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Applied and Environmental Microbiology, April 2004, p. 2240-2244, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2240-2244.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Prevalence of the Genes Encoding Propionicin T1 and Protease-Activated Antimicrobial Peptide and Their Expression in Classical Propionibacteria

Therese Faye,^1* Dag Anders Brede,¹ Thor Langsrud,¹ Ingolf F. Nes,¹ and Helge Holo^1,2

Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, N-1432 Ås,¹ Norwegian Dairies Association, Oslo, Norway²

Received 19 December 2003/ Accepted 22 December 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
The purpose of this study was to investigate the frequency of production of the bacteriocin propionicin T1 and the protease-activated antimicrobial peptide (PAMP) and their corresponding genes in 64 isolates of classical propionibacteria. This study revealed that these genes are widespread in Propionibacterium jensenii and Propionibacterium thoenii but absent from the remaining species of classical propionibacteria that were studied. The pro-PAMP-encoding gene (pamA) was found in 63% of the P. jensenii strains and 61% of the P. thoenii strains, and all of these strains displayed PAMP activity. The propionicin T1-encoding gene (pctA) was present in 89% of the P. thoenii strains and 54% of the P. jensenii strains. All P. thoenii strains containing the pctA gene exhibited antimicrobial activity corresponding to propionicin T1 activity, whereas only 38% of the pctA-containing P. jensenii strains displayed this activity. Sequencing of the pctA genes revealed the existence of two allelic variants that differed in a single nucleotide in six strains of P. jensenii; in these strains the glycine at position 55 of propionicin T1 was replaced by an aspartate residue (A variant). No strains harboring the A variant showed any antimicrobial activity against propionicin T1-sensitive bacteria. An open reading frame (orf2) located immediately downstream from the pctA gene was absent in three strains containing the G variant of propionicin T1. Two of these strains showed low antimicrobial activity, while the third strain showed no antimicrobial activity at all. The protein encoded by orf2 showed strong homology to ABC transporters, and it has been proposed previously that this protein is involved in the producer immunity against propionicin T1. The limited antimicrobial activity exhibited by the strains lacking orf2 further suggests that this putative ABC transporter plays an important role in propionicin T1 activity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antimicrobial peptides are important components of the innate defenses in all species of organisms (8, 21). Antimicrobial peptides produced by bacteria are generally referred to as bacteriocins, which include the posttranslationally modified lantibiotics. Bacteriocins and bacteriocin-like peptides are usually ribosomally synthesized, even though it has been shown that some antimicrobial peptides from bacteria are formed by degradation of larger proteins (5, 24, 25).

The classical propionibacteria are gram-positive bacteria with a long history of safe use in dairy fermentations, especially in the production of Swiss-type cheeses, where they are responsible for the formation of flavor and the characteristic eyes. It has also been proposed that propionibacteria may function as probiotic organisms for humans and animals (2, 17).

A variety of antimicrobial compounds are produced by propionibacteria; these compounds include propionic acid, acetic acid, and diacetyl in addition to the antimicrobial peptides (9). Although a number of antimicrobial activities of propionibacteria have been reported, only three antimicrobial peptides from the classical propionibacteria have been characterized so far at the molecular level (6, 7, 14, 15, 18, 22, 26). Antimicrobial peptides from propionibacteria may have potential as natural preservatives since these organisms are considered generally recognized as safe. Propionibacteria capable of producing antimicrobial peptides may therefore be useful for safeguarding dairy products from food-borne pathogens and spoilage bacteria. Antimicrobial compounds from propionibacteria may also contribute to accelerated ripening of cheeses (6). Furthermore, bacteriocins and bacteriocin-like peptides from classical propionibacteria might function as selective weapons against the pathogenic cutaneous propionibacteria (1).

In this study we screened 64 independent isolates of classical propionibacteria in order to determine the frequencies of bacteriocin production and the corresponding genes coding for propionicin T1 (6) and the protease-activated antimicrobial peptide (PAMP) (5).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and media.
The propionibacteria used in this study are shown in Table 1. The propionibacteria were propagated anaerobically in 10 ml of sodium lactate broth (SLB) (16) at 30°C for approximately 48 h. The indicator strain Lactobacillus sakei NCDO 2714 was propagated anaerobically in 10 ml of MRS (Oxoid) at 30°C for 24 to 48 h.

View this table: [in this window] [in a new window]

TABLE 1. Production of the antimicrobial peptides propionicin T1 and PAMP and distribution of their genes in a strain collection of classical propionibacteria

Screening for the production of propionicin T1 in agar plate assays.
Strains of propionibacteria were spotted as colonies on SLB agar plates and incubated for 5 or 12 days depending on the growth rate of the strain. Five-milliliter portions SLB soft agar mixed with 0.5-ml cultures of the indicator bacterium Propionibacterium acidipropionici ATCC 4965 in the late logarithmic growth phase (approximately 10⁸ CFU/ml) were then poured over the plates. After incubation for 48 h at 30°C, the plates were examined for zones of growth inhibition (in millimeters) surrounding the colonies.

Screening for the production of PAMP in agar plate assays.
One-microliter portions of a solution containing proteinase K (20 mg/ml) were spotted at the borders of colonies of potential pro-PAMP-producing bacteria. The agar plates were then incubated for 1 to 2 h at 30°C before 5 ml of soft agar mixed with a 0.5-ml culture of the indicator bacterium L. sakei NCDO 2714 was poured over each plate. After incubation for 24 h at 30°C, the agar plates were examined for zones of growth inhibition (in millimeters).

DNA sequence analysis.
Total DNA from propionibacteria was obtained by a freezing-heat shock method. Aliquots (1 ml) of a bacterial culture in the late logarithmic or early stationary growth phase were centrifuged for 10 min at 9,300 x g at room temperature. The cells were then resuspended in 100 µl of 1 mM Tris-HCl (pH 7.5)-0.1 mM Na₂EDTA (pH 8.0) and placed at –80°C for 15 min; this was followed by exposure to 100°C for 15 min. The cultures were then vortexed and frozen until they were used. Three microliters of an aliquot of lysed cells was used as the template in a PCR.

PCRs were carried out with a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, Conn.), and Taq polymerase was used as recommended by the manufacturer (Qiagen, Crawley, West Sussex, United Kingdom). The reactions (volume, 100 µl) were performed with 2.5 U of Taq polymerase and 100 pmol of each primer. The DNA primers used for amplification of the species-specific sequences in the 16S-23S rRNA intergenic spacer region (28), for the amplification and partial sequencing of the 16S rRNA genes (27), and for amplification and sequencing of the genes encoding propionicin T1 or pro-PAMP are shown in Table 2. The PCR conditions used for amplification of DNA fragments containing the propionicin T1 and pro-PAMP genes, the 16S rRNA genes, or the species-specific sequences in the 16S-23S rRNA intergenic spacer region included a hot start at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58 or 60°C for 30 s, and polymerization at 72°C for 2 or 3 min.

View this table: [in this window] [in a new window]

TABLE 2. Specific primers used for PCR amplification of the 16S rRNA genes, the species-specific sequences in the 16S-23S rRNA intergenic spacer region, and the propionicin T1- and pro-PAMP-encoding genes

PCR fragments were isolated by using a Qiagen PCR purification kit (Qiagen) or by agarose gel electrophoresis followed by extraction with a QIAgen gel extraction kit (Qiagen). The isolated PCR products were sequenced by using an ABI Prism dye terminator cycle sequencing ready reaction kit and an ABI PRISM 377 DNA sequencer (Perkin-Elmer, Applied Biosystems, Foster City, Calif.). Analyses of DNA and protein sequences were performed by using the OMIGA 2.0 DNA and protein sequence analysis software (Oxford Molecular, Oxford, United Kingdom).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Taxonomic classification of a culture collection of propionibacteria.
The members of a culture collection consisting of propionibacteria obtained from various sources (Table 1) were identified to the species level by PCR amplification of the 16S-23S rRNA intergenic spacer region with species-specific primers (28) or by partial sequencing of the 16S rRNA gene and detection of species-specific sequences (27) (Table 1). The majority of the strains had up to that time been identified by conventional phenotypic classification methods. Of the 64 strains in the collection, 14 were reclassified at the species level by using the molecular methods described above (Table 1).

Distribution of the genes encoding propionicin T1 and pro-PAMP.
All strains of propionibacteria in our collection were screened for the presence of the genes encoding propionicin T1 (pctA) and pro-PAMP (pamA) by performing PCRs with specific primers (Table 1). No strains of Propionibacterium acidipropionici or Propionibacterium freudenreichii harbored the genes encoding propionicin T1 or pro-PAMP. However, these genes were widely distributed among the strains of Propionibacterium jensenii and Propionibacterium thoenii. Thirteen of the 24 strains (54%) of P. jensenii contained the gene encoding propionicin T1, while 15 strains (63%) carried the pro-PAMP-encoding gene. Of the 18 strains classified as P. thoenii, 16 (89%) contained pctA and 11 (61%) contained pamA.

The structural gene of propionicin T1 in P. thoenii 419 and LMGT 2792 is followed by an open reading frame (orf2) that encodes a putative ABC transporter (6). All propionibacteria containing the pctA gene were tested for the presence of this gene by performing PCR with orf2-specific primers. With the exception of two strains of P. jensenii (INF P316 and INF P324) and one strain of P. thoenii (LMGT 2871), the expected PCR product was obtained from all strains containing pctA.

Expression of the propionicin T1 and PAMP activities.
The antimicrobial activities of all propionibacteria in our collection were measured by the agar plate assay, and none of the strains without the pctA gene and without the pamA gene exhibited antimicrobial activity against the corresponding indicator organisms.

All strains carrying the pamA gene showed protease-dependent antimicrobial activity against L. sakei NCDO 2714 in overlay assays in the presence of proteinase K. No significant differences in antimicrobial activity were observed among the strains harboring pamA. In contrast, the antimicrobial activities against the propionicin T1-sensitive indicator organism P. acidipropionici ATCC 4965 exhibited by the pctA-containing propionibacteria were highly variable (Table 1). Whereas all 16 strains of P. thoenii harboring the pctA gene showed antimicrobial activity against P. acidipropionici ATCC 4965, only five strains of P. jensenii containing the gene inhibited this indicator organism (Table 1). Variation in the expression of the antimicrobial activities of propionicin T1-positive strains was also observed. The inhibition zones produced by colonies of different strains varied in size (0.5 to 12 mm) and time of production. Some strains were incubated for only 5 days before inhibition zones were noticed in the overlay assay, while other strains had to grow for 12 days before any antimicrobial activity was observed (data not shown). The remaining strains did not produce any zones of inhibition even after 20 days of growth.

Molecular analysis of the gene encoding propionicin T1.
Due to the variability of the antimicrobial activities exhibited by propionibacteria containing the pctA gene, this gene and its promoter region were amplified and sequenced from all these strains. The sequences revealed the existence of two allelic variants that differ in a single nucleotide (G or A) at position 257 of the pctA gene. This difference results in replacement of glycine by aspartate at position 55 in mature propionicin T1. The propionicin T1 gene that resulted in the aspartate substitution (A variant) was found only in strains of P. jensenii, and these strains did not exhibit any antimicrobial activity against the standard indicator strain (Table 1).

Previous sequencing has shown that the structural gene encoding propionicin T1 is followed by a second open reading frame (orf2), which encodes a putative ABC transporter. This open reading frame is most likely cotranscribed with the pctA gene (6). In order to detect any correlation between the presence of orf2 and the antimicrobial activity corresponding to propionicin T1, this region was amplified by PCR with specific primers for all strains containing one of the two variants of the propionicin T1 gene.

One of the strains (P. jensenii INF P324) that exhibited no propionicin T1 activity although it contained the G variant of the gene gave no orf2-specific PCR product. Two additional strains with low levels of propionicin T1 activity, P. jensenii INF P316 and P. thoenii LMGT 2871, also lacked the orf2 gene (Table 1).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Only a few antimicrobial peptides of the classical propionibacteria have been biochemically and genetically characterized (5, 6, 18). However, these antimicrobial compounds have several biochemical features in common, such as inhibitory spectrum, stability, hydrophobicity, and cationic nature (9).

In order to investigate the prevalence of antimicrobial peptides in propionibacteria, a collection of 64 strains was screened for the presence of the genes encoding propionicin T1 and pro-PAMP. In contrast to the gene encoding propionicin SM1 (18), these genes are probably chromosomal since the original producing strains contain no plasmids. The antimicrobial activities corresponding to these genes were also examined.

Both genes occurred at high frequencies in both P. jensenii and P. thoenii, but they were totally absent in P. acidipropionici and P. freudenreichii. Only 6 of the 42 strains belonging to P. jensenii and P. thoenii did not carry any of the genes, and only 10 strains of these species did not exhibit any antimicrobial activity. In comparison, a study in which the presence of the nisin structural gene in a collection of Lactococcus lactis subsp. lactis strains was investigated showed that about 10% of the strains carried the nisin gene (19).

All strains of P. thoenii containing the pctA gene exhibited antimicrobial activity against P. acidipropionici ATCC 4965, an activity that was attributed to expression of propionicin T1 (Table 1). However, only 5 of the 13 strains of P. jensenii containing the propionicin T1-encoding gene exhibited antimicrobial activity corresponding to this bacteriocin (Table 1). The presence of silent or nonfunctional bacteriocin genes has also been reported in sakacin P-negative strains of L. sakei (10) and nisin-negative strains of L. lactis subsp. lactis (19).

The variations in the production of propionicin T1 observed might simply reflect differences in growth rates between the producing strains. However, previous results also showed that propionicin T1 was produced under different growth conditions by two strains of P. thoenii (6). While P. thoenii 419 produced the bacteriocin in the late logarithmic growth phase at an incubation temperature of 30°C, propionicin T1 production by P. thoenii LMGT 2792 could be detected only in the stationary growth phase after incubation at 22°C (6).

To determine if the observed differences in propionicin T1 production among the pctA-containing propionibacteria were due to variations in the DNA sequences, the pctA gene from all potential producer strains was sequenced. The results of this sequencing revealed that six strains P. jensenii with no propionicin T1 activity harbored an allelic variant (A variant) of the pctA gene (Table 1). Since no differences were found in the predicted promoter regions of the allelic variants, the A variant of propionicin T1 is probably inactive or has a different antimicrobial activity spectrum.

Propionicin T1 is produced with an N-terminal leader sequence typical of peptides processed and secreted by the sec-dependent pathway (20, 23). The presence of a sec leader indicates that the limited antimicrobial activity of strains lacking orf2 is not due to deficient peptide secretion. It has been proposed previously that the putative ABC transporter encoded by orf2 may be implicated in propionicin T1 immunity (6). The low levels of production of propionicin T1 by the strains lacking orf2 further support the idea that orf2 is important for efficient production of this bacteriocin.

All of the strains of both P. jensenii and P. thoenii containing the pamA gene exhibited the corresponding antimicrobial activities. No significant differences in activity were observed, indicating that all strains had the same features related to the production and regulation of pro-PAMP. Previous results have shown that pro-PAMP is expressed constitutively in the producer strain P. jensenii LMGT 3032 (5). The activation of the exported proprotein by an external protease is an unusual feature, as most bacteriocins and antimicrobial peptides are processed and activated concomitant with export from the cell (11). However, it is possible that PAMP is the result of proteolytic degradation of a protein with a different biological function.

Phylogenetic studies have shown that P. jensenii and P. thoenii are more closely related than other dairy propionibacteria (4, 13). The strains harboring the pctA and pamA genes were obtained from different environments in Europe, the United States, and South Africa, indicating that the ability to produce these antimicrobial compounds is widely distributed in the two species. The only two strains of P. thoenii tested which did not contain the propionicin T1-encoding genes were isolated from sludge in South Africa (T. Langsrud, personal communication). This habitat is quite different than the habitats of most strains of propionibacteria, which are isolated mainly from various dairy sources (3, 4) (Table 1).

The inhibitory spectrum of propionicin T1 and PAMP is restricted to propionibacteria and lactobacilli (6) that occupy the same habitats as the producer bacteria. Bacteriocins are generally thought to be weapons in the competition for space and nutrients among bacteria living in the same ecological niche. Likewise, the production of propionicin T1 and PAMP might provide a selective advantage to the producing propionibacteria.

Although different species exhibit diverse inhibitory spectra in colony assays, their antimicrobial peptides can in fact be identical (12). Interestingly, we found that the producer of jenseniin G, P. thoenii ATCC 4872 (7), and the producer of propionicin PLG-1, P. thoenii ATCC 4874 (14, 15), carry both the pctA and pamA genes (Table 1). These strains also exhibited antimicrobial activities corresponding to propionicin T1 and PAMP activities, indicating that both genes were expressed. A comparison of the studies reviewed by Holo et al. (9) and the results presented here suggests that some of the previously reported antimicrobial activities of propionibacteria may be attributed to some of the peptides that have been identified already. The results described in this paper show the importance of screening potential bacteriocin producers for bacteriocin genes that already have been characterized and for the corresponding bacteriocin production in order to avoid duplication of research efforts.

 
T. Faye was funded by a grant from the Norwegian Research Council. D. A. Brede was funded by grant P98089 from The Nordic Industrial Fund.

 
* Corresponding author. Mailing address: Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, P.O. Box 5003, N-1432 Ås, Norway. Phone: 47 64 94 85 33. Fax: 47 64 94 77 20. E-mail: therese.faye{at}ikbm.nlh.no.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, April 2004, p. 2240-2244, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2240-2244.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Brede, D. A., Faye, T., Stierli, M. P., Dasen, G., Theiler, A., Nes, I. F., Meile, L., Holo, H. (2005). Heterologous Production of Antimicrobial Peptides in Propionibacterium freudenreichii. Appl. Environ. Microbiol. 71: 8077-8084 [Abstract] [Full Text]  
• Brede, D. A., Faye, T., Johnsborg, O., Odegard, I., Nes, I. F., Holo, H. (2004). Molecular and Genetic Characterization of Propionicin F, a Bacteriocin from Propionibacterium freudenreichii. Appl. Environ. Microbiol. 70: 7303-7310 [Abstract] [Full Text]  

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