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Keywords:

  • Lactobacillus fermentum;
  • lysogenic strain;
  • prophage-cured strain;
  • restriction patterns;
  • temperate bacteriophage;
  • 16S rDNA

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  The aim of this study was to investigate the properties of temperate bacteriophage of Lactobacillus fermentum, based on its morphology, restriction patterns, protein profile and the impact on the growth of host strain.

Methods and Results:  With Mitomycin C, seven temperate phages were induced from Lactobacilli derived from Chinese yogurt. The temperate phages induced belong to the most common Bradley's group B, having hexagonal head and long, noncontractile tail. They were furthermore confirmed to be the same bacteriophage by identical restriction patterns. SDS–PAGE profile showed that the phage studied had one major structure protein about 31·9 kDa. The presence of the prophage influenced the cell shape and colony size of its lysogenic strain.

Conclusions:  The phage obtained had similar, but not complete identical properties with other L. fermentum phages reported. It influenced the growth behaviour of its lysogenic strain.

Significance and Impact of the Study:  This study provides some information about bacteriophages occurring in the Chinese yoghurt manufacture and contributes to our knowledge on the bacteriophage diversity in the dairy industry.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Lactic acid bacteria are industrially important microbes that are used all over the world in a large variety of industrial food fermentations. They have the ability to contribute to the product characteristics such as flavour, texture and nutrition (Adolfsson et al. 2004). However, one of the greatest hazards of the technological process is phage attacking (Foschino et al. 2001). Contamination by virulent phages may result in the lysis of the starter strains in fermentation, which causes slow fermentations or even complete failure with consequent loss of the product. The economic impact has led to intensive research on phage attacking lactic acid bacteria (Desiere et al. 2000). It has been known that temperate phages exist widely in starter cultures (Altermann et al. 1999), but they are often ignored owing to their little influence on stability of dairy products.

Traditionally, mesophilic starters including most Lactococcus lactis strains are very susceptible to phage infection, whereas thermophilic cultures composed of Lactobacillus delbrueckii and Streptococcus thermophilus are rarely reported to be the subject of phage attack. A probable reason is that such cultures are widely used in yogurt manufacture, a process that does not generate whey. It is well known that whey treatment is an important aspect of phage control in cheese plant. However, in recent years, phage infection of thermophilic cultures used in yogurt and cheese production has become more frequent because of increased plant scale and heavier production schedules. In China, yogurt has become one of the most popular dairy products as it circumvents the lactose-intolerance problem for the majority of Chinese people. Little is known about the economic loss suffered from phage attacking the starters for yogurt production. On the other hand, many yogurts are processed on a household or small industrial scale in China, and consequently suffering from inconsistent product quality including texture and flavour.

In this paper, an attempt was made to find the reason resulting in the instability in yogurt production in China. Several strains, identified to be Lactobacillus fermentum by 16S rDNA sequence, were isolated from yogurt products and found to be lysogenic. The lysogenic phages present in the different L. fermentum strains isolated from Chinese yogurt were characterized to obtain thorough understanding on the role of phages in the yogurt manufacturing process.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strain isolation and culture conditions

Twenty commercial yogurt samples were purchased from different cities in China. To isolate Lactobacillus strains from these products, a loopful of each product was streaked on the acidified MRS agar with pH 6·0 as previously described (Champagne and Gardner 1995). The agar plates were incubated at 37°C for 48 h. Each isolate from the acidified MRS agar was further confirmed to be Lactobacillus sp. by phenotypic morphology: rod cell, Gram-positive stain and catalase-negative reaction. Lactobacillus strains were propagated at 37°C in MRS medium. Agar was added at 0·5% and 1·0% to make a soft-agar or solid medium. If necessary, the medium was supplemented with CaCl2 10 mmol l−1 (MRS–Ca). Frozen cultures were maintained at −20°C in MRS broth with 30% glycerol.

Induction of bacteriophage

Lactobacillus strains were incubated at 37°C for 8–10 h in MRS–Ca medium. Subsequently, 0·1 ml of each bacterial culture was transferred into 5-ml fresh MRS–Ca broth. Induction of temperate phages was performed by addition Mitomycin C at a final concentration of 0·5 μg ml−1 for about 2 h after inoculation (Kilic et al. 1996). The presence of Lactobacillus bacteriophages was indicated by a clear lysis of the turbid culture after the addition of Mitomycin C. Lysates obtained were centrifugated at 8000 g for 10 min to remove cell debris, and the supernatant was filtered through a 0·45 μm membrane and stored at 4°C.

To determine the induction efficiency, Mitomycin C was applied at a final concentration of 0·1, 0·4, 1·0 and 1·5 μg ml−1, respectively. Absorbance of the cultures was monitored at 610 nm (A610) (Auad et al. 1997).

PCR amplification and sequencing of 16S rDNA

Lactobacillus sp. carrying temperate phage was incubated in MRS medium at 37°C for 10 h. Then 0·5 ml of bacteria culture was heated at 100°C for 10 min. After centrifugation at 8000 g for 10 min, supernatant was kept at −20°C and used as DNA template for PCR amplification. The primers used for PCR amplification were 5′-AGA GTT TGA TCC TGG CTC AG-3′ (forward primer, corresponding to position 8–27 relative to Escherichia coli numbering), and 5′-AAGGAGGTGATCCAGCCGCA-3′ (reverse primer, to position 1523–1504 relative to E. coli 16S rDNA). Reaction mixtures were prepared according to the instruction of the manufacture in a total volume of 50 μl. PCR amplification was carried out as follows: at 94°C for 4 min, followed by 30 cycles of 30 s at 94°C, 30 s at 58°C, and 2 min at 72°C. The PCR was ended with an extra incubation of 7 min at 72°C. PCR product was visualized by ethidium broidine UV fluorescence on 1% agar gel. After purified with the agarose gel DNA purification kit ver. 2·0 as the instruction of manufacture (TaKaRa, Japan), the DNA fragment amplified was cloned into the pGEM-T vector and sequenced by ABI 3730 DNA analyzer (Applied Biosystem, Foster City, CA, USA) according to the manufacture's instruction.

Concentration and purification of bacteriophage

The filtrate from stock was treated with DNase and RNase A at a final concentration of 1 μg ml−1 at 37°C for 2 h to degrade nucleotide acid residue from the bacterial cells. Phage particles were then precipitated from supernatant by incubating with 0·5 mol−1 NaCl and 10% (wt/vol) polyethylene glycol 8, 000 for 1 h on ice (Yamamoto et al. 1970). The pellet, obtained by centrifugation for 20 min at 12 000 g, was resuspended in SM buffer (0·58% NaCl, MgSO4·7H2O 0·2%, 1 mol−1 pH 7·5 Tris–HCl). Phage particles were purified through CsCl2 density gradient centrifugation at 80 000 g for 2 h at 5°C. The phage band was collected. The pellets purified were used for morphology observation by electron microscope or DNA analysis by agarose gels electrophoresis.

Confirming the presence of bacteriophage

The presence of phages was investigated by spot test and turbidity test as reported with slight modification (Svensson and Christiansson 1991). Briefly, in the spot test, soft agar (MRS broth with 0·7% agar, 3 ml) was seeded with 0·1 ml of exponential phase cultures of purified strains, mixed gently, and poured into MRS agar plates. After solidification, 10 μl of phage lysate was spotted on the lawn of Lactobacillus strains. These plates were dried for 20 min prior to incubate at 37°C for 16–18 h. A clear zone formed on the plate, indicating the presence of phages, as a result of the lysis of host cells.

In turbidity test, tubes containing 5 ml of MRS–Ca broth were inoculated with 0·2 ml of fresh overnight cultures of strains and 0·2 ml of the filtrate suspected of containing phages. MRS–Ca broth only seeded with Lactobacillus strains was used as control. Tubes were incubated at 37°C for 10 h, and compared in turbidity with the control tubes.

Electron microscopy

Bacteriophage morphology was observed by transmission electron microscopy. A drop of viral suspension concentrated to 100 folds was placed on 300 mesh copper grids coated with carbon film; the grids were then dipped in phosphotungstic acid (2% w/v, pH 4·5) for 30 s to negatively stain the viral particles. After drying, the preparation was examined on a JEM-100CX11 electron microscope (JEOL, Tokyo, Japan) at different magnitudes. Bacteriophage configuration and dimensions (capsid diameter, tail length) were recorded.

DNA manipulation and analysis

Phage particles, resuspended in 2 ml SM buffer, were treated with 20 μl 10% SDS and 20 μl 0·5 mol −1 EDTA, then heated at 68°C for 15 min. Phage DNA was extracted by phenol–chroloform–isoamyl alcohol. An equal volume of isopropanol was used to precipitate DNA. Phage DNA was quantified by electrophoresis on agarose (0·7%, w/v) gel. The visualization by ethidium bromide staining was performed according to the standard protocols (Sambrook et al. 1989).

Bacteriophage DNA was digested with restriction enzymes (BamH, EcoRI, XhoI, PstI and HindIII) as recommended by the suppliers. DNA fragments generated were subjected to electrophoresis on a 1% w/v agarose gel at 5 mv cm−1 in Tris–Acetate–EDTA buffer. Gels were stained with ethidium bromide and photographed under UV illumination. DNA standard fragments of HindIII Lambda marker were used to calculate the size of DNA fragments. The genome size of phage was estimated by the sum of molecular weights of DNA fragments generated by digestion. Data were the average of several measurements carried out on at least two different gels.

Analysis of phage structure proteins

SDS–PAGE was performed for detecting the size and numbers of the protein of phage particles. A quantity of 0·1 ml of purified sample was heated with 25 μl loading buffer at 100°C for 5 min to ensure denaturation before loading on a gel for electrophoresis. Electrophoresis was carried out with 4% stacking layer and 12·5% separating gel according to the method described previously (Laemmli 1970). Standard proteins were used as mass standard to calculate the size of phage proteins. When electrophoresis was ended, gel was stained with Coomassie brilliant blue R-250, destained with 7·5% glacial acetic acid and 5% ethanol.

Phage-cured strain

The prophage-cured experiment was performed according to the method described previously with slight modification (Raya et al. 1989; Neve et al. 2003). The lysogenic strain was induced with Mitomycin C at the exponential phase of growth in MRS–Ca broth, followed by incubation at 37°C overnight. A quantity of 0·1 ml of the overnight culture was plated on MRS agar, and plates were incubated at 37°C for 24 h. Single colonies were picked to inoculate MRS broth and incubated for 24 h. The culture was then tested for Mitomycin C induction. The colonies not induced by Mitomycin C were regarded as prophage-cured derivatives.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation and identification of strains

Twenty strains were isolated from 20 yogurt samples, which were confirmed to be Lactobacillus sp. by cell morphology, Gram-positive stain and catalase-negative reaction, and were designated YB1 to YB20, respectively. All of the isolates were treated with Mitomycin C in MRS–Ca broth to detect their lysogenic status. The results showed that seven cultures became clear after addition of Mitomycin C. These lysates were further tested to see whether they could form plaque (Lillehaug 1997). However, none of the seven lysates could form plaques on MRS–Ca agar when the 20 isolated strains were used as indicator strain in a spot test. By electron microscopy observation and DNA analysis, bacteriophages were found to be present in the seven lysates, indicating that there were seven lysogenic strains among the 20 purified strains. The seven bacteriophages were designated φPYB2, φPYB3, φPYB4, φPYB5, φPYB7, φPYB9 and φPYB11, respectively.

16S rDNA from Lactobacillus sp. YB4 and YB5 were sequenced for strains identification. The result showed that the two sequences were identical. The comparison of the 16S rDNA nucleotide sequences with the sequences from GenBank database indicated that the 16S rRNA genes were 98%∼99% identical to those of L. fermentum (AY373589·1, AF302116·1), respectively. Therefore, the lysogenic Lactobacilli YB4 and YB5 were regarded as L. fermentum (accession number DQ208913).

Induction of phages

To investigate the optimal concentration of Mitomycin C for induction, different concentration of Mitomycin C was applied as described above. The result showed that the concentration used in this study did have clearly lysis function (see Fig. 2). The growth rate of the strain treated with lower concentration of Mitomycin C faster slightly than that of the strain treated with higher concentration of Mitomycin C. And accordingly, the former cultures became clear slightly earlier.

image

Figure 2.  Effect of Mitomycin C on growth of Lactobacillus strain in MRS broth. Symbols: ◊, 0 μg ml−1; bsl00001, 0·1 μg ml−1; bsl00084, 0·4 μg ml−1; ×, 1·0 μg ml−1; inline image, 1·5 μg ml−1.

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Bacteriophage morphology

Electron micrographs showed that all phages induced had the same morphology with hexagonal heads, noncontractile tails and several fibres (Fig. 1), belonging to Bradley's group B of the International Committee on Taxonomy of Viruses (Matthews 1982). There is no significant difference among these induced phages as for head diameters and tail lengths of the phages, which were 40 ± 1 nm and 130 ± 3 nm, respectively.

image

Figure 1.  Electron micrograph of Lactobacillus bacteriophage induced from yogurt samples.

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Characterization of phage genome

The restriction patterns of phage DNA were determined with restriction endonucleases BamHI, EcoRI, XhoI, PstI and HindIII, respectively. The restriction patterns of the seven phages were found to be identical (Fig. 3), which suggested that they might be the same phage. However, the restriction patterns of these phages were distinct from other phages active on L. fermentum reported previously (Foschino et al. 2001) (data not shown). The genome of the phages induced ranged from 32 to 34 kb estimated by distinct restriction patterns. When packing sites were investigated, phages induced presented pac-type because heating the restriction digestions (digested with BamHI and PstI) at 74°C for 10 min did not alter their restriction patterns on agarose gel (data not shown) (Lakshmidevi et al. 1988).

image

Figure 3.  Agarose gel electrophoresis of DNA from the temperate bacteriophages induced, digested with BamHI-(A), XhoI-(B). Lane 1–7, bacteriophage φPYB2, φPYB3, φPYB4, φPYB5, φPYB7, φPYB9 and φPYB11. Lane M, HindIII/bacteriophage Lambda DNA marker.

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SDS–PAGE profile of phage induced

Because the seven bacteriophages had identical genetic properties, structure protein profile of bacteriophageφPYB5, representing this group bacteriophages, was examined by SDS–PAGE (Fig. 4). Protein profile showed only one major band with molecular weight 31·9 kDa, one inferior band of about 25·8 kDa and several other faint bands of about 45·0, 62·0, 65·4 and 79·6 kDa, respectively.

image

Figure 4.  SDS–PAGE of structural protein from bacteriophageφPYB5. Lane 1, φPYB5; Lane M, molecular weight standard.

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The impact of prophage on the growth of host strain

The impact of prophage on the growth of host strain was performed with strain YB5. A prophage-cured derivative from lysogenic strain was isolated and named YB5-A. The biological properties of YB5 and YB5-A including colonies size, cell morphology and growth behaviour were determined. The results showed that the prophage-cured strain YB5-A formed larger colonies than that of the lysogenic strain YB5; cells of the prophage-cured strain YB5-A appeared longer and are arranged in chains while cells of the lysogenic strain YB5 was smaller and tended to distribute in clusters (Fig. 5). The growth curve of the two strains demonstrated that the growth rate of lysogenic strain YB5 was slightly slower than that of the prophage-cured strain YB5-A while the acid production rate of lysogenic strain YB5 was almost equal to that of prophage-cured YB5-A strain. However, the lysogenic strain YB5 seemed to accumulate more acid.

image

Figure 5.  Cell morphology of the prophage-cured strain YB5-A (a) and the lysogenic strain YB5 (b).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Currently, many small yogurt factories in China did not use direct-to-vat starter culture and easily suffered from the quality instability of the fermentation dairy product. To our knowledge, the reason has not been investigated and reported in China. In this study, an attempt was made to analyse the possibility of Lactobacillus infection by phage. From 20 isolated Lactobacillus strains, seven of them were found to be lysogenic. By sequence analysis of the 16S rDNA of strain YB4 and YB5, both of them were identified to be L. fermentum. The result indicated that some starter cultures for yogurt fermentation in China also contained other lactic acid bacteria besides Lactobacillus bulgaricus and S. thermophilus. Whether these other strains discovered in starters could influence the stability of fermented dairy products needs to be studied further.

Seven temperate phages were induced from their lysogenic strains with Mitomycin C. By morphologic observation, restriction patterns as well as protein profiles analysis, they were identified as the same phage. As the same phage was induced from these seven isolated strains, the starter cultures involved may be purchased from the same company. Another possible reason for the limited diversity of lactic acid bacteria phages could be the few number of fermented dairy products (Brüssow et al. 1998).

The temperate phage induced has the same hexagonal head and long, noncontractile tail as most lactic acid bacteria phages, thus most probably belonged to the most common Bradley's group B (Matthews 1982). However, it had a head only 40 ± 1 nm diameter and a tail 130 ± 1 nm long. Foschino et al. (2001) reported that seven of nine L. fermentum bacteriophages belonged to B1 group in Siphoviridae family. Their head diameters were approx. 50–59 nm, and the shortest tail was 169·0 ± 5·3 nm. The phage studied in this paper seems much smaller compared with those phages. Moreover, its restriction pattern (Fig. 3) determined with different restriction endonucleases was distinct from that of other L. fermentum phage described by Foschino.

In the prophage-cured experiments, we found that the lysogenic strain and the prophage-cured strain showed different growth behaviour. The prophage-cured strain YB5-A showed larger colonies than that of the lysogenic strain. Cells of prophage-cured strain YB5-A were longer and were arranged in chains, while the lysogenic strain YB5 preferred to distribute in clusters. This phenotypic difference was coincident with characteristics of lysogenic conversion (Cluzel et al. 1987). It was inferred that the integration of prophage DNA into chromosome increased the load of host strains. However, the increased load did not influence the acid production of the host strain, which means that bacteriophage-bacterium interaction was not a simply parasite-host relationship, while a co-evolution of viral and bacterial genome might be plausible.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank the School of Environmental Science and Engineering for taking electron micrograph of phages and Dr Weifeng Liu for reading the manuscript critically. This work was supported by the National Science Foundation of China (NSFC project number 30370035) and the open project of State Key Lab of Microbial Technology.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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