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Applied and Environmental Microbiology, December 2006, p. 7644-7651, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01428-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Expression of the Immunity Protein of Plantaricin 423, Produced by Lactobacillus plantarum 423, and Analysis of the Plasmid Encoding the Bacteriocin{triangledown}

C. A. Van Reenen, W. H. Van Zyl, and L. M. T. Dicks*

Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa

Received 21 June 2006/ Accepted 18 October 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Plantaricin 423 is a class IIa bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer. It has been previously determined that plantaricin 423 is encoded by a plasmid designated pPLA4, which is now completely sequenced. The plantaricin 423 operon shares high sequence similarity with the operons of coagulin, pediocin PA-1, and pediocin AcH, with small differences in the DNA sequence encoding the mature bacteriocin peptide and the immunity protein. Apart from the bacteriocin operon, no significant sequence similarity could be detected between the DNA or translated sequence of pPLA4 and the available DNA or translated sequences of the plasmids encoding pediocin AcH, pediocin PA-1, and coagulin, possibly indicating a different origin. In addition to the bacteriocin operon, sequence analysis of pPLA4 revealed the presence of two open reading frames (ORFs). ORF1 encodes a putative mobilization (Mob) protein that is homologous to the pMV158 superfamily of mobilization proteins. Highest sequence similarity occurred between this protein and the Mob protein of L. plantarum NCDO 1088. ORF2 encodes a putative replication protein that revealed low sequence similarity to replication proteins of plasmids pLME300 from Lactobacillus fermentum and pYIT356 from Lactobacillus casei. The immunity protein of plantaricin 423 contains 109 amino acids. Although plantaricin 423 shares high sequence similarity with the pediocin PA-1 operon, no cross-reactivity was recorded between the immunity proteins of plantaricin 423 and pediocin PA-1.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacteriocins produced by lactic acid bacteria (LAB) have received considerable attention as food preservatives and as potential replacements of antibiotics (28). These peptides are classified based on their primary structure, and one of the largest groups, class II, containing small unmodified heat-stable proteins, is further divided into four subgroups (20). The pediocin-like bacteriocins of class IIa have a strong inhibitory effect on Listeria spp. and share a conserved sequence motif in their N termini (9). Bacteriocin genes may be present on the chromosome or on plasmids and usually occur in one or two operons encoding a structural protein, an immunity protein that protects the producer from its own bacteriocin, and several translocation genes.

Plantaricin 423, a class IIa pediocin-like bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer, has been characterized at a protein and genetic level (31, 32). The genetic determinants for this bacteriocin are located on an 8,135-bp plasmid. The genes encoding plantaricin 423 are arranged in an operon with four open reading frames (ORFs) encoding the structural protein PlaA, putative immunity protein PlaB, and two putative membrane translocation proteins, PlaC and PlaD (31, 32). The first 31 amino acid residues in the N terminus of the prepeptide PlaA, which includes an 18-amino-acid leader peptide and 13 amino acid residues of the mature peptide (32), are identical to that of coagulin, pediocin PA-1, and pediocin AcH (22, 23, 25). PlaC and PlaD are 99 to 100% similar to the translocation proteins found in the operons of coagulin (CoaC and CoaD), pediocin PA-1 (PedC and PedD), and pediocin AcH (PapC and PapD). The C terminus of PlaA and the entire putative immunity protein PlaB differ completely from that described for coagulin and pediocins PA-1 and AcH (32). No sequence similarity was recorded between the putative immunity protein of plantaricin 423 and the immunity proteins of coagulin and pediocins PA-1 and AcH (22, 23, 25, 32).

Furthermore, a DNA fragment in the plantaricin 423 operon between plaB and plaC is identical to the DNA encoding the last 58 amino acids of the C terminus of PedB, the immunity protein of pediocin PA-1/AcH/coagulin. This DNA fragment is not part of any ORF within the plantaricin 423 operon.

In this paper, we describe the immunity protein of plantaricin 423 and report the complete sequence of plasmid pPLA4.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Microbial strains and media.
The strains used in this study are listed in Table 1. All LAB were cultured in MRS medium (Biolab Diagnostics, Midrand, South Africa) at 30°C. Escherichia coli DH5{alpha} transformed with pGEM-T Easy Vector (Promega, Madison, WI) was cultured at 37°C in Luria-Bertani medium (3) with ampicillin (100 µg ml–1). Escherichia coli DH5{alpha} transformed with pMG36e (30) was cultured in brain heart infusion medium (Biolab Diagnostics) supplemented with erythromycin (200 µg ml–1). Lactobacillus sakei and Pediococcus pentosaceus transformants were cultured in MRS broth containing 10 µg ml–1 erythromycin.


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  		TABLE 1. Bacterial strains and plasmids

 
DNA isolation and manipulation.
DNA resolution, restriction enzyme digests, ligations, E. coli transformations, and E. coli plasmid isolations were performed by using standard techniques (3). Enzymes used for restriction analysis and ligations were supplied by Roche Molecular Chemicals, Mannheim, Germany. Plasmid pPLA4 was isolated from L. plantarum 423, as described previously (4), and purified by CsCl density gradient centrifugation (3). DNA fragments generated by restriction enzyme digests or PCR were purified from agarose gels with the QIAquick PCR purification kit (QIAGEN Inc., Valencia, CA).

Primers used for amplification of the immunity gene (pedB) and different putative plaB genes are listed in Table 2. In three independent experiments, the immunity gene of pediocin PA-1 and two different DNA sequences of the putative immunity gene of plantaricin 423 were inserted directly after the P32 promoter and ribosome binding site on the E.coli/LAB shuttle vector pMG36e by using overlap PCR.


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  		TABLE 2. List of PCR primers for amplification of immunity proteins

 
Plasmid pPED1e was constructed as follows. Using pMG36e as template, primer pair 36e5' and 36ePedIF, which contained the first 18 bp of pedB (Table 2), was used to amplify promoter P32 and the start of pedB. This PCR product was used as a forward primer and PedIR (Table 2) was used as reverse primer, with pSRQ220 (Table 1) as template, to amplify a product that consisted of P32 and the pediocin immunity gene. This second PCR product was ligated into pGEM-T Easy Vector and transformed into E. coli DH5{alpha}. From this plasmid, a 520-bp fragment (P32-PED) was generated using EcoRI and PstI that was cloned into pMG36e.

To determine which section of DNA encodes the plantaricin 423 immunity protein, two plasmids were constructed. Plasmid pPLA1e was constructed to determine whether the plantaricin 423 immunity gene starts at the first ATG site after the end of plaA (Fig. 1A). Using pMG36e as template, a forward primer for the second PCR was amplified using primer pair 36e5' and 36ePlaIF (Table 2). This product and PedIR (reverse primer) with pPLA4 as template was used to amplify a product that also included the DNA sequence encoding the last 53 amino acid residues of PedB and which occurs in the operon of plantaricin 423. This second PCR product was ligated into pGEM-T Easy Vector and transformed into E. coli DH5{alpha}. From this resultant plasmid, a 731-bp fragment (P32-PLA/PED) was generated using EcoRI and PstI that was cloned into pMG36e.


Figure 1
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  		FIG. 1. A. DNA and putative protein sequence of the plantaricin 423 immunity protein. The superscript and subscript numbers in the protein sequence indicate the following: 1, boldface indicates the end of the plantaricin 423 structural gene; 2a and 2b, putative immunity protein in pPLA2e; 3a and 3b, putative immunity protein in pPLA1e; 4, partial sequence of PedB in the operon of plantaricin 423 (in italics); 5, boldface indicates the start of PlaC. B. Schematic comparison of genes on pPLA4 encoding plantaricin 423 (A), pSMB74 encoding pediocin AcH (25, 26) (B), the 5,595-bp fragment of the partially sequenced plasmid pSRQ11 encoding pediocin PA-1 (23) (C), and the 6,455-bp fragment of the partially sequenced plasmid pI4 encoding coagulin (22) (D). The images were drawn to scale using LBDraw for Windows (Lynnon Biosoft, Quebec, Canada).

 
Plasmid pPLA2e was constructed to determine whether plaB started at the first TTG site after plaA. A forward primer was first amplified using 36e5' and 36eTTG (Table 2) with pMG36e as template. This product was used as forward primer, with PlaIR (Table 2) as reverse primer and pPLA4 as template (Fig. 1A), to amplify a second possible immunity protein. This PCR product was ligated into pGEM-T Easy Vector and transformed into E. coli DH5{alpha}. From the resultant plasmid, a 528-bp fragment (P32-TTG/PLA) was generated using EcoRI and XbaI and was cloned into pMG36e.

PCR conditions.
DNA was amplified using a GeneAmpPCR Instrument System 9700 (Applied Biosystems, Foster City, CA). An initial denaturation step of 94°C for 4 min was used, followed by 35 cycles of 94°C for 1 min, 50°C for 30 s, and 72°C for 1 min. A final extension step at 72°C for 7 min was added, and samples were kept at 4°C until analyzed. Reaction mixtures (50 µl) containing TaKaRa Ex Taq (Takara Bio Inc., Shiga, Japan) together with the supplied 10x Ex Taq buffer and deoxynucleoside triphosphate mixture were used at concentrations recommended by the manufacturer. Final primer concentrations and MgCl2 were 1 µM and 3 mM, respectively.

Plasmid isolations from LAB transformants.
Transformants were grown overnight in 10 ml MRS supplemented with 10 µg ml–1 erythromycin. Cells from 4 ml were collected by centrifugation and resuspended in 200 µl solution A containing 20% sucrose, 10 mM Tris (pH 8), 10 mM EDTA, 50 mM NaCl, 20 mg ml–1 lysozyme (Roche), and 250 U ml–1 mutanolysin (Sigma-Aldrich, St. Louis, MO). The suspension was incubated at 37°C for 2 h, the last hour on a shaker. Solution B containing 200 mM NaOH plus 1% sodium dodecyl sulfate (400 µl) was added. To the clear lysate, 300 µl cold (4°C) solution C (5 M potassium acetate, pH 4.8) was added, and the samples were incubated on ice for 10 min.

The samples were centrifuged for 10 min at 10,000 x g, and the top phase was extracted twice with 750 µl phenol:chloroform:isoamylalcohol (25:24:1) and centrifuged for 3 min at 10,000 x g. After a final extraction with chloroform:isoamylalcohol (24:1) and centrifugation, 500 µl of the upper phase was precipitated with 2.5 volumes of cold 96% ethanol for 15 min at –80°C, centrifuged at 13,000 x g for 15 min, and washed. The pellet was resuspended in 20 µl Tris-EDTA buffer and 1 mg ml–1 (final concentration) RNase and incubated at 37°C for 15 min. Samples were analyzed on an 8% (wt/vol) agarose gel containing ethidium bromide (0.4 µg ml–1 final concentration).

Transformation of Lactobacillus sakei.
To prepare cells for electroporation, 100 ml of filter-sterilized MRS containing 1% (wt/vol) glycine and 40 mM threonine was inoculated with L. sakei DSM 20017T to an optical density at 600 nm (OD600) of 0.25 and grown to an OD600 of 0.6. Cells were collected by centrifugation at 1,500 x g for 5 min and washed twice in 100 ml SM buffer (952 mM sucrose and 3.5 mM MgCl2). The cells were resuspended in 1 ml SM buffer, and 100-µl aliquots were pipetted into sterile Eppendorf tubes. After addition of 5 µl DNA, the cells were transferred to 2-mm electroporation cuvettes. The Gene Pulser (Bio-Rad Laboratories, Hercules, CA) was set at 7.5 kV cm–1, 25 µF, and 800 {Omega}. Immediately after electroporation, cells were resuspended in 1 ml warm (30°C) medium containing, per 100 ml, 10 g MRS, 0.5 M sucrose, and 0.1 M MgCl2. Samples were incubated at 30°C for 2 h and plated onto MRS agar containing 10 µg ml–1 erythromycin. Plasmid isolations were performed on transformants.

Transformation of Pediococcus pentosaceus.
Five hundred milliliters MRS containing 0.5 M sucrose, 1% (wt/vol) glycine, and 40 mM threonine (prewarmed to 37°C) was inoculated (2%, vol/vol) with an overnight culture of P. pentosaceus PPE1.2. Cells were grown at 37°C without aeration to an OD600 of 0.2, collected by centrifugation, resuspended in cold 0.5 M sucrose and 10% (vol/vol) glycerol, and incubated on ice for 1 h. Cells were washed three times with 20 to 50 ml of the same ice-cold solution and resuspended in 0.5 ml cold buffer (1 mM K2HPO4-KH2PO4, 1 mM MgCl2, 0.5 M sucrose, pH 7.0). DNA (approximately 1 µg dissolved in sterile distilled water) was added to 100 µl cells and electroporated in 2-mm cuvettes at 25 µF, 2.2 kV, and 200 {Omega}. Cells were immediately transferred to 1 ml MRS containing 0.5 M sucrose, 20 mM MgCl2, and 2 mM CaCl2 and left on ice for 5 min. After addition of 1 ml of the same medium (prewarmed to 37°C), cells were incubated at 37°C for 2 to 4 h without aeration and then plated onto MRS agar with 10 µg ml–1 erythromycin.

Screening of bacteriocin activity.
Bacteriocin activity was determined using the spot-on-lawn (31), well diffusion, and microtiter plate (10) methods. For the spot-on-lawn and well diffusion methods, 10-ml volumes of MRS agar (0.7%) supplemented with 10 µg ml–1 erythromycin were inoculated with overnight cultures of L. sakei and P. pentosaceus, each transformed with pMG36e, pPED1e, pPLA1e, or pPLA2e. The cell suspensions were poured onto MRS agar (1.6%, wt/vol) containing 10 µg ml–1 erythromycin. For the spot-on-lawn method, 10 µl of twofold serial dilutions of plantaricin 423, pediocin PA-1, and mundticin ST (a class IIa bacteriocin produced by Enterococcus mundtii isolated from soya beans in our laboratory [unpublished data] similar to mundticin CRL 35 [29]), respectively, were spotted onto the surface. The plates were incubated at 30°C for 20 h and checked for inhibition zones. For the well diffusion tests, wells were punched into the agar and 30 µl undiluted plantaricin 423, pediocin PA-1, and mundticin ST was spotted into the wells. The plates were incubated at 30°C for 20 h and checked for inhibition zones. Lactobacillus sakei DSM 20017T and Pediococcus pentosaceus PPE1.2 transformed with pMG36e (with no immunity genes) and pPED1e (containing the known pediocin PA-1 immunity gene) were used as controls.

The microtiter assay was performed as previously described (10). For each bacteriocin, four rows of seven wells each of a microtiter plate were filled with 40 µl supernatant from the bacteriocin producer strain at twofold dilutions (undiluted to 1/64). An eighth well contained 40 µl inactivated supernatant of the appropriate bacteriocin. Bacteriocins were inactivated by incubating 1 ml of supernatant with 1 mg ml–1 (final concentration) of proteinase K (Roche) for 1 h at 37°C, followed by 3 min of boiling. To each row of eight wells, 160 µl MRS supplemented with 10 µg ml–1 erythromycin, containing cells at an OD595 of 0.1 of L. sakei transformed with pMG36e, pPED1e, pPLA1e, or pPLA2e, was added. Plates were incubated at 30°C, and OD595 readings were recorded every 3 h using a model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA). This experiment was done in triplicate.

DNA sequencing and analysis.
DNA was sequenced on an automatic sequencer (ABI Genetic Analyzer 3130Xl; Applied Biosystems SA [Pty] Ltd.) using big-dye terminator chemistry (Biosystems, Warrington, England). All immunity constructs were sequenced. The complete sequence of pPLA4 was determined by primer walking. Primers were obtained from UCT (Cape Town, South Africa) and Operon Biotechnologies (Cologne, Germany). All DNA fragments were sequenced at least twice. Computer alignment and BLAST (basic local alignment search tool) analysis of sequences were obtained using DNAMAN for Windows (Lynnon Biosoft, Quebec, Canada), BLAST (2), and Glimmer 2.02 (7).

Nucleotide sequence accession number.
The sequence of pPLA4 was submitted to GenBank (Los Alamos, NM) under accession no. AF304384.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the immunity gene of plantaricin 423.
Lactobacillus sakei DSM 20017T and P. pentosaceus PPE1.2 are sensitive to plantaricin 423, pediocin PA-1, and mundticin ST. Cells transformed with the shuttle vector pMG36e remained sensitive to all three bacteriocins. Twofold serial dilutions of Lactobacillus sakei transformed with pMG36e indicated sensitivity to the bacteriocins up to a dilution of 1/64 (plantaricin 423) and 1/128 (pediocin PA-1 and mundticin ST), while Pediococcus pentosaceus transformed with pMG36e was sensitive to the bacteriocins up to a dilution of 1/4. Lactobacillus sakei DSM 20017T and P. pentosaceus PPE1.2 transformed with pPED1e containing the gene coding for the pediocin PA-1 immunity protein (Table 1) were inhibited by plantaricin 423 but not by pediocin PA-1 or mundticin ST, indicating that the immunity protein of pediocin PA-1 did not protect the cells against plantaricin 423.

Two potential start codons occur after the plaA gene (Fig. 1A). Plasmid pPLA1e contained a sequence between plaA, from the first ATG codon, and plaC (from bp 48 to 596 in Fig. 1A), including the sequence homologous to the partial sequence of pedB, the immunity gene of pediocin PA-1, which occurs on pPLA4. Plasmid pPLA2e contained a DNA sequence starting with the first TTG codon (bp 27 in Fig. 1A) up to bp 377, the end of the ORF as predicted by the computer-generated translation of the sequence of pPLA4 (Fig. 1A). Lactobacillus sakei DSM 20017T and P. pentosaceus PPE1.2 transformed with pPLA1e and pPLA2e were resistant to plantaricin 423 but sensitive to pediocin PA-1 and mundticin ST, suggesting that the functional plantaricin 423 immunity gene, encoding a 109-amino-acid protein, was present in both constructs. Loss of zones of inhibition by the transformants are shown in Fig. 2. Similar results were recorded for Pediococcus pentosaceus transformants (data not shown), although zones of inhibition were smaller. Loss of inhibition as determined by microtiter plate assays for L. sakei transformants is depicted in Fig. 3.


Figure 2
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  		FIG. 2. Zones of inhibition on plates with overlays of (A) L. sakei transformed with pMG36e, (B) L. sakei transformed with pPED1e, (C) L. sakei transformed with pPLA1e, and (D) L. sakei transformed with pPLA2e. Wells were filled with 30 µl undiluted supernatant (corrected to pH 7) of plantaricin 423 (423), pediocin PA-1 (PA-1), and mundticin ST producers, respectively. Similar results were observed for P. pentosaceus transformants (data not shown).

 

Figure 3
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  		FIG. 3. Effect of (A) plantaricin 423, (B) pediocin PA-1, and (C) mundticin ST on growth of L. sakei transformed with pMG36e, pPED1e, pPLA1e, and pPLA2e, respectively, for 24 h, as determined by microtiter plate assays. Experiments were done in triplicate. Results represent growth in the presence of bacteriocin diluted 1/16. Similar results were observed for bacteriocins undiluted and diluted 1/2, 1/4, and 1/8.

 
Although no sequence similarity was recorded with DNA sequences in GenBank, significant sequence similarity was found with the immunity proteins of leucocin A (14), mesentericin Y105 (12), divercin V41 (24), enterocin A (27), OrfY (8), and the immunity protein of L. plantarum WCFS1 lacking a cognate bacteriocin (21) with BLAST analysis of the translated PlaB product (Fig. 4).


Figure 4
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  		FIG. 4. Comparison of the plantaricin 423 immunity protein PlaB to the immunity proteins of L. plantarum WCFS1 (21), leucocin A (LcaB) (14), mesentericin Y105 (MesI) (12), enterocin A (EntI) (27), divercin V41 (DvnI) (24), sakacin (OrfY) (8), and pediocin PA-1 (PedB) (23). Black shading indicates 100% homology, while gray shading indicates more than 75% sequence similarity.

 
Further analysis of pPLA4 revealed that, apart from the bacteriocin operons, the DNA sequence of plasmid pSMB74 (pediocin AcH [26]; accession no. NC 004832) and sections of pI4 (coagulin [22]; accession no. AF300457) and pSRQ11 (pediocin PA-1 [23]; accession no. M83924) do not show any similarity to the DNA sequence of pPLA4. Two further ORFs were detected in the sequence of pPLA4 encoding a putative conjugative mobilization (Mob) protein (ORF1), also known as a plasmid recombination enzyme (Pre), while ORF2 encodes a putative protein that initiates plasmid replication (Rep). The putative mobilization gene of pPLA4 shows extensive sequence similarity to the pMV158 superfamily of small mobilizable plasmids (Table 3).


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  		TABLE 3. Genes and their products on pPLA4

 
Although analysis of the pPLA4 Rep protein sequence revealed conserved domains, the percentage of sequence similarity to other Rep proteins was low (Table 3). BLAST analysis indicated that this protein is present in a wide range of gram-positive and gram-negative bacteria. Low levels of sequence similarity with other lactic acid bacteria were recorded (Table 3).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Identification of the immunity gene of plantaricin 423.
An immunity protein protects a bacteriocin-producing cell from its own bacteriocin (10). Loss of sensitivity by these organisms to the bacteriocin after transformation with an immunity protein gene indicates expression of the immunity protein.

Immunity proteins of bacteriocins range in size from 81 to 115 amino acid residues and vary between 5 and 85% in sequence similarity (9). Although the exact mode of action of immunity proteins is unknown (9, 10, 15, 16, 17), several studies have indicated that these proteins, which recognize and confer immunity only to their cognate bacteriocin or closely related bacteriocins, may act by disturbing bacteriocin aggregation (10) or pore formation (10) or by the interaction between a bacteriocin and a possible membrane-located receptor (10, 15, 16, 17).

Pediocin-like bacteriocins have recently been divided into three groups based on common sequence motifs in the C terminus, the occurrence of a disulfide bridge in the C terminus, and overall length of the peptide (10). Group 1 contains the bacteriocins enterocin A, divercin V41, pediocin PA-1, and sakacin P; group 2 contains leucocin A, mesentericin Y105, and plantaricin C19; and group 3 contains curvacin A, carnobacteriocin BM1, and enterocin P. Based on this classification, plantaricin 423 is a combination of groups 1 and 2. Homology to the first 13 amino acid residues of the N-terminal sequence of pediocin PA-1 and a putative disulfide bridge in the C-terminal part classifies plantaricin 423 as a member of group 1. However, the 24-amino-acid sequence in the C-terminal part of plantaricin 423 is similar in length and amino acid sequence to that recorded for bacteriocins in group 2.

Immunity proteins are classified into three different groups with large differences in protein sequences. The immunity proteins of group 2 bacteriocins and immunity proteins of bacteriocins that contain a C-terminal disulfide bridge, such as that recorded for leucocin A, mesentericin Y105, divercin V41, enterocin A, sakacin (OrfY), and pediocin PA-1, are classified as group A (10). Immunity proteins of group 1 bacteriocins and immunity proteins of bacteriocins lacking disulfide bridges in the C terminus are classified as group B. Immunity proteins of group 3 bacteriocins are classified in group C.

Cross-immunity for bacteriocins belonging to different groups does occur but only if their immunity proteins are of the same group (10). Even though the immunity proteins of pediocin PA-1 and plantaricin 423 belong to the same group (group A), the two proteins share no similarity or cross-reactivity.

The origin of pPLA4.
ORF1 and ORF2 in the pPLA4 sequence encode a putative mobilization protein and a putative replication initiation protein, respectively. These genes have been described on plasmids of a wide range of bacteria, including L. plantarum. Although several genes have been described on the plasmids pSMB74, encoding pediocin AcH (26), pSRQ11, encoding pediocin PA-1 (23), and plasmid pI4, encoding coagulin (22), low sequence similarity was detected with non-bacteriocin operon ORFs on pPLA4. The putative mobilization gene suggests that pPLA4 may be transferred to other bacterial hosts, provided that all conditions for conjugation are met (11). Small mobilizable plasmids are classified into four superfamilies, i.e., MOBQ, ColEI, pMV158, and CloDF13 (11). BLAST analysis of the gene product encoded by ORF1 indicates that this protein belongs to the pMV158 superfamily. Three motifs have been identified in these proteins (11). Motif I (HxxR) and motif II [NY(D/E)L] are located near the N terminus. A third motif is HxDExxPHMHxxxxP. The mobilization region usually consists of the relaxase gene and an upstream origin-of-transfer (oriT) site that overlaps the relaxase promoter. The oriT sites of the pMV158 superfamily are similar and include an inverted repeat with a stem consisting of 7 to 10 nucleotides and a 6-nucleotide loop. The Mob protein of pMV158 cleaves DNA at the nic site, which is located in the pMV158 loop (11). The three motifs and the potential nic site are located in the putative Mob protein on pPLA4. The percentage of sequence similarity of the putative Mob protein of pPLA4 with other Mob proteins is shown in Table 3. Highest similarity was observed with sequences recorded for other strains of L. plantarum. A mobilization protein has been identified on pI4, but it shares low sequence similarity with the putative Mob protein of pPLA4 (Table 3; Fig. 1B).

Circular plasmids may replicate by the rolling circle method or theta method, and plasmids using these methods are widespread (5, 19). Plasmids such as pLAB1000, pPB1, and pRS4 with Mob proteins homologous to the putative Mob protein of pPLA4 (Table 3) also contain sequences encoding Rep proteins (1, 6, 18). However, these proteins revealed no sequence similarity with the Rep protein of pPLA4. Plasmid LME300 has been tentatively classified as a theta-replicating plasmid (13). The Rep protein of pPLA4 shows highest sequence similarity to the Rep protein of pLME300. Further theta-like properties have also been identified on pPLA4, such as four 22-bp direct repeats 172 bp upstream of the rep gene. Replication (rep) regions are often not related, and plasmids may contain more than one (11).

Proteins RepB, OrfX, and Orf264 identified on pSMB74 (Fig. 1B) are involved in plasmid replication and are frequently found on plasmids replicating by the theta method. PemK and PemI are plasmid maintenance system proteins (26). These proteins are not homologous to any of the putative ORFs in pPLA4. BLAST analysis of the partial sequence of pSRQ11 also indicated the presence of PemK (Fig. 1B).

In conclusion, the immunity protein of plantaricin 423 consists of 109 amino acids and occurs within the putative operon encoding plantaricin 423. pPLA4, the plasmid carrying this operon, appears to be a mobilizable theta-replicating plasmid derived from L. plantarum.


   ACKNOWLEDGMENTS
 
We thank D. E. Rawlings, Department of Microbiology, University of Stellenbosch, South Africa, for critical assessment of the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa. Phone: 27-21-8085849. Fax: 27-21-8085846. E-mail: lmtd{at}sun.ac.za. Back

{triangledown} Published ahead of print on 20 October 2006. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, December 2006, p. 7644-7651, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01428-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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