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Abstract Background Endolysins produced by bacteriophages lyse bacteria, and are thus considered a novel type of antimicrobial agent. Several endolysins from Bacillus phages or prophages have previously been characterized and used to target Bacillus strains that cause disease in animals and humans. B. thuringiensis phage BtCS33 is a Siphoviridae family phage and its genome has been sequenced and analyzed. In the BtCS33 genome, orf18 was found to encode an endolysin protein (PlyBt33). Results Bioinformatic analyses showed that endolysin PlyBt33 was composed of two functional domains, the N-terminal catalytic domain and the C-terminal cell wall binding domain. In this study, the entire endolysin PlyBt33, and both the N- and C-termini,were expressed in Escherichia coli and then purified. The lytic activities of PlyBt33 and its N-terminus were tested on bacteria. Both regions exhibited lytic activity, although PlyBt33 showed a higher lytic activity than the N-terminus. PlyBt33 exhibited activity against all Bacillus strains tested from five different species, but was not active against Gram-negative bacteria. Optimal conditions for PlyBt33 reactivity were pH 9.0 and 50°C. PlyBt33 showed high thermostability, with 40% of initial activity remaining following 1 h of treatment at 60°C. The C-terminus of PlyBt33 bound to B. thuringiensis strain HD-73 and Bacillus subtilis strain 168. This cell wall binding domain might be novel, as its amino acid sequence showed little similarity to previously reported endolysins. Conclusions PlyBt33 showed potential as a novel antimicrobial agent at a relatively high temperature and had a broad lytic spectrum within the Bacillus genus. The C-terminus of PlyBt33 might be a novel kind of cell wall binding domain.

The coevolution between phage and host bacterium is an important force that drives the evolution of the microbial community, yet the coevolution mechanisms have still not been well analyzed. Here, by analyzing the interaction between a Bacillus phage vB_BthS_BMBphi and its host bacterium, the coevolution mechanisms of the first-generation phage-resistant bacterial mutants and regained-infectivity phage mutants were studied. The phage-resistant bacterial mutants showed several conserved mutations as a potential reason for acquiring phage resistance, including the mutation in flagellum synthesis protein FlhA and cell wall polysaccharide synthesis protein DltC. All the phage-resistant bacterial mutants showed a deleted first transmembrane domain of the flagellum synthesis protein FlhA. Meanwhile, the regain-infectivity phage mutants all contained mutations in three baseplate-associated phage tail proteins by one nucleotide, respectively. A polymorphism analysis of the three mutant nucleotides in the wild-type phage revealed that the mutations existed before the interaction of the phage and the bacterium, while the wild-type phage could not infect the phage-resistant bacterial mutants, which might be because the synchronized mutations of the three nucleotides were essential for regaining infectivity. This study for the first time revealed that the synergism mutation of three phage baseplate-associated proteins were essential for the phages' regained infectivity. Although the phage mutants regained infectivity, their storage stability was decreased and the infectivity against the phage-resistant bacterial mutants was reduced, suggesting the phage realized the continuation of the species by way of "dying to survive". [ABSTRACT FROM AUTHOR]

Phages, the parasites of bacteria, are considered as a new kind of antimicrobial agent due to their ability to lyse pathogenic bacteria. Due to the increase of available phage isolates, the newly isolated phage showed increasing genomic similarities with previously isolated phages. In this study, the novel phage vB_BthS_BMBphi, infecting the Bacillus thuringiensis strain BMB171, is isolated and characterized together with its endolysin. This phage is the first tadpole-like phage infecting the Bacillus strains. Genomic analysis shows that the phage genome is dissimilar to all those of previously characterized phages, only exhibiting low similarities with partial regions of the B. thuringiensis prophages. Phylogenetic analysis revealed that the phage was distant from the other Bacillus phages in terms of evolution. The novel genome sequence, the distant evolutionary relationship, and the special virion morphology together suggest that the phage vB_BthS_BMBphi could be classified as a new phage lineage. The genome of the phage is found to contain a restriction modification system, which might endow the phage with immunity to the restriction modification system of the host bacterium. The function of the endolysin PlyBMB encoded by the phage vB_BthS_BMBphi was analyzed, and the endolysin could lyse all the tested Bacillus cereus group strains, suggesting that the endolysin might be used in controlling pathogenic B. cereus group strains. The findings of this study enrich the understanding of phage diversity and provide a resource for controlling the B. cereus group pathogenic bacteria. [ABSTRACT FROM AUTHOR]