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Vol. 180, Issue 13, 3400-3404, July 1, 1998

Expression of the bglH Gene of Lactobacillus plantarum Is Controlled by Carbon Catabolite Repression

Rosangela Marasco1, Lidia Muscariello2, Mario Varcamonti3dagger , Maurilio De Felice3, and Margherita Sacco2*

1 Dipartimento di Chimica, Università degli Studi di Salerno, 84084 Fisciano (Sa),1 2 Istituto Internazionale di Genetica e Biofisica, Consiglio Nazionale delle Ricerche, 80125 Naples,2 and 3 Sezione di Microbiologia, Dipartimento di Fisiologia Generale ed Ambientale, Università Federico II, 80134 Naples,3 Italy

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

A newly identified bglH gene coding for a phospho-beta -glucosidase of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed an open reading frame encoding a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The bglH gene was shown to be expressed on a monocistronic transcriptional unit. Its transcription was repressed 10-fold in L. plantarum cells grown on glucose compared to the beta -glucoside salicin as a sole carbon source. A catabolite-responsive element (CRE) spanning from -3 to +11 with respect to the transcriptional start point was found, and its functionality was assessed by mutational analysis. In vitro and in vivo DNA binding experiments suggested the occurrence of a DNA-protein complex at the CRE site, which would mediate glucose repression of bglH expression.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Much effort has been devoted in most recent years to the understanding of regulation of carbon metabolism in gram-positive bacteria (11, 16, 20, 26, 28-31, 36). Carbon catabolite repression (CCR) has been extensively studied in Bacillus subtilis, and it has been shown to be different from the well known CCR mechanism operating in Escherichia coli and other gram-negative bacteria, where the catabolite gene activator protein in complex with cyclic AMP activates transcription from catabolite repression-responsive operons (27). CCR in B. subtilis involves a negative regulatory mechanism characterized by the cis-acting catabolite-responsive element (CRE) and the CcpA trans-acting element belonging to the GalR-LacI family of bacterial regulatory proteins (13, 14, 17, 39, 40). Mutations in CRE sequences occurring in various carbon catabolite-responsive operons result in loss of glucose repression (17, 18). It has been recently reported that the negative control underlying catabolite repression in B. subtilis might represent a global regulatory mechanism for gram-positive bacteria (16, 29, 30). Immunological cross-reactivity experiments have recently shown the presence of the catabolite control protein CcpA in many gram-positive bacteria (20). Very recently CcpA-mediated catabolite repression has been reported also for Lactobacillus casei (25).

In this paper we report the cloning and expression in E. coli of the phospho-beta -glucosidase-encoding bglH gene of Lactobacillus plantarum, one of the most widespread lactic acid bacteria in the environment and also widely used in fermented-food technology, where beta -glucosidase activity is responsible for reduction of bitter flavor (10, 38). The bglH gene complemented a bglB mutation of E. coli. It was shown to be homologous to the bglH gene of B. subtilis, encoding a phospho-beta -glucosidase active on aryl-beta -glucosides such as salicin and arbutin and belonging to the bglPH operon, whose expression is regulated by a CcpA-mediated carbon catabolite repression (18, 19, 21). Our results indicate that bglH expression is negatively regulated by glucose and suggest that this control might be exerted by a CcpA-like mechanism.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacterial strains and culture conditions. L. plantarum B21, isolated from a brining stage of naturally ripened olives (5), was used throughout this study. L. plantarum was grown in MRS medium (Oxoid) supplemented with 2% glucose, 1% ribose, or 0.4% salicin. E. coli FA31 (ara rbs115 xyl7 lacY1 mglP1 bglB208 bglY202) (6) was grown in TY or M9 minimal salts supplemented with 0.4% glucose or 0.4% salicin.

DNA cloning and sequencing. Total DNA from L. plantarum B21 was prepared as described previously (22), partially digested with Sau3A1, and size fractionated through a sucrose gradient as described previously (32). DNA fragments of 5 kb (average size) were ligated into the BamHI site of plasmid pKK232 (4), and the ligation mixture was used to transform E. coli FA31 competent cells. Transformants were plated on TY medium supplemented with 50 µg of ampicillin ml-1 and screened for complementation of the bglB208 mutation of E. coli FA31 on minimal medium supplemented with 0.4% salicin. Sequencing analysis of one positive clone was performed by the method of Sanger and coworkers (33).

Primer extension analysis. Total RNA from L. plantarum cells grown to mid-exponential phase was isolated as described by Leong-Morgenthaler and coworkers (23). Primer extension products were obtained by using oligonucleotide SP1 (5'-GCCACCTGGTAACCGATCCGC-3'), corresponding to the complement of nucleotide positions 351 to 331 (Fig. 1). The oligonucleotide was end labeled with [gamma -32P]ATP and T4 polynucleotide kinase, and the reverse transcriptase reaction was performed as described previously (32). The extension products were separated by electrophoresis on 6% polyacrylamide urea sequencing gels (32) and were visualized and quantified by autoradiography on phosphor storage plates (PhosphorImager; Molecular Dynamics). As a reference, sequencing reactions were performed by the dideoxy-chain termination method (33) by using the same primer as in the primer extension experiments.


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  		Fig. 1.   Nucleotide sequence of the nontranscribed strand of the bglH gene and inferred BglH amino acid sequence. The vertical arrow corresponds to the position of the 5' terminus of bglH mRNA. Putative -10 and -35 recognition sequences and ribosome-binding site (RBS) are underlined. Dashed arrows indicate an inverted repeat sequence corresponding to the transcription termination site. The CRE sequence overlapping the +1 position is in bold.

Assay of beta -glucosidase activity. L. plantarum cells (100 ml) were grown to mid-exponential phase in MRS supplemented with either 2% glucose or 0.4% salicin, washed twice with 150 mM NaCl, and resuspended in 1 ml of 50 mM phosphate buffer (pH 6.2). Appropriate aliquots of cell suspensions were added to 800 µl of 30 mM salicin in phosphate buffer. After 20 min of incubation at 30°C, the enzymatic reaction was stopped by adding 500 µl of 1 M Na2CO3. The production of saligenin from salicin was detected as described previously (34).

Gel retardation analysis. Gel retardation experiments were performed as already described (37). Cell extracts were obtained by resuspending exponential cultures in buffer containing 20 mM Tris HCl (pH 8.0), 100 mM KCl, 1 mM EDTA (pH 8.0), 0.5 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride, followed by grinding with glass beads. Binding was carried out for 15 min at room temperature in 20 mM Tris HCl (pH 8.0)-100 mM KCl-5 mM MgCl2-1 mM EDTA (pH 8.0)-0.5 mM dithiothreitol-10% glycerol-0.02% Nonidet P-40, 2 µg of bovine serum albumin-1 µg of poly(dI-dC). A synthetic oligonucleotide called CRE1 (5'-GGAGGCGACTTGTTGTAAGGGCTATCATTATTAGCGGACG-3') and its complement CRE2 were annealed to form a 40-bp fragment (CREa) containing the bglH sequence spanning from nucleotides (nt) 184 to 211 (Fig. 1). Oligonucleotide CRE3 (5'-GGAGGCGACTTGTTCTATGGGCTATCTTTATTAGCGGACG-3') and its complement CRE4 were used to construct a 40-bp fragment (CREb) containing three mismatches in the CRE sequence (underlined).

In vivo footprinting. L. plantarum cells were grown overnight in MRS medium supplemented with 0.4% salicin or 2% glucose. Cells were then diluted 1:100 and grown to mid-exponential phase. Methylation was performed by adding freshly diluted dimethyl sulfate (DMS; Aldrich) to a final concentration of 0.1% for 3 min at 30°C with shaking. The methylation reaction was stopped by adding an equal volume of ice-cold saline phosphate buffer (150 mM NaCl, 40 mM K2HPO4, 22 mM KH2PO4 [pH 7.2]). Cells were harvested by centrifugation at 10,000 × g for 10 min and washed twice with saline phosphate buffer. Chromosomal DNA was purified as described previously (22). Contaminating RNA was removed by treatment with RNases A and T1, followed by polyethylene glycol 6000 precipitation (32).

Breakage points of the modified DNAs were revealed by a primer extension method adapted from that of Brewer and coauthors (3) as follows. A linear PCR using Taq polymerase was performed on chromosomal DNAs. Primers SP1 (see above) and SP2 (5'-GCGGTATGGCTTCATCTATGTCG-3'), corresponding to nucleotide positions 22 to 44 (Fig. 1), were used to probe the top and bottom strands, respectively. End labeling was performed with [gamma -32P]ATP and T4 polynucleotide kinase as described previously (32). Primer extension reactions were carried out in a volume of 20 µl containing 150 ng of chromosomal DNA, 0.5 pmol of 32P-end-labeled oligonucleotide, 2 µl of 10× Taq polymerase reaction buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, 20 mM MgCl2, 0.2% [wt/vol] gelatin), and each deoxynucleoside triphosphate at a final concentration of 200 µM. The DNA was denatured by incubating the samples at 95°C for 5 min, followed by addition of 1 U of Taq polymerase. A program of 40 cycles, each consisting of 1 min of denaturation at 94°C, 5 min of annealing at 63°C, and 2 min of chain elongation at 72°C, was used for the amplification procedure. Four microliters of formamide dye mixture was added to the samples, and the extension products were separated by electrophoresis on 6% polyacrylamide urea sequencing gels.

Nucleotide sequence accession number. The sequence shown in Fig. 1 has been deposited in the DDBJ/EMBL/GenBank databases under accession no. Y15954.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning and sequencing of the bglH gene from L. plantarum B21. Total DNA from L. plantarum B21 was partially cleaved with Sau3A1 and fractionated through a sucrose gradient, and fragments with an average size of 5 kb were ligated to the pKK232 vector in the BamHI restriction site. Competent cells of E. coli FA31 (unable to grow on salicin as a sole carbon source due to the bglB208 mutation) were transformed with the ligation mixture, and up to 2,500 recombinant clones were obtained on nutrient ampicillin plates. The whole library was screened on minimal medium supplemented with salicin, and two positive clones, able to complement the E. coli bglB208 mutation, were selected. The two inserts of 5.8 and 2.7 kb, contained in plasmids pRM10 and pRM11, respectively, showed an overlapping restriction map (data not shown), indicating that the same gene was present on both fragments.

Sequence analysis of the 2.7-kb fragment of pRM11 showed the presence of an open reading frame hereafter called bglH (Fig. 1), coding for a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The transcriptional start point, located at nt -42 relative to the putative translational start, and the promoter sequence (Fig. 1) were identified by primer extension analysis performed on total RNA from L. plantarum cells grown on salicin (Fig. 2, lane 2). A putative terminator structure was found downstream of the stop codon (Fig. 1). A bglH-specific transcript of 1.5 kb was detected by Northern analysis (data not shown), indicating that the bglH gene is expressed on a monocistronic transcriptional unit.


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  		Fig. 2.   Primer extension analysis of bglH mRNA. Primer extension products were obtained by using oligonucleotide SP1 and total RNA extracted from L. plantarum cells during exponential growth on ribose (lane 1), salicin (lane 2), or glucose (lane 3). As a reference, sequencing reactions were performed by using the same primer.

A beta -glucosidase activity able to hydrolyze salicin only in the presence of a phosphorylating system was found in extracts of both L. plantarum and the E. coli strain carrying plasmid pRM11, thus showing that bglH encodes an aryl phospho-beta -glucosidase (data not shown).

Glucose repression of bglH expression. A primer extension analysis was performed on total RNA from L. plantarum cells grown in MRS medium supplemented with ribose (Fig. 2, lane 1), salicin (lane 2), or glucose (lane 3). The relative amount of the extension products showed a 10-fold reduction of the bglH-specific mRNA in cells grown on glucose, as quantified by autoradiography on phosphor storage plates. No detectable difference in the amount of the bglH-specific mRNA was found in cells grown on ribose or salicin.

Enzyme data were in agreement with the results of the primer extension. beta -Glucosidase activity was analyzed by assaying the hydrolysis of salicin in whole cells of L. plantarum grown in MRS medium supplemented with 2% glucose or 0.4% salicin. The presence of glucose in the growth medium caused a 60-fold decrease in the ability of cells to take up and hydrolyze salicin compared to cells grown in the presence of salicin. The specific activities were 7.6 and 464 mmol of product formed per min per 1010 cells grown in the presence of glucose and salicin, respectively.

Computer analysis of the bglH sequence. A computer search showed the presence of three putative CRE sequences within the bglH gene (Fig. 1). The CRE sequence overlapping the transcriptional start site, and spanning from nt 190 to nt 203 (Fig. 1), contained one mismatch with respect to the consensus sequence (17), while the other two, spanning from nt 1215 to 1228 and from nt 1549 to 1562 (Fig. 1), showed two mismatches each.

Using the FASTA program, we found that the predicted BglH protein has significant sequence identity (60%) with the BglH protein of B. subtilis, a phospho-beta -glucosidase active on aryl-beta -glucosides such as salicin and arbutin (21), and with the BglB protein of E. coli (53%).

Recognition of the bglH CRE sequence by L. plantarum protein extracts. Gel retardation experiments were performed to check whether the CRE sequence is involved in the binding of a putative CcpA-like protein. A 40-bp DNA fragment, containing the bglH CRE sequence overlapping the transcriptional start site, was used as a probe in binding assays with protein extracts from L. plantarum cells grown in catabolite-repressing conditions (2% glucose). Figure 3 shows the appearance of a retarded complex when 0.5 µg of protein extract was added to the 32P-labeled CREa fragment (see Materials and Methods) (lane 2). This binding was specifically counteracted by addition of increasing amount of unlabeled CREa fragment (Fig. 3, lanes 3 and 4). In contrast, the occurrence of a retarded complex obtained with the CREb fragment (see Materials and Methods), containing three mismatches in the CRE sequence, was fourfold less efficient and was not specifically counteracted by addition of unlabeled CREb fragment, as quantified by autoradiography on phosphor storage plates (Fig. 3, lanes 5 to 8).


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  		Fig. 3.   Gel retardation analysis of the bglH CRE sequence. Lanes 1 to 4, 1 ng of 40-bp DNA fragment containing the wild-type CRE sequence of bglH (CREa); lanes 5 to 8, 1 ng of 40-bp DNA sequence containing the bglH CRE sequence with three mismatches (CREb) (see Materials and Methods); lanes 2 to 4 and 6 to 8, 0.5 µg of proteins from L. plantarum crude extract; lanes 3 and 4, 1 and 10 ng of unlabeled CREa fragment, respectively; lanes 7 and 8, 1 and 10 ng of unlabeled CREb fragment, respectively. The presence of an inverted repeat in CREa, which is disrupted in CREb, seems to affect the band mobility of the free 40-bp fragments (the four single-strand fragments have the same mobility [data not shown]).

In vivo analysis of the CRE sequence. An in vivo footprinting analysis was performed to test whether the CRE sequence overlapping the transcription start site is involved in the regulation of bglH expression. Chromosomal DNA of L. plantarum cells grown on glucose or on salicin was methylated with DMS during exponential growth. The analysis was focused on G residues protected from DMS attack during growth in the presence of glucose. Figure 4 shows the occurrence of protection of the G residue in position 191 of the top strand (Fig. 4A, lane 2) and of the G residue in position 202 of the bottom strand (Fig. 4B, lane 2). Both G residues protected from the DMS attack belong to the CRE sequence.


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  		Fig. 4.   In vivo footprinting analysis of the bglH regulatory region. L. plantarum cells were grown in MRS medium supplemented with salicin (lane 1) or glucose (lane 2). G, A, T, and C indicate the nucleotide sequencing reactions for the strand being extended (G's in the methylated strand correspond to C's in the sequencing lane). (A) Top strand; (B) bottom strand. Oligonucleotides used for primer extension were SP1 (A) and SP2 (B). The arrows point to G residues in positions 191 (top strand; A) and 202 (bottom strand; B), respectively.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have identified and characterized the L. plantarum phospho-beta -glucosidase-encoding bglH gene by complementation of an E. coli strain carrying a mutation in the bglB gene. The predicted BglH protein showed significant sequence identity with BglH of B. subtilis (60%) and BglB of E. coli (53%), two phospho-beta -glucosidases active on aryl-beta -glucosides such as salicin and arbutin. Northern analysis indicated that bglH is transcribed on a monocistronic unit, while in E. coli and in B. subtilis the genes coding for BglB and BglH are organized in operons coding also for specific permeases (21, 24). The expression of these operons responds to transcriptional antiterminator-mediated regulation (15, 19, 35), whose cis element is the so-called RAT (ribonucleic antiterminator) sequence, highly conserved among gram-positive and gram-negative bacteria (2, 28). A BglR protein, belonging to the BglG family of transcriptional antiterminators, has been found in Lactococcus lactis (1). A computer search for RAT sequences within the bglH sequence gave negative results, which, together with the lack of substrate induction, suggests that bglH may not be regulated by an antitermination mechanism.

The beta -glucosidase activity was repressed 60-fold in cells grown in the presence of glucose compared to salicin, as measured by enzyme activity in whole L. plantarum cells, while bglH transcription was repressed 10-fold. The different degree of repression by glucose on bglH transcription and on beta -glucosidase activity may be explained if a catabolite-repressed permease exists in L. plantarum. The presence of other catabolite-repressed bgl genes might also account for the difference observed.

The glucose effect on bglH expression together with the presence of a CRE sequence suggested the occurrence of a CcpA-like-mediated carbon catabolite repression of the gene, a control mechanism described for various genes in gram-positive bacteria (7, 9, 12, 14). The positional distribution of CREs has been reported to be within ±200 bp of the translational start site, with a distinct peak at the translational start site (17). In agreement with this and other more recent reports (8, 18, 26), we found that the bglH CRE sequence overlaps the transcriptional start site. Only one deviation from the 14-bp consensus sequence was found, a G in place of C at position 7 of the consensus (nt 196 in Fig. 1) (17). The functional CRE sequence overlapping the bglPH promoter of B. subtilis also shows one deviation from the consensus (19).

We performed in vitro and in vivo experiments aimed at finding a CRE sequence-function correlation for bglH expression. Gel retardation experiments showed a more efficient and specific formation of retarded complexes with DNA fragments carrying the wild-type bglH CRE sequence compared to a mutated sequence, containing three deviations from the consensus, introduced to create mismatches in the partial inverted repeat present in CRE (8, 17, 26). In vivo footprinting experiments showed that two G residues were protected in cells grown on glucose versus cells grown on salicin. These G's are in positions 2 and 13 in the top and bottom strands, respectively, of the 14-bp CRE sequence.

While Miwa and coworkers (26) have shown previously that substitution by site-directed mutagenesis of the C residue in position 13 of the CRE affected carbon catabolite repression of the B. subtilis gnt operon, to our knowledge the result shown here represents the first in vivo protection data obtained for a CRE sequence and suggests that these fully conserved G residues might be functional in the DNA-protein interaction.

    ACKNOWLEDGMENTS

We thank A. La Volpe for helpful discussion, S. Gargano and E. Patriarca for critical reading of the manuscript, M. Valenzi for computer assistance, and C. Sole and P. Villano for technical assistance.

This work was supported by MIRAAF, Piano Nazionale Biotecnologie Vegetali. Minor support was also obtained from EU contract ERBBIO4CT960439.

    FOOTNOTES

* Corresponding author. Mailing address: Istituto Internazionale di Genetica e Biofisica, Consiglio Nazionale delle Ricerche, via G. Marconi 10, 80125 Naples, Italy. Phone: 39 81 7257219. Fax: 39 81 5936123. E-mail: sacco{at}iigbna.iigb.na.cnr.it.

dagger Present address: Department of Genetics, University of Groningen, 9751 NN Haren, The Netherlands.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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