Search

The t protein of bacteriophage T4 shares with other holins the ability to cause the formation of a lethal membrane lesion which allows the phage endolysin to attack the peptidoglycan. Moreover, T, like other holins, acts in a saltatory manner at a precisely programmed time in the vegetative cycle. Unlike other holins, however, T has the unique ability to postpone its lethal function in response to a secondary infection by a T-even phage during the vegetative cycle. A signal transduction system that responds to the secondary infection is thought to be encoded by some of the numerous r genes, defined by mutations that abolish this lysis-inhibition (LIN) response. The primary structure of T differs from two main structural patterns found in more than 30 orthologous groups of holins. Genetic approaches were taken to probe the t sequence for features involved in membrane localization, functional timing and LIN regulation. Gene fusion analysis indicates that T has a single TMD near the N-terminus, with the bulk of the protein residing in the periplasm. Mapping and phenotypic analysis of deletion and point mutations in t indicates that the periplasmic domain of T is the major determinant of the timing mechanism and is involved in the LIN response.

Lactobacillus plantarum phage φ g1e has two consecutive cell lysis genes hol–lys ( Oki et al., 1996b ). In the present study, functional and structural properties of the hol protein (Hol) were characterized in Escherichia coli . Electron microscopic examinations showed that hol under p lac in E. coli XL1-Blue injured the inner membrane to yield empty ghost cells with the bulk of the cell wall undisturbed. Northern blot analysis indicated that hol–lys genes under p lac were co-transcribed, although the amount of hol transcript was larger than that of lys , ceasing via an apparently ρ -independent terminator just downstream of hol . However, deletion and/or fusion experiments suggested that: (1) the N-terminal half of φ g1e Hol composed of three putative transmembrane domains may be responsible for interaction with membrane; (2) the N-terminal end (five amino acids) seems nonessential; and (3) the C-terminal half containing charged amino acids appears to be involved in proper hol function. These results suggest that φ g1e Hol is a member of the lambdoid holin family, but divergent in several properties from lambda holin.

The lysis genes of a Lactobacillus phage Fgle were cloned, sequenced, and expressed in Escherichia coli . Nucleotide sequencing of a 3813-bp Fgle DNA revealed five successive open reading frames (ORF), Rorf50, Rorf118, hol , and lys and Rorf175 , in the same DNA strand. By comparative analysis of the DNA sequence, the putative hol product (holin) has an estimated molecular weight is 14.2 kDa, and contains two potential transmembrane helices and highly charged N- and C-termini, resembling predicted holins (which are thought to be a cytoplasmic membrane-disrupting protein) encoded by other phages such as mvl from Lactobacillus bulgaricus , Fadh from Lactobacillus gasseri , as well as monocins from Listeria . On the other hand, the putative Fgle lys product (lysin) of 48.4 kDa shows significant similarity with presumed muramidase, known as a cell wall peptidoglycandegrading enzyme, encoded by the Lactobacillus phage mvl and Fadh, the Lactococcus lactis phage FLC3, and the Streptococcus pneumoniae phages Cp-1, Cp-7 and Cp-9. When expressed in E. coli , the Fgle lysin and/or holin decreased the cell turbidity significantly, suggesting that the Fgle hol-lys system is involved in cytolytic process.

A Bacillus licheniformis DNA fragment which exhibits homology with the upstream region of the cell-wall hydrolase-encoding gene, cwlL, was cloned into Escherichia coli (Ec). Nucleotide sequencing indicated that there are two open reading frames (tentatively designated as xpaG1 and xpaG2) which encode polypeptide of 89 and 88 amino acids (aa) (10044 and 9764 Da, respectively). Ec cells harboring two compatible plasmids (pMWB1 and pHSGKH) containing the Bacillus subtilis cell-wall hydrolase-encoding gene, cwlA, and xpaG1–G2, respectively, exhibited higher extracellular cell-wall hydrolase activity than did cells harboring pMWB1 and a control plasmid, pHSG398. The aa sequence homology of XpaG2 with other polypeptides indicated that xpaG2 is a holin-encoding gene. Moreover, Ec C600 harboring a plasmid containing xpaG1-xpaG2 led to leakage of β-galactosidase into the extracellular fraction.

A defective prophage vector, phi 105MU331, for high-level protein overproduction in Bacillus subtilis, was derived by random insertion of a lacZ reporter gene. The site of insertion not only provided efficient inducible transcription of heterologous genes, but also prevented lysis of the host cell. The region of the insertion in phi 105MU331 lies close to the right cohesive end of phi 105. DNA sequence analysis revealed that this region of phi 105 somewhat resembles the lysis cassette of various phages, including lambda. The site of insertion lies in a possible 'holin' gene, which could explain the block in host cell lysis. Dual promoters apparently responsible for the strong inducible transcription lie in an untranslated region just upstream from the putative holin gene. This region is probably equivalent to the site of the major late promoter and antiterminator of the lambdoid phages. The sequence features could, thus, account for the useful properties of the phi 105MU331 vector system.

Nonlysogenic Escherichia coli K cells exhibit a delay in lysis when infected by T4 rII phage termed lysis inhibition (LIN). E. coli K cells expressing λ rexB from either a prophage defective for rexA , or a multicopy plasmid supported T4 rII infection, but prevented the establishment of LIN. In addition, E. coli null mutations in either the periplasmic “tail-specific protease” tsp , or the 10Sa RNA ssrA , completely blocked the establishment of LIN following T4 infections. The expression of rexB in the absence of rexA resulted in several cellular phenotypes, including aberrant cell surface morphology, the partial to near complete suppression of mutations of λ S and T4 t holin genes, and lysis by cells aging on plates or growing with high rexB expression at elevated temperatures. These activities of RexB were impeded in the presence of RexA.

The nucleotide sequence of a DNA fragment previously shown to contain the attachment site (attP) of Oenococcus oeni phage fOg44 (. Arch. Virol. 143, 523-536) has been determined. Sequence analysis indicated that this 6226bp EcoRI fragment harbours an integrase gene, in the vicinity of a direct repeat rich region defining attP, as well as genes encoding a muramidase-related lysin (Lys) and a holin polypeptide (Hol). Transcriptional studies suggested that lys and hol are mainly co-expressed, late in the lytic cycle, from a promotor located upstream of lys. Between the lytic cassette and the phage integration elements three additional open reading frames were found: orf217 and orf252 of unknown function and orf72, the putative product of which bears 32% identity with acidic excisionases from other Gram positive phages. We have established that the first two orfs, as well as the predicted promotor of orf72, are included in a 2143-bp DNA segment missing from the genome of the deletion mutant fOg44Delta2. Although lysogens of fOg44 and fOg44Delta2 exhibited similar properties, each phage produced two distinguishable types of lysogenic strains, differing in inducibility and immunity to other oenophages.