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Applied and Environmental Microbiology, April 2005, p. 2061-2069, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2061-2069.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of a Mobile clpL Gene from Lactobacillus rhamnosus

Aki Suokko, Kirsi Savijoki, Erja Malinen, Airi Palva, and Pekka Varmanen^*

Division of Microbiology and Epidemiology, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland

Received 6 August 2004/ Accepted 15 November 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two genes encoding ClpL ATPase proteins were identified in a probiotic Lactobacillus rhamnosus strain, E-97800. Sequence analyses revealed that the genes, designated clpL1 and clpL2, share 80% identity. The clpL2 gene showed the highest degree of identity (98.5%) to a clpL gene from Lactobacillus plantarum WCFSI, while it was not detected in three other L. rhamnosus strains studied. According to Northern analyses, the expression of clpL1 and the clpL2 were induced during heat shock by >20- and 3-fold, respectively. The functional promoter regions were determined by primer extension analyses, and the clpL1 promoter was found to be overlapped by an inverted repeat structure identical to the conserved CIRCE element, indicating that clpL1 belongs to the HrcA regulon in L. rhamnosus. No consensus binding sites for HrcA or CtsR could be identified in the clpL2 promoter region. Interestingly, the clpL2 gene was found to be surrounded by truncated transposase genes and flanked by inverted repeat structures nearly identical to the terminal repeats of the ISLpl1 from L. plantarum HN38. Furthermore, clpL2 was shown to be mobilized during prolonged cultivation at elevated temperature. The presence of a gene almost identical to clpL2 in L. plantarum and its absence in other L. rhamnosus strains suggest that the L. rhamnosus E-97800 has acquired the clpL2 gene via horizontal transfer. No change in the stress tolerance of the ClpL2-deficient derivative of E-97800 compared to the parental strain was observed.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria have evolved a complex network of stress response pathways to promote their survival during environmental challenges. As a consequence of stress, a particular set of proteins, including molecular chaperones and proteases, is expressed (18, 22). Molecular chaperones can function under both physiological and stress conditions by preventing premature folding or by assisting the proper folding of proteins, while proteases dispose those which cannot be correctly folded (19, 21, 26). The most extensively studied bacterial ATP-dependent protease is the Clp protease complex of gram-negative Escherichia coli (20). It consists of a proteolytic subunit, ClpP, on which substrate specificity is mediated through association with Clp ATPase subunits ClpA, ClpC, and ClpX. These regulatory subunits, which alone can function as chaperones, are members of the ubiquitous Hsp100 family (54, 71). The Clp proteases have been found in several bacteria, and in some bacteria their importance for survival during stress has been reported (reviewed in reference 44). There is also growing evidence indicating the role of ClpP in virulence of common pathogens, such as Salmonella enterica serovar Typhimurium (27), Staphylococcus aureus (14), Listeria monocytogenes (15), and Yersinia enterocolitica (43). Furthermore, the ClpX, ClpB, ClpC, ClpE, and ClpL ATPases have also been frequently linked to virulence of bacteria (13, 14, 33, 34, 38, 39, 47, 48, 54).

Lactobacilli are commonly found in the gastrointestinal tracts (GIT) of humans and animals and widely used in manufacturing of various foods and feed products. Certain strains of Lactobacillus, i.e., probiotic lactobacilli, are increasingly studied due to their health-promoting benefits (46, 49, 52). To be classified as probiotic, a bacterium must fulfill many criteria; adhesion to intestinal mucosa is considered to be the most important (42). In addition, probiotics must withstand a variety of challenges associated with industrial processes and storage as well as the harsh conditions of the GIT. However, the mechanisms the probiotic bacteria employ for their survival or colonization of the intestinal mucosa are still poorly understood. Some stress-inducible genes of Lactobacillus, such as htrA and groESL of Lactobacillus helveticus (5, 60), groESL of Lactobacillus johnsonii (69), dnaK and the rrp (response regulator protein) operon of Lactobacillus sakei (35, 55), the csp-like genes of Lactobacillus bulgaricus (57), and the atpBEFHAGDC operon of Lactobacillus acidophilus (32), have been characterized. However, their significance in vivo, with the exception of the rrp genes encoding response regulators in L. sakei (35), has remained obscure.

Acquisition of genetic material via transposable elements is one of the strategies bacteria may employ to increase their stress tolerance, the emergence of multidrug resistance among pathogenic bacteria being one of the well-known consequences of such transmission (3, 6, 11, 23, 56, 63). The human or animal GIT is considered to be the site where the gene transmission is claimed to occur due to the conditions that promote gene transfer (50, 56). It has also been reported that mobilization of some transposable elements can be induced following exposure to a variety of stress conditions, including microaerobic environment, starvation, temperature upshift, UV light, erythromycin, and sodium acetate (8, 12, 17, 24, 37, 61, 64). Lactobacilli are known to carry a number of transposable elements (16, 41, 58, 59, 61, 68, 72), and the role of stress in triggering mobilization of such elements has recently been demonstrated in a Lactobacillus crispatus strain (61).

In this study, we report that the probiotic L. rhamnosus E-97800 carries two genes capable of encoding ClpL proteins. We further show that both of the clpL genes are heat induced and that the mobilization of the clpL2 gene can be triggered during prolonged incubation at elevated temperature.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains, media, and culture conditions.
Bacterial strains and plasmids used in this study are listed in Table 1. E. coli was grown in Luria-Bertani broth (Difco) supplemented with ampicillin (50 µg/ml) where appropriate. The Lactobacillus strains were routinely grown in MRS (9) at 37°C, and Lactococcus lactis was grown in M17 (62) supplemented with 0.5% glucose at 30°C without stirring.

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TABLE 1. Bacterial strains and plasmids used in this study

clpL1 and clpL2 expression was studied under different stress conditions which cause damage to proteins (high temperature and presence of ethanol, salt, and hydrogen peroxide [H₂O₂]). Also, the effect of low temperature, bile, and DNA damage on clpL1 and clpL2 expression was tested. Shortly, L. rhamnosus E-97800 cells were grown in MRS medium at 37°C until the optical density at 600 nm (OD₆₀₀) reached 0.4 to 0.5, at which point stress conditions were applied. The effect of temperature was studied by transferring the cells from 37 to 50 or 10°C. The effects of ethanol (7%, vol/vol), NaCl (4%, wt/vol; water activity, 0.98), bile (0.5%, wt/vol), H₂O₂ (0.002%, vol/vol) and the DNA-damaging agent mitomycin C (3 µM) were tested by adding each corresponding chemical to cells growing at 37°C. Samples of 1 ml were withdrawn before and 10 min after the heat, ethanol, NaCl, bile, and H₂O₂ treatments, and 30 min after the cold shock and mitomycin C treatments. For analysis of clpL1 and clpL2 expression as a function of growth, an overnight culture of L. rhamnosus E-97800 was diluted 1:1,000 in fresh MRS, followed by incubation at 37°C and withdrawal of 1-ml samples at different phases of growth. After centrifugation (18,000 x g) for 5 min at +4°C, the cell pellets were stored at –70°C until used for isolation of RNA and Northern blot analysis.

The genetic stability of clpL2 was studied as follows. A single colony of L. rhamnosus E-97800 grown on MRS agar was inoculated in 10 ml of MRS broth and incubated overnight at 37°C. The overnight culture (OD₆₀₀, 5) was diluted 1:100 in MRS broth, and three aliquots (10 ml each) were incubated overnight at 30, 37, or 45°C followed by seven serial passages and withdrawal of 1-ml samples. Samples were mixed with glycerol (15%, vol/vol) and stored at -70°C. The frozen stocks were streaked onto MRS agar plates, and the presence of the clpL1, clpL2, and pepX genes was tested by colony PCR.

Recombinant DNA techniques.
Standard recombinant DNA techniques were performed essentially as described by Sambrook and Russell (51). Restriction enzymes, DyNAzyme polymerase, and Smartladder DNA molecular weight marker were obtained from Promega (Madison, Wisc.), Finnzymes (Espoo, Finland), and Eurogentec (San Diego, Calif.), respectively. Plasmids were isolated from E. coli with the Wizard Plus DNA purification system (Promega). To prepare total DNAs (chromosome and plasmid) from Lactobacillus strains, 1 ml of stationary-phase cultures was pelleted by centrifugation and treated with mutanolysin (1 U µl^–1; Sigma-Aldrich Corp., St. Louis, Mo.) and lysozyme (5 µg µl^–1) in 20 mM Tris-HCl, 5 mM EDTA, 10 mM dithiothreitol, and 1 mM CaCl₂ (pH 7.5) at 37°C for 30 min followed by the addition of sodium dodecyl sulfate (0.5%) and incubation for an additional 30 min at 37°C. Total DNAs were purified by two phenol-chloroform/isoamylalcohol (25:24:1) extractions and resuspended in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

Cloning and sequence analyses of the clpL2 gene.
Degenerated oligonucleotides p1 and p2 (29), designed according to the conserved regions of the Clp ATPase-encoding genes, were used for amplification of 550-bp DNA fragments. The PCR products were cloned using pCR2.1-TOPO vector in E. coli TOP10F', and the resulting clones were sequenced. The entire clpL2 region was obtained using the Vectorette library kit according to the instructions provided by the manufacturer (Sigma-Aldrich Corp.). Sequencing was carried out using the ABI Prism dye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, Calif.) and the ABI Prism 310 sequencer (Applied Biosystems). Sequences were assembled and analyzed using the Sequencher 3.0 software (Applied Biosystems).

Database searches were performed using the search tool (BLAST) programs (2) from the National Center for Biotechnology Information's BLAST website (http://www.ncbi.nlm.nih.gov/BLAST/).

Southern and dot blot analyses.
For Southern blotting the total DNAs were digested with HindIII, separated by gel electrophoresis, and transferred to a nylon membrane (Hybond-XL; Amersham Biosciences, Piscataway, N.J.). For dot blotting, total DNAs (300 and 600 ng) were blotted onto a positively charged nylon membrane (Roche, Basel, Switzerland). The 850-bp probe DNA for clpL2 was amplified with primers p3 and p4 (Table 2) from L. rhamnosus E-97800, and the 550-bp clpL1 probe was obtained after EcoRI digestion of the plasmid pKTH5182 (Table 1). Primers p5 and p6 (Table 2) were used to amplify a 450-bp region including the IS1480-like sequence located upstream of clpL2 (Fig. 1). The probe DNA for the pepX gene (66) was amplified from L. rhamnosus E-97800 by PCR with primers p7 and p8 (Table 2) and used as a control probe for L. rhamnosus. The ISLpl1-specific probe was obtained from Lactobacillus plantarum HN38 (41) by PCR amplification with primer p9 that anneals to both arms of ISLpl1. The DNA probes were labeled with [-³³P]ATP (>92.5 TBq mmol^–1) using the Megaprime DNA labeling system (Amersham Biosciences) following the instructions provided. After hybridization the membranes were washed and scanned using the Molecular Imager GS-525 system (Bio-Rad). The membranes were stripped and reprobed according to the instructions provided by the manufacturer of the membrane (Amersham Biosciences).

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TABLE 2. Primers used in this study

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FIG. 1. Schematic presentation of the clpL2 gene region in L. rhamnosus E-97800. (A) Partial restriction map of the clpL gene and its flanking region. Horizontal arrows on both sides of clpL2 indicate the location and orientation of the inverted repeat sequences (IRL and IRR). Double-headed arrows indicate the probe target sequences (P1 and P2) for the putative truncated transposase and the ClpL2-encoding genes. (B) Partial nucleotide sequence with the predicted amino acid sequence of the clpL2 gene. The putative ribosome binding site (RBS) and the transcriptional start point are indicated by a dashed line and a vertical arrow, respectively. The promoter regions, –10 and –35, preceding the clpL2 gene are underlined. The inverted repeats (IRL and IRR) are shown with horizontal arrows, and the translation start and stop codons are boxed.

RNA methods.
Total RNA from L. rhamnosus E-97800 was obtained by disrupting the cell samples with glass beads ( 106 µm; Sigma) in a homogenizer (Vibrogen-Zellmühle; Edmund Bühler, Tübingen, Germany) for 4 min at +4°C. The RNA was purified using an RNeasy mini kit according to the instructions of the manufacturer (QIAGEN, Valencia, Calif.). Separation and transfer of RNA for Northern analysis were carried out by using a Latitude precast gel (1.25% Seakem Gold gel; Cambrex, Rockland, Maine) and a Hybond-XL (Amersham Pharmacia Biotech) nylon membrane according to the instructions provided. The DNA probes for clpL1 and clpL2 were labeled with ³³P as described above (Southern and dot blot analyses). The RNA molecular weight marker (0.28 to 6.58 kb) was obtained from Promega. Northern hybridization conditions were as described previously (65). The membranes were scanned and quantified using the Molecular Imager GS-525 system (Bio-Rad). The RNA amounts on the membrane were corrected after probing and quantifying the membrane with a 16S rRNA-specific probe obtained by PCR using primers p10 (70) and p11 (25) (Table 2).

The primer extensions were performed with total RNA and analyzed essentially as described earlier (36, 66) using the ALF Express DNA sequencer. The antisense Cy5-labeled primers p12 and p13 (Table 2) were used in primer extension, complementary to nucleotides located 180 to 201 and 193 to 217 bp downstream from the start codons of clpL1 and clpL2, respectively. Reverse transcription-PCR was carried out as follows. Total RNA (10 µg) isolated from cells withdrawn at the mid-exponential phase of growth and the primers p12 and p13 (Table 2) were used for cDNA synthesis of clpL1 and clpL2, respectively. The cDNA synthesis was carried out using a reverse transcription system (Promega) according to instructions provided by the manufacturer. The cDNA products were analyzed using an ALF Express sequencer (Amersham Biosciences) in parallel with sequencing reactions obtained with the same primers.

Colony PCR.
The presence of clpL and pepX in Lactobacillus strains was screened by colony PCR with primers p14 and p15 specific for the clpL1 gene, p16 and p17 for the clpL2 gene, and p7 and p8 for the pepX amplicon. The reaction mixtures (25 µl) containing cells picked from an L. rhamnosus colony were incubated at 95°C for 5 min before the amplification reaction was performed. The PCR conditions were 29 cycles of amplification consisting of denaturation at 95°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 1 min and 30 s, followed by one cycle at 72°C for 5 min. Amplification was performed using DyNAzyme polymerase (Finnzymes) and a DNA Engine (PTC-200) thermal cycler (MJ Research, Watertown, Mass.).

Physiological characterization of the L. rhamnosus strains.
Physiological characteristics of the parental L. rhamnosus E-97800 strain and its derivative devoid of the clpL2 gene (strain GRL1056) were studied as follows. The Bioscreen C monitoring system (Transgalactic Ltd.) was used to compare the growth rates and final cell densities at 37 and 44°C and in the presence of 0.15% (wt/vol) and 0.5% (wt/vol) bile at 37°C. Each well contained 300 µl of fresh growth medium inoculated with 0.1% of an overnight culture grown at 37°C. The colony-forming ability of the strains at 14 and 16°C was tested by plating appropriate dilutions of the exponential-phase cultures (MRS broth at 37°C) onto MRS plates, followed by incubation of the plates for 1 to 5 days under anaerobic conditions at the indicated temperatures. The colony-forming ability of the strains in the presence of puromycin (15 to 20 µg/ml), NaCl (4 to 5%, wt/vol), and rifampin (50 to 75 µg/ml) was tested by plating appropriate dilutions of the exponential-phase cultures onto MRS agar containing the corresponding chemicals at the indicated concentrations, followed by incubation of the plates under anaerobic conditions for 1 to 5 days at 37°C. The sensitivity of the strains to H₂O₂ was tested by seeding the MRS agar plates with the exponentially growing cells (OD₆₀₀, 0.5) and then placing the antibiotic disks loaded with 200 µl of 0.3% H₂O₂ in the center of each plate. Plates were incubated overnight at 37°C under anaerobic conditions. The relative sensitivity of the strains to H₂O₂ was investigated by comparing the sizes of growth inhibition zones surrounding the disks.

Nucleotide sequence accession numbers.
The sequence data of the L. rhamnosus E-97800 clpL2 gene have been assigned GenBank database accession number AJ749818. The sequence data of the L. rhamnosus E-97800 clpL1 promoter region have been assigned GenBank database accession number AJ749817.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of the clpL2 gene region.
Degenerate primers previously utilized to obtain partial coding regions of the clpC, clpB, and clpE genes in L. lactis (29) were used for amplification of a PCR product from the L. rhamnosus strain E-97800. The 550-bp PCR product obtained was subcloned using pCR2.1-TOPO vector (Invitrogen) in E. coli TOP10F', and 29 clones with an insert were analyzed by sequencing. As a result, 15 clones were found to carry an insert with homology to the Clp ATPase-encoding genes (data not shown). One of the plasmid clones was found to contain an insert sharing 96% identity with a corresponding fragment of the ClpL-encoding gene, here designated clpL1, in L. rhamnosus strain RW-9595M (45), whereas the rest of the clones were shown to carry inserts with 98% identity to a clpL gene previously identified in L. plantarum (30). The complete nucleotide sequence of this clpL gene, here designated clpL2, and its flanking regions was obtained using the Vectorette library kit.

Sequence analysis of the clpL2 region of L. rhamnosus E-97800 revealed the presence of a 2,112-bp open reading frame. The clpL2 gene was found to be flanked by repeats with high sequence homology (>90%) to terminal inverted repeats, inverted repeat left (IRL) and inverted repeat right (IRR), of the recently characterized IS30-related functional insertional element ISLpl1 of L. plantarum HN38 (41) (Fig. 1). A BLAST search conducted at http://www.ncbi.nlm.nih.gov/BLAST using the deduced amino acid sequence of ClpL2 revealed 98% identity to the L. plantarum WCFS1 clpL gene product (30) and 81% identity to the plasmid-encoded ClpL of L. lactis (28). The clpL2 gene region in E-97800 is surrounded by sequence elements similar to different transposable elements, including ISLpl1, the first functional IS element characterized in L. plantarum (41) and IS1480 from Xanthomonas campestris (NC_003902.1) (Fig. 1).

Detection of clpL1, clpL2, ISLpl1, IS1480, and pepX by Southern analyses.
Total DNAs isolated from L. rhamnosus E-97800, L. rhamnosus GG, L. rhamnosus ATCC 7469, L. rhamnosus 1/6, L. plantarum ATCC 14917, and Lactobacillus paracasei ATCC 25302 were digested with HindIII and analyzed by Southern hybridization with probes derived from the clpL1, clpL2, IS1480-like, ISLpl1, and pepX sequences (Fig. 2). The clpL2-specific probe hybridized to 4.0- and 6.0-kb fragments of the HindIII-digested DNA samples from L. rhamnosus E-97800 and L. plantarum ATCC 14917, respectively, while no signals were obtained in samples from other strains. The clpL1-specific probe was found to hybridize to DNA fragments of 4.0, 1.6, and 0.6 kb in samples from L. rhamnosus E-97800. The clpL1-specific probe was found to contain one HindIII restriction site, and according to the sequence analysis of the published clpL1 sequence of L. rhamnosus RW-9595M (45), the probe was expected to hybridize to HindIII fragments of 585 bp and 1.582 kb in samples from L. rhamnosus strains. The 4.0- and 6.0-kb signals obtained with the clpL1-specific probe in samples from L. rhamnosus E-97800 and L. plantarum ATCC 14917, respectively, presumably result from unspecific binding of the clpL1 probe to clpL2-containing fragments. The probe derived from the IS1480-like sequence hybridized only to samples from L. rhamnosus 1/6 and L. rhamnosus E-97800, whereas the ISLpl1-probe hybridized to samples from L. paracasei ATCC 25302, L. rhamnosus E-97800, and L. plantarum ATCC 14917. The pepX-derived control probe was found to hybridize to samples from the L. rhamnosus strains.

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FIG. 2. Distribution of two clpL genes and transposase-related elements in Lactobacillus strains. (A) Ethidium bromide-stained agarose gel of HindIII-digested total DNAs isolated from L. rhamnosus E-97800 (lane 2), L. rhamnosus GG (lane 3), L. rhamnosus ATCC 7469 (lane 4), L. rhamnosus 1/6 (lane 5), L. plantarum ATCC 14917 (lane 6), and L. paracasei ATCC 25302 (lane 7) prior to blotting. A Smartladder DNA molecular weight marker (lane 1) was applied to determine the sizes of the hybrids. (B) Southern blots of HindIII-digested total DNAs with clpL1, clpL2, IS1480-like, ISLpl1, and pepX probes.

Expression analyses of clpL1 and clpL2.
The expression of clpL1 and clpL2 in response to different stress conditions was studied by Northern blotting (Fig. 3). The clpL1 and clpL2 probes detected single transcripts of 2.4 and 2.3 kb, respectively, which were in good agreement with the size predicted from the DNA sequences of the clpL1 gene of L. rhamnosus RW-9595M (45) and the clpL2 gene of E-97800, thus indicating that both genes form a monocistronic transcriptional unit. The clpL1 transcript was detected only in RNA samples isolated from heat-shocked cells (Fig. 3B), indicating that the expression of clpL1 is strictly controlled. The amount of clpL2 transcript appeared to be more constant at most of the growth conditions investigated; however, 3- and 2.5-fold increases in the amount of clpL2 transcript were observed after heat shock and addition of hydrogen peroxide, respectively (Fig. 3A). The mean values for the relative induction factor of the clpL2 expression by 10 min of heat shock and H₂O₂ treatments calculated from at least two independent experiments were 2.6 (±0.39) and 2.25 (±0.25), respectively (data not shown). Differences in the relative amounts of the clpL1 and clpL2 transcripts during growth are shown in Fig. 3C. The clpL1 transcript was only weakly detected in samples harvested at earlier phases of growth, whereas the clpL2 transcript was detected at all growth phases (Fig. 3C). The different expression patterns obtained for clpL1 and clpL2 indicate that the probes used were specific for each clpL gene under Northern hybridization conditions. The Northern analyses of clpL1 and clpL2 expression at different time points following heat shock (Fig. 4) revealed that clpL2 transcription is only moderately (2.6-fold) induced and is slowly rerepressed 20 min after application of stress. In contrast, the expression of clpL1 was more strongly induced (>20-fold), the maximum expression level being reached 30 min after shifting the cells to 50°C (Fig. 4).

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FIG. 3. Expression of the clpL1 and clpL2 genes in L. rhamnosus E-97800. Northern blot analyses with (A) clpL1- and (B) clpL2-specific probes. Total RNAs were isolated from cells growing in MRS before (lane 1) and after exposure to heat stress (lane 2), ethanol (lane 3), NaCl (lane 4), cold shock (lane 5), bile (lane 6), H2O2 (lane 7), and mitomycin C (lane 8). (C) Growth curve of L. rhamnosus E-97800 and Northern blot analyses of RNA samples using the clpL1- and clpL2-specific probes. Cell samples for RNA isolation are indicated by filled circles and numbered 1 to 7. The sizes of mRNAs were estimated according to an RNA molecular weight marker (Promega) which was run in parallel with RNA samples, cut off from the gel, and stained with ethidium bromide.

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FIG. 4. Northern blot analyses of clpL2 (A) and clpL1 (B) expression in L. rhamnosus E-97800 during heat stress. Total RNAs were isolated from cells growing in MRS before (0') and 5, 10, 20, and 30 min after application of heat stress at 50°C. The bar graph indicates the relative expression levels at each time point calculated by dividing the clpL-specific signal by the 16S rRNA-specific signal. Values and error bars represent averages and standard errors of results from two independent experiments.

Primer extension.
Primer extension analyses using the primers p12 and p13, complementary to the 5' end of clpL1 and 5' end of clpL2, respectively, were used to locate the 5' ends of the clpL1- and clpL2-specific transcripts of mid-exponential-phase growing cells before and after heat shock (+50°C, 10 min). The clpL2 extension product in both the control and heat-stressed cells (data not shown) corresponded to the location of a promoter resembling the prokaryotic consensus –35 and –10 sequences (Fig. 1). No clear extension products were obtained in the RNA samples isolated from the nonstressed cells with the primer complementary to the clpL1 5' end (data not shown). However, the clpL1 extension product obtained from RNA isolated from heat-stressed cells (data not shown) corresponded to the location of a promoter resembling the prokaryotic consensus –35 and –10 sequences (Fig. 5).

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FIG. 5. Schematic presentation of the clpL1 5' region. The promoter region (–35 and –10) is underlined, and the ribosomal binding sequence (RBS) is indicated by a dashed line. The transcription start point and the inverted repeats resembling the conserved CIRCE element are shown with vertical and horizontal arrows, respectively.

Genetic stability of the clpL genes and location of clpL2 in L. rhamnosus E-97800.
Southern analyses with clpL1-, clpL2-, and pepX-specific probes indicated that in total DNA samples isolated from L. rhamnosus E-97800 culture grown for several generations (seven growth cycles) at 45°C, the intensity of the clpL2-specific signal was clearly weaker than the signal obtained from culture grown at 37°C, whereas the clpL1-and pepX-specific signals were at the same level (data not shown). To confirm the instability of clpL2 at high temperature, we plated appropriate dilutions of the parental culture (grown overnight at 37°C) and the cultures grown for seven (7-day culture) serial growth cycles at 30, 37, and 45°C on MRS agar plates and investigated the individual clones for the presence of clpL1, clpL2, and pepX by PCR (data not shown). Analyses of the PCR products revealed the presence of the three genes in all six colonies tested from the parental and 7-day cultures grown at 30 and 37°C, and the presence of clpL1 and pepX genes in all six colonies tested from the 7-day culture grown at 45°C, while three colonies from the 7-day culture grown at 45°C were found to be devoid of clpL2. One of the colonies devoid of clpL2 was designated GRL1056 and subjected to Southern and dot blot hybridization to confirm the loss of clpL2 (Fig. 6).

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FIG. 6. DNA-DNA hybridization analyses of the clpL2 curing from L. rhamnosus E-97800 and GRL1056. (A) Southern blot of the HindIII-digested total DNAs isolated from the wild-type L. rhamnosus E-97800 (lane 1) and from the clpL2 cured L. rhamnosus GRL1056 (lane 2) with the clpL2- and pepX-specific probes. (B) Dot blot analyses of total DNAs (300 and 600 ng) isolated from L. rhamnosus E-97800 (1) and GRL1056 (2) with the clpL2- and pepX-specific probes.

Since the clpL2 gene appears to be mobilized in a relatively short time period at high temperature and its orthologue is borne by a plasmid in L. lactis (28), we asked whether clpL2 is plasmid borne. Several attempts to isolate plasmid DNA from E-97800 grown at different temperatures (30 to 45°C) resulted in isolation of a single 14-kb cryptic plasmid that according to Southern analysis does not carry clpL2 (data not shown). Thus, we conclude that clpL2 is chromosomally located.

Physiological characterization of strain GRL1056.
The growth characteristics of GRL1056 and the parental strain (E-97800) were compared by measuring the generation times of both strains using the Bioscreen C monitoring system. The generation times of GRL1056 and E-97800 calculated from six parallel cultures growing at 37°C were 98 (±4) and 121 (±6) min, respectively (data not shown), while the generation times for GRL1056 and E-97800 growing at 44°C were 120 (±6) and 131 (±2) min, respectively (data not shown), indicating that GRL1056 has a faster growth rate than E-97800 at 37 and 44°C. Based on these results, ClpL2 was found to be nonessential for L. rhamnosus E-97800 during growth at moderate or high temperature.

The effects of various physiological conditions and/or chemical agents on the colony-forming abilities of the parental E-97800 strain and the GRL1056 strain were also studied (see Materials and Methods). Based on the results obtained, no significant differences in the stress tolerance between the two strains were observed (data not shown). Thus, the physiological condition by which ClpL2 adds a selective advantage to L. rhamnosus E-97800 remains to be found.

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In this work, we have identified two genes, clpL1 and clpL2 encoding members of the ubiquitous AAA(+) superfamily of ATPases (53) in the probiotic L. rhamnosus E-97800. The sequence similarity search revealed that the clpL2 gene is almost identical to a gene present in the genome of L. plantarum WCFS1 (30), while the corresponding gene from three other L. rhamnosus strains was not found by Southern analyses. To our knowledge, there are no published reports of other Lactobacillus strains with multiple clpL genes. The attempts to isolate other members of the Clp ATPase family were not successful, as cloning of the PCR products in E. coli resulted in 15 clones, of which 14 were found to carry an insert specific to clpL2, while only one clone represented a sequence specific to clpL1. This biased result could be due to more efficient binding of the primers to clpL2 than to clpL1 or indicate that the copy number of the clpL2 gene is higher than that of the clpL1 gene. In an L. lactis strain, the clpL gene located on a cryptic plasmid (28) shares over 90% identity with the clpL2 gene from E-79800, which, together with the fact that clpL2 is lost at high temperature, led us to speculate that clpL2 may be located on a cryptic plasmid also in L. rhamnosus E-79800. However, according to Southern analysis, the clpL2 gene is not plasmid borne.

According to the transcriptional analyses, the expression of the clpL2 gene is induced by heat shock and also by treatment with hydrogen peroxide in E-97800, whereas the expression of clpL1 was induced only by heat stress. Additionally, the relative amount of clpL1 transcript was found to be more dependent on growth phase than that of clpL2. According to Northern blot analysis, the highest level of clpL1 transcript was found at the end of the exponential growth phase. In Streptococcus pneumoniae, the ClpL protein has been shown to function as a chaperone and to be important under heat stress conditions and for virulence gene expression (33). Furthermore, the expression of clpL of S. pneumoniae has been shown to be regulated by CtsR (7). Orthologs of CtsR have been identified in a number of gram-positive bacteria, and CtsR has been shown to bind to well conserved DNA binding sites present in the promoter regions of its target genes (10, 31). In L. lactis, genes encoding the Clp ATPase family proteins and ClpP have been demonstrated to belong to the CtsR regulon (65). The presence of CtsR in L. rhamnosus E-97800 has been supported by the presence of a consensus CtsR binding site in the promoter region of clpP in this strain (A. Suokko et al., unpublished results). However, while no conserved CtsR binding site could be identified in the upstream regions of clpL1 or clpL2, an inverted repeat structure containing the conserved bacterial CIRCE sequence (TTAGCACTC-N9-GAGTGCTAA) (40) was localized in the clpL1 promoter region (Fig. 5). Since the CIRCE element is the well-established operator sequence for HrcA repressor, it is tempting to speculate that clpL1 belongs to the HrcA regulon in L. rhamnosus. To our knowledge, this is the first report indicating a direct role of HrcA in regulating the expression of a gene encoding the Clp/HSP100 family of ATPases in a gram-positive organism. In Oenococcus oeni, the clpL gene was recently shown to be part of a dicistronic operon with clpP located upstream (4). In this organism the expression of clpL was found to be regulated by two promoters; one CtsR-regulated promoter was located upstream of clpP while another, CtsR-independent, promoter was located in the intergenic region of clpP-clpL (4). Furthermore, the amount of clpL mRNA in O. oeni was also found to be regulated at the level of mRNA stability (4). The difficulty of detecting the clpL1-specific transcripts by Northern or primer extension analyses in other than heat shock conditions indicates that the expression of clpL1 is strictly controlled, and possibly, besides the HrcA-dependent pathway, may involve other mechanisms as well. Indeed, when mapping the 5' ends of the clpL1 mRNA isolated from nonstressed cells we obtained some weak primer extension signals indicating shorter extension products than those obtained from the heat-shocked cells (data not shown). This may imply that these shorter primer extension products present the 5' ends of the processed mRNA or, alternatively, result from premature termination of the reverse transcription elongation reaction. Interestingly, the 5' region of clpL1 (83% identity over a 71-nucleotide overlap) resembles the intergenic region of glnRA-pepX that is suggested to be a site where mRNA processing occurs (66). The secondary structure analysis revealed that the 5' end of the clpL1 transcript is able to form a stable (–18.2-kcal/mol) stem and loop structure similar to the glnRA-pepX intergenic region. In the case of clpL2, it appears that HrcA and CtsR are not directly involved in regulation of the gene expression, as no consensus DNA binding sites for these, or any other known regulators, could be found in the promoter region of clpL2.

The clpL2-like gene has previously been found as part of a structure resembling a transposon on a plasmid in L. lactis (28). The codon usage of the clp-like genes and the IS element in this lactococcal plasmid suggests that the transposition event occurred many generations ago and that they both give a phenotypic advantage to their host (28). The low G+C content (0.40%) of clpL2 compared to clpL1 (0.49%) and to the average L. rhamnosus entries at GenBank (0.48%) together with the high identity of clpL2 to the L. plantarum clpL gene suggests that the clpL2-carrying element has been transposed quite recently. It should, however, be noted that the clpL2 counterpart in L. plantarum WCFS1 (30) appears not to be part of a transposable element (data not shown). Among questions remaining to be answered are the function of the conserved clpL2 flanking inverted repeats and whether they play any role in transposing events or participate in regulatory functions. While we could not amplify the regions located upstream and downstream of the flanking repeats from the clpL2-negative clones after the 7-day passages at 45°C (data not shown) by PCR, we suggest that the mobilized unit has been larger than clpL2 and the flanking repeats. The clpL2 flanking repeats are almost identical to those genetically linked to the TraISLpl1 transposase-encoding gene, which is part of the first functional IS element characterized in L. plantarum (41). Southern hybridization with the ISLpl1-specific probe (Fig. 2) results in two signals in L. rhamnosus strain E-97800, one most probably corresponding to the 3.9-kb HindIII fragment sequenced in this study. It remains to be studied whether the other signal indicates the presence of a functional copy of the ISLpl1 in L. rhamnosus E-97800. However, these repeats appear to be well conserved in a number of lactobacilli (41), suggesting that they are able to withstand selective pressure and may be functional also in L. rhamnosus E-97800. Nevertheless, the E-97800 strain appears to have acquired the clpL2 gene as part of a mobile element, presumably from another Lactobacillus strain and possibly to increase its evolutionary fitness in response to environmental challenges. Whatever the conditions that have resulted in acquisition of such determinants are and what the physiological role of the ClpL2 in this particular L. rhamnosus strain is remain to be studied.

 
We gratefully acknowledge I. Palva for valuable discussions. We thank our project partners J. Mättö and M. Saarela for collaboration and helpful discussions. We thank L. Paulin and S. Suomalainen for running of the ALF Express sequencer and for help with the primer extension analyses. Bacterial strain L. plantarum HN38 was kindly provided by F. Bringel.

This work was supported by the Academy of Finland (project no. 53908), TEKES (40079/01), Oy Fazer Ab, Oy Sinebrychoff Ab, and an ABS graduate school scholarship to Aki Suokko.

 
* Corresponding author. Mailing address: Division of Microbiology and Epidemiology, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, 00014 University of Helsinki, Helsinki, Finland. Phone: (358) 9 19 15 70 57. Fax: (358) 9 19 15 70 33. E-mail: pekka.varmanen{at}helsinki.fi.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, April 2005, p. 2061-2069, Vol. 71, No. 4
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