Characterization of a (p)ppApp Synthetase Belonging to a New Family of Polymorphic Toxin Associated with Temperate Phages.

Polymorphic toxins (PTs) are a broad family of toxins involved in interbacterial competition and pathogenesis. PTs are modular proteins that are comprised of a conserved N-terminal domain responsible for its transport, and a variable C-terminal domain bearing toxic activity. Although the mode of transport has yet to be elucidated, a new family of putative PTs containing an N-terminal MuF domain, resembling the Mu coliphage F protein, was identified in prophage genetic elements. The C-terminal toxin domains of these MuF PTs are predicted to bear nuclease, metallopeptidase, ADP-ribosyl transferase and RelA_SpoT activities. In this study, we characterized the MuF-RelA_SpoT toxin associated with the temperate phage of Streptococcus pneumoniae SPNA45. We show that the RelA_SpoT domain has (p)ppApp synthetase activity, which is bactericidal under our experimental conditions. We further determine that the two genes located downstream encode two immunity proteins, one binding to and inactivating the toxin and the other detoxifying the cell via a pppApp hydrolase activity. Finally, based on protein sequence alignments, we propose a signature for (p)ppApp synthetases that distinguishes them from (p)ppGpp synthetases.

The multidomain architecture of a bacteriophage endolysin enables intramolecular synergism and regulation of bacterial lysis.

Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage (phage) replication cycle to lyse the host cell. Endolysins in Gram-positive phages come in a variety of multimodular forms that combine different catalytic and cell wall binding domains. However, the reason why phages adopt endolysins with such complex multidomain architecture is not well understood. In this study, we used the Streptococcus dysgalactiae phage endolysin PlySK1249 as a model to investigate the role of multidomain architecture in phage-induced bacterial lysis and lysis regulation. PlySK1249 consists of an amidase (Ami) domain that lyses bacterial cells, a nonbacteriolytic endopeptidase (CHAP) domain that acts as a dechaining enzyme, and a central LysM cell wall binding domain. We observed that the Ami and CHAP domains synergized for peptidoglycan digestion and bacteriolysis in the native enzyme or when expressed individually and reunified. The CHAP endopeptidase resolved complex polymers of stem-peptides to dimers and helped the Ami domain to digest peptidoglycan to completion. We also found that PlySK1249 was subject to proteolytic cleavage by host cell wall proteases both in vitro and after phage induction. Cleavage disconnected the different domains by hydrolyzing their linker regions, thus hindering their bacteriolytic cooperation and possibly modulating the lytic activity of the enzyme. PlySK1249 cleavage by cell-wall-associated proteases may represent another example of phage adaptation toward the use of existing bacterial regulation mechanism for their own advantage. In addition, understanding more thoroughly the multidomain interplay of PlySK1249 broadens our knowledge on the ideal architecture of therapeutic antibacterial endolysins.

Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal.

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.

Bactericidal synergism between antibiotics and phage endolysin Cpl-711 to kill multidrug-resistant pneumococcus.

AIM: To test the synergistic effect of Cpl-711 endolysin and antibiotics for antipneumococcal activity. MATERIALS & METHODS: A combination of Cpl-711 and different antibiotics (amoxicillin, cefotaxime, levofloxacin and vancomycin) was tested in a checkerboard assay against several multidrug-resistant Streptococcus pneumoniae strains. Mouse and zebrafish models of pneumococcal sepsis were used to confirm the in vitro data. RESULTS: The activity of Cpl-711 combined with amoxicillin or cefotaxime was synergistic in the bactericidal effect against a serotype 23F multiresistant clinical isolate of S. pneumoniae. Synergy between Cpl-711 and cefotaxime was validated using both mouse and zebrafish models. CONCLUSION: Combination of Cpl-711 and cefotaxime may help in the treatment of diseases caused by multiresistant pneumococcal strains.

A novel prophage lysin Ply5218 with extended lytic activity and stability against Streptococcus suis infection.

Streptococcus suis (S. suis) is an emerging zoonotic agent that exhibits high level resistance to classic antibiotics and a heavy burden in the swine industry. Therefore alternative antibacterial agents need to be developed. A novel endolysin derived from the S. suis temperate phage phi5218, termed Ply5218, was identified. The minimum inhibitory concentration (MIC) of Ply5218 was 2.5 µg ml(-1) against S. suis strain HA9801, an activity many times greater than the lysins reported previously (MIC of LY7917 and Ply30 against HA9801 were 80 and 64 µg ml(-1), respectively). Ply5218 at 10 µg ml(-1) in vitro exerted broad antibacterial activities against S. suis strains with OD600 ratios decreased from 1 to <0.2 within 1 h. Moreover, Ply5218 showed favorable thermal stability. It was stable at 50°C >30 min, 4°C >30 days, -80°C >7 months, and >60% of the enzyme activity remained after 5 min pre-incubation at 70°C. In vivo, a 0.2 mg dose of Ply5218 protected 90% (9/10) of mice after infection with S. suis HA9801. Finally, Ply5218 maintained high antibacterial activity in some bio-matrices, such as culture media and milk. The data indicate that Ply5218 has all the characteristics to be an effective therapeutic agent against multiple S. suis infections.

A bacteriophage endolysin that eliminates intracellular streptococci.

PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB-PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities.

A novel endolysin disrupts Streptococcus suis with high efficiency.

Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen that exhibits high-level resistance and multi-drug resistance to classic antibiotics and causes serious human casualties and heavy economic losses in the swine industry worldwide. Therefore, alternative therapies or novel antibacterial agents need to be developed to combat this pathogen. A novel endolysin derived from the S. suis temperate phage phi7917, termed Ly7917, was identified, which had broad lytic activity against S. suis type 1, 2, 7 and 9. Ly7917 consisted of an N-terminal cysteine, histidine-dependent amidohydrolases/peptidase catalytic domain and C-terminal SH3b cell wall binding domain. The endolysin maintained activity at high pH and its catalytic activity could be improved by addition of 10 µM 1.5 mM Ca(2+). In animal studies, 90% of BALB/c mice challenged with typical virulent strain HA9801 of S. suis 2 were protected by Ly7917 treatment. The bacterial load in the blood of HA9801-challenged mice was efficiently reduced almost 50% by Ly7917 while that of penicillin-G-treated mice kept almost unchanged. Our data suggest that Ly7917 may be an alternative therapeutic agent for infections caused by virulent S. suis strains.

Synergistic streptococcal phage λSA2 and B30 endolysins kill streptococci in cow milk and in a mouse model of mastitis.

Bovine mastitis results in billion dollar losses annually in the USA alone. Streptococci are among the most relevant causative agents of this disease. Conventional antibiotic therapy is often unsuccessful and contributes to development of antibiotic resistance. Bacteriophage endolysins represent a new class of antimicrobials against these bacteria. In this work, we characterized the endolysins (lysins) of the streptococcal phages λSA2 and B30 and evaluated their potential as anti-mastitis agents. When tested in vitro against live streptococci, both enzymes exhibited near-optimum lytic activities at ionic strengths, pH, and Ca(2+) concentrations consistent with cow milk. When tested in combination in a checkerboard assay, the lysins were found to exhibit strong synergy. The λSA2 lysin displayed high activity in milk against Streptococcus dysgalactiae (reduction of CFU/ml by 3.5 log units at 100 µg/ml), Streptococcus agalactiae (2 log), and Streptococcus uberis (4 log), whereas the B30 lysin was less effective. In a mouse model of bovine mastitis, both enzymes significantly reduced intramammary concentrations of all three streptococcal species (except for B30 vs. S. dysgalactiae), and the effects on mammary gland wet weights and TNFα concentrations were consistent with these findings. Unexpectedly, the synergistic effect determined for the two enzymes in vitro was not observed in the mouse model. Overall, our results illustrate the potential of endolysins for treatment of Streptococcus-induced bovine mastitis.

A novel chimeric phage lysin with high in vitro and in vivo bactericidal activity against Streptococcus pneumoniae.

OBJECTIVES: Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide and new antimicrobials are urgently needed. Our aim was new chimeric phage endolysins, or lysins, with improved bactericidal activity by swapping the structural components of two pneumococcal phage lysozymes: Cpl-1 (the best lysin tested to date) and Cpl-7S. METHODS: The bactericidal effects of four new chimeric lysins were checked against several bacteria. The purified enzymes were added at different concentrations to resuspended bacteria and viable cells were measured after 1 h. Killing capacity of the most active lysin, Cpl-711, was tested in a mouse bacteraemia model, following mouse survival after injecting different amounts (25-500 µg) of enzyme. The capacity of Cpl-711 to reduce pneumococcal biofilm formation was also studied. RESULTS: The chimera Cpl-711 substantially improved the killing activity of the parental phage lysozymes, Cpl-1 and Cpl-7S, against pneumococcal bacteria, including multiresistant strains. Specifically, 5 µg/mL Cpl-711 killed ≥7.5 log of pneumococcal R6 strain. Cpl-711 also reduced pneumococcal biofilm formation and killed 4 log of the bacterial population at 1 µg/mL. Mice challenged intraperitoneally with D39_IU pneumococcal strain were protected by treatment with a single intraperitoneal injection of Cpl-711 1 h later, resulting in about 50% greater protection than with Cpl-1. CONCLUSIONS: Domain swapping among phage lysins allows the construction of new chimeric enzymes with high bactericidal activity and a different substrate range. Cpl-711, the most powerful endolysin against pneumococci, offers a promising therapeutic perspective for the treatment of multiresistant pneumococcal infections.

A highly active and negatively charged Streptococcus pyogenes lysin with a rare D-alanyl-L-alanine endopeptidase activity protects mice against streptococcal bacteremia.

Bacteriophage endolysins have shown great efficacy in killing Gram-positive bacteria. PlyC, a group C streptococcal phage lysin, represents the most efficient lysin characterized to date, with a remarkably high specificity against different streptococcal species, including the important pathogen Streptococcus pyogenes. However, PlyC is a unique lysin, in terms of both its high activity and structure (two distinct subunits). We sought to discover and characterize a phage lysin active against S. pyogenes with an endolysin architecture distinct from that of PlyC to determine if it relies on the same mechanism of action as PlyC. In this study, we identified and characterized an endolysin, termed PlyPy (phage lysin from S. pyogenes), from a prophage infecting S. pyogenes. By in silico analysis, PlyPy was found to have a molecular mass of 27.8 kDa and a pI of 4.16. It was active against a majority of group A streptococci and displayed high levels of activity as well as binding specificity against group B and C streptococci, while it was less efficient against other streptococcal species. PlyPy showed the highest activity at neutral pH in the presence of calcium and NaCl. Surprisingly, its activity was not affected by the presence of the group A-specific carbohydrate, while the activity of PlyC was partly inhibited. Additionally, PlyPy was active in vivo and could rescue mice from systemic bacteremia. Finally, we developed a novel method to determine the peptidoglycan bond cleaved by lysins and concluded that PlyPy exhibits a rare d-alanyl-l-alanine endopeptidase activity. PlyPy thus represents the first lysin characterized from Streptococcus pyogenes and has a mechanism of action distinct from that of PlyC.

Bactericidal synergism between daptomycin and the phage lysin Cpl-1 in a mouse model of pneumococcal bacteraemia.

Combination therapy may improve the outcome of Streptococcus pneumoniae-induced bacteraemia. Here we tested the combination of two antipneumococcal agents, daptomycin and Cpl-1 (the pneumococcal Cp-1 bacteriophage lysin), in a mouse model of pneumococcal bacteraemia. Mice were challenged intraperitoneally (i.p.) with 10(6)CFU of the extremely virulent serotype 2 S. pneumoniae D39 isolate. Subtherapeutic doses of daptomycin (0.4mg/kg) and Cpl-1 (0.4mg/kg and 1mg/kg) were administrated i.p. either alone or in combination by a single bolus injection 1h after bacterial challenge. Survival rates of animals were followed over a period of 7 days. Daptomycin (0.4mg/kg) in combination with Cpl-1 (0.4mg/kg) significantly increased the percentage of surviving mice at Day 7 (80%) compared with the untreated control (0%) and daptomycin or Cpl-1 monotherapy (35% and 0%, respectively). Whilst increasing the concentration of Cpl-1 to 1.0mg/kg did not improve survival when injected alone, its combination with 0.4mg/kg daptomycin further increased the survival rate to 95%. Thus, it was found that the combination of daptomycin with Cpl-1 was synergistic and bactericidal against S. pneumoniae in a mouse model of pneumococcal bacteraemia. To our knowledge, this is the first report of synergism between daptomycin and a phage lysin demonstrated in vivo. Such a combination could represent an interesting alternative therapy for the treatment of pneumococcal bacteraemia/sepsis and possibly other severe pneumococcal infections.

Improving the lethal effect of cpl-7, a pneumococcal phage lysozyme with broad bactericidal activity, by inverting the net charge of its cell wall-binding module.

Phage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny. Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from -14.93 to +3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity against Streptococcus pneumoniae (including multiresistant strains), Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-µg dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected with S. pneumoniae or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules.

Novel bacteriophage lysin with broad lytic activity protects against mixed infection by Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes (group A streptococcus [GrAS]) cause serious and sometimes fatal human diseases. They are among the many Gram-positive pathogens for which resistance to leading antibiotics has emerged. As a result, alternative therapies need to be developed to combat these pathogens. We have identified a novel bacteriophage lysin (PlySs2), derived from a Streptococcus suis phage, with broad lytic activity against MRSA, vancomycin-intermediate S. aureus (VISA), Streptococcus suis, Listeria, Staphylococcus simulans, Staphylococcus epidermidis, Streptococcus equi, Streptococcus agalactiae (group B streptococcus [GBS]), S. pyogenes, Streptococcus sanguinis, group G streptococci (GGS), group E streptococci (GES), and Streptococcus pneumoniae. PlySs2 has an N-terminal cysteine-histidine aminopeptidase (CHAP) catalytic domain and a C-terminal SH3b binding domain. It is stable at 50 °C for 30 min, 37 °C for >24 h, 4°C for 15 days, and -80 °C for >7 months; it maintained full activity after 10 freeze-thaw cycles. PlySs2 at 128 µg/ml in vitro reduced MRSA and S. pyogenes growth by 5 logs and 3 logs within 1 h, respectively, and exhibited a MIC of 16 µg/ml for MRSA. A single, 2-mg dose of PlySs2 protected 92% (22/24) of the mice in a bacteremia model of mixed MRSA and S. pyogenes infection. Serially increasing exposure of MRSA and S. pyogenes to PlySs2 or mupirocin resulted in no observed resistance to PlySs2 and resistance to mupirocin. To date, no other lysin has shown such notable broad lytic activity, stability, and efficacy against multiple, leading, human bacterial pathogens; as such, PlySs2 has all the characteristics to be an effective therapeutic.

Export of the pneumococcal phage SV1 lysin requires choline-containing teichoic acids and is holin-independent.

Streptococcus pneumoniae bacteriophages (phages) rely on a holin-lysin system to accomplish host lysis. Due to the lack of lysin export signals, it is assumed that holin disruption of the cytoplasmic membrane allows endolysin access to the peptidoglycan. We investigated the lysis mechanism of pneumococcal phage SV1, by using lysogens without holin activity. Upon phage induction in a holin deficient background, phage lysin was gradually targeted to the cell wall, in spite of lacking any obvious signal sequence. Our data indicate that export of the phage lysin requires the presence of choline in the teichoic acids, an unusual characteristic of pneumococci. At the bacterial surface, the exolysin remains bound to choline residues without inducing lysis, but is readily activated by the collapse of the membrane potential. Additionally, the activation of the major autolysin LytA, which also participates in phage-mediated lysis, is equally related to perturbations of the membrane proton motive force. These results indicate that collapse of the membrane potential by holins is sufficient to trigger bacterial lysis. We found that the lysin of phage SV1 reaches the peptidoglycan through a novel holin-independent pathway and propose that the same mechanism could be used by other pneumococcal phages.

Thermal stability of Cpl-7 endolysin from the streptococcus pneumoniae bacteriophage Cp-7; cell wall-targeting of its CW_7 motifs.

Endolysins comprise a novel class of selective antibacterials refractory to develop resistances. The Cpl-7 endolysin, encoded by the Streptococcus pneumoniae bacteriophage Cp-7, consists of a catalytic module (CM) with muramidase activity and a cell wall-binding module (CWBM) made of three fully conserved CW_7 repeats essential for activity. Firstly identified in the Cpl-7 endolysin, CW_7 motifs are also present in a great variety of cell wall hydrolases encoded, among others, by human and live-stock pathogens. However, the nature of CW_7 receptors on the bacterial envelope remains unknown. In the present study, the structural stability of Cpl-7 and the target recognized by CW_7 repeats, relevant for exploitation of Cpl-7 as antimicrobial, have been analyzed, and transitions from the CM and the CWBM assigned, using circular dichroism and differential scanning calorimetry. Cpl-7 stability is maximum around 6.0-6.5, near the optimal pH for activity. Above pH 8.0 the CM becomes extremely unstable, probably due to deprotonation of the N-terminal amino-group, whereas the CWBM is rather insensitive to pH variation and its structural stabilization by GlcNAc-MurNAc-l-Ala-d-isoGln points to the cell wall muropeptide as the cell wall target recognized by the CW_7 repeats. Denaturation data also revealed that Cpl-7 is organized into two essentially independent folding units, which will facilitate the recombination of the CM and the CWBM with other catalytic domains and/or cell wall-binding motifs to yield new tailored chimeric lysins with higher bactericidal activities or new pathogen specificities.

X-ray crystal structure of the streptococcal specific phage lysin PlyC.

Bacteriophages deploy lysins that degrade the bacterial cell wall and facilitate virus egress from the host. When applied exogenously, these enzymes destroy susceptible microbes and, accordingly, have potential as therapeutic agents. The most potent lysin identified to date is PlyC, an enzyme assembled from two components (PlyCA and PlyCB) that is specific for streptococcal species. Here the structure of the PlyC holoenzyme reveals that a single PlyCA moiety is tethered to a ring-shaped assembly of eight PlyCB molecules. Structure-guided mutagenesis reveals that the bacterial cell wall binding is achieved through a cleft on PlyCB. Unexpectedly, our structural data reveal that PlyCA contains a glycoside hydrolase domain in addition to the previously recognized cysteine, histidine-dependent amidohydrolases/peptidases catalytic domain. The presence of eight cell wall-binding domains together with two catalytic domains may explain the extraordinary potency of the PlyC holoenyzme toward target bacteria.

Combined antibacterial activity of phage lytic proteins holin and lysin from Streptococcus suis bacteriophage SMP.

Development of novel antibacterial agents is required to control infection with multidrug-resistant Streptococcus suis. HolSMP and LySMP, the holin and lysin of S. suis serotype 2 bacteriophage, named SMP, are responsible for lysis of host cells and release of progeny phage. HolSMP and LySMP expressed in Escherichia coli BL21(DE3) exerted efficient activity at 37 °C, pH 5.2, with addition of 0.8 % ß-mercaptoethanol. Lytic spectra of purified HolSMP, LySMP or HolSMP + LySMP mixture were investigated. HolSMP, exhibiting a narrow lytic spectrum, was effective against Staphylococcus aureus and Bacillus subtilis, which were insensitive to LySMP. Moreover, HolSMP was identified as a promising antibacterial agent which was able to extend the spectrum of LySMP. The data suggest that combined use of holin and lysin could be a candidate strategy for resolution of drug resistance.

Application of a bacteriophage lysin to disrupt biofilms formed by the animal pathogen Streptococcus suis.

Bacterial biofilms are crucial to the pathogenesis of many important infections and are difficult to eradicate. Streptococcus suis is an important pathogen of pigs, and here the biofilm-forming ability of 32 strains of this species was determined. Significant biofilms were completely formed by 10 of the strains after 60 h of incubation, with exopolysaccharide production in the biofilm significantly higher than that in the corresponding planktonic cultures. S. suis strain SS2-4 formed a dense biofilm, as revealed by scanning electron microscopy, and in this state exhibited increased resistance to a number of antibiotics (ampicillin, amoxicillin, ciprofloxacin, kanamycin, and rifampin) compared to that of planktonic cultures. A bacteriophage lysin, designated LySMP, was used to attack biofilms alone and in combination with antibiotics and bacteriophage. The results demonstrated that the biofilms formed by S. suis, especially strains SS2-4 and SS2-H, could be dispersed by LySMP and with >80% removal compared to a biofilm reduction by treatment with either antibiotics or bacteriophage alone of less than 20%; in addition to disruption of the biofilm structure, the S. suis cells themselves were inactivated by LySMP. The efficacy of LySMP was not dose dependent, and in combination with antibiotics, it acted synergistically to maximize dispersal of the S. suis biofilm and inactivate the released cells. These data suggest that bacteriophage lysin could form part of an effective strategy to treat S. suis infections and represents a new class of antibiofilm agents.

A phage protein that binds φC31 integrase to switch its directionality.

The serine integrase, Int, from the Streptomyces phage φC31 mediates the integration and excision of the phage genome into and out of the host chromosome. Integrases usually require a recombination directionality factor (RDF) or Xis to control integration and excision and, as φC31 Int only mediates integration in the absence of other phage proteins, we sought to identify a φC31 RDF. Here we report that the φC31 early protein, gp3 activated attL x attR recombination and inhibited attP x attB recombination. Gp3 binds to Int in solution and when Int is bound to the attachment sites. Kinetic analysis of the excision reaction suggested that gp3 modifies the interactions between Int and the substrates to form an active recombinase. In the presence of gp3, Int assembles an excision synaptic complex and the accumulation of the integration complex is inhibited. The structure of the excision synaptic complex, like that of the hyperactive mutant of Int, IntE449K, appeared to be biased towards one that favours the production of correctly joined products. The functional properties of φC31 gp3 resemble those of the evolutionarily unrelated RDF from phage Bxb1, suggesting that these two RDFs have arisen through convergent evolution.

[Development of Streptococcus pyogenes cells inactivation for turbidimetric determination of bacteriolytic activity of phage-associated enzyme].

AIM: Development a method of treatment of Streptococcus pyogenes bacteria that does not disrupt the integrity of surface structure of cell and provides optimal reproducibility of enzyme preparation activity test results. MATERIALS AND METHODS: Museum cultures of S. pyogenes M29 and S. pyogenes T1 were used, as well as standard strain S.pyogenes T5 (ATCC) and 3 phage-associated lysine PlyC preparations (enzybiotics): 2 isolated from phage C1, third--recombinant enzyme obtained by cloning phage C1 DNA. 3 methods of S. pyogenes cells treatment were used: inactivation by chloroform, antibiotics and heating. RESULTS: Treatment of S. pyogenes cells by rifampicin and gentamicin allows simultaneous turbidimetric determination of enzyme preparations activity and streptococci lysis effectiveness with a good reproducibility of test results. Comparison of kinetic curves of streptococci lysis killed by heating with curves of live culture lysis showed that heat treatment of cells results in a decrease oflysis depth and a reduction of enzyme activity. Pattern and effectiveness of lysis of cells incubated with chloroform approached curve of live streptococci lysis, however this method did not exclude lysis of part of cells and required presence of equipment for work with chemical substances. CONCLUSION. S. pyogenes test culture inactivation method by 2-step treatment of culture with antibiotics that does not disrupt the integrity of surface structure of cells and provides optimal reproducibility of enzyme preparation activity test results was developed.