The N-terminal region of the Oenococcus oeni bacteriophage fOg44 lysin behaves as a bona fide signal peptide in Escherichia coli and as a cis-inhibitory element, preventing lytic activity on oenococcal cells

The function of the N-terminal region of the Oenococcus oeni phage fOg44 lysin (Lys44) as an export signal was investigated. We observed that when induced in Escherichia coli, Lys44 was cleaved between residues 27 and 28 in a SecA-dependent manner. Lys44 processing could be blocked by a specific signal peptidase inhibitor and was severely reduced by modification of the cleavage site. The lethal effect of Lys44 expression observed in E. coli was ascribed to the presence of its N-terminal 27-residue sequence, as its deletion resulted in the production of a nontoxic, albeit active, product. We have further established that lytic activity in oenococcal cells was dependent on Lys44 processing. An active protein with the molecular mass expected for the cleaved enzyme was detected in extracts from O. oeni-infected cells. The temporal pattern of its appearance suggests that synthesis and export of Lys44 in the infected host progress along with phage maturation. Overall, these results provide, for the first time, experimental evidence for the presence of a signal peptide in a bacteriophage lysin. Database searches and alignment of protein sequences support the prediction that other known O. oeni and Lactococcus lactis phages also encode secretory lysins. The evolutionary significance of a putative phage lysis mechanism relying on secretory lytic enzymes is tentatively discussed, on the basis of host cell wall structure and autolytic capacity.     

Construction and properties of a ribosome-binding site mutation in gene E of phi X174 bacteriophage.

Oligodeoxyribonucleotide mutagenesis has been used to produce a G----A mutation at nucleotide 557 of the phi X174 genome. This changes the ribosome-binding sequence GAGG of gene E to GAAG without affecting the amino acid, glutamine, encoded by the overlapping gene D. The phi X174rb(E)557 mutant does not lyse infected Escherichia coli C and therefore results in the accumulation of a large number intracellular mature phage particles. Thus, the mutation inactivates production of the gene E lytic product, presumably by blocking translation of gene E, without affecting other phage functions.     

Lactococcus lactis lytic bacteriophages of the P335 group are inhibited by overexpression of a truncated CI repressor

Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage phi31 encodes a genetic switch region of cI and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative cI repressor gene of phage phi31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length cI gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the cI gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage phi31 was inhibited to an efficiency of plaquing (EOP) of 10(-6) in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the phi31 EOP to <10(-7). Phage phi31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the phi31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.     

Identification and characterization of an endolysin encoded by the<Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> phage μ1/6

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.     

Nucleotide sequence of the right early region of Bacillus phage phi 15 and comparison with related phages: reorganization of gene 17 during evolution

The rightmost 2016 bp of the Bacillus subtilis phage phi 15 genome were sequenced. The nucleotide sequence was compared with the homologous regions of the related phages PZA and phi 29. There are six open reading frames (ORFs) in this region of the phi 15 genome; all of them are present in the PZA and phi 29 genomes. One of the ORFs was assigned to gene 17, which is involved in the replication of the phage DNA. Gene 17 has undergone reorganization during the evolution of this phage family. Comparison of the nucleotide sequence of its mRNA-like strand in phi 15, PZA and phi 29 showed that deletions in its central and 3'-end-proximal parts are tolerated and do not interfere with the gene 17 product function. It seems that the only portion of gene 17 that has to be conserved to encode the functional product is its 5'-end-proximal part.     

DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu.

We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth.     

A triggered-suicide system designed as a defense against bacteriophages.

A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.     

Nucleotide sequence of the genome of the bacteriophage alpha 3: interrelationship of the genome structure and the gene products with those of the phages, phi X174, G4 and phi K.

The complete nucleotide sequence of the genome of the circular single-stranded DNA (isometric) phage alpha 3 has been determined and compared with that of the related phages phi X174 and G4. The alpha 3 genome consists of 6087 nucleotides, which is 701 nucleotides longer than the nucleotide sequence of the phi X174 genome and 510 nucleotides more than that of the G4 genome. The results demonstrated that the three phage species have 11 homologous genes (A, A*, B, C, K, D, E, J, F, G and H), the order of which is fundamentally identical, suggesting that they have evolved from a common ancestor. The sequence of some genes and untranslated intergenic regions, however, differs significantly from phage to phage: for example, the degree of amino acid sequence homology of the gene product is averaged at 47.7% between alpha 3 and phi X174 and 46.9% between alpha 3 and G4, and alpha 3 has a remarkable longer intergenic region composed of 758 nucleotides between the genes H and A compared with the counterparts of phi X174 and G4. Meanwhile, in vivo experiments of genetic complementation showed that alpha 3 can use none of the gene products of phi X174 and G4, whereas the related phage phi K can rescue alpha 3 nonsense mutants of the genes B, C, D and J. These sequencing and in vivo rescue results indicated that alpha 3 is closely related to phi K, but distantly remote from phi X174 or G4, and supported an evolutional hypothesis which has been so far proposed that the isometric phages are classified into three main groups: the generic representatives are phi X174, G4 and alpha 3.     

Lysogenic conversion caused by phage phi 80. III. The mapping of the conversion gene and additional characterization of the phenomenon

The cor gene specifies lysogenic conversion caused by the lambdoid phage phi 80. The cor gene product inhibits tonA function in infected and lysogenic cells. The cells harboring pBR322 plasmid with the cloned cor gene of phi 80 became resistant to the phages T1 and phi 80 (TonA phenotype). The cor gene was mapped between 24 and 13 genes on the phi 80 phage genetic map. It is not essential for phage lytic growth. Its presence in lysogens leads up to accumulation of tonA mutants in a cell population.     

Tail-associated structural protein gp61 of Staphylococcus aureus phage phi MR11 has bifunctional lytic activity.

A tailed bacteriophage, phi MR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1-150] and the lysozyme (aa 401-624) domains but not the linker domain (aa 151-400) caused efficient lysis of S. aureus. Immunoelectron microscopy localized gp61 to the tail tip of the phi MR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of phi MR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with phi MR11 was also lysed by both proteins. Staphylococcus aureus strains on which phi MR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.     

Comparison of the amino acid sequence of the lytic enzyme from broad-host-range bacteriophage PRD1 with sequences of other cell-wall-peptidoglycan lytic enzymes

The gene for the lytic enzyme of the lipid-containing, broad-host-range bacteriophage PRD1 codes for a protein of 149 amino acids (17271 Da). The sequence of the protein is unique when compared to other lytic enzymes sequenced. However, three regions of weak similarity with other phage lytic enzymes were observed. The C-terminal region shared seven amino acids in common with phage P22 lysozyme at a site which is conserved in phage-type lysozymes.     

Molecular analysis of the lysis protein Lys encoded by Lactobacillus plantarum phage phig1e

The putative cell-lysis gene lys of Lactobacillus plantarum G1e phage phig1e encodes for a 442 amino-acids protein Lys. The N-terminal region (about 80 amino acids) of Lys consists of two discrete regions (the signal-peptide-like domain and the DE domain containing putative active sites of endolysin). To elucidate functions of the regions of Lys, mutational (random, site-directed, and/or fusion) analysis was performed. The plasmid pNdEHL, expressing the wild type Lys protein under promoter of lacZ' gene in Escherichia coli, was constructed. Two molecular species (44 kDa; referred to as pre-Lys, and 42 kDa; mature-Lys) from the protein extract of XL1-Blue/pNdEHL were detected on a sodium dodecyl sulfate gel and zymogram with L. plantarum G1e cells. Based on the N-terminal amino acid sequences, the two molecules were determined as; pre-Lys (the amino acid position deduced from lys gene, 1-7) MKLKNKL, mature-Lys (27-33) QTLSSQS. The mature Lys was hardly detected in the cells treated with sodium azide.These results suggested that the N-terminal 26 amino acids region of Lys precursor form is possibly processed posttranslationally, by a SecA-dependent manner at least in E. coli.Analysis of the point mutants (pLD36A, pLE39A, pLE55A, pLE67A and pLD71A), indicated that the acidic residues (aspartic acids at position 36, 71 and glutamic acids at position 39, 55) of N-terminal region and the serine at the position 48 of phig1e Lys are essential for the lytic activity.