Characterization of the N-Terminal Catalytic Domain of Lytµ1/6, an Endolysin from Streptomyces aureofaciens Phage µ1/6.

Previous characterization of Lytµ1/6, an endolysin from Streptomyces aureofaciens phage µ1/6, suggested that the N-terminal domain is responsible for the catalytic activity of Lytµ1/6. Mutational analyses (deletions and site-directed mutagenesis) demonstrated that lytic activity of Lytµ1/6 relies on the N-terminal part of about 200 amino acid residues. Various C-terminally truncated versions of Lytµ1/6 failed to cause lysis, indicating the necessity of the CBD for full enzyme activity. Functional analysis of the point mutants suggested that the residues K27, H31, E109, H176, and D184 were essential for lytic activity of the µ1/6 endolysin. Further characterization of the purified Lytµ1/6 revealed that this endolysin is an N-acetylmuramoyl-L-alanine amidase which seems to be unrelated to any of the known conserved catalytic domains of phage endolysins or bacterial autolysins.

Bacteriophage endolysin Lyt µ1/6: characterization of the C-terminal binding domain.

The gene product of orf50 from actinophage µ1/6 of Streptomyces aureofaciens is a putative endolysin, Lyt µ1/6. It has a two-domain modular structure, consisting of an N-terminal catalytic and a C-terminal cell wall binding domain (CBD). Comparative analysis of Streptomyces phage endolysins revealed that they all have a modular structure and contain functional C-terminal domains with conserved amino acids, probably associated with their binding function. A blast analysis of Lyt µ1/6 in conjunction with secondary and tertiary structure prediction disclosed the presence of a PG_binding_1 domain within the CBD. The sequence of the C-terminal domain of lyt µ1/6 and truncated forms of it were cloned and expressed in Escherichia coli. The ability of these CBD variants fused to GFP to bind to the surface of S. aureofaciens NMU was shown by specific binding assays.

Analysis of the site-specific integration system of the Streptomyces aureofaciens phage µ1/6.

The bacteriophage µ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the µ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The µ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3' end of a putative tRNA(Thr) gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing µ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The µ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified µ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.

The lysis system of the Streptomyces aureofaciens phage mu1/6.

Previously, two genes, designated as lyt and hol, were identified in the lysis module of phage mu1/6. They were cloned and expressed in Escherichia coli. An additional candidate holin gene, hol2, was found downstream from the hol gene based on one predicted transmembrane domain and a highly charged C-terminal sequence of the encoded protein. Expression of hol or hol2 in E. coli was shown to cause cell death. The concomitant expression of lambda endolysin (R) and mu1/6 holin resulted in cell lysis. Similarly, the coexpression of the endolysin and holin of phage mu1/6 led to lysis, apparently due to the ability of mu1/6 endolysin to hydrolyze the peptidoglycan layer of this bacterium. In contrast, the simultaneous expression of mu1/6 hol2 and the endolysin gene (lambdaR or mu1/6 lyt) did not cause detectable lysis of the host cells. Demonstration of the holin function in streptomycetes was achieved by providing for the release of mu1/6 endolysin to the periplasm and subsequent cleavage of the peptidoglycan, which strongly suggested that the holin produces lesions in the streptomycete membrane.

Complete genome sequence and analysis of the Streptomyces aureofaciens phage mu1/6.

The entire double-stranded DNA genome of the Streptomyces aureofaciens phage mu1/6 was sequenced and analyzed. Its size is 38.194 kbp with an overall molar G+C content of 71.2 %. Fifty-two potential open reading frames (orfs) were identified, divided into two oppositely transcribed regions. In the left arm of the mu1/6 genome, an identified putative integrase and possible regulation proteins were identified. The rightwards transcribed region contains genes organized into apparently four functional units responsible for: (i) replication, (ii) DNA packaging and head assembly, (iii) tail morphogenesis, and (iv) lysis. Putative functions were assigned to twelve orfs based on bioinformatic analysis or experimental substantiation. Comparative analysis with three complete genomes of streptomycete phages revealed resemblance with respect to the organization of their genes into functional modules. Closer relationship was observed only between mu1/6 and S. venezuelae phage VWB.

Identification of a holin encoded by the Streptomyces aureofaciens phage micro1/6; functional analysis in Escherichia coli system.

An open reading frame encoding an 88 amino acid protein was present downstream of the previously characterized endolysin of Streptomyces aureofaciens phage micro1/6. Structural analysis of its sequence revealed features characteristic for holin. This open reading frame encoding the putative holin was amplified by polymerase chain reaction and cloned into the expression vector pET-21d(+). Synthesis of the holin-like protein resulted in bacterial cell death but not lysis. The holmicro1/6 gene was able to complement the defective lambda S allele in the nonsuppressing Escherichia coli HB101 strain to produce phage progeny, This fact suggests that the proteins encoded by both phage genes have analogous function, i.e. the streptomycete holin induces nonspecific lesions in the cytoplasmic membrane, through which the lambda endolysin gains an access to its substrate, the cell wall. The concomitant expression of both S. aureofaciens holmicro 1/6 and lambda endolysin in E. coli resulted in abrupt cell lysis. This result provided further evidence that the product of holmicro 1/6 gene is a holin.

Identification and characterization of an endolysin encoded by the Streptomyces aureofaciens phage mu 1/6.

An open reading frame homologous to the genes encoding several cell-wall hydrolyzing enzymes was identified on the genome of actinophage mu 1/6. This open reading frame encoding the putative endolysin was amplified by polymerase chain reaction and cloned into the expression vector pET-21a. This gene consisted of 1182 bp encoding a 393 amino acid polypeptide with a molar mass of 42.1 kDa. The gene product was overexpressed in Escherichia coli, and then the lytic enzyme was purified by a two-step chromatographic procedure. When applied exogenously, the endolysin of phage mu 1/6 was active against all tested Streptomyces strains but did not affect other bacteria. The amino acid sequence showed a high homology with a putative amidase of the Streptomyces phase phi C31. Downstream of the endolysin gene, an open reading frame encoding an 88 amino acid protein was identified. Structural analysis of its sequence revealed features characteristics for holin.

Characterization of a XhoI isoschizomer in Streptomyces aureofaciens after actinophage infection.

After infection of tetracycline producing strains of S. aureofaciens with actinophages mu 1/6 and B1 some phage resistant colonies were obtained in each experiment. These colonies expressed a new restriction-modification (RM) system of type II, which was different from the common RM system (SauLPI) of these strains recognizing the sequence GCCGGC. This new RM system was not detected before in parental strains. The new endonuclease was purified from a phage resistant strain of S. aureofaciens B96, using two step column chromatography to the grade without non-specific nucleolytic activity. SauLPII endonuclease recognized and cleaved the palindromic hexanucleotide sequence 5'-C/TCGAG-3', thus it was a true isoschizomer of XhoI.