Bacteriophages for lactococci
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- Written by Michael Mullan
This section provides links to articles on the discovery, biology, lysins, industrial significance, control, isolation, propagation, storage and enumeration (assay) of lactococcal bacteriophages on this website. Information on other phages is also covered.
It is important to note that while bacteriophage, usually abbreviated to phage, infection is a major cause of poor growth and acid production by starter cultures, that these bacteria may also be inhibited by added substances including antibiotics, sterilant and detergent residues, or free fatty acids produced by or as a result of the growth of microorganisms, and natural often called indigenous antimicrobial proteins. Additionally use of incorrect temperatures during fermentation processes can also cause problems.
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Dr Hugh Whitehead and his colleagues at the New Zealand Dairy Research Institute realised that if the dairy industry in that country was to produce close-textured cheese, free from taste and body defects and manufactured within a consistent time period that it would be necessary to use standardised starter cultures. They also realised that they needed to prevent problems arising from the growth of 'wild' lactic acid bacteria and spoilage organisms in the raw milk and introduced pasteurisation of milk for cheese manufacture.
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The major functions of starters in dairy fermentations are shown in table 1. See the section on starters also.
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Infection and replication of bacteriophages:
Phage adsorption
The infection of a growing bacterial culture with phage is initiated by the adsorption of the phage to the host cell. The specificity of adsorption of lactococcal phages and the location of phage receptor substances have been studied and has been reviewed (Lawrence et. al., 1976).
Lactococci have been shown to have different receptor sites that may reside in both the cell wall and plasma membrane. Some phages may be relatively strain specific attacking only one or two strains but others show less specificity and may even attack strains of different subspecies, i.e. a phage may attack strains of Lc. lactis, Lc. lactis subsp. cremoris and Lc. lactis biovariant diacetylactis. Although many strains are resistant to a specific phage because the phage cannot adsorb to receptor sites some strains which allow adsorption are also phage resistant . The resistance of the latter has been attributed to lysogenic immunity or to the operation of a modification/restriction (M/R) system .
It is generally recognised that the ionic environment is an important factor in adsorption. In particular, inorganic salts must be present (Cherry and Watson, 1949) and probably act by neutralising the net negative charges on the host cell and its phage so that initial contact is facilitated. Calcium which is required for the multiplication of most lactococcal phages (Shew, 1949; Potter and Nelson, 1952) is not a specific requirement for adsorption since monovalent cations are apparently just as effective (Reiter and Møller-Madsen, 1963); note some lactic phages do not require calcium for replication. The calcium-dependant requirement for replication has been exploited in the development of phage inhibitory media for phage-free bulk starter production.
The adsorption of some phages to lactococci has been reported to be reduced by rennet, increased temperature or by growth on a different host (Keogh, 1973). Although the differences were often small, the combination of rennet with elevated temperature (37°C) substantially reduced adsorption for several phages.
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Morphology
Bradley (1967), in a classic review paper, summarised the principles of phage morphology and outlined six basic morphological types (fig. 1). The tailed phages, Bradley's groups A-C account for some 96% of all phages isolated to date (2012) and as discussed below belong to the order Caudovirales. Only phages in Group A have contractile tails. All tailed bacteriophages have a nucleic acid core surrounded by a protein coat. Phages active against lactic acid bacteria are approximately tadpole or sperm shaped and have a distinct head terminating in a tail with a hollow core. Phages attacking lactic acid bacteria belong to Groups A, B and C and contain double stranded DNA. Phages in Groups D and F contain single stranded DNA, however, Group E phages contain single-stranded RNA.
Filamentous phages (filament-like or rod-like) (Bradley's Group F) have been well documented for E. coli and Pseudomonas spp. Several reports of filamentous phages for Gram positive bacteria including Propionibacterium freudenreichii have been reported. Propionibacterium freudenreichii is used in Swiss cheese manufacture and a phage designated B6 (below) has been characterised by Chopin et al. (2002).
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The basic principles of phage control in commercial plants have been known since the early 1940s and the pioneering work of Dr Hugh Whitehead and his colleagues in New Zealand. The review by Whitehead and Hunter (1945)* on the measures that were being used in New Zealand to control slow acid production due to phage infection is still of relevance to factory managers today. The 1945 review focused on whey as the vehicle for phage transmission, and on work designed to break the cycle of phage infection. Even in 1945 they recognised the challenge of keeping phage concentrations low in the environment, the need for special facilities to produce phage-free bulk starter in a potentially phage-infected environment, the possibility of raw milk being contaminated with phage because the cans, tankers today, carrying whey, and also used to carry raw milk, could contaminate raw milk. They were also aware that whey separators and 'splashes' of whey produced aerosols and that these would enable phage to become airborne. Off course they were not aware of lysogency and the possibility of phage arising from the starter or lactococci in raw milk.
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Phage release, the final stage in the phage-life cycle, has been extensively studied and is caused, at least in part, by the action of phage-induced hydrolytic or lytic enzymes.
The presence of phage-induced, cell-wall degrading enzymes in lysates of phage-infected bacteria has now been described for an extensive range of bacteria including Escherichia coli, Staphylococcus aureus, Azotobacter agilis, Aerobacter cloacae, Bacillus megaterium, Micrococcus lysodeikticus, Bacillus stearothermophilus, Klebsiella pneumoniae, Pseudomonas putida, Ps. aeruginosa and for lactic acid bacteria including lactococci, leuconstocs and lactobacilli.
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Because phage lysin has a much broader lytic range than phage, infection of paired and multi-strain cultures with a lysin-producing phage has the potential to cause fermentation failure, dead-vats, and consequent economic loss.
The effect of infecting paired-strain cultures with ØC2 (W) is shown in table 2. Acid production was markedly inhibited for six of the eight combinations. Extensive replication of ØC2 (W) occurred in all phage-infected cultures. Phage infection did not inhibit acid production when the component strains were inoculated singly (in the absence of ØC2) and infected in control experiments. Cell numbers in the inhibited cultures were low at 6 h incubation and ranged from 0.57-30 x 106 colony forming units (CFU/mI). In control cultures, counts ranged from 1-3 x 109 CFU/ml.
Table 2. Effect of infecting paired phage-unrelated single-strain starters with ØC2 (W)*
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While phage lysin has long been suspected of having an important part in phage lysis it has taken techniques using molecular biology to clarify its in vivo role.
For many phages, their release from the infected cell has been shown to require the action of two phage-encoded proteins. The first protein is called holin; this is a small transmembrane protein that creates lesions or 'holes' in the cell-wall membrane. These lesions function as pores or holes and allow the passage of a second protein, phage lysin through the cell membrane and access to the cell wall. Holin production is essential since it is only when pores have been created in the cytoplasmic membrane that phage lysins can reach their substrate and lyse the cell wall. There have been very considerable developments in both the molecular biology and biochemistry of phage release-see reviews by Gasson (1996) and Sable and Lortal (1995).
How to cite this article
Mullan, W.M.A. (2003) .
[On-line]. Available from: http://dairyscience.info/index.php/bacteriophages-for-lactococci.html . Accessed: 18 May, 2013.
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- Written by Michael Mullan
The activity of phage lysins can be determined using several methods; turbidimetry or the determination of the change in concentration of some solubilised cell wall component are frequently used.
Turbimetry, where a standardised suspension of cells in buffer is mixed with a sample of lysin-containing material is widely used. The lysin causes lysis of the cell suspension and a reduction in optical density (OD). Enzyme activity can be calculated from the decrease in OD with time.
