Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Supplemental material 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 Denou, E. Articles by Brüssow, H. Search for Related Content PubMed PubMed Citation Articles by Denou, E. Articles by Brüssow, H.

 Previous Article  |  Next Article 

Journal of Bacteriology, September 2008, p. 5806-5813, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.01802-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Role of Prophage for Genome Diversification within a Clonal Lineage of Lactobacillus johnsonii: Characterization of the Defective Prophage LJ771 ^,

Emmanuel Denou,^1,2, Raymond David Pridmore,^1, Marco Ventura,^3, Anne-Cécile Pittet,¹ Marie-Camille Zwahlen,¹ Bernard Berger,¹ Caroline Barretto,¹ Jean-Michel Panoff,² and Harald Brüssow^1*

Nestlé Research Center, Nestec Ltd., P.O. Box 44, CH-1000 Lausanne 26, Switzerland,¹ Laboratoire de Microbiologie Alimentaire, IBFA-ISBIO, Université de Caen, F-14032 Caen cedex, France,² Department of Genetics, Anthropology and Evolution, University of Parma, Parco Area delle Scienze 11/a, 43100 Parma, Italy³

Received 14 November 2007/ Accepted 22 May 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Two independent isolates of the gut commensal Lactobacillus johnsonii were sequenced. These isolates belonged to the same clonal lineage and differed mainly by a 40.8-kb prophage, LJ771, belonging to the Sfi11 phage lineage. LJ771 shares close DNA sequence identity with Lactobacillus gasseri prophages. LJ771 coexists as an integrated prophage and excised circular phage DNA, but phage DNA packaged into extracellular phage particles was not detected. Between the phage lysin gene and attR a likely mazE ("antitoxin")/pemK ("toxin") gene cassette was detected in LJ771 but not in the L. gasseri prophages. Expressed pemK could be cloned in Escherichia coli only together with the mazE gene. LJ771 was shown to be highly stable and could be cured only by coexpression of mazE from a plasmid. The prophage was integrated into the methionine sulfoxide reductase gene (msrA) and complemented the 5' end of this gene, creating a protein with a slightly altered N-terminal sequence. The two L. johnsonii strains had identical in vitro growth and in vivo gut persistence phenotypes. Also, in an isogenic background, the presence of the prophage resulted in no growth disadvantage.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Microarray analyses have demonstrated that there is substantial diversity within bacterial species with respect to gene content. Consequently, a bacterial "species" has to be defined within the framework of population genetics, and the bacterial clone takes a cornerstone position in the discussion of bacterial evolution (24). Associating the different genotypes with specific phenotypes is now an important task for understanding the ecological physiology in a given bacterial species (13). A clear-cut clonal population structure has been documented for some pathogenic bacteria. As much of medical epidemiology deals with the wax and wane of bacterial clones over short time scales (decades) (47), lateral gene transfer by mobile DNA elements like plasmids, transposons, and particularly prophages (5, 7) has shaped the evolution of bacterial virulence.

Prophages have been intensively studied in pathogenic lactic acid bacteria (LAB). For example, Streptococcus pyogenes prophages encode tissue-degrading enzymes that contribute to the spread of infection and superantigens that paralyze the immune system by overstimulation. Without exaggeration it can be argued that the pathogenic potential of S. pyogenes is the result of prophage-encoded virulence factors (6). However, prophages not only are found frequently in pathogenic LAB (9) but also are found in starter bacteria, like Lactococcus lactis used in cheese fermentation (10), and in gut commensals. Lactobacillus species isolated from human subjects, like Lactobacillus johnsonii (41), Lactobacillus plantarum (39), Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei (38), all contained numerous prophages. Microarray analyses of lactobacilli (2, 29) revealed that prophages contributed up to one-half of the variable gene content (8, 40). Interestingly, the presence of prophage has been associated with a growth phenotype switch in Streptococcus thermophilus, an important yogurt starter (31). In contrast, not much is known about the role of prophages in Lactobacillus commensals isolated from the alimentary tract. The gut microbiota is a very complex community that is characterized by very high numbers of cells (10¹² cells per g of colon contents) and great species diversity (currently estimate, >400 different bacterial phylotypes) (14, 19). Despite this complexity, the gut ecosystem is very stable in terms of its bacterial composition, and it is impossible for probiotic organisms and difficult for pathogens to become established (20). When the gut microbiota is disturbed by antibiotics, the same commensal gut microbiota becomes established again. The selection pressure in the gut is therefore likely to be immense.

Prophage integration can confer new phenotypic traits to a lysogenic host via positive or negative lysogenic conversion. The latter can occur by inactivation of bacterial genes due to prophage integration. The former is achieved by expression of phage genes in the lysogen that can positively affect the fitness of the host. Along with a few genes from the lysogeny module (repressor and immunity genes), prophages from LAB frequently express extra genes located between the phage lysin gene and the right attachment site, attR (38, 41, 42). In contrast to findings for pathogenic LAB, the extra phage genes from commensal LAB so far do not have informative annotations (8).

In the present study we investigated two closely related but independent isolates of the probiotic (17, 21, 22, 46) gut commensal L. johnsonii (32) to examine genotypic and phenotypic differences. The strains differed mainly in prophage content. The prophage behaved as a selfish DNA element elaborating a toxin-antitoxin system to ensure its maintenance but otherwise had only small phenotypic consequences for the lysogen.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Bacterial strains and culture conditions. L. johnsonii NCC533 and NCC2761 are independent stool isolates from healthy adults and were obtained from the Nestlé Culture Collection. They were grown in MRS (Difco, Basel, Switzerland) with 2% glucose as the carbon source, to which filter-sterilized cysteine (final concentration, 0.05% [wt/vol]) was added after autoclaving. Cultures were incubated at 37°C under anaerobic conditions using AnaeroGen (Oxoid, Basingstoke, United Kingdom) and anaerobic jars. L. lactis MG1363 was grown at 30°C in M17-glucose, and Escherichia coli strains EC101 and TOP 10 (Invitrogen) were grown in brain heart infusion or LB medium at 37°C with shaking.

Antibiotics were used at the following concentrations: erythromycin (Em), 10 µg/ml for Lactobacillus and Lactococcus and 150 µg/ml for E. coli; and chloramphenicol (Cm), 10 µg/ml for Lactobacillus and Lactococcus and 25 µg/ml for E. coli.

Molecular techniques. General molecular cloning techniques, restriction enzyme analysis, and transformation of E. coli by CaCl₂-induced competence were performed as described previously (33). Plasmid DNA from E. coli and L. lactis were isolated with a QIAprep spin miniprep kit (Qiagen). Recombinant DNA molecules were introduced into L. lactis (37) and Lactobacillus by electrotransformation. Genomic DNA was isolated as described previously (2) from cells which were grown in 10 ml of MRS at 37°C until the optical density at 600 nm (OD₆₀₀) was 1.0. The cells were suspended in 1 ml of Tris-EDTA (10:1) supplemented with lysozyme (1 mg/ml) and mutanolysin (25 µg/ml) at 37°C for 30 min with shaking (200 rpm). RNase A (Sigma) was added at a concentration of 5 µg/ml, and this was followed by incubation for 30 min and then by addition of 1% sodium dodecyl sulfate, 50 mM EDTA, and 14 µg/ml proteinase K (Merck) and incubation at 55°C for 60 min. The genomic DNA was extracted and purified by using phenol-chloroform extraction. Pulsed-field gel electrophoresis was performed as described previously (42). Southern and Northern blot hybridizations were performed using previously described protocols (41). All of the primers used in this study are described Table S1 in the supplemental material, and plasmids were constructed as described in Table S2 in the supplemental material.

Comparison of NCC533 and NCC2761 genomes. A partial genome sequence of NCC2761, comprising about 10⁶ bp, was obtained by using the Sanger sequencing technique with large DNA fragments digested with rare-cutting restriction enzymes cloned into the pUC19 vector and primer walking. The contigs were associated with a Paracell computer. Nucleotide differences were compared using the EMBOSS diffseq program, and comparison at the nucleotide level was performed by alignment with the "Artemis Comparison Tool" (http://www.sanger.ac.uk/Software/ACT/). Comparative genomic hybridization was performed as described previously (2).

Prophage LJ771 was sequenced using inverted PCR fragments, with walking from the phage-genome borders until the gap could be closed by long-range PCR. The total sequence was compiled and analyzed using the Lion bioSCOUT bioinformatics package. Nucleotide and predicted amino sequences were compared with sequences in public sequence databases (GenBank, EMBL, PIR-Protein, Swiss-Prot and PROSITE) using BLAST (1) at the NCBI and FASTA (25). PCRs specific for all phage and bacterial attachment sites of prophage LJ771 from L. johnsonii NCC2761 were performed using primers described in Table S1 in the supplemental material.

Integration plasmid construction and integration. The LJ771 int gene and attB were amplified from strain NCC2761 chromosomal DNA using the Expand high-fidelity (HiFi) PCR system (Roche Molecular) and primers I1 and I2, and the amplicon was digested with restriction enzymes EcoRI and PstI and ligated into plasmid pDP600 (13) digested with the same enzymes to obtain plasmid pDP818. Plasmid DNA was isolated from strain MG1363 and used to transform strain NCC533, and transformants were selected at 32°C (permissive temperature for plasmid replication) on MRS plates containing Cm. Plasmid integration was achieved by cultivating the strain in MRS broth containing Cm at 37°C, which yielded strain NCC533::pDP818 (NCC533-Cm^r). The integration was proven by PCR.

Em resistance gene integration in LJ771. To create a derivative of NCC2761 genetically marked in prophage LJ771 for stability assays, we exchanged the LJ771_042 gene (LJ771 genes are designated LJ771_001, LJ771_002, etc.) encoding the minor tail protein by using the pDP600-Ery allele exchange replacement system (13) (see Table S2 in the supplemental material). Plasmid DNA was isolated and used to transform L. johnsonii NCC2761, and transformants were selected on MRS plates containing Em at 32°C. Allele exchange was performed by growing a culture at 37°C for five passages and then plating it to obtain single colonies on MRS plates containing Em. Replica streaking of colonies onto Em- and Cm-containing plates resulted in identification of an erythromycin-resistant, chloramphenicol-sensitive strain, which was designated NCC9322 (NCC2761-Em^r).

Expression of the mazE and pemK genes in E. coli. A bidirectional terminator situated between the LJ1125 and LJ1126 genes was amplified using primers AT1 and AT2 introducing HindIII and XhoI restriction sites, respectively. The amplicon was digested with HindIII plus XhoI and ligated into pNZ124-Sph (pNZ124 with an expanded cloning array containing the BglII, PvuII, BamHI, MluI, PstI, EcoRI, BsrGI, SphI, NheI, SacI, HindIII, and XhoI restriction sites) previously digested with the same enzymes to obtain plasmid pNZ124-term. The L. lactis pgiA promoter was amplified using primers AT3 and AT4 introducing SphI and BglII restriction sites, respectively. The amplicon was digested with SphII plus BglII and ligated into pNZ124-term to obtain plasmid pDP778. The mazE gene (LJ771_055) was HiFi amplified using primers AT5 and AT6 introducing SphI and SacI restriction sites, respectively. Likewise, the pemK gene (LJ771_056) was amplified using primers AT7 plus AT8, and both the mazE and pemK genes were amplified using AT5 plus AT8. The amplicons were digested with SphI plus SacI and ligated into pDP778 digested with the same enzymes. A clone of the mazE gene in pDP778 whose sequence was verified was designated pDP830.

Expression of antitoxin and LJ771 curing. NCC9322 was transformed with pDP830, and colonies were selected at 37°C on MRS plates containing Cm. A preculture was prepared in MRS broth supplemented with Em plus Cm at 37°C, cultivated for 10 serial passages and in MRS broth containing Cm, and plated to obtain single colonies. An Em-sensitive colony was cured of plasmid pDP830, and the NCC2761 derivative cured of prophage LJ771 was designated NCC9323.

Functional analysis of the pemK gene. The predicted toxin gene was cloned under the control of the tightly regulated promoter in pBAD/HisA (Invitrogen). The pemK gene was HiFi amplified using primers AT7 and AT8 introducing NcoI and KpnI restriction sites, respectively. The amplicon was digested with NcoI plus KpnI and ligated into pBAD/HisA digested with the same enzymes to obtain pDP981. The sequence of plasmid pDP981 was verified, and this plasmid was used to transform E. coli strain TOP 10. A culture of TOP 10(pDP981) was inoculated (1%) into fresh LB broth containing ampicillin and incubated at 37°C. At an OD₆₀₀ of approximately 0.2 arabinose was added to a concentration of 0.2%, the cultivation was continued, and the OD₆₀₀ and cell viability were monitored.

Overexpression of MsrA. N-terminal poly(His)-tagged forms of MsrA (NCC533) and MsrA' (NCC2761) were obtained by overexpression in E. coli (see Tables S1 and S2 in the supplemental material).

Growth properties. L. johnsonii NCC2761, NCC9322, or NCC9323 or a 1:1 mixture of NCC9322 and NCC9323 was grown in a Sixfors fermentor system. Growth curves were obtained by using four individual 500-ml vessels (Infors, Bottmingen, Switzerland). Anaerobic conditions were obtained by bubbling the medium with CO₂ overnight prior to inoculation. All fermentations were carried out at 37°C with constant agitation (150 rpm), and all preparations were inoculated to obtain an OD₆₀₀ of approximately 0.07. The optical density was determined, and the pH was kept constant at pH 6.5 by addition of 1 M NaOH. The 1:1 mixture of NCC9322 and NCC9323 was also plated at time zero and at 7.5 and 24 h to obtain single colonies on MRS plates, and 200 individual colonies were streaked onto MRS plates containing Em to distinguish between the two strains.

Gut persistence experiments. In order to monitor the bacteria in mice, we used an NCC533 derivative with an integrated Cm^r marker, NCC533::pDP818 (see above), and the NCC2761 derivative NCC9322 labeled with an Em^r marker in prophage LJ771 (see above). The strains were grown overnight at 37°C, harvested by centrifugation at 3,000 x g for 10 min, washed with fresh MRS, and suspended at a concentration of 5 x 10¹⁰ CFU/ml. Fresh bacterial suspensions were prepared daily. Conventional female C3H mice whose average age was 8 weeks were given access to a sterilized standard diet (UAR 03-40; Villemoisson, France) and water ad libitum. L. johnsonii NCC2761 or NCC533 was administrated by intragastric gavage to groups of five animals for three successive days. Fecal samples were collected daily, weighed, suspended in 0.5 ml of Ringer solution, and mechanically homogenized. The colonization dynamics of strains were determined by culture of fecal pellets. Dilutions were plated on selective media described above (supplemented with Cm or Em) and incubated at 37°C for 2 days before enumeration. The plating procedure was repeated twice for each fecal sample.

Nucleotide sequence accession number. The GenBank accession number for the L. johnsonii LJ771 prophage sequence is NC_010179.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Prophage LJ771 genomics. L. johnsonii strains NCC533 and NCC2761 had identical pulsed-field gel electrophoresis restriction patterns except for the shift of a 70-kb SmaI fragment in NCC533 to a 110-kb SmaI fragment in NCC2761 (43) (data not shown). The size difference was shown to be the result of acquisition of a 40,876-bp Sfi11-like prophage by strain NCC2761, which was designated LJ771 (Fig. 1B). The closest relatives of LJ771 are the L. gasseri prophages LgaI (38) and K5Ca (GenBank accession number NQ320509 [S. Pavlova and L. Tao, Oral Biology, University of Illinois at Chicago]) (Fig. 1A). At the DNA sequence level, prophage LJ771 could be aligned over 59% of its length with the genome of prophage LgaI (see Fig. S1 in the supplemental material). The two phages shared 89% DNA identity over the aligned genome segments.

View larger version (49K): [in this window] [in a new window]

FIG. 1. Genome analysis of L. johnsonii prophage LJ771. (A) Alignment of the genome maps of L. gasseri prophages K5CA and Lga1 with that of LJ771. The open reading frames are indicated by arrows. Open reading frames whose products share significant (E value, <10–11) amino acid sequence identity are linked by different colors; the color scale is shown at the top (the highest level of identity is indicated by yellow). In each case the top number indicates the amino acid sequence identity (expressed as a percentage), and the bottom number indicates the length of the alignment (in amino acids). The vertical lines indicate the positions of tRNA genes. (B) Annotated genome map of prophage LJ771. The suggested modular structure is indicated by colors (red, lysogeny genes; orange, DNA replication genes; green, DNA packaging and head genes; yellow, head-to-tail genes; blue, tail genes; pink, tail fiber genes; violet, lysis genes). Selected genes are annotated for orientation. (C) Positions of the probes used in the Northern blots (Fig. 2), indicated by black lines. The red lines indicate the positions of in vitro-transcribed NCC2761 genes as revealed by microarray analysis.

Prophages LJ771 and LgaI are the most closely related pair of phages currently known in gram-positive bacteria which are derived from two clearly separate host species (2). In comparison, the two L. gasseri prophages could be aligned over 73% of their genome lengths (Fig. 1A) and exhibited 93% sequence identity for the aligned DNA.

The alignment of prophages LJ771 and LgaI was especially good for the structural gene module, but it was relatively poor for the genes located between the DNA replication and DNA packaging modules (Fig. 1A). Directly downstream of the DNA replication module, LJ771 had more genes, including a gene duplication (orf14 and orf16, whose products share 81% amino acid identity). Next to the four tRNA genes (the Glu, Glu, Phe, and Cys genes) LJ771 had fewer genes than the L. gasseri prophages.

Prophage gene expression. Total mRNA from broth-grown uninduced NCC2761 was analyzed by Northern blot hybridization. From mitomycin C-induced cells we obtained only poor-quality mRNA preparations that were unsuitable for Northern blot analysis. We used 10 different probes covering the lysogeny, DNA replication, and DNA packaging regions, two regions of the structural module, and the lysis cassette (Fig. 1C). Only prophage genes next to the left and right attachment sites were expressed. Prominent 0.9- and 1.8-kb transcripts were detected with an LJ771_002-specific probe (Fig. 2). orf2 encodes a lipoprotein found in Lactobacillus and Streptococcus phages, and its position suggests that it is a superinfection exclusion gene (sie) (27). As the LJ771_001 (integrase int) gene gave only a very weak 0.9-kb signal and the LJ771_004 (repressor cI) gene gave a weak 1.8-kb signal (data not shown), we tentatively placed the 1.8-kb mRNA at orf4, orf3 (encoding a conserved phage protein, a putative repressor), and orf2 (length, 1.6 kb). Primer extension experiments showed that a clear start site was located ahead of LJ771_002 at bp 2141 (Fig. 2); the 900 bp signal, therefore, corresponds mainly to an LJ771_002 transcript (length, 800 bp). Phage genes sie and cI are normally expressed during the lysogenic cycle (42).

View larger version (58K): [in this window] [in a new window]

FIG. 2. (Top) Northern transcription analysis of prophage LJ771. Northern blot hybridization was performed with total RNA from in vitro-grown L. johnsonii NCC2761 isolated at OD600 of 0.05. 0.1, 0.7, and 1.2 (lanes 1 to 4, respectively). The blots were probed with radiolabeled PCR products covering orf2, the putative phage lysin gene (orf54), and the putative antitoxin-toxin genes (orf55 and orf56). The molecular sizes calculated for the hybridization signals are indicated on the right. (Bottom) Primer extension experiments were performed with RNA extracted from L. johnsonii NCC2761 using oligonucleotides targeted to the 5' end of orf2 and the mazE-pemK genes.

Likewise, two strong transcripts with estimated lengths of 0.5 and 0.7 kb (Fig. 2) were detected with probes from open reading frames located between the phage lysin gene (lys) and the right attachment site (attR) (Fig. 1). The product of the first gene downstream of lys contained a MazE domain of a growth regulator; the closest database match was with a suppressor of the growth-inhibiting protein ChpA from Erwinia (32% amino acid identity; E value, 10^–5). The second gene encoded a PemK domain known to be a growth inhibitor of L. plantarum plasmid p256. PemK belongs to a toxin-antitoxin system that ensures that low-copy-number extrachromosomal DNA is not lost from a cell (36). The closest database match was with ChpA from Bacillus annotated as a ppGpp-regulated growth inhibitor (44% amino acid identity; E value, 10^–15). The L. gasseri prophages lacked this putative toxin-antitoxin system.

A weak primer extension signal indicated that a transcription start site was located 18 bp ahead of mazE at bp 40,509 (Fig. 2). Since no signal was obtained with a lys-specific probe (Fig. 2) and since the combined length of the mazE-like and pemK-like genes is 0.65 kb, we tentatively located the transcript over mazE and pemK.

Microarray analysis showed that there was expression of LJ771_001 and LJ771_002, as well as mazE and pemK, during in vitro growth of NCC2761 (data not shown).

Prophage integration and excision. Using PCR, all possible attachment sites could be amplified from NCC2761 DNA, including the unoccupied bacterial DNA (attB), the nonintegrated circular prophage DNA (attP), and the two prophage-bacterium transition zones (attR and attL) from the lysogen (Fig. 3). In contrast, only attB was amplified from NCC533 DNA (Fig. 3). The coexistence of the four attachment sites in NCC2761 suggests that for prophage LJ771 there is concomitant excision from and integration into the bacterial chromosome.

View larger version (32K): [in this window] [in a new window]

FIG. 3. Prophage integration. (A) Schematic diagram of the four different phage attachment sites, showing the locations of the primers used for the PCR and the predicted lengths of the amplification products (indicated on the right). Bacterial DNA is indicated by a straight line, and phage DNA is indicated by a dashed line. The primers used are described in the primer list. (B) Agarose gel analysis of the PCR amplification products for the specified att sites of the L. johnsonii strains indicated. Lanes M contained 400- and 200-bp molecular size markers.

However, phage DNA packaging into extracellular phage particles could not be detected in NCC2761. Potential phage particles from the cell-free supernatant of bacterial cultures were concentrated by sedimentation using an ultracentrifuge. No phage particles were visualized by negative staining electron microscopy. As this method is not particularly sensitive (there must be more than 10⁶ PFU/ml to observe virions), we used PCR as a more sensitive detection technique. The "virion pellet" was treated with DNase to digest free, nonencapsidated phage DNA, and "phage DNA" was extracted from the "phage" pellet after extraction with phenol-chloroform. A primer pair prepared from the structural gene module of the LJ771 prophage did not amplify DNA (data not shown).

To test whether prophage LJ771 has an unusually excision-proficient integrase, we cloned the phage integrase gene together with the adjacent attP site. A plasmid containing this PCR fragment was correctly integrated into the attB site from NCC533, as demonstrated by the amplification of the attL and attR sites in NCC533::pDP818 (Fig. 3). However, neither attP nor attB sites could be amplified from the cells, suggesting that the phage integrase was able to integrate but was unable to excise the plasmid from the chromosome. In this respect the prophage LJ771 integrase resembles that of L. lactis prophage TP901-1 (4, 11). As the integrated plasmid contained an antibiotic resistance marker, we could trace the stability of the construct. After 10 serial passages (corresponding to about 100 generations) in the absence of antibiotic, all 300 colonies tested were still Cm^r. Apparently, an additional gene is needed for efficient excision. Notably, the excisionase gene from L. lactis prophage TP901-1 was located downstream of the Cro repressor gene (4). However, none of the LJ771 genes in that location gave an excisionase match.

Prophage integration disrupts the msrA gene. Alignment of the NCC533 and NCC2761 sequences showed that prophage LJ771 had integrated into the 5' end of the msrA gene, which encodes a peptide methionine sulfoxide reductase (15). The role of this enzyme in E. coli has been described as essential, because it protects the cell against oxidative stress by reducing oxidized methionine residues in proteins (16, 30, 34). The msrB gene is one of the three Lactobacillus reuteri genes specifically induced during gut colonization (45). Strains with an inactivated msrB gene were also less competitive than the wild-type bacterium in the mouse gut (44).

An alignment of all four attachment sites revealed two perfectly conserved DNA regions that were 54 and 33 bp long (Fig. 4A). This is an unusual situation since alignments of att sites normally reveal only one conserved region, the core site. In fact, LJ771 used the 33-bp segment as a core site for the homologous recombination between phage and bacterial DNA. Prophage integration thus disrupts the msrA gene. On the attL side, the sequence starts with the authentic 5' upstream sequence of msrA, and it is followed by codons for the correct 28 N-terminal amino acids of MsrA; however, it is then interrupted by a stop codon when it enters the prophage DNA. On the attR side, the 54-bp duplication of the bacterial DNA sequence on the phage ensures that there is a short correct 5' upstream sequence for the msrA gene, followed by a coding region whose product differs from the NCC533-encoded MsrA by five amino acid changes in the N-terminal part of the protein, resulting in the variant MsrA' protein (Fig. 4B). We expressed both the MsrA and MsrA' genes from a heterologous promoter in E. coli (but not in L. johnsonii), which yielded stable proteins that were the expected molecular weight (data not shown). Since an E. coli MsrA protein with a truncated N terminus was still able to reduce methionine sulfoxide (3), we suspected, but did not verify experimentally, that both forms were enzymatically active. Notably, under various in vitro growth conditions the msrA gene was not detectably transcribed in NCC533 (12). Furthermore, when the NCC533 promoter from msrA was inserted upstream of an indicator gene (β-galB), no expression was detected under conditions known to induce msrA expression, like exposure to H₂O₂, paraquat, or 10 g/liter NaCl. These experiments were conducted both with E. coli and with L. johnsonii (data not shown). Obviously, insertion of a prophage into a protein-encoding gene is selection neutral if the gene is not expressed.

View larger version (23K): [in this window] [in a new window]

FIG. 4. Integration site of prophage LJ771. (A) Sequence alignment of the attL, attR, attB, and attP sites obtained from the sequenced PCR products identified the 33-bp core sequence and a 54-bp repeat region. (B) Deduced N-terminal part of the authentic MsrA protein (attB), the modified MsrA' protein (attR), and the truncated MsrA' protein (attL).

Genotypic and phenotypic comparison of NCC533 and NCC2761. In addition to prophage LJ771, strains NCC533 and NCC2761 differed in a short DNA inversion, in a shift in the position of a transposase gene, in a DNA rearrangement involving two genes, and in changes in less than 0.1% of the base pairs (see Fig. S2 in the supplemental material).

The two strains showed similar growth and acidification properties in MRS medium under aerobic and anaerobic conditions (data not shown). Also, competition experiments with the two strains revealed that after 20 serial passages the ratio of the strains was unchanged (data not shown). To test for an effect of lysogeny in vivo, we force-fed NCC533-Cm^r or NCC2761-Em^r separately to groups of five mice either with or without concomitant antibiotic administration. No significant difference in the gut persistence phenotypes of the L. johnsonii strains was seen (data not shown). Finally, four mice received a mixture of the two strains without resistance markers, and the ratio of the strains was determined by a strain-specific PCR (data not shown). No difference in the relative proportions was observed during this competition experiment.

LJ771 stability and curing by mazE complementation. We then investigated prophage LJ771 stability by introducing an Em resistance cassette into the prophage to monitor the prophage stability. After approximately 100 generations of growth in the absence of antibiotic selection, 300 colonies were tested for Em resistance. All colonies were Em resistant, demonstrating that prophage LJ771, even when it is present in its circular form, is surprisingly stable.

We therefore considered the possibility that the mazE and pemK homologues represent a selfish antitoxin-toxin maintenance system for the circular prophage DNA. We transformed NCC9322 with a plasmid containing the cloned mazE ("antitoxin") gene in plasmid pDP830. When NCC9322 was then subcultured in the presence of Cm (plasmid selection), we observed that after 10 passages 10% of the colonies were Em^s. The resulting strain, NCC9323, also lacked prophage LJ771, as verified with a prophage-specific PCR (Fig. 3C). Apparently, when provided in trans with the "antitoxin" MazE, prophage LJ771 could be cured from NCC2761.

The prophage curing allowed us to test the phenotypic effect of the presence of prophage on an isogenic background. The growth and acidification kinetics of NCC9322 and NCC9323 were indistinguishable when the strains were grown separately. When both strains were grown in the same fermentor starting with a 1:1 ratio at the time of inoculation, we observed no change in the proportion over a 24-h observation period in three independent experiments (data not shown).

Toxin-antitoxin systems. To substantiate the role of the LJ771 mazE and pemK genes as a prophage maintenance system, we cloned the genes singly or together using the expression plasmid pDP778 and expression from the L. lactis pgiA promoter. We obtained sequence-validated clones for mazE (pDP830) and mazE plus pemK, but for pemK cloned alone all plasmids contained deletions in the pgiA promoter region. These data suggest that pemK expression was counterselected and deleterious for E. coli. We also cloned pemK under the control of the tightly arabinose-regulated promoter in pBAD/HisA. Addition of arabinose led to growth arrest and a 10-fold decrease in cell viability, which, however, was not attenuated by LJ771 mazE coexpression (data not shown). Whatever the precise role of the two prophage proteins, the available evidence suggests that they are used as a maintenance device like proteins in low-copy-number plasmids (18). It is surprising that only one other phage which contains a toxin-antitoxin gene cassette has been described (namely, E. coli phage P1). It is perhaps significant that P1 persists as a nonintegrated extrachromosomal circular prophage DNA in the lysogen (26).

Many putative toxin-antitoxin modules have been identified on bacterial chromosomes, and they were interpreted either as short-term metabolic stress managers in the case of amino acid starvation or oxidative stress (18) or as programmed-cell-death effectors that respond to crowding (23). However, the physiological role of the genes is not yet clear, since E. coli strains containing and lacking them (including one prophage-encoded cassette) did not differ in in vitro growth (35).

Conclusion. The major objective of the present study was to evaluate the role of prophages in lactobacilli. Lactobacilli are full of prophages, and these prophages contribute substantially to the strain-specific genes, but their physiological importance has not been defined yet. The findings reported in the present study suggest that some of the phages that we found in genome sequences of lactobacilli may represent genetic noise and do not cause harm. This conclusion does not exclude the possibility that other prophages may be beneficial. Prophage LJ771 has several peculiar characteristics, and its interaction with the bacterial host cannot be classified easily as beneficial coevolution, an arms race, or a neutral relationship. As it is not advisable to draw general conclusions from observations made with a single phage-bacterium system, more studies of the physiological role of prophages are needed to elucidate the role of prophage genes in phenotypic traits of commensal bacteria.

 
We thank Deborah Moine for her help with testing the in vitro growth properties of the prophage-cured NCC2761 derivative.

 
* Corresponding author. Mailing address: Nestlé Research Centre, Nutrition and Health Department/Food and Health Microbiology, CH-1000 Lausanne 26 Vers-chez-les-Blanc, Switzerland. Phone: 41 21 785 8676. Fax: 41 21 785 8544. E-mail: harald.bruessow{at}rdls.nestle.com

Published ahead of print on 30 May 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

E.D., R.D.P., and M.V. contributed equally to this work.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

1
1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
2
2. Berger, B., R. D. Pridmore, C. Barretto, F. Delmas-Julien, K. Schreiber, F. Arigoni, and H. Brüssow. 2007. Similarity and differences in the Lactobacillus acidophilus group identified by polyphasic analysis and comparative genomics. J. Bacteriol. 189:1311-1321.[Abstract/Free Full Text]
3
3. Boschi-Muller, S., S. Azza, and G. Branlant. 2001. E. coli methionine sulfoxide reductase with a truncated N terminus or C terminus, or both, retains the ability to reduce methionine sulfoxide. Protein Sci. 10:2272-2279.[CrossRef][Medline]
4
4. Breüner, A., L. Bronsted, and K. Hammer. 1999. Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1. J. Bacteriol. 181:7291-7297.[Abstract/Free Full Text]
5
5. Brüssow, H. 2007. Bacteria between protists and phages: from antipredation strategies to the evolution of pathogenicity. Mol. Microbiol. 65:583-589.[CrossRef][Medline]
6
6. Brüssow, H. 2007. The impact of phages on the evolution of bacterial pathogenicity, p. 267-300. In M. Pallen, K. E. Nelson, and G. M. Preston (ed.), Pathogenomics. ASM Press, Washington, DC.
7
7. Brüssow, H., C. Canchaya, and W. D. Hardt. 2004. Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol. Mol. Biol. Rev. 68:560-602.[Abstract/Free Full Text]
8
8. Canchaya, C., G. Fournous, and H. Brüssow. 2004. The impact of prophages on bacterial chromosomes. Mol. Microbiol. 53:9-18.[CrossRef][Medline]
9
9. Canchaya, C., C. Proux, G. Fournous, A. Bruttin, and H. Brüssow. 2003. Prophage genomics. Microbiol. Mol. Biol. Rev. 67:238-276.[Abstract/Free Full Text]
10
10. Chopin, A., A. Bolotin, A. Sorokin, S. D. Ehrlich, and M.-C. Chopin. 2001. Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations. Nucleic Acids Res. 29:644-651.[Abstract/Free Full Text]
11
11. Christiansen, B., L. Bronsted, F. K. Vogensen, and K. Hammer. 1996. A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1. J. Bacteriol. 178:5164-5173.[Abstract/Free Full Text]
12
12. Denou, E., B. Berger, C. Barretto, J.-M. Panoff, F. Arigoni, and H. Brüssow. 2007. Gene expression of commensal Lactobacillus johnsonii strain NCC533 during in vitro growth and in the murine gut. J. Bacteriol. 189:8109-8119.[Abstract/Free Full Text]
13
13. Denou, E., R. D. Pridmore, B. Berger, J.-M. Panoff, F. Arigoni, and H. Brüssow. 2008. Identification of genes associated with the long gut persistence phenotype of the probiotic Lactobacillus johnsonii strain NCC533 using a combination of genomics and transcriptome analysis. J. Bacteriol. 190:3161-3168.[Abstract/Free Full Text]
14
14. Eckburg, P. B., E. M. Bik, C. N. Bernstein, E. Purdom, L. Dethlefsen, M. Sargent, S. R. Gill, K. E. Nelson, and D. A. Relman. 2005. Diversity of the human intestinal microbial flora. Science 308:1635-1638.[Abstract/Free Full Text]
15
15. Etienne, F., D. Spector, N. Brot, and H. Weissbach. 2003. A methionine sulfoxide reductase in Escherichia coli that reduces the R enantiomer of methionine sulfoxide. Biochem. Biophys. Res. Commun. 300:378-382.[CrossRef][Medline]
16
16. Ezraty, B., R. Grimaud, M. El Hassouni, D. Moinier, and F. Barras. 2004. Methionine sulfoxide reductases protect Ffh from oxidative damages in Escherichia coli. EMBO J. 23:1868-1877.[CrossRef][Medline]
17
17. Fukushima, Y., S. Miyaguchi, T. Yamano, T. Kaburagi, H. Iino, K. Ushida, and K. Sato. 2007. Improvement of nutritional status and incidence of infection in hospitalised, enterally fed elderly by feeding of fermented milk containing probiotic Lactobacillus johnsonii La1 (NCC533). Br. J. Nutr. 98:969-977.[Medline]
18
18. Gerdes, K., S. K. Christensen, and A. Lobner-Olesen. 2005. Prokaryotic toxin-antitoxin stress response loci. Nat. Rev. Microbiol. 3:371-382.[CrossRef][Medline]
19
19. Gill, S. R., M. Pop, R. T. Deboy, P. B. Eckburg, P. J. Turnbaugh, B. S. Samuel, J. I. Gordon, D. A. Relman, C. M. Fraser-Liggett, and K. E. Nelson. 2006. Metagenomic analysis of the human distal gut microbiome. Science 312:1355-1359.[Abstract/Free Full Text]
20
20. Hooper, L. V., and J. I. Gordon. 2001. Commensal host-bacterial relationships in the gut. Science 292:1115-1118.[Abstract/Free Full Text]
21
21. Humen, M. A., G. L. De Antoni, J. Benyacoub, M. E. Costas, M. I. Cardozo, L. Kozubsky, K. Y. Saudan, A. Boenzli-Bruand, S. Blum, E. J. Schiffrin, and P. F. Perez. 2005. Lactobacillus johnsonii La1 antagonizes Giardia intestinalis in vivo. Infect. Immun. 73:1265-1269.[Abstract/Free Full Text]
22
22. Inoue, R., A. Nishio, Y. Fukushima, and K. Ushida. 2007. Oral treatment with probiotic Lactobacillus johnsonii NCC533 (La1) for a specific part of the weaning period prevents the development of atopic dermatitis induced after maturation in model mice, NC/Nga. Br. J. Dermatol. 156:499-509.[CrossRef][Medline]
23
23. Kolodkin-Gal, I., R. Hazan, A. Gaathon, S. Carmeli, and H. Engelberg-Kulka. 2007. A linear pentapeptide is a quorum-sensing factor required for mazEF-mediated cell death in Escherichia coli. Science 318:652-655.[Abstract/Free Full Text]
24
24. Lan, R., and P. R. Reeves. 2001. When does a clone deserve a name? A perspective on bacterial species based on population genetics. Trends Microbiol. 9:419-424.[CrossRef][Medline]
25
25. Lipman, D. J., and W. R. Pearson. 1985. Rapid and sensitive protein similarity searches. Science 227:1435-1441.[Abstract/Free Full Text]
26
26. Lobocka, M. B., D. J. Rose, G. Plunkett, M. Rusin, A. Samojedny, H. Lehnherr, M. B. Yarmolinsky, and F. R. Blattner. 2004. Genome of bacteriophage P1. J. Bacteriol. 186:7032-7068.[Abstract/Free Full Text]
27
27. Lucchini, S., F. Desiere, and H. Brüssow. 1999. Similarly organized lysogeny modules in temperate Siphoviridae from low GC content Gram-positive bacteria. Virology 263:427-435.[CrossRef][Medline]
28
28. Miller, J. H., K. Ippen, J. G. Scaife, and J. R. Beckwith. 1968. The promoter-operator region of the lac operon of Escherichia coli. J. Mol. Biol. 38:413-420.[CrossRef][Medline]
29
29. Molenaar, D., F. Bringel, F. H. Schuren, W. M. de Vos, R. J. Siezen, and M. Kleerebezem. 2005. Exploring Lactobacillus plantarum genome diversity by using microarrays. J. Bacteriol. 187:6119-6127.[Abstract/Free Full Text]
30
30. Moskovitz, J., M. A. Rahman, J. Strassman, S. O. Yancey, S. R. Kushner, N. Brot, and H. Weissbach. 1995. Escherichia coli peptide methionine sulfoxide reductase gene: regulation of expression and role in protecting against oxidative damage. J. Bacteriol. 177:502-507.[Abstract/Free Full Text]
31
31. Neve, H., W. Freudenberg, F. Diestel-Feddersen, R. Ehlert, and K. J. Heller. 2003. Biology of the temperate Streptococcus thermophilus bacteriophage TP-J34 and physical characterization of the phage genome. Virology 315:184-194.[CrossRef][Medline]
32
32. Pridmore, R. D., B. Berger, F. Desiere, D. Vilanova, C. Barretto, A. C. Pittet, M. C. Zwahlen, M. Rouvet, E. Altermann, R. Barrangou, B. Mollet, A. Mercenier, T. Klaenhammer, F. Arigoni, and M. A. Schell. 2004. The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc. Natl. Acad. Sci. USA 101:2512-2517.[Abstract/Free Full Text]
33
33. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
34
34. Tamburro, A., N. Allocati, M. Masulli, D. Rotilio, C. Di Ilio, and B. Favaloro. 2001. Bacterial peptide methionine sulphoxide reductase: co-induction with glutathione S-transferase during chemical stress conditions. Biochem. J. 360:675-681.[CrossRef][Medline]
35
35. Tsilibaris, V., G. Maenhaut-Michel, N. Mine, and L. Van Melderen. 2007. What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome? J. Bacteriol. 189:6101-6108.[Abstract/Free Full Text]
36
36. Tsuchimoto, S., Y. Nishimura, and E. Ohtsubo. 1992. The stable maintenance system pem of plasmid R100: degradation of PemI protein may allow PemK protein to inhibit cell growth. J. Bacteriol. 174:4205-4211.[Abstract/Free Full Text]
37
37. Vaughan, E. E., R. D. Pridmore, and B. Mollet. 1998. Transcriptional regulation and evolution of lactose genes in the galactose-lactose operon of Lactococcus lactis NCDO2054. J. Bacteriol. 180:4893-4902.[Abstract/Free Full Text]
38
38. Ventura, M., C. Canchaya, V. Bernini, E. Altermann, R. Barrangou, S. McGrath, M. J. Claesson, Y. Li, S. Leahy, C. D. Walker, R. Zink, E. Neviani, J. Steele, J. Broadbent, T. R. Klaenhammer, G. F. Fitzgerald, P. W. O'Toole, and D. van Sinderen. 2006. Comparative genomics and transcriptional analysis of prophages identified in the genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei. Appl. Environ. Microbiol. 72:3130-3146.[Abstract/Free Full Text]
39
39. Ventura, M., C. Canchaya, M. Kleerebezem, W. M. de Vos, R. J. Siezen, and H. Brüssow. 2003. The prophage sequences of Lactobacillus plantarum strain WCFS1. Virology 316:245-255.[CrossRef][Medline]
40
40. Ventura, M., C. Canchaya, D. Pridmore, B. Berger, and H. Brüssow. 2003. Integration and distribution of Lactobacillus johnsonii prophages. J. Bacteriol. 185:4603-4608.[Abstract/Free Full Text]
41
41. Ventura, M., C. Canchaya, R. D. Pridmore, and H. Brüssow. 2004. The prophages of Lactobacillus johnsonii NCC 533: comparative genomics and transcription analysis. Virology 320:229-242.[CrossRef][Medline]
42
42. Ventura, M., S. Foley, A. Bruttin, S. Chibani-Chennoufi, C. Canchaya, and H. Brüssow. 2002. Transcription mapping as a tool in phage genomics: the case of the temperate Streptococcus thermophilus phage Sfi21. Virology 296:62-76.[CrossRef][Medline]
43
43. Ventura, M., and R. Zink. 2002. Specific identification and molecular typing analysis of Lactobacillus johnsonii by using PCR-based methods and pulsed-field gel electrophoresis. FEMS Microbiol. Lett. 217:141-154.[CrossRef][Medline]
44
44. Walter, J., P. Chagnaud, G. W. Tannock, D. M. Loach, F. Dal Bello, H. F. Jenkinson, W. P. Hammes, and C. Hertel. 2005. A high-molecular-mass surface protein (Lsp) and methionine sulfoxide reductase B (MsrB) contribute to the ecological performance of Lactobacillus reuteri in the murine gut. Appl. Environ. Microbiol. 71:979-986.[Abstract/Free Full Text]
45
45. Walter, J., N. C. Heng, W. P. Hammes, D. M. Loach, G. W. Tannock, and C. Hertel. 2003. Identification of Lactobacillus reuteri genes specifically induced in the mouse gastrointestinal tract. Appl. Environ. Microbiol. 69:2044-2051.[Abstract/Free Full Text]
46
46. Yamano, T., H. Iino, M. Takada, S. Blum, F. Rochat, and Y. Fukushima. 2006. Improvement of the human intestinal flora by ingestion of the probiotic strain Lactobacillus johnsonii La1. Br. J. Nutr. 95:303-312.[CrossRef][Medline]
47
47. Zo, Y. G., I. N. Rivera, E. Russek-Cohen, M. S. Islam, A. K. Siddique, M. Yunus, R. B. Sack, A. Huq, and R. R. Colwell. 2002. Genomic profiles of clinical and environmental isolates of Vibrio cholerae O1 in cholera-endemic areas of Bangladesh. Proc. Natl. Acad. Sci. USA 99:12409-12414.[Abstract/Free Full Text]

---

Journal of Bacteriology, September 2008, p. 5806-5813, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.01802-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Brussow, H. (2009). The not so universal tree of life or the place of viruses in the living world. Phil Trans R Soc B 364: 2263-2274 [Abstract] [Full Text]  
• Romero, P., Croucher, N. J., Hiller, N. L., Hu, F. Z., Ehrlich, G. D., Bentley, S. D., Garcia, E., Mitchell, T. J. (2009). Comparative Genomic Analysis of Ten Streptococcus pneumoniae Temperate Bacteriophages. J. Bacteriol. 191: 4854-4862 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Supplemental material 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 Denou, E. Articles by Brüssow, H. Search for Related Content PubMed PubMed Citation Articles by Denou, E. Articles by Brüssow, H.