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Journal of Bacteriology, May 1999, p. 3288-3292, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Regulation of -Galactosidase Expression in Bacillus megaterium DSM319 by a XylS/AraC-Type Transcriptional Activator

Jan Strey, Klaus D. Wittchen, and Friedhelm Meinhardt^*

Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany

Received 7 December 1998/Accepted 15 March 1999

	ABSTRACT

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The -galactosidase-encoding bgaM gene of Bacillus megaterium DSM319 and the divergently orientated bgaR operon were isolated and sequenced. Both traits are subject to catabolite repression. A set of single-gene replacement mutants was generated and used to analyze gene function. BgaR was found to be a XylS/AraC-type positive transcriptional regulator of bgaM; a potential regulator binding site overlaps the bgaM promoter. A mechanism for regulation of -galactosidase expression in B. megaterium is proposed.

	TEXT

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The lactose utilization system of Escherichia coli encoded by the well-known lac operon serves as a paradigm for regulated gene expression in bacteria (reviewed in reference 20). Investigations of lac regulation provided deep insights into gene regulation mechanisms enabling bacteria to respond to changing concentrations of metabolizable compounds. Usually, sugar utilization systems are subject to carbon catabolite repression, the mechanism of which is clearly different in bacilli and enteric bacteria, as no cyclic AMP and cyclic AMP receptor protein homologue was found in Bacillus species (2, 32). Carbon catabolite repression in bacilli and other gram-positive bacteria is mediated by negative regulation (for reviews, see references 13 and 22). Studying Bacillus subtilis mutants relieved from catabolite repression led to the identification of cis-acting catabolite-responsive elements (CRE) (27) and a gene encoding the corresponding trans-acting catabolite control protein CcpA (12), a member of the GalR/LacI family of negative transcriptional regulators (28).

Recently, it has been shown that the -galactosidase-encoding mbgA gene of Bacillus megaterium ATCC 14581 is subject to catabolite repression. However, little is known for B. megaterium about genetics of -galactosidase induction by -galactosides such as lactose (25). Here, we report on the isolation and characterization of the lactose utilization gene of B. megaterium DSM319 (bgaM) and its positive regulation by a XylS/AraC-type transcriptional regulator encoded by the bgaR gene located immediately upstream with a polarity opposite to that of bgaM.

Cloning and characterization of the -galactosidase and its regulator. Bacterial strains, plasmids, and phage vectors used in this study are listed in Table 1. A -EMBL4-based genomic library of B. megaterium DSM319 (18) was used for cloning the -galactosidase-encoding region. The lac-negative E. coli strain JM107 was infected and plated on 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)-containing medium. Positive clones were obtained as blue plaques. Subcloning a 6.8-kb internal XbaI fragment into plasmid vector pUCBM20 (pUCGal3 [Table 1]) gave rise to dark blue colonies. Sequence analysis revealed four complete and two incomplete open reading frames (ORFs) (Fig. 1).

View this table: [in this window] [in a new window]   TABLE 1.   Bacterial strains, bacteriophages, and plasmids used in this study

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the -galactosidase-encoding genomic region of B. megaterium DSM319 wild-type and mutant strains. ORFs are indicated as arrows, the direction of which corresponds to transcription. Truncated ORFs are represented by 4' and 5'. The same designation was chosen for genes inactivated by insertional mutagenesis. Potential Rho-independent terminators are marked by hairpins. bgaM, -galactosidase gene; bgaR, XylS/AraC-type regulator gene; bglM, -1,3-1,4-glucanase gene of P. macerans; nprM, neutral protease gene. bgaR lacks 263 nt of the coding region. Restriction enzymes are abbreviated as follows: A, AflII; E, EcoRV; H, HincII; S, SalI; X, XbaI. The positions of probes used in Northern blot analyses are depicted from the top. Designations of corresponding strains are indicated on the right side.

The predicted polypeptide encoded by ORF bgaM (1,034 amino acids [aa]) exhibits striking similarities to -galactosidases of several bacteria, such as Clostridium acetobutylicum (45% identity [11]), Streptococcus thermophilus (40% [24]), E. coli (36% [15]), and Klebsiella pneumoniae (36% [6]). The recently published sequence of the -galactosidase of B. megaterium ATCC 14581 is almost identical (97% [25]). Accordingly, as for strain ATCC 14581, disruption of the chromosomal copy by insertional mutagenesis resulted in a -galactosidase-deficient strain (MS981 [Fig. 1]). The gene was disrupted by applying a previously established single-copy replacement system (29). For insertional mutagenesis of bgaM, the following steps were performed. A 3.14-kb HindIII fragment of plasmid pUCGal3 was cloned into HindIII-digested pUCTV2, resulting in pUCGalD. As the insert, a 2.38-kb chromosomal fragment containing the entire nprM gene of B. megaterium (18) was amplified with oligonucleotides 5'-GTTTTTATCAGTCGACTTGAAGAATTATT-3' and 5'-GTAATGAAGTCGACCCAGTTTCCCTTTTATTAC-3', cut with SalI, and ligated to SalI-digested pUCGalD; the resulting disruption vector was designated pDGal2. The genetic organization of the bgaM mutant strain MS981 is outlined in Fig. 1.

The polypeptide (275 aa) predicted for the divergently orientated locus bgaR exhibits obvious similarities to members of the XylS/AraC family of regulators. Within the highly conserved C terminus is situated the family profile, spanning aa 173 to 272 (entry PS01124 in PROSITE database). As for other XylS/AraC proteins, which are in most cases approximately 250 to 300 aa in size, the polypeptide has two helix-turn-helix DNA-binding motifs located within the family profile. Furthermore, the gene has a polarity opposite to that of the trait that is regulated as frequently observed for members of this family, the vast majority of which are positive transcriptional regulators (reviewed in references 9 and 10).

Gene products of ORF2, ORF3 (158 and 160 aa), and ORF5 resemble hypothetical proteins of B. subtilis (16). The deduced amino acid sequence of truncated ORF4 was found to contain an ATP-binding cassette transporter family signature (for a review, see reference 23).

Two potential Rho-independent transcriptional terminators were identified downstream of bgaM and ORF2, respectively (Fig. 1), and two overlapping CRE were found within the short intergenic region (129 nucleotides [nt]) of the -galactosidase gene and bgaR (see Fig. 3).

Construction of bgaR and ORF2 mutant strains. -Galactosidase expression in B. megaterium is subject to catabolite repression mediated by cis-acting CRE and the CcpA protein (25) but is in addition inducible by -galactosides (Fig. 2A) (17). Because of the similarities of BgaR to XylS/AraC transcriptional regulators, it was tempting to disrupt the gene and thereby determine its function in -galactosidase expression. We applied the already-mentioned gene replacement system (29) to generate a set of B. megaterium mutants, the genetic structures of which are schematically presented in Fig. 1. A bgaR mutant strain, MS970, was generated by using the pUCTV2 derivative pDbgaR containing a 1.76-kb chromosomal fragment amplified with oligonucleotides 5'-CCATGACGGAATTCCCCAGCTG-3' and 5'-GAAGTTATATCGAATTCAGCTGGAG-3'; in addition, it carries the -1,3-1,4-glucanase gene (bglM) of Paenibacillus macerans (5), which was amplified from plasmid pBCmac with oligonucleotides 5'-ACCCAGGTAAAAGCTTCCAACACCG-3' and 5'-GGACTAAAAAGCTTATGCCCAGCTGC-3'. The glucanase gene was subsequently ligated into the EcoRV site of the bgaR locus. Since we could not exclude a polar effect of the integrated bglM gene on expression of ORF2, which appeared to be part of the bgaR transcriptional unit (Fig. 2B), we also generated a bgaR deletion mutant (MS971 in Fig. 1). Deletion was achieved with vector pD2bgaR, which carries the above-mentioned bgaR locus, from which 263 nt between HincII and EcoRV was removed.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   (A) -Galactosidase activities for wild-type and mutant B. megaterium strains. Strain genotypes are depicted in Fig. 1. Hatched columns correspond to enzyme activities measured without additional sugars (LB medium), black columns represent activities of cultures grown with lactose, and white columns refer to cultures to which lactose and glucose were added simultaneously. Values given in Miller units (19) are the means of two experiments. Compared to LB medium without sugar, glucose addition resulted in lowered -galactosidase activity of all strains (catabolite repression). Lactose as the sole carbon source caused a clear increase in enzyme activity in wild-type strain DSM319 and the ORF2 mutant MS972, whereas in mutant strains MS970 and MS971, in which bgaR was inactivated, inducibility is lacking. (B) Transcript analyses of the bgaM gene and the bgaR operon. Total RNA isolated from cultures of the wild-type strain DSM319 and bgaR deletion mutant MS971 grown in LB broth without sugars (lanes 1), with lactose (lanes 2), and with glucose and lactose (lanes 3) was subjected to Northern blot analysis. On the left, sizes of hybridizing transcripts (in kilobases) and positions of 16S and 23S rRNAs are indicated. Sizes of the bgaM and bgaR transcripts, 3.1 and 1.5 kb, respectively, correspond to the length of the putative transcriptional units outlined in Fig. 1. Transcripts of bgaR mutant MS971 are shortened as expected from the degree of deletion. The occurrence of minor transcripts may be due to degradation or displacement caused by rRNAs.

For checking a potential influence of ORF2 on bgaM expression, mutant strain MS972 (Fig. 1) was constructed by employing disruption vector pDORF2, which carries a 1.72-kb chromosomal fragment generated with oligonucleotides 5'-TTTTTTCATGCGAATTCTTTCAGCCAC-3' and 5'-CAATTTATGAATTCGGGAAAAATCGTCC-3'. The bglM gene of P. macerans was integrated into the internal AflII site. Amplification of bglM was done from plasmid pBCmac with primers 5'-GGGGGGCTTAAGTAAAATATTCCAACACCGTGG C-3' and 5'-CCTGGAGTGGACTTAAGAGCTCATGCCCAGCTG-3'.

Comparative analysis of wild-type and mutant strains. The role of bgaR and ORF2 in regulation of -galactosidase expression was examined by measuring -galactosidase activities in wild-type and mutant strains. Strains were grown in Luria-Bertani (LB) medium to which sugars were added during mid-log phase. Samples were collected after cultivation for an additional hour and assayed for -galactosidase activity by measuring hydrolysis of the chromogenic substrate o-nitrophenyl--D-galactopyranoside (ONPG) at 31°C (21).

In all strains examined, similar levels of enzyme activity were obtained when no sugar was added to LB broth irrespective of the genetic background. Likewise, in glucose-lactose-grown cultures enzyme activities were lowered to comparable levels in all strains, reflecting catabolite repression of the -galactosidase. As for the wild-type B. megaterium DSM319, lactose caused a clear increase in -galactosidase activity in the ORF2::bglM mutant MS972. Thus, the predicted ORF2 polypeptide does not play a crucial role in -galactosidase induction. In contrast, both bgaR mutants, MS970 and MS971, completely lack inducibility. However, inducibility was restored when the amplified bgaR gene (5'-TATGACCTAGATAAAACCCG-3' and 5'-CTCATTTCTAGAGTATTAGGTTATTAG-3') was integrated into the SmaI site of pSVB2 (30) and introduced into mutants (data not shown).

To confirm the influence of the bgaR gene product on transcription of bgaM, both the deletion mutant MS971 and the wild-type strain DSM319 were subjected to comparative Northern blot analyses. An internal 1.75-kb HindIII/SalI fragment served as a bgaM-specific probe. Additionally, for gathering information on regulation of bgaR expression, we included a bgaR-specific probe, synthesized by PCR (5'-TATGACCTAGATAAAACCCG-3' and 5'-CTCATTTCTAGAGTATTAGGTTATTAG-3'). Both probes, positions of which are indicated in Fig. 1, were digoxigenin labeled by in vitro transcription with the digoxigenin RNA labeling kit (Boehringer Mannheim, Mannheim, Germany). Total RNA from cultures grown under the conditions described for enzyme assays was isolated (30). Following gel electrophoresis in 1.5% (wt/vol) formaldehyde agarose gels, Northern blot hybridization was carried out at 55°C and chemiluminescence detection was performed (3). The results of these experiments are shown in Fig. 2B.

As for DSM319, low-level transcription of the bgaM gene occurs in MS971 in LB medium without any sugar added. Due to catabolite repression, bgaM transcripts were not detectable in cultures supplied with glucose and lactose. However, addition of lactose caused a clear increase of bgaM transcription only on the wild-type background. In the bgaR deletion mutant MS971, no accumulation of bgaM transcripts occurred. These findings correspond to those of the enzymatic assays summarized in Fig. 2A and are in accordance with positive transcriptional regulation of bgaM by BgaR.

In medium lacking glucose, bgaR transcription could readily be observed in both the wild-type strain and mutant MS971 irrespective of lactose addition. As depicted in Fig. 2B, transcripts of the bgaR operon in mutant MS971 are shortened by approximately 300 nt, which accords well with the 263-bp deletion of bgaR. Remarkably, transcription of bgaR isas for bgaMalso subject to catabolite repression; no transcript was detected when glucose was added along with lactose.

Based on our results, we propose a model for regulation of -galactosidase expression in B. megaterium as outlined in Fig. 3. The transcriptional start point (data not shown) as well as the structure of the bgaM gene and its promoter region (Fig. 3) is almost identical to that published for strain ATCC 14581. A 27-bp inverted repeat overlaps the bgaM promoter containing two partially overlapping CRE. Base substitutions within both overlapping CRE caused partial relief of catabolite repression, and that is why it was suggested that the inverted repeat may serve as the binding site for the catabolite repressor proteins (25). The trans-acting catabolite control protein CcpA is effective by binding to CRE, which need not necessarily overlap the respective promoter but can be located at a considerable distance upstream or downstream (14). Thus, though we did not determine the transcriptional start of the bgaR operon, the localization of the CRE within the intergenic region spanning only 129 nt probably facilitates negative regulation by CcpA of both the bgaM gene and the opposite bgaR operon.

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Regulation of -galactosidase expression in B. megaterium. The bgaM gene and loci of the bgaR operon are represented by arrows indicating transcriptional orientation (gene signature as in Fig. 1). Transcriptional terminators are sketched as hairpins. The sequence of the intergenic region between bgaM and bgaR is written on top with start codons marked by small arrows. Potential ribosomal binding sites (S/D) were found at appropriate distances. 35 and 10 regions of the bgaM promoter are boxed. The corresponding start point of the bgaM transcript (TS) is indicated by a dot. Two overlapping CRE are located within the promoter region. The arrows underneath indicate direct imperfect repeats (DR) constituting a putative regulator binding site for BgaR. In the presence of glucose, phosphorylated HPr causes CcpA repressor binding to CRE, which facilitates catabolite repression of both divergently orientated transcriptional units. Absence of glucose leads to weak transcription of bgaM but clear transcription of the bgaR operon. When lactose is added exclusively, it acts as an effector of BgaR, eventually resulting in transcriptional activation of bgaM.

For B. megaterium ATCC 14581, the bgaR gene apparently corresponds to a locus that was incompletely sequenced and designated ORF1 by Shaw et al. (25). The predicted polypeptide of the latter comprising 33 aa is almost identical to the corresponding N terminus of the bgaR gene product of DSM319; only two residues are different.

Concerning positive regulation of -galactosidase expression, we searched for a putative BgaR regulator binding site within the intergenic region of bgaM and bgaR. We identified two imperfect direct repeats again in both DSM319 and ATCC 14581 (DR in Fig. 3). As for the bgaM promoter, such sites for XylS/AraC regulators proximal to the RNA polymerase binding region have been found in most cases to overlap or abut the 35 region of the gene or operon that is regulated (10). Gel mobility shift assays using purified bgaR gene products should help to elucidate the role of these imperfect direct repeats in binding the regulator.

Regulation of -galactosidase expression appears to be different in B. subtilis. Besides catabolite repression by CcpA, induction of enzyme expression was found to be negatively controlled by another repressor protein belonging to the LacI/GalR regulator family (7).

To our knowledge, positive gene regulation of -galactosidase expression by a member of the XylS/AraC family has been reported only for Staphylococcus xylosus, in which, however, formation of the regulator is not subject to catabolite repression but is obviously constitutive (1).

Catabolite repression of the regulator BgaR makes the B. megaterium genetic system controlling -galactosidase expression extraordinarily economic and elegant likewise since it links synthesis of the transcriptional regulator to glucose consumption.

Nucleotide sequence accession number. The sequence of the 6.8-kb chromosomal XbaI fragment of B. megaterium DSM319 containing the genes bgaR and bgaM has been submitted to the EMBL database under accession no. AJ000733.

	ACKNOWLEDGMENTS

J.S. was supported by grants from the Max-Buchner-Forschungsstiftung, Germany.

We thank R. Borriss for kindly providing plasmid pBCmac.

	FOOTNOTES

^* Corresponding author. Mailing address: Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstr. 3, 48149 Münster, Germany. Phone: 49 251 83-39825. Fax: 49 251 83-38388. E-mail: meinhar{at}uni-muenster.de.

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Journal of Bacteriology, May 1999, p. 3288-3292, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Strey, J. Articles by Meinhardt, F. Search for Related Content PubMed PubMed Citation Articles by Strey, J. Articles by Meinhardt, F.