PepR1, a CcpA-like transcription regulator of Lactobacillus delbrueckii subsp. lactis

Joachim Schick1, Beate Weber1, Jürgen R. Klein1 and Bernhard Henrich1

Universität Kaiserslautern, Fachbereich Biologie, Abteilung Mikrobiologie, PO Box 3049, D-67653 Kaiserslautern, Germany1

Author for correspondence: Bernhard Henrich. Tel: +49 631 2052347. Fax: +49 631 2053799. e-mail: henrich{at}rhrk.uni-kl.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The PepR1 protein from Lactobacillus delbrueckii subsp. lactis DSM 7290 shares extensive homology with catabolite-control proteins from various Gram-positive bacteria. Expression of the subcloned pepR1 gene allowed for partial complementation of a ccpA defect in Staphylococcus xylosus. The influence of PepR1 on transcription of the prolidase gene pepQ, which is located adjacent to pepR1, was examined by use of lacZ reporter gene fusions in Escherichia coli. PepR1 stimulated transcription initiation at the pepQ promoter about twofold, and this effect required the integrity of a 14 bp palindromic cre-like sequence located 74 nt upstream of pepQ. In gel-mobility-shift assays, PepR1 specifically interacted with the pepQ promoter region and also with DNA fragments covering the promoters of the pepX, pepI and brnQ genes of Lb. delbrueckii subsp. lactis, which encode two additional peptidases and a branched-chain amino acid transporter, respectively. cre-like elements were identified in each of these DNA fragments. Catabolite control of PepQ was demonstrated in Lb. delbrueckii subsp. lactis. During growth with lactose the enzyme activity was twofold higher than in the presence of glucose, and corresponding differences were also detected in the level of pepQ transcription.

Keywords: Lactobacillus delbrueckii, PepR1 transcriptional regulator, peptidase Q, cre sequence, catabolite control


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Thermophilic lactic acid bacteria of the species Lactobacillus delbrueckii are involved in the fermentation of various dairy products, especially in the production of Italian-type hard cheeses (Olson, 1990 ). Due to numerous amino acid auxotrophies, growth of these bacteria in milk depends on the effective utilization of milk proteins (Juillard et al., 1995 ). This is achieved by the coordinate action of a cell-envelope-associated proteinase, various peptidases, and some peptide and amino acid transport systems (Mierau et al., 1997 ). We have previously isolated a series of 10 genes of the proteolytic system of Lb. delbrueckii subsp. lactis DSM 7290, a strain originally isolated from Emmental cheese. These genes, which code for peptidases with different specificities and an amino acid transporter, have been cloned and sequenced, and biochemical properties of most of the respective proteins have been established (Klein et al., 1995 , 1997 ). From an economic point of view, it seems reasonable that the expression or activities of the various components of the proteolytic system are controlled in response to specific external or internal stimuli. Little is known, however, about the underlying mechanisms and the genetic functions mediating them.

In the vicinity of pepQ (Stucky et al., 1995a ), one of the cloned peptidase genes of Lb. delbrueckii subsp. lactis, we recently detected an open reading frame (pepR1) which was suspected to encode a catabolite-responsive regulatory protein (Stucky et al., 1996 ). The pepR1 gene and its flanking regions were sequenced, and transcriptional start points were determined. The 152 bp intergenic region between the divergently running pepQ and pepR1 genes contains a 14 bp palindromic sequence which partly overlaps the -35 region of the pepR1 promoter. This palindrome is a highly conserved copy of the catabolite-responsive element cre, which is suspected to be involved in catabolite control of a number of genes in Gram-positive bacteria. The 37 kDa PepR1 protein has a typical helix–turn–helix DNA-binding motif and shares homology with a catabolite-control protein (CcpA), which is involved in the control of carbohydrate metabolism in Gram-positive bacteria. cre elements can be identified in the promoters of many CcpA-regulated genes, suggesting direct interactions of CcpA with these sequences (Hueck et al., 1994 ). Recently, in Streptococcus mutans (Simpson & Russell, 1998 ), Lactococcus lactis (Luesink et al., 1998 ) and Lb. delbrueckii subsp. bulgaricus (Morel et al., 1999 ), two genes encoding CcpA and PepQ homologues were found to be arranged similarly to pepR1 and pepQ of Lb. delbrueckii subsp. lactis.

In the present communication we show that PepR1 fulfils a CcpA-like physiological function, that it modulates transcription from the pepQ promoter, and that it specifically binds to DNA sequences derived from several genes of the proteolytic system.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. Media used were LB medium (1% tryptone, 0·5% yeast extract, 0·5% NaCl) for Escherichia coli, B medium (1% peptone, 0·5% yeast extract, 0·5% NaCl, 0·1% K2HPO4) for Staphylococcus xylosus and CDM (chemically defined medium; Ledesma et al., 1977 ) or MRS medium (De Man et al., 1960 ) for Lb. delbrueckii. Antibiotics and supplements were added to the media as required: kanamycin at 40 µg ml-1, ampicillin at 200 µg ml-1, tetracycline at 30 µg ml-1, chloramphenicol at 20 µg ml-1, erythromycin at 2·5 µg ml-1 and thymidine at 100 µg ml-1.


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Table 1. Bacterial strains and plasmids

 
Recombinant DNA techniques and DNA sequencing.
Standard techniques were applied for the cloning, PCR amplification and Southern blotting of DNA, and for purification of DNA fragments from gels (Sambrook et al., 1989 ). Plasmid DNA was prepared by the method of Birnboim & Doly (1979) except that, in the case of Sta. xylosus, lysostaphin (0·1 mg ml-1 final concentration) was added to the lysis mixture. Restriction endonucleases, nucleic acid modifying enzymes (Boehringer Mannheim and New England Biolabs) and ULTma DNA polymerase (Perkin Elmer) were used as recommended by the manufacturers. Escherichia coli and Sta. xylosus were transformed by electroporation (Dower et al., 1988 ; Brückner, 1997 ) using a Gene Pulser (Bio-Rad). Primers for PCR and nucleotide sequencing were purchased from MWG-Biotech. Automated sequencing with an Applied Biosystems model 373A sequencer was performed on both strands of the respective DNA segment. Synthetic primers were designed from the known sequences of the vector and the DNA insert. Nucleotide and amino acid sequences were analysed by the Microgenie (Beckman), Husar (Geniusnet) and PC/Gene (IntelliGenetics) programs.

Plasmid construction.
To subclone pepR1, the gene, together with 102 nt upstream and 229 nt downstream sequence, was excised from pUR1 as a 1330 bp KpnI–BamHI fragment and inserted between the BamHI and KpnI sites of the shuttle vector pRB473. The resulting plasmid, pR1473, initially identified in transformants of E. coli DH5{alpha}, was introduced into Sta. xylosus C2a and TX154.

Transcriptional fusions of the pepQ promoter region (PpepQ) with a promoterless lacZ reporter gene were constructed by cloning a 123 bp fragment, covering the first 18 nt of the pepQ coding sequence and 105 upstream nt (see Fig. 3b) and a variant of this fragment (PpepQ*) with a CG->AT exchange at positions 81/82 upstream of the pepQ start codon, into the SmaI site of the pMC1871 vector, leading to the plasmids pMCPQ2 and pMCPQ3. The 123 bp fragments were amplified by PCR from plasmid pMS1, using the primers 5'-TAATTTGTCTAAATTCATGGCAAAATTCTC-3' and 5'-CATGAGAACATTTTTTGTGCAATCG(->TA)CTTACAG-3' (underlined nucleotides were exchanged in the primer used for construction of pMCPQ3).



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Fig. 3. (a) Gel retardation of promoter-containing DNA fragments by PepR1. Digoxigenin-labelled DNA fragments (2 ng), covering the assigned promoters of pepQ (lanes 3, 4), pepX (lanes 5, 6), pepI (lanes 7, 8) and brnQ (lanes 9, 10) were mixed with 2 ng of a labelled negative-control fragment (‘bla’) and incubated at room temperature for 20 min in the absence (lanes 3, 5, 7, 9) or in the presence (lanes 4, 6, 8, 10) of purified PepR1 protein (1·6 µg). As a control, the ‘bla’ fragment alone (2 ng) was incubated without (lane 1) or with (lane 2) PepR1 (1·6 µg). After 6% polyacrylamide gel electrophoresis, the bands were blotted to nylon membranes and visualized by chemiluminescence. (b) cre-like sequences in promoter-containing DNA fragments. The nt sequences of sections of the DNA fragments shown in (a) were aligned by the previously determined transcription-start sites (indicated by the angled arrow) of the pepQ, pepX, pepI and brnQ genes (Matern et al., 1994 ). Numbers of the first and last residues of the entire fragments used in gel retardation assays (a) are given. Numbering refers to the respective GenBank entries Z54205 (pepQ), Z14230 (pepX), Z26948 (pepI) and Zw48676 (brnQ). For comparison, the corresponding upstream section of the malRA genes of Sta. xylosus (X78853) (Egeter & Brückner, 1996 ) is also shown. Potential cre sequences are displayed in black boxes; -10 and -35 regions are indicated with open boxes; translation-initiation codons and putative ribosome-binding sites are underlined.

 
To construct a pepR1::malE gene fusion (di Guan et al., 1988 ), a 1019 bp DNA fragment including the pepR1 coding sequence together with 8 upstream and 12 downstream nt was amplified by PCR from plasmid pUR1 by using the primers 5'-cgggatccATGAATAAGCAAGATGTAACCATTTACG-3' and 5'-aaactgcagTTACTTGTTCAAGGTACTTTGCTTCTTG-3' (nucleotides in lower case were added to introduce the underlined BamHI and PstI sites) and, after BamHI/PstI digestion, was ligated to BamHI/PstI-cut plasmid pMAL-c2 (New England Biolabs). The integrity of the fusion gene in the resulting construct, pR1Mal, was examined by sequencing a segment of the resulting plasmid spanning the entire pepR1 coding region and its upstream and downstream junctions with the vector. pR1Mal allows for transcriptional control of the pepR1::malE gene fusion by the tac promoter and for cleavage of its hybrid protein product by the factor Xa protease (Nagai & Thogersen, 1987 ).

Enzyme assays.
To determine the activity of {alpha}-glucosidase in strains of Sta. xylosus, bacteria were grown in B medium supplemented with appropriate sugars, and cell-free extracts were prepared and assayed as previously described by Egeter & Brückner (1996) . The reactions contained 15–600 µg total soluble protein, as determined by the method of Spector (1978) . Specific enzyme activities were calculated as nmol nitrophenol released from the respective chromogenic substrates (mg protein)-1 min-1.

To assay PepQ, cell-free extracts were prepared from Lb. delbrueckii subsp. lactis after centrifugation, washing the cells with 50 mM Tris/HCl, pH 7·5, and resuspending them in two cell-wet-weight equivalents of the same buffer. After mixing with glass beads (5 cell-wet-weight equivalents, 0·17 mm diameter), cells were disrupted by agitation in a Vibrogen cell mill (Sauer) for 15 min at 4 °C and maximum speed. The extracts, obtained after removal of debris by centrifugation (15 min, 4 °C, 15000 g), contained about 10 mg total soluble protein ml-1, as determined by the method of Spector (1978) . PepQ activities in the extracts were measured with Leu-Pro as a substrate and ninhydrin reagent, according to Morel et al. (1999) . The units (U) of specific PepQ activity were defined as nmol proline liberated (mg total protein)-1 min-1. Transformants of E. coli were assayed for ß-galactosidase (Miller, 1972 ) after washing the cells once with 0·9% NaCl.

Purification of the PepR1 protein.
Expression of the pepR1::malE fusion was induced in a growing culture of E. coli TB1(pR1Mal) by the addition of IPTG (0·3 mM final concentration) at OD600 0·5. Two hours after induction, bacteria were harvested by centrifugation (20 min, 4 °C, 3000 g), washed in buffer A (20 mM Tris/HCl, pH 7·4, 200 mM NaCl, 1 mM EDTA), resuspended in 10 ml of the same buffer per gram of cells (wet weight), and disrupted by ultrasonication. Debris was removed by centrifugation for 30 min at 4 °C and 10000 g, and the supernatant, after adjusting its protein content to 2·5 mg ml-1 (Spector, 1978 ), was subjected to affinity chromatography through an amylose resin (New England Biolabs). The column (2·5x10 cm, 15 ml bed volume, equilibrated with buffer A) was loaded with the extract and washed with 8 vols buffer A. The fusion protein was eluted with 10 mM maltose in the same buffer at a flow rate of 1 ml min-1 and, of 30 2 ml fractions collected, those containing the protein peak were pooled. The resulting solution, containing 15 mg protein at a concentration of 1·1 mg ml-1, was treated with 0·5 mg factor Xa for 14 h at 22 °C to cleave the PepR1–MalE fusion. The sample was dialysed against 20 mM Tris/HCl, pH 8·0, 25 mM NaCl, and subjected to ion-exchange chromatography on a Q-Sepharose Fast Flow (Pharmacia Biotech) column (1x10 cm, 5 ml bed volume), equilibrated with buffer B (10 mM Tris/HCl, pH 8·0, 25 mM NaCl). After application of the protein solution, the column was washed with 5 vols buffer B and the proteins were eluted with a gradient of 25–500 mM NaCl (25 ml each) in 20 mM Tris/HCl, pH 8·0. Fractions (1 ml) containing the separated PepR1 protein were identified by analysing aliquots (5 µl) on SDS-polyacrylamide gels.

Preparation of promoter-containing DNA fragments.
DNA fragments covering the promoter regions of the pepQ gene (positions -153 to +17 from the first nt of the start codon), the pepX gene (positions -288 to -5 from the first nt of the start codon), the pepI gene (positions -127 to +59 from the first nt of the start codon), and the brnQ gene (positions -171 to +12 from first nt of the start codon) were amplified by PCR from the plasmids pMS1 (Stucky et al., 1995a ), pJK433 (Meyer-Barton et al., 1993 ), pJK502 (Klein et al., 1994 ) and pKS1 (Stucky et al., 1995b ) by using the primer pairs 5'-AATAACAGCCCTACCTATCTAAATAC-3'/5'-TAATTTGTCTAAATTCATGGCAAAATTCTC, 5'-GTGTTCAATTTATTCTTGCAA-3'/5'-TACCTCATCTTTATATCTTTACATCTATGG-3', 5'-ACCAGTTAAAGTCTGTTTCA-3'/5'-CAGTAGGTTTGCCAATTT CC-3', and 5'-GCCTGGTACTTTTCAAAAGGGG-3'/5'-GCTTTTCTTTCATGTTGATCC-3', respectively. As a control, an internal fragment of the bla gene (positions +586 to +732 from the first nt of the start codon) was amplified from pBR322 by using the primers 5'-CTACTTACTCTAGCTTCCCGGC-3' and 5'-CAATGATACCGCGAGACCCACG-3'. PCR products were non-radioactively labelled as follows. To generate 3'-recessed ends, the DNA samples (1 µg) were combined with 1 U T4 DNA polymerase in 20 µl reaction volumes, containing 0·1 mM digoxigenin-11-dUTP (Boehringer Mannheim), 1 mM Tris/HCl, pH 7·5, 1 mM MgCl2, 5 mM NaCl and 0·1 mM DTT, and incubated for 10 min at 37 °C. After addition of 1 µl 1 mM (each) dATP, dCTP and dGTP, DNA ends were filled in during a further incubation at 37 °C for 10 min. Reactions were stopped by heating at 75 °C for 10 min, and the DNA was precipitated with ethanol and redissolved in 20 µl water.

Gel-retardation assays.
Of the individual promoter-containing DNA fragments, 2 ng were incubated with 1·6 µg purified PepR1 protein for 20 min at room temperature in 20 µl reaction volumes containing 20 mM Tris/HCl, pH 8·0, 20% (v/v) glycerol, 1 mM EDTA, 200 mM KCl, 1 mM DTT, 2 µg BSA and 1 ng sonicated herring sperm DNA. The samples were loaded at 100 V on 6% polyacrylamide gels in 40 mM Tris/HCl, pH 8·0, 20 mM acetic acid, 2 mM EDTA, 3% (v/v) glycerol and run at 150 V for 2·5 h. After blotting the DNA to positively charged nylon membranes, bands were visualized by chemiluminescence using a commercially available digoxigenin detection kit (Boehringer Mannheim).

Northern analysis.
Total RNA (10 µg), prepared from culture aliquots of Lb. delbrueckii subsp. lactis according to Chomczynski (1994) , was separated on 1·2% agarose, 1·1% formaldehyde gels and blotted to positively charged nylon membranes (Boehringer Mannheim) (Sambrook et al., 1989 ). A pepQ-specific probe of 1107 bp was amplified by PCR from plasmid pMS1 by using the primers 5'-ATGAATTTAGACAAATTACAAAACTGGCTGCAGGAAAACGG-3' and 5'-TTATTCCTTAACTGGCAGAACCTTCAATTCCTTGCTA-3'. After random-primed labelling with [{alpha}32P]dCTP (High Prime kit; Boehringer Mannheim), the probe was used in Northern hybridizations as described by Church & Gilbert (1984) .


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
PepR1 complements a ccpA mutation
On the basis of sequence comparisons, PepR1 of Lb. delbruecki subsp. lactis DSM 7290 falls into the LacI/GalR family of transcription regulators (Weickert & Adhya, 1992 ). PepR1 shares significant sequence identity with CcpA proteins from Lactobacillus casei ATCC 393 (50·5%) (Monedero et al., 1997 ), Bacillus megaterium (44·8%) (Hueck et al., 1995 ), Sta. xylosus (44·7%) (Egeter & Brückner, 1996 ), Bacillus subtilis (43·9%) (Henkin et al., 1991 ), and with the CcpA homologue RegM from Str. mutans (49·6%) (Simpson & Russell, 1998 ). To check the functional significance of these similarities, we subcloned the pepR1 gene into the E. coli/Bacillus shuttle vector pRB473. The resulting plasmid, pR1473, was used to perform complementation studies in a ccpA knock-out mutant of Sta. xylosus TX154, and expression of the malRA operon, known to be subject to CcpA-dependent catabolite repression in this organism (Egeter & Brückner, 1996 ), was examined by assaying the {alpha}-glucosidase activity of MalA. A wild-type strain, Sta. xylosus C2a, showed about fivefold repression of {alpha}-glucosidase in the presence of 25 mM glucose; this repression was completely abolished in the ccpA mutant (Table 2). Expression of the cloned pepR1 gene from plasmid pR1473 in the ccpA mutant was sufficient to relieve the deregulation of {alpha}-glucosidase to an extent that allowed for almost threefold repression of {alpha}-glucosidase by glucose. This clearly indicated that PepR1 of Lb. delbrueckii subsp. lactis DSM 7290 can interact in a CcpA-like manner with elements of the signal-transduction chain, which mediate catabolite control in Gram-positive bacteria (Hueck & Hillen, 1995 ). It is therefore possible that PepR1 is not only operative in Sta. xylosus but that it might be the functional equivalent of CcpA in Lb. delbrueckii. The fact that PepR1 achieved only partial complementation of the CcpA defect in Sta. xylosus, is most probably related to structural differences between the two proteins. These differences may account for less specific interactions of PepR1 with the HPr component of the phosphotransferase system (Deutscher et al., 1995 ) and with DNA target sites (cre sequences) (Fujita et al., 1995 ) in the heterologous Sta. xylosus host. In Fig. 3b, the upstream sequence of Sta. xylosus malRA, including the putative CcpA interaction site cre (Egeter & Brückner, 1996 ), is compared with upstream regions of Lb. delbrueckii genes that were found to specifically bind PepR1 (see below).


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Table 2. Complementation of a ccpA defect in Sta. xylosus by pepR1 of Lb. delbrueckii subsp. lactis

 
PepR1 regulates pepQ expression
From the fact that the pepR1 and pepQ genes are closely linked, we previously suspected that PepR1 might exert a regulatory effect on the expression of pepQ (Stucky et al., 1996 ). This was supported by the observation that in initial attempts to identify pepQ in a plasmid library of the DSM 7290 genome, only plasmids which, in addition to pepQ, contained at least the 5' part of pepR1 (nt 1–470) were able to complement a prolidase mutation in E. coli (Stucky et al., 1995a ). This part covers the region (nt 19–84) that encodes the predicted DNA-binding motif of PepR1. To investigate a possible influence of PepR1 on pepQ transcription, we fused a DNA fragment (position -77 to +45 from the transcription-start site; see Stucky et al., 1995a ), carrying the assigned pepQ promoter (PpepQ), with the promoterless lacZ gene of the vector pMC1871. Transcription of the PpepQ::lacZ fusion from the resulting construct pMCPQ2 was monitored by measuring ß-galactosidase activity in transformants of E. coli expressing pepR1 from its own promoter in the compatible plasmid pFNP13 (Stucky et al., 1996 ). Fig. 1 shows that PepR1 activated the expression of the PpepQ::lacZ fusion about twofold in this system, as compared with a control culture containing the unmodified vector pFN476 instead of pFNP13. This effect remained nearly constant throughout the growth of the bacteria and therefore appeared to be independent of the growth phase. To check the significance of the cre-like palindrome in the pepQ promoter as a site of potential PepR1–DNA interaction, we constructed a pMCPQ2 analogue, pMCPQ3, in which the central dinucleotide 5'-CG-3' of the original cre-like sequence was changed to 5'-AT-3'. Due to this exchange, the modified sequence differed in 3 of 14 nt from the cre consensus, 5'-TGWNANCGNTNWCA-3', established by Weickert & Chambliss (1990) . In the lacZ reporter-gene assay, this exchange almost completely abolished transcription activation from the modified promoter region PpepQ* by PepR1. From these results, we concluded that PepR1 modulates the initiation of transcription at the pepQ promoter and that the cre-like element plays an essential role in this interaction. In addition, cre may also be involved in some kind of autoregulation of PepR1, since the element was found to overlap the assigned -35 region of the pepR1 promoter (Stucky et al., 1996 ).



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Fig. 1. Influence of PepR1 on transcription from variants of the pepQ promoter. Transformants of E. coli CM89 ({Delta}lac), carrying pMCPQ2 (PpepQ::lacZ) and pFN476 ({blacksquare}), pMCPQ2 (PpepQ::lacZ) and pFNP13 (pepR1) ({square}), pMCPQ3 (PpepQ*::lacZ) and pFN476 ({bullet}), or pMCPQ3 (PpepQ*::lacZ) and pFNP13 (pepR1) ({circ}), were grown at 37 °C in LB broth supplemented with ampicillin and tetracycline. Transcription from PpepQ and PpepQ* was monitored by assaying ß-galactosidase in aliquots of the cultures.

 
PepR1 interacts with promoter regions
To investigate whether PepR1 can directly interact with susceptible promoters or genes, we purified the protein by constructing an N-terminal fusion with MalE (di Guan et al., 1988 ) (Fig. 2). Verification of the fusion by sequencing the respective region of the hybrid plasmid pR1Mal revealed a T at position 212 of the pepR1 coding sequence instead of the previously reported C (Stucky et al., 1996 ). After cleavage of the fusion protein and removal of the MalE moiety (Fig. 2), purified PepR1 was used to perform gel-mobility-shift assays with a 172 bp DNA fragment containing the pepQ promoter region. This DNA–protein complex was almost completely retarded in the gel, whereas an arbitrary control DNA fragment was not shifted (Fig. 3a).



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Fig. 2. Purification of the PepR1 protein. Purification steps were (lane 1) preparation of crude extract of E. coli TB1, carrying the pepR1::malE fusion plasmid pR1Mal, (lane 2) affinity chromatography on amylose resin, (lane 3) cleavage with factor Xa, and (lanes 4 and 5) ion exchange chromatography on Q-Sepharose (lane 4, purified PepR1; lane 5 purified MalE). After each individual step, samples were subjected to SDS 12% polyacrylamide gel electrophoresis. Sizes of protein standards (lane M) are given in kDa.

 
Since CcpA proteins of other Gram-positive bacteria are global regulators, modulating the expression of numerous genes or operons (Hueck et al., 1995 ), we also checked the affinity of PepR1 for the upstream and 5'-terminal regions of other genes of the proteolytic system of Lb. delbrueckii subsp. lactis DSM 7290. In gel-retardation assays (Fig. 3a), PepR1 was found to specifically interact with promoter-containing fragments of two additional peptidase genes, pepX and pepI (Meyer-Barton et al., 1993 ; Klein et al., 1994 ), and of the brnQ gene, which encodes a branched-chain amino acid transporter (Stucky et al., 1995b ). Under the conditions used, PepR1 showed distinct affinity for the pepI and brnQ fragments whereas the pepX fragment was only partially shifted.

Interaction of PepR1 with multiple promoter regions from the chromosome of Lb. delbrueckii strongly suggested that the protein is involved in the coordinate transcriptional control of a set of susceptible genes. This set seems to include various functions related to the production and uptake of protein cleavage products. Searching for a physical basis for specific PepR1–DNA recognition, we found cre-like sequences in all of the PepR1-binding DNA fragments (Fig. 3b). They match the cre consensus for B. subtilis (Weickert & Chambliss, 1990 ) in at least 10 out of 14 positions and are located at distances of 16, 39 and 22 nt upstream of the assigned -35 regions of the pepQ, pepI and brnQ promoters, or between the -35 and -10 regions of the pepX promoter. An additional cre-like element overlaps the translation-start codon of pepI. Together with the clear effect of altering the cre-like sequence upstream of pepQ on PepR1-dependent control of the pepQ promoter (see Fig. 1), the occurrence of potential cre sites in additional PepR1-binding DNA sequences indicates that cre is directly involved in PepR1–DNA interactions. Differences between the primary sequences of individual cre elements and between their locations relative to the transcription-start sites of the respective genes may contribute to determining the efficiency of PepR1 binding and action. In general, cre sites of genes activated by CcpA proteins are located upstream of the promoters, whereas for genes repressed by CcpA they are found within the promoters or at the 5' end of the respective genes or operons (Henkin, 1996 ). The location of a cre-like sequence upstream of the pepQ promoter (Fig. 3a) and the stimulating effect of PepR1 on pepQ transcription (Fig. 1) are in agreement with this general principle. From this, one might expect that pepX (where cre is within the promoter) would be repressed whereas brnQ (where cre is upstream of the promoter) would be activated by PepR1.

PepQ is subject to catabolite control
To examine whether the expression or the activity of pepQ or its product are effectively modulated by the nature of the carbon source, we cultivated Lb. delbrueckii subsp. lactis DSM 7290 in different media in the presence of either glucose or lactose (Fig. 4a). Fig. 4b shows the specific PepQ activities measured in cell-free extracts prepared at different times during growth in chemically defined medium supplemented with 2% Casamino acids (Difco). During the exponential, late-exponential and stationary phases, the specific PepQ activities determined in the presence of glucose were 1·8-, 2- and 1·7-fold higher than those measured with lactose. This effect was reproducible in a number of experiments, and it was similar when other culture media (chemically defined medium without Casamino acids or MRS medium) were used. As estimated from Northern blots (Fig. 4c), the amount of pepQ transcripts correspondingly varied in response to the carbon source used. This indicated that the observed differences in PepQ activity were at least party due to a modulation of pepQ transcription. In the exponential and stationary phases, the catabolite effects on pepQ transcription and PepQ activity, however, were not of the same magnitude, suggesting that post-transcriptional steps might be involved in regulation of PepQ expression.



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Fig. 4. Catabolite effects on PepQ activity and expression. (a) Lb. delbrueckii subsp. lactis DSM 7290 was grown at 42 °C in chemically defined medium supplemented with 2% Casamino acids and 2% glucose (solid line) or lactose (broken line), respectively. (b) At the times indicated by arrows, specific PepQ activities were determined in cells harvested from the glucose-grown (dotted bars) and the lactose-grown (hatched bars) cultures. (c) RNA prepared from the respective culture aliquots was used in Northern hybridizations with a pepQ probe.

 
It is therefore tempting to speculate that catabolite control of PepQ activity in Lb. delbrueckii is linked to the observed interaction of PepR1 with the pepQ promoter and to the glucose repression that PepR1 mediated in Sta. xylosus. Definitive proof that PepR1 is directly involved in catabolite effects in Lb. delbrueckii, however, might have to await the construction of appropriate pepR1 mutants.


   ACKNOWLEDGEMENTS
 
We are grateful to Reinhold Brückner and Oliver Egeter for the gift of Sta. xylosus strains and plasmids and for valuable advice and discussions during the experimental work with their materials. We also thank the group of Wolfgang Hillen for helpful discussions and Ulrike Klein for expert technical assistance. This work was supported by the BIOTECH G (contract BIOT-CT94-3055) and STARLAB (contract ERBBIO4CT960016) projects of the European Community.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 19 July 1999; accepted 30 July 1999.