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Applied and Environmental Microbiology, August 2002, p. 4132-4135, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.4132-4135.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Inducible Promoter-Repressor System from the Lactobacillus casei Phage {phi}FSW

Bernhard Binishofer,1 Isabella Moll,1 Bernhard Henrich,2 and Udo Bläsi1*

Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, 1030 Vienna, Austria,1 Department of Biology, University of Kaiserslautern, 67653 Kaiserslautern, Germany2

Received 20 December 2001/ Accepted 29 April 2002


   ABSTRACT
Top
 Abstract
Introduction
 Isolation of a promoter...
 Characterization of the promoter...
 Characterization of the cognate...
 References
 
With the aim to extend the presently available inducible gene expression systems for lactobacilli, we have isolated a thermoinducible promoter-repressor cassette from the temperate Lactobacillus casei phage {phi}FSW-TI in Escherichia coli. The {phi}FSW-TI promoter fragment was abutted to the plasmid-borne promoterless ß-glucuronidase (gusA) reporter gene and shown to direct its transcription in L. casei. In addition, the functionality of the promoter-repressor system was verified in the L. casei {phi}FSW-TI lysogen by showing that the gusA reporter gene, controlled by the isolated {phi}FSW-TI promoter, was repressed at 28°C and expressed at 42°C. Moreover, a homology search revealed that the C terminus of the isolated {phi}FSW repressor shows a high similarity to the small mutS-related domain of the MutS2 protein family that is unprecedented for phage-encoded repressor proteins.


   INTRODUCTION
Top
 Abstract
 Introduction
Isolation of a promoter...
 Characterization of the promoter...
 Characterization of the cognate...
 References
 
Given the economic importance of lactic acid bacteria, and more recently their prospective use as safe vaccines, a number of physiological traits as well as food-grade expression tools of lactobacilli and lactococci have been studied (9). Various inducible gene expression systems have been developed for Lactococcus lactis (reviewed in reference 3). In general, these transcriptional induction systems require the exogenous supply of inducers (18, 19, 21). In addition, a heat-inducible homologous expression system has been developed for L. lactis based on a genetically engineered thermolabile repressor of the lactococcal phage r1t (17). In lactobacilli, studies on the regulation of gene expression have focused mainly on carbon catabolism pathways, and an inducible-repressible expression system derived from the lactose operon of Lactobacillus casei has been described (5 and references therein).

The temperate phage {phi}FSW-TI was isolated from L. casei strain Shirota (S1) after chemical mutagenesis of a lysogen followed by heat induction (22, 24). Since the mutation responsible for the thermoinducibility was shown to reside in the phage DNA, it seemed likely that it could affect the stability of the phage repressor at the elevated temperature. In this study, we have characterized a promoter and a cognate repressor of {phi}FSW-TI with the potential to be exploited in a thermoinducible food-grade expression system for L. casei.


   Isolation of a promoter and a cognate heat-labile repressor of {phi}FSW-TI in Escherichia coli.
Top
 Abstract
 Introduction
 Isolation of a promoter...
Characterization of the promoter...
 Characterization of the cognate...
 References
 
The following strategy was used to isolate the thermoinducible promoter-repressor cassette of phage {phi}FSW-TI. The {phi}FSW-TI DNA was first shotgun cloned into the E. coli promoter test vector pKK232-8 (Amersham Pharmacia) carrying a promoterless cat gene. Chloramphenicol (Cm)-resistant clones were cotransformed with a {phi}FSW-TI library generated in the compatible plasmid pK194 (7). The DNA sequence encoding the protein with apparent repressor activity and its cognate promoter sequence were then obtained from clones that were sensitive and resistant to the antibiotic at 28 and 42°C, respectively.

Phage {phi}FSW-TI DNA was prepared as described earlier (23). The DNA was cleaved with either HaeIII, RsaI, HincII, or EcoRV, and the corresponding fragments were cloned into the SmaI site of the promoter test vector pKK232-8. The E. coli strain MC4100F' (25) was transformed with the ligation mix, and 51 candidate clones were selected on Luria-Bertani agar plates (14) containing 50 µg of Cm/ml.

To isolate the cognate repressor gene, {phi}FSW-TI DNA was partially digested with the same enzymes used for the procedure described above. The restriction fragments were cloned into the SmaI site of plasmid pK194, and the ligation mix was transformed into E. coli strain MC4100F'. From the pool of the transformants obtained on Luria-Bertani agar supplemented with 50 µg of kanamycin (Km)/ml, a batch plasmid isolation was performed. Then each of the 51 candidate promoter clones was transformed with the batch plasmid preparation. Cotransformants were selected on Cm/Km plates at 42°C, a temperature at which the putative repressor was predicted to be inactive, and thus, transcription of the cat gene in plasmid pKK232-8 was not supposed to be repressed. One hundred and fifty cotransformants were replica plated on Cm/Km at 28°C. The recombinant plasmids of five cotransformants unable to grow at 28°C were isolated from the corresponding colonies on the master plate and retransformed into MC4100F' in order to obtain colonies containing the individual plasmids. The inserts of two corresponding plasmid pairs were sequenced using an Applied Biosystems DNA sequencer. Both pairs contained the same core sequences. The plasmids with the shortest inserts were designated pKK232-8P (524-bp insert containing the putative promoter) and pK194R-1 (1,557-bp insert containing the putative repressor gene) and were used in further experiments.


   Characterization of the promoter region.
Top
 Abstract
 Introduction
 Isolation of a promoter...
 Characterization of the promoter...
Characterization of the cognate...
 References
 
The DNA sequence of the 524-bp insert (National Center for Biotechnology Information [NCBI] accession no. AF458097) present in plasmid pKK232-8P was analyzed for putative promoter sequences using the promoter search program available on the website http://www.dot.imgen.bcm.tmc.edu:9331/cgi-b. The highest score was obtained for a putative promoter spanning bp 275 (-35 hexamer) to bp 309 (predicted transcriptional start). To determine the transcriptional start site, primer extension was performed as described earlier (6) with total RNA prepared from E. coli MC4100F' harboring plasmid pKK232-8P containing the sequenced 524-bp insert with the putative promoter. The oligonucleotide V10, 5'-GACATGGGAGGCACCAGC-3', which binds 58 bp downstream of the putative -10 region, was used as a primer for the reverse transcriptase reaction. As shown in Fig. 1A, the primer extension analysis revealed that the transcript starts with the G located 6 bp downstream of the -10 hexamer. The alignment with the previously defined Lactobacillus consensus sequence (20) showed five matches for both the -35 and -10 hexamers, which are separated by 18 bp (Fig. 1B). The distance between the -10 hexamer and the transcriptional start site is 5 bp (Fig. 1B), which is within the range found for other Lactobacillus promoters (13).



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  		FIG. 1. (A) Determination of the transcriptional start site of the isolated {phi}FSW promoter ({phi}FSW-P). Lane 1, the oligonucleotide V10 complementary to the sequence downstream of the predicted start point of {phi}FSW-P was used to prime cDNA synthesis of RNA isolated from strain MC4100F'(pKK232-8P). The 3'-terminal nucleotide of the cDNA complementary to the 5' end of the transcript assigned to a G residue is marked by an arrow. The -10 hexamer is shaded. Reference sequencing reactions were performed with the same primer on double-stranded plasmid pKK232-8P as template. (B) Comparison of {phi}FSW-P with the promoter consensus sequence for lactobacilli. Boldface type indicates identity with consensus (20). The transcriptional start site (TS) is underlined. Dots are used for the alignment of the -35 and -10 hexamer sequences.

 
The functionality of the isolated {phi}FSW promoter was verified in L. casei S1 (23) by transforming this strain with a plasmid in which the promoter (including upstream and downstream sequences: bp -99 to +35 with regard to the transcriptional start [+1]) was abutted to the gusA reporter gene. The selected {phi}FSW promoter was cloned 5' of the gus gene in plasmid pNZ8008 (18) as follows: the promoter fragment was obtained by PCR amplification using plasmid pKK232-8P and the forward primer 5'-TATCTGCAGTG-AATCAAAGCCGTGGCGTTA-3' together with the reverse primer 5'-CCGAATTCGCGGCCAGCGCAATGAGCTT-3' containing a PstI site and an EcoRI site, respectively. The purified PCR fragment was cleaved with PstI and EcoRI and cloned into the corresponding sites of plasmid pNZ8008, resulting in plasmid pNZFSWP. The plasmid was transformed in L. casei S1 as described earlier (2). Colonies of L. casei S1 transformed with pNZFSWP showed ß-glucuronidase activity, whereas colonies of L. casei S1 transformed with the parental vector pNZ8008 did not, demonstrating that the {phi}FSW promoter was directing transcription of the gus reporter gene in the L. casei strain (not shown).


   Characterization of the cognate repressor.
Top
 Abstract
 Introduction
 Isolation of a promoter...
 Characterization of the promoter...
 Characterization of the cognate...
References
 
As mentioned above, plasmid pK194R-1 contained a 1,557-bp insert of {phi}FSW-TI DNA comprising five open reading frames (ORFs) (Fig. 2). To identify the repressor gene, four different restriction fragments (Fig. 2) were isolated from plasmid pK194R-1 and recloned into plasmid pK194. Plasmid pK194R-2 contained the 3' end of the 1,557-bp fragment covering ORF4 and ORF5, whereas plasmid pK194R-3 contained ORF3 in addition to ORF4 and ORF5. Plasmid pK194R-4 harbored the 5'-proximal reading frames ORF1 to ORF3, while plasmid pK194R-5 contained only ORF1. These four plasmids were individually transformed into E. coli strain MC4100F'(pKK232-8P). The presence of either plasmid pK194-R-4 or pK194R-5 in strain MC4100F'(pKK232-8P) conferred the same levels of Cm resistance at 42°C (expression of the cat gene due to thermal inactivation of the repressor) and resulted in Cm sensitivity at 28°C (repression of the cat gene due to the presence of active repressor protein). The repressor gene was therefore assigned to ORF1.



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  		FIG. 2. Identification of the ORF encoding the repressor by deletion analysis. As described in the text, plasmid pK194R-1 bears a 1,557-bp DNA insert of {phi}FSW containing five putative ORFs. The restriction sites used for isolation of the different subfragments and for reinsertion into plasmid pK194 are indicated on top. The restriction sites are indicated by capital letters: H, HindIII; B, BamHI; X, XmaI; E, EcoRI; and S, SmaI. The lengths of the DNA fragments present in plasmids pK194R-2 to -5 are schematically shown. + and - signs signify repressor activity (i.e., Cm sensitivity at 28°C) in strain MC4100F'(pKK232-8P) cotransformed with either of the depicted plasmids. ORF1 (indicated by a black bar) encodes the Smr-homologous {phi}FSW repressor.

 
To test qualitatively whether the identified {phi}FSW-TI promoter is controllable as well by the {phi}FSW-TI repressor in L. casei, the L. casei {phi}FSW-TI lysogen was transformed with plasmid pNZFSWP bearing the gusA reporter gene under transcriptional control of the isolated {phi}FSW-TI promoter. Since no ß-glucuronidase activity was detected at 28°C, whereas thermal induction at 42o resulted in expression of the reporter gene (not shown), we concluded that the thermoinducible promoter-repressor system is functional in L. casei.

As revealed by a PSI-Blast search available on the website http://www3.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-psi_blast, the isolated 113-amino-acid (aa) {phi}FSW-TI repressor protein (NCBI accession no. AF406970) shows a high similarity (E = 2 x 10-18) to the small mutS-related (Smr) consensus motif (15). The Smr protein in turn has significant homology to the C terminus of MutS2 proteins (Fig. 3), which are presumably involved in DNA mismatch repair (4, 15). Smr homologs have been found in a number of proteobacteria. The apparent absence of Smr proteins in all species that have mutS2 genes and their presence in species that have mutS1-homologous genes have led to the speculation that the Smr protein may play a role in mismatch repair through an interaction with MutS1 (15). Malik and Henikoff (12) have suggested that the Smr domain in MutS2 proteins and Smr protein linked via MutL to MutS might act as a nicking endonuclease, thus replacing the E. coli MutH nuclease.



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  		FIG. 3. Primary sequence of the Smr-homologous repressor of phage {phi}FSW ({phi}FSWR) and multiple alignment of the sequence shared by the repressor and the carboxy-terminal domain (Smr domain) of the MutS-like or MutS2 proteins from Streptococcus pyogenes (AE006610; 776 aa), Streptococcus pneumoniae (AE007352; 778 aa), L. lactis (AE006395; 776 aa), Bacillus halodurans (AP001517; 785 aa), Staphylococcus aureus (AP003132; 782 aa), and the Smr-like protein of Listeria monocytogenes (AJ133006; 131 aa). Given in parentheses are the NCBI accession number and the length of the corresponding proteins in number of amino acids. The Smr consensus sequence is shown at the bottom. The homologous regions of the protein domains were identified by BlastP 2.2.1 (1). Multiple alignments were done with CLUSTALW (26) and created using BOXSHADE. Numbers at the left correspond to distances (in number of amino acids) from the N terminus of the respective proteins. Identical amino acids are highlighted in black, whereas amino acids in grey symbolize conservative exchanges.

 
Several E. coli phage {lambda}-homologous repressors have been identified from temperate phage of lactobacilli (8, 10) and L. lactis (16). In general, these repressor proteins undergo self-cleavage upon DNA damage via a RecA-mediated pathway, which results in prophage induction (11). In contrast, the {phi}FSW prophage was not inducible by either UV irradiation or mitomycin treatment (22), underlining that the prophage status of {phi}FSW-TI is not controlled by a canonical repressor. To our knowledge this is the first time that a repressor activity has been attributed to an Smr-homologous protein. Since the Smr protein and/or domain has apparent DNA binding activity, it is conceivable that the phage has acquired from its host the smr gene, which may then have gained repressor function. It remains to be seen whether the isolated {phi}FSW-TI repressor represents the founding member of a new family of repressors containing a region of homology to Smr proteins.


   ACKNOWLEDGMENTS
 
We are grateful to M. Shimizu-Kadota for providing phage {phi}FSW-TI and L. casei strain S1, to O. P. Kuipers for providing plasmid pNZ8008, and to A. Witte for computer help.

This work was supported by grant 10006 from the Austrian Science Fund (FWF) and grant 6170 from the Austrian National Bank to U.B.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Microbiology and Genetics, Vienna Biocenter, Dr. Bohrgasse 9, 1030 Vienna, Austria. Phone: 43-1-4277-54609. Fax: 43-1-4277-9546. E-mail: Udo.Blaesi{at}univie.ac.at. Back


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Applied and Environmental Microbiology, August 2002, p. 4132-4135, Vol. 68, No. 8
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.8.4132-4135.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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