Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Polígono de la Coma s/n, Apartado de correos (PO Box) 73, 46100-Burjassot, Valencia, Spain1
Author for correspondence: Gaspar Pérez-Martínez. Tel: +34 96 3900022. Fax: +34 96 3636301. e-mail: gaspar.perez{at}iata.csic.es
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ABSTRACT |
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Keywords: L. casei, lactose induction, carbon catabolite repression, mutagenesis, phosphorylation
Abbreviations: CCR, carbon catabolite repression; P-ß-gal; phospho-ß-galactosidase; PRD, PTS regulation domain; PTS, phosphoenol-pyruvate-dependent phosphotransferase system; RAT, ribonucleic antiterminator
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INTRODUCTION |
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Antitermination activity has been extensively studied in homologous proteins, such as BglG of Escherichia coli (Mahadevan & Wright, 1987 ; Mahadevan et al., 1987
; Schnetz et al., 1987
; Schnetz & Rak, 1988
, 1990
; Amster-Choder et al., 1989
; Houman et al., 1990
; Amster-Choder & Wright, 1993
; Görke & Rak, 1999
) and SacT, SacY, LicT and GlcT of Bacillus subtilis (Zukowski et al., 1990
; Crutz et al., 1990
; Débarbouillé et al., 1990
; Arnaud et al., 1992
, 1996
; Crutz & Steinmetz, 1992
; Le Coq et al., 1995
; Krüger & Hecker, 1995
; Schnetz et al., 1996
; Rutberg, 1997
; Stülke et al., 1997
; Tortosa et al., 1997
; Bachem & Stülke, 1998
; Idelson & Amster-Choder, 1998
). In lactic acid bacteria, only two other antiterminator proteins have been reported: BglR in Lactococcus lactis and, recently, BglG in Lactobacillus plantarum (Bardowski et al., 1994
; Marasco et al., 2000
). Antitermination activity requires binding of these proteins to a ribonucleic antiterminator (RAT) sequence in the mRNA, stabilizing a stemloop structure that prevents the formation of the transcriptional terminator located in the leader fragment preceding the coding regions (Aymerich & Steinmetz, 1992
). The antiterminators show a modular organization, with an RNA-binding region and two conserved PTS regulation domains (PRD-I and PRD-II), which, as demonstrated for BglG, LicT, SacT and SacY, contain phosphorylatable histidyl residues (two in each domain) that render them susceptible to regulation by PTS elements (Stülke et al., 1998
; Tortosa et al., 1997
). It has been proposed that PRD-I is negatively regulated by the sugar-specific PTS elements, EII, and PRD-II could be subject to a positive control by the general PTS protein, HPr (Stülke et al., 1998
). Biochemical and genetic studies allowed the phosphorylation sites in different regulators to be identified. However, despite the fact that they are well conserved, they have different regulatory roles. The antiterminators SacT, GlcT and LicT from Bacillus subtilis are negatively controlled by EII-dependent phosphorylation of one histidyl residue in PRD-I (His-97, His-104 and His-100, respectively) (Arnaud et al., 1992
, 1996
; Le Coq et al., 1995
; Bachem & Stülke, 1998
). However, in the PRD-containing activators LicR and LevR of B. subtilis, and MtlR of Bacillus stearothermophilus, the phosphorylation sites involved in this regulatory mechanism are located in the EIIA-like domain and PRD-II (Martin-Verstraete et al., 1998
; Tobisch et al., 1999
; Henstra et al., 2000
). In addition, some of these regulators (SacT, LicT and LevR) are activated by HPr-dependent phosphorylation at different sites (Martin-Verstraete et al., 1995
, 1998
; Arnaud et al., 1996
; Lindner et al., 1999
). In E. coli, the antiterminator BglG regulates the expression of the bgl operon involved in ß-glucoside transport. EIIBgl transports and phosphorylates ß-glucosides but it also modulates BglG activity by phosphorylation. The His-208 of this antiterminator, located in PRD-II, was identified as the phosphorylation site (Chen et al., 1997
, 2000
). On the other hand, it has been recently been shown that HPr also phosphorylates BglG, but the presumptive sites remain unidentified (Görke & Rak, 1999
).
The aim of this work was to investigate the role of the conserved histidyl residues (His-101, His-159, His-210 and His-273) of LacT in the mechanisms that control antiterminator activity in Lactobacillus casei. For this purpose, the potential phosphorylation sites of LacT were replaced by alanine or aspartate by site-directed mutagenesis. The effect of these mutations on the expression of the lac operon was evaluated by determining P-ß-gal activity from cultures grown under different induction/repression conditions.
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METHODS |
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Construction of a lacT mutant.
Deletion of lacT was obtained by Campbell-like recombination using the integrative plasmid pRV300 (Leloup et al., 1997 ). Two fragments of 500 and 543 bp spanning the regions upstream and downstream of lacT, respectively, were obtained by PCR using total DNA of L. casei as template. The primers used were lac50 (5'-GAGCGGTACCATCAGTAGA-3') and lac51 (5'-GTTGTCATCCCCTCCCAG-3') for one fragment and lac52 (5'-CGCGCTCGCAGTCGTAGA-3') and lac25 (5'-CGATATGAGCTCAGATC-3') for the other fragment. The two PCR products were ligated and the ligation mix used as template in a PCR with lac50 and lac25. These two oligonucleotides carried KpnI and SacI sites (underlined), respectively, allowing the cloning of the last PCR product into KpnI/SacI-digested pRV300, giving pRV
lacT. This plasmid was used to transform L. casei. A Lac+ and Er+ transformant was grown for 200 generations without antibiotic in order to allow the second recombination event, which would excise the vector, giving Lac- Er- colonies. The correct first and second recombination events were confirmed by PCR. One of the lacT mutants was selected and the strain named BL195. In this strain the lacT gene was totally deleted, while keeping the lac promoter, including all the initiation signals and regulatory sites unaltered upstream of the remaining genes of the lac operon.
Plasmid constructions and site-directed mutagenesis.
To obtain the plasmid pLacT, lacT was amplified with primers lac53 (5'-GCACTGGGAGGGGATGACAA-3') and lac54 (5'-TTGGAAGCTTGCTTCAAAGCC-3') using L. casei DNA as template, and the PCR product was cloned into the SmaI site of pUC18 using a Sureclone kit (Amersham Pharmacia Biotech). The lac54 primer had two substitutions (boldface) to introduce a new HindIII site (underlined). pLacT was used as template in the site-directed mutagenesis to replace the triplets encoding histidyl residues of LacT by alanine or aspartate (H101A, H101D, H159A, H210A, H210D and H273A). These amino acids (Ala and Asp) mimic non-phosphorylated and phosphorylated forms of the residue. The overlap extension method applied allows introduction of site-specific mutations and generation of a mutant gene in two PCR steps (Ho et al., 1989 ). In the first PCR step, two fragments of lacT were independently amplified using outside primers (lac53 or lac54) and the appropiate oligonucleotide of the following mutagenic primers (lac59 5'-TGAGTGATGCTATTTACGA and lac60 5'-TCGTAAATAGCATCACTCA, lac61 5'-TGAGTGATGATATTTACGA and lac62 5'-TCGTAAATATCATCACTCA, lac63 5'-TGGCGTTGGCCTTTATCAA and lac64 5'-TTGATAAAGGCCAACGCCA, lac65 5'-TTATGATTGCTCTCCAGTA and lac66 5'-TACTGGAGAGCAATCATAA, lac67 5'-TTATGATTGATCTCCAGTA and lac68 5'-TACTGGAGATCAATCATAA, lac69 5'-TCATCATCGCCATTCAGCG and lac70 5'-CGCTGAATGGCGATGATGA) (Fig. 1
). The mutated triplets are in boldface. These pairs of primers are complementary, hence the strands of the fragments obtained overlapped and they could be extended in a subsequent reaction using lac53 and lac54 primers. The products of these second PCRs were cloned into pUC18/SmaI. The resulting plasmids were named pLacH101A, pLacH101D, pLacH159A, pLacH210A and pLacH210D (Fig. 1
). To replace His-273 by an Ala residue, lac2 (5'-CAACGATATAAGCGCAGATC) was used as external primer. The strategy was followed as described above and the plasmid obtained was designated pLacH273A (Fig. 1
). The orientation of the inserts in pUC18 vector was checked by digestion with the endonuclease HindIII. The fragments were subsequently sequenced to exclude PCR artifacts using a Perkin-Elmer Abi Prism 310 automated sequencer. Plasmids carrying wild-type and mutated genes were digested with BamHI and HindIII and the resulting fragment cloned into BamHI/HindIII-digested pGAL9 (Pérez-Martínez et al., 1992
), generating pGALT, pGALTH101A, pGALTH101D, pGALTH159A, pGALTH210A, pGALTH210D and pGALTH273A. In these pGAL9-derived plasmids lacT variants are under control of the constitutive SPO2/AL9 promoter. These plasmids were used to transform L. casei CECT5275, the
lacT strain (BL195) and the ccpA mutant (C. D. Esteban, unpublished data) (Fig. 1
). The L. casei CECT5275(pGAL9) transformant was named PL20.
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RESULTS |
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The involvement of His-101 and His-159 in the induction mechanism of the lac operon was studied in vivo by determining P-ß-gal activity in BL195(pGALT), BL195(pGALTH101A), BL195(pGALTH101D), BL195(pGALTH159A) and wild-type strains, when grown on ribose (non-inducing sugar), lactose (inducer) and glucose plus lactose (CCR) (Table 2). In BL195(pGALT), the expression of lac operon was restored in lactose-grown cells, although P-ß-gal activity was lower than that found in the wild-type strain (PL20). Both strains, BL195(pGALT) and PL20, showed the same pattern of P-ß-gal expression: induction by lactose and repression by glucose. This suggested that PRD-I from LacT could be dephosphorylated by EIILac even when it is overexpressed. In the absence of lactose (ribose-grown cells), transformants with Ala replacements [BL195(pGALTH101A) and BL195(pGALTH159A)] showed about half the activity detected on lactose, suggesting an additive effect of these mutations. Alternatively, if each of the Ala replacements in PRD-I could render a fully active LacT, the structural instability provoked by these mutations may be the cause for the reduced activity detected. In contrast, no, or negligible, activity was detected in BL195(pGALTH101D) grown on any of the three sugars used (Table 2
), indicating that the phosphorylation of this residue may inactivate LacT, which would complement the results obtained with the Ala replacements.
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To study the involvement of PRD-II (His-210 and His-273) in CCR, a ccpA mutant (BL190) was transformed with pGAL9, pGALT, pGALTH210A, pGALTH210D and pGALTH273A. All the transformants showed P-ß-gal activity in lactose-grown cells due to the normal expression of the chromosomal lac operon in the host strain BL190. However, any effect on the expression of the lac operon in BL190 on glucose-plus-lactose medium would be exclusively due to LacT-mediated CCR. Expression of LacT from a multicopy plasmid (pGALT) in the ccpA mutant conferred glucose insensitivity, possibly due to dephosphorylation of PRD-II when the inducer was present and in conditions of LacT molar excess. Expression of the mutation H210D in BL190 resulted in the activation of the operon in the presence of glucose and the inducer, lactose, indicating that this mutation was partially active and insensitive to glucose repression in the ccpA mutant. However, Ala replacements did not give an active form of antiterminator protein, since the repressing effect of glucose on P-ß-gal activity remained in BL190(pGALH210A) and BL190(pGALH273A) (Table 2
).
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DISCUSSION |
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BL195 (lacT) had a complete deletion of lacT, and therefore it had a Lac- phenotype. The operator, RAT sequence and terminator structure remained functional, as demonstrated by the fact that constitutive expression of LacT from pGALT partially restored growth on lactose and gave the same expression pattern as the wild-type (glucose repression and induction by lactose). However, in other works reporting overproduction of antiterminators, such as LicT, substrate induction was eliminated (Krüger & Hecker, 1995
; Krüger et al., 1996
).
Antiterminator activity of all members of the BglG family of antiterminators has been shown to be subject to substrate induction through phosphate exchange with the sugar-specific EII PTS elements (Stülke et al., 1998 ). In L. casei, transcriptional studies of the lac operon in lacE and lacF mutants showed an inducer-independent antiterminator activity, indicating a negative control of these elements (Gosalbes et al., 1999
). According to the model of PTS-mediated regulation of antiterminators (Stülke et al., 1998
), EIILac would dephosphorylate LacT PRD-I in the presence of lactose to yield its active form. EII-dependent negative control has been demonstrated to involve at least one of two conserved histidines of PRD-I in LicT, SacY, SacT and GlcT (His-101, His-99, His-97 and His-104, respectively) (Schnetz et al., 1987
; Débarbouillé et al., 1990
; Arnaud et al., 1992
, 1996
; Stülke et al., 1997
; Bachem & Stülke, 1998
). PRD-I alignment of different antiterminators located highly conserved histidines at positions His-101 and His-159 in LacT. In this work, the replacement of His-101 or His-159 by Ala rendered LacT active in the absence of lactose (ribose-grown cells), although the P-ß-gal activity observed in these strains [BL195(pGALTH101A) and BL195(pGALTH159A)] was lower than that detected on lactose-grown cells. It could be proposed that both residues were involved in the lactose-induction process and one or both of them should be dephosphorylated to obtain full LacT activity. These replacements of His-101 and His-159 by Ala yielded the same phenotype of thermosensitive LacT, suggesting that these histidyl residues may interact and be playing a role in the conformational stability of PRD-I in LacT, as has recently been proposed from the resolved structure of PRDs in LicT (van Tilbeurgh et al., 2001
).
LacT activity has also been shown to be sensitive to the presence of glucose, which caused the already reported CcpA-independent CCR of the L. casei lac operon (Monedero et al., 1997 ; Gosalbes et al., 1999
; Viana et al., 2000
). In homologous antiterminators, this event apparently required phosphorylation of PRD-II by HPr-His-P, although this effect varied slightly among them. Whereas SacT and LicT are subject to a positive control by HPr-dependent phosphorylation, SacY and GlcT activities do not require it. LicT can be phosphorylated by HPr at three sites, one in PRD-I (His-159) and the other two in PRD-II (His-207 and His-269). Furthermore, His-207 is preferentially phosphorylated, indicating a fine modulation of LicT activity (Lindner et al., 1999
). In contrast, HPr-dependent phosphorylation at the equivalent sites (His-207 and His-269) in SacY does not affect its activity (Tortosa et al., 1997
). The total loss of activity in BL195(pGALTH210A) and BL195(pGALTH273A) when grown on lactose suggested that both His residues (His-210 and His-273) should be phosphorylated to obtain the active form of LacT. All the mutants and the wild-type showed a remarkable glucose repression effect, as expression of the lac operon is also repressed by CcpA-dependent CCR in this genetic background (
lacT). Thus, we studied the involvement of His-210 and His-273 in LacT-mediated CCR using a ccpA mutant. Strikingly, a derepressed phenotype was observed in BL190(pGALT), possibly due to the overexpression of LacT from a constitutive promoter in a high-copy-number plasmid. Also, a likely expression of the lac operon was found when the mutant protein H210D was expressed in a ccpA background, although the activity was lower than expected if the operon in this mutant was fully activated or insensitive to the glucose effect, as observed in LicT (van Tilbeurgh et al., 2001
). Although this mutation was not temperature sensitive, it may introduce conformational changes or difficulties in dimer formation, which is apparently required for activity in E. coli BglG (Amster-Choder & Wright, 1992
, 1993
; Boss et al., 1999
). In E. coli it has recently been proposed that HPr-dependent phosphorylation of PRD-II is necessary for formation of the dimer (active) form of BglG (Görke & Rak, 1999
), and a similar model was initially suggested for LicT (Krüger & Hecker, 1995
; Krüger et al., 1996
). However, recent data obtained from the crystallized regulatory domains of LicT (van Tilbeurgh et al., 2001
) showed that phosphorylated histidines in PRDs face inwards, hidden from interactions with the paired molecule in the dimer. Hence, the negative charge introduced in LacT(H210D) might introduce structural changes that indirectly alter the dimer interface. In BL190(pGALTH210D), the overproduced LacT(H210D) may be forming heterodimers with the chromosomally encoded LacT with weak antiterminator activity. In contrast, in BL195 (
lacT) only an unstable homodimer would be formed from pGALTH210D. Nevertheless, the role of the PRD-II histidines in glucose inactivation of LacT has been confirmed with the H210A and H273A mutants as they lack activity in any background.
Further studies are required to clarify the mechanism by which phosphorylation regulates dimerization and activity of LacT. Furthermore, in vitro phosphorylation experiments with the different purified forms of LacT will be necessary to confirm the proposed phosphorylation processes.
In conclusion, we have determined the in vivo effect of site-directed mutations of the LacT antiterminator on lac operon expression in L. casei. Dephosphorylated PRD-I histidyl residues, His-101 and His-159, are indispensable for full induction of lac operon, whilst His-210 and His-273 of PRD-II play a role in CcpA-independent carbon catabolite repression. The results obtained suggest that there could be differences in the affinity of different phosphotransfer elements for PRD-I and PRD-II histidines.
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ACKNOWLEDGEMENTS |
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Received 10 July 2001;
revised 15 October 2001;
accepted 13 November 2001.