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
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ABSTRACT |
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Keywords: Lactobacillus delbrueckii, PepR1 transcriptional regulator, peptidase Q, cre sequence, catabolite control
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INTRODUCTION |
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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 helixturnhelix 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.
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METHODS |
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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 KpnIBamHI 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, 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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Enzyme assays.
To determine the activity of -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 15600 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 PepR1MalE 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 25500 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 [
32P]dCTP (High Prime kit; Boehringer Mannheim), the probe was used in Northern hybridizations as described by Church & Gilbert (1984)
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RESULTS AND DISCUSSION |
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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 PepR1DNA 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 PepR1DNA 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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ACKNOWLEDGEMENTS |
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Received 19 July 1999;
accepted 30 July 1999.