoded BglF derivatives to phosphorylate and transport this sugar which is then cleaved by the product of the unlinked locus bglA. Utilization of the β-glucoside salicin is prohibited in these strains due to the polarity of the mutation in the chromosomal bglF gene on the adjacent bglB gene, whose product preferentially cleaves phosphosalicin (Mahadevan et al., 1987). We used several bglF strains which are wild-type for crr and nagE (Mahadevan et al., 1987), and also isogenic strains defective in the crr and nagE genes which we have constructed (see Materials and methods). The results are presented in Table I. While the control wild-type bglF plasmids (pMN5 and pCQ-F) complemented all the bglF strains to Arb+ (growth on minimal arbutin and red colonies on MacConkey arbutin), a mutation in site 2 abolished the ability of the plasmid-encoded BglF to complement any of these strains (no growth on minimal arbutin and white colonies on MacConkey arbutin in all strains containing pCQ-F2). The site 1 mutant (encoded by pCQ-F1) showed no complementation in bglF strains defective in the crr gene. However, bglF strains carrying the wild-type crr gene were complemented by the site 1 mutant and grew on minimal arbutin. They also led to the formation of red colonies on MacConkey arbutin, though paler in some cases than the same strains containing a plasmid which encodes wild-type BglF. Table 1. Plasmid-encoded BglF mutated in site 1, but not in site 2, can complement bglF strains and enable β-glucoside utilization Plasmid Plasmid-encoded BglF derivative Complementation of bglF mutant strainsa MA231 AE304-1 AE304-2 AE304-4 PPA543 (IIAglc−, IInag−)b pBR322 − − − − − − pMN5 wild-type + + + + + pCQ-F wild-type + + + + + pCQ-F1 H547R + +c +c +c − pCQ-F2 C24S − − − − − a Complementation was indicated by two alternative methods: (+) growth on minimal arbutin plates and red colonies on MacConkey arbutin plates; (−) no growth on minimal arbutin plates and white colonies on MacConkey arbutin plates. Complementation of strain MA231 was assayed only on MacConkey arbutin plates. b The crr and nag genes of strain PPA543 were mutated (see Materials and methods). This strain is thus deficient for the IIAglc and IInag proteins. Strains PPA546 and PPA547 that also carry mutations in these genes behaved as PPA543 (not shown). c The colour on MacConkey arbutin plates was pale red but the number of colonies on minimal arbutin plates was the same as for other plasmids. Thus, β-glucoside utilization can be restored in bglF strains by a plasmid-encoded BglF mutated in the first phosphorylation site, provided that the strain produces IIAglc. The slight difference between the effect of the wild-type BglF and the site 1 mutant, observed with some strains (all originating from the same parental strain) in one of the complementation tests, i.e. colour on MacConkey arbutin, can be explained by the more efficient phosphoryl transfer from site 1 to site 2 when both sites are present on the same molecule than in the heterologous system (which necessitates phosphoryl flow from IIAglc to site 2 of BglF). The other test, growth on minimal arbutin, is not sensitive to this difference. Also, strain MA231, which gives bright red colonies on MacConkey arbutin when transformed with pCQ-F1, might have a slighty higher level of IIAglc which compensates for the intramolecular phosphoryl transfer. Based on the results presented in this section, it can be concluded that our mutants behave as expected with regard to phosphotransfer of β-glucosides into the bacterial cell. The effect of wild-type and mutant BglF proteins on BglG activity as a transcriptional antiterminator BglF was shown before to exert its negative effect on operon expression by phosphorylating BglG, blocking its action as an antiterminator (Amster-Choder et al., 1989). To establish which phosphorylation site on BglF is responsible for BglG negative regulation by phosphorylation, we tested the effect of the mutations in the two phosphorylation sites of BglF on the protein's ability to negatively regulate BglG. To address this question, we made use of strain MA200-1, whose chromosome carries a bgl′-lacZ fusion (a fusion of the bgl promoter and transcription terminator to lacZ) and a mutation in the bglF gene (Mahadevan et al., 1987). Due to the mutation in the chromosomal bglF gene, BglG is not negatively regulated in this strain and therefore enables constitutive expression of the lacZ gene. Expression of plasmid-encoded wild-type BglF protein in MA200-1 renders lacZ expression inducible, i.e. β-galactosidase is produced only upon addition of β-glucosides to the growth medium. The β-galactosidase levels measured in MA200-1-containing plasmids which encode the various BglF derivatives, pCQ-F1 and pCQ-F2, in the absence and presence of β-glucosides, are given in Table II. BglF mutated in site 1 (H547R) behaved like wild-type BglF, allowing lacZ expression only upon addition of β-glucosides (two types of β-glucosides were used in this assay, salicin or β-methyl glucoside). Mutation in site 2 (C24S) abolished the ability of BglF to negatively regulate BglG and could not prevent constitutive expression of lacZ. Table 2. BglF mutated in site 1, but not in site 2, negatively regulates BglG transcription antitermination activity Straina Plasmid Plasmid-encoded BglF derivative β-galactosidase activity (U) βMGb Salicinc − + − + MA200-1 pMN5 Wild-type 6 50 6 128 pCQ-F1 H547R 6 43 9 133 pCQ-F2 C24S 30 36 30 59 PPA546 (crr, nagE) pMN5 wild-type 5 100 3 324 pCQ-F1 H547R 34 80 82 148 pCQ-F2 C24S 68 152 73 133 PPA547 (crr, nagE) pMN5 wild-type 4 82 5 283 pCQ-F1 H547R 60 172 179 340 pCQ-F2 C24S 85 180 70 138 a MA200-1 is Bgl+ and it carries a bgl-lacZ transcriptional fusion. PPA546 and PPA547 are derivatives of MA200-1 but their crr and nagE genes were mutated. b 10 mM β-methylglucoside (βMG) were added to the growth medium when indicated. c
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