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.
These enzymes have been designated using various names including phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, muralysin, muramidase and virolysin. Note that some of these designations describe enzymes with similar substrate specificity.All phage lysins are characterised by their ability to hydrolyse specific bonds in the murein or pepidoglycan layer of the cell wall. This is the layer responsible for the rigidity and mechanical strength of bacterial-cells.
There are several types of lytic enzyme as defined by the cell wall linkage cleaved, figure 1. These are either glucosaminidases, hydrolysing the glycosidic bonds between the aminosugars (N-acetylmuramic acid and N-acetylglucosamine) of the peptidoglycan, amidases, which cleave the N-acetylmuramoyl-L-alanine amide linkage between the glycan strand and the cross-linking peptide, or endopeptidases that hydrolyse the interpeptide bridge linkage.
Muramidases produced by phages for lactic acid bacteria, one of two classes of glucosaminidase enzymes, also called endoacetylmurmidases have been well characterised particularly for lactococci. These enzymes hydrolyse the N-acetylmuramyl-N-acetylglucosamine linkage releasing muramic acid; they have the same substrate specificity as hen-egg lysozyme.
Most of the lactococcal phage lysins characterised todate have been produced by phages with prolate morphology; these seem to 'overproduce' lysin compared with isometric-phages.
Characterisation of phage lysins
Lysins are basic enzymes
Characterisation of partly purified lysins produced by lactococcal phages revealed that they were basic enzymes, having high isoelectric points, e.g. Tourville and Tokuda (1967) reported that a ØC10 lysin (produced by an isometric phage) had an isoelectric point of >8.6. This mean that lysins have a net positive charge at pH values below their isoelectric point. Because lysins are positively charged they can be purified using weak cation exchange resins e.g. Amberlite CG50.
Effect of cations
Lactococcal and other LAB phage lysins are generally activated by cations. Activation of ØC2(W) lysin occurred with both monovalent and divalent cations. Maximum lytic activity was found with 0.1 M concentrations of either Na + or K+. Concentrations of Na+ or K+ higher than 1 M gave reduced activity. A sharp peak of lytic activity was found for Mn2+, Ca2+ and Co2+ at ionic concentrations 3, 5 and 8 mM respectively. Complete inhibition of lytic activity was found with 30 mM Co2+. With Mg2+ a broad response curve was obtained which exhibited maximum lytic activity at a concentration of 5 mM. In contrast to the effect of cations, anions had little influence on the lytic reaction.
Sensitivity of phage and lysin to thermal inactivation
Phage lysins, particularly those of lactococcal phages, are much more susceptible to thermal inactivation than phage. In the case of ØC2(W) lysin, rapid inactivation occurred in buffer at 47°C whereas the virus showed little change in titre under similar conditions at 60°C.
Q10 and activation energies of lactococcal phage lysins
Relatively few studies reporting the effect of temperature on lysin activity have been reported.
ØC2 (W) lysin had a Q10 (range 22°-32°C) of 2.5 (Mullan and Crawford, 1985b), compared with 3.9 for ØC10 (Tourville and Tokuda, 1967) and 4.9 for ØML3 lysins (Oram and Reiter, 1965).
ØML3 lysin (Oram and Reiter, 1965) was found to have a high activation energy (eA), around 28.0 kcal mole -1(117.0 kJ mole -1 ) compared with 69.2 kJ mole -1 for ØC2(W) lysin (Mullan and Crawford, 1985b).
Since Q10 and Ea values are enzyme and not substrate dependent, they have utility in differentiating between phage lysins.
Optimal pH and temperature values for lytic activity
Lactococcal lysins demonstrate optimal lytic activity over pH 6-7 and at 37°C.
Requirement for reduced sulphydryl groups for activity
Lactococcal phage lysins have been shown to require reduced suphydryl (-SH) groups for lytic activity. Complete inactivation of ØC2(W) lysin was found using a 10-5 M solution of p-chloromercuriobenzoate. This inhibition can be reversed by cysteine.
Lytic spectrum of phage lysins compared with particulate phage.
Pette (1953) first established that lactococcal phage lysins had a broader lytic range than phage. Naylor and Czulak (1956) confirmed this finding and Oram & Reiter (1965) further extended this work.
The sensitivity of some lactic acid bacteria to ØC2(W) lysin is shown in table 1, Mullan and Crawford (1985b).
All strains of the three species of group N streptococci (now lactococci) studied were lysed. Marked strain-dependent differences in sensitivity, however, was apparent. Str. cremoris strains R6, 1249 and Str. lactis C10 were particularly sensitive whereas Str. cremoris strains US3, AM2 and Str. lactis subsp. diacetylactis DRC2 were relatively insensitive. All strains of the four species of group D streptococci tested were lysed ( classification now changed). Strain-dependent differences in sensitivity were also apparent. Strains of Str. dysgalactiae(2), Str. thermophilus(2), L. bulgaricus, L. fermentum, Leuc. lactic(2), Leuc. dextranicum(2), Leuc. cremoris(2), E. coli and Micrococcuslysodeikticus were not lysed.
Table 1. Sensitivity of lactic acid bacteria to ØC2(W) lysin
From Mullan and Crawford (1985b)
ApurifiedÆ C2(W) lysin preparation was assayed against log-phase test cultures1
×Relative activity = activity towards strain´100 activity with lyophilised C2 cells
Lysis of group D streptococci by group N phage lysin was first reported by Oram and Reiter (1965). The wide lytic range of phage lysin compared with the relatively narrow lytic spectrum of particulate phage provides additional evidence for the role of lysins in the nascent phage phenomenon generally (see also Tourville & Tokuda, 1967) and the involvement of lysin in the inhibition of acid production experienced when certain paired-strain cultures were infected with ØC2(W) (Mullan & Crawford, 1985a).
Unlike ØC10 lysin (Tourville & Tokuda, 1967),ØC2(W) and ØML3 lysins (Oram & Reiter, 1965) failed to lyse M. lysodeikticus. The lytic reaction of lactococcal phage lysins towards this organism may prove useful in their differentiation and, indirectly, in the differentiation of lysin-producing phages.
The chemical and/or stereochemical factors which determine whether a particular phage lysin lyses one bacterial species while not lysing another are not fully understood. Differences in peptidoglycan structure are likely to be important. Although two different types of peptidoglycan have been reported for group N streptococci (Schleifer & Kandler, 1972) it is probable that the peptidoglycan type with the D-isoasparagine crosslinkage represents peptidoglycan present in lactococci. Of the strains tested, with the exception of the lactococci, only Str. faecium and Str. faecium subsp. duransStr. faecalis which has a different peptidoglycan structure was lysed whereas Str. thermophilus, which was not lysed, possesses a peptidoglycan of identical chemical structure to Str. faecalis (Schleifer & Kandler, 1972). The nature of the non-peptidoglycan components may also influence sensitivity of a strain to lysin (Brumfitt et al. 1958; Krause & McCarty, 1961; Mandelstam & Strominger, 1961). possessed this type of peptidoglycan (Schleifer & Kandler, 1972). Since strains of these species were lysed, a relationship between the chemical structure of a peptidoglycan and its susceptibility to lysis may exist. However,
Molecular weight of phage lysins
Limited data on the molecular weight of phage lysins for lactic acid bacteria have been reported. Recent studies have predicted molecular weight based on the translation of the DNA sequence. Older studies have used molecular sieving techniques such as gel filtration using columns calibrated with protein standards.
The catalytically active form of any enzyme may consist of two or more monomers that combine to form diamers or heterodimers. Gel filtration would be expected to give the molecular weight of the catalytically active form of a lysin. Interestingly, John Oram's PhD thesis (Oram, 1965) indicated that the catalytically active form of ØML3 lysin is >200,000 daltons ( gel filtration). Tourville and Tokuda (1967) reported a sedimentation rate of 7S for ØC10 lysin suggesting a molecular weight for the active form of the enzyme >200,000 daltons.
Using gel filtration the molecular weight of ØC2 (W) lysin has been estimated as 46,000 ( Mullan and Crawford, 1985b). Using sequence analysis the molecular weight of ØvML3 lysin was found to be 25,352 ( Shearman et al., 1994).
Mechanism of inhibitory effects of Ø C2 (W) on paired phage unrelated cultures
The activity of a ØC2 (W) lysate towards freshly harvested strains of lactococci and lyophilized cells of C2 is shown in figure 2.
A marked decrease in absorbance with time was observed for all strains studied. No significant decrease occurred over a 4 min period for control cell suspensions in which lysate was not included or when heat-treated lysate (90 °C for 5 min) was used. Lyophilization markedly increased the sensitivity of C2 to lysis. Cell suspensions, incubated with lysate for 1 h at 37°C and examined by phase contrast microscopy, revealed extensive cellular lysis, confirming that the decrease in absorbance was due to cell lysis, Cells in the process of lysis were also observed. Rapid dissolution of chains into collections of single cocci was also a characteristic of the action of ØC2 (W) lysates on cell suspensions.
The action of lactococcal and other phage lysins on bacteria has been studied using transmission electron microscopy. The effect of ØC2 (W) lysin on Lc. lactis C10 is shown in figure 2; click on the figure for a higher resolution image. Control cells had clearly defined walls (Fig. 3)
but the cell membrane was difficult to see clearly. After 150 s incubation, cells virtually free of cell wall material were visible and the cell membrane was apparent (figure 4); click on the figure for a higher resolution image. These results show that the cell wall is the site of action of the ØC2 (W)-induced lysin and that it is removed as a result of lysin action.
Phage lysins because of their ability to hydrolyse cell-wall linkages are being used to produce protoplasts for genetic and other studies.