A Cytoplasmic Antiholin Is Embedded In Frame with the Holin in a Lactobacillus fermentum Bacteriophage.

In double-stranded DNA bacteriophages, infection cycles are ended by host cell lysis through the action of phage-encoded endolysins and holins. The precise timing of lysis is regulated by the holin inhibitors, named antiholins. Sequence analysis has revealed that holins with a single transmembrane domain (TMD) are prevalent in Lactobacillus bacteriophages. A temperate bacteriophage of Lactobacillus fermentum, ϕPYB5, has a two-component lysis cassette containing endolysin Lyb5 and holin Hyb5. The hyb5 gene is 465 bp long, encoding 154 amino acid residues with an N-terminal TMD and a large cytoplasmic C-terminal domain. However, the N terminus contains no dual-start motif, suggesting that Hyb5 oligomerization could be inhibited by a specific antiholin. Two internal open reading frames in hyb5, hyb5157-465 and hyb5209-328, were identified as genes encoding putative antiholins for Hyb5 and were coexpressed in trans with lyb5-hyb5 in Escherichia coli Surprisingly, host cell lysis was delayed by Hyb5157-465 but accelerated by abolishment of the translation initiation site of this protein, indicating that Hyb5157-465 acts as an antiholin to holin Hyb5. Moreover, deletion of 45 amino acid residues at the C terminus of Hyb5 resulted in early cell lysis, even in the presence of Hyb5157-465, implying that the interaction between Hyb5157-465 and Hyb5 occurs at the C terminus of the holin. In vivo and in vitro, Hyb5157-465 and Hyb5 were detected in the cytoplasmic and membrane fractions, respectively, and pulldown assays confirmed direct interaction between Hyb5157-465 and Hyb5. All the results suggest that Hyb5157-465 is an antiholin of Hyb5 that is involved in lysis timing.IMPORTANCE Phage-encoded holins are considered to be the "molecular clock" of phage infection cycles. The interaction between a holin and its inhibitor antiholin precisely regulates the timing of lysis of the host cells. As a prominent biological group in dairy processes, phages of lactic acid bacteria (LAB) have been extensively genome sequenced. However, little is known about the antiholins of LAB phage holins and the holin-antiholin interactions. In this work, we identified an in-frame antiholin against the class III holin of Lactobacillus fermentum phage ϕPYB5, Hyb5, and demonstrated its interaction with the cognate holin, which occurred in the bacterial cytoplasm.

Functional analysis of the N-terminal region of endolysin Lyb5 encoded by Lactobacillus fermentum bacteriophage φPYB5.

Lactobacillus fermentum temperate bacteriophage φPYB5 uses endolysin Lyb5 and holin Hyb5 to burst the host cell. Previous results showed that expression of Lyb5 in Escherichia coli caused host cell lysis slowly, leading us to suppose that Lyb5 could pass the cytoplasmic membrane partly. In this work, the function of a putative signal peptide (SPLyb5) at the N-terminal of Lyb5 was investigated. In E. coli, the cell adopted a spherical shape during induction of Lyb5 protein, while morphological changes were not observed during expression of the SPLyb5 truncation, indicating that the SPLyb5 motif may serve as a functional signal peptide. However, SPLyb5 was not proteolytically cleaved at the predicted site during the translocation of Lyb5, and the expressed Lyb5 protein appeared in the cytoplasm, cytoplasmic membrane and periplasm fractions with the same molecular mass. Similar results were obtained using Lactococcus lactis as a host to express Lyb5. These results indicated that SPLyb5 could direct Lyb5 to the periplasm in a membrane-tethered form, and then release it as a soluble active enzyme into the periplasm. In addition, SPLyb5 could also drive the fused NucleaseB protein to the extracytoplasm environment in E. coli as well as in L. lactis. We proposed that in Gram-negative and Gram-positive hosts SPLyb5 acted as a signal-anchor-release domain, which was firstly identified here by experimental evidences in lactic acid bacteria phages. The application of signal-anchor-release domain for endolysin export in bacteriophages infecting Gram-positive and Gram-negative hosts was discussed.

Complete genomic sequence of the Lactobacillus temperate phage LF1.

Bacteriophage LF1, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild type Lactobacillus fermentum, was found to contain a double-strand DNA of 42,606 base pairs (bp) with a G+C content of 45%. Bioinformatic analysis of the phage genome revealed 57 putative open reading frames (ORFs). The predicted protein products of ORFs were determined and described. According to morphological analysis by transmission electron microscopy (TEM), LF1 has an isometric head and a non-contractile tail, indicating that it belongs to the family Siphoviridae. The temperate phage LF1 has a good genetic mosaic relationship with ΦPYB5 in the packaging module. To our knowledge, this is first report of genomic sequencing and characterization of temperate phage LF1 from wild-type L. fermentum isolated from Kimchi in Korea.

Genome analysis of Lactobacillus fermentum temperate bacteriophage ФPYB5.

The complete genomic sequence of Lactobacillus fermentum temperate bacteriophage ФPYB5 was determined. The phage possesses a linear, double-stranded DNA genome of 32,847 bp with a G+C content of 45.21%. A total of 46 putative open reading frames (ORFs) were identified. On the basis of homology comparisons, 25 ORFs could be assigned putative functions. The genome of bacteriophage ФPYB5 is highly modular with functionally related genes clustered together. Genome DNA of temperate bacteriophage ФPYB5, induced from heterofermentative lactic acid bacteria, showed to be closely related to that of the prophage of heterofermentative Lactobacillus reuteri 100-23 and heterofermetative Leuconostoc oenos bacteriophage 10MC in an evolutionary aspect.

Isolation and characterization of a Lactobacillus fermentum temperate bacteriophage from Chinese yogurt.

AIMS: The aim of this study was to investigate the properties of temperate bacteriophage of Lactobacillus fermentum, based on its morphology, restriction patterns, protein profile and the impact on the growth of host strain. METHODS AND RESULTS: With Mitomycin C, seven temperate phages were induced from Lactobacilli derived from Chinese yogurt. The temperate phages induced belong to the most common Bradley's group B, having hexagonal head and long, noncontractile tail. They were furthermore confirmed to be the same bacteriophage by identical restriction patterns. SDS-PAGE profile showed that the phage studied had one major structure protein about 31.9 kDa. The presence of the prophage influenced the cell shape and colony size of its lysogenic strain. CONCLUSIONS: The phage obtained had similar, but not complete identical properties with other L. fermentum phages reported. It influenced the growth behaviour of its lysogenic strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides some information about bacteriophages occurring in the Chinese yoghurt manufacture and contributes to our knowledge on the bacteriophage diversity in the dairy industry.