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Functions involved in bacteriophage P2-induced host cell lysis and identification of a new tail gene.

ABSTRACT: Successful completion of the bacteriophage P2 lytic cycle requires phage-induced lysis of its Escherichia coli host, a process that is poorly understood. Genetic analysis of lysis-deficient mutants defined a single locus, gene K, which lies within the largest late transcription unit of P2 and maps between head gene L and tail gene R. We determined and analyzed the DNA sequence of a ca. 2.1-kb EcoRV fragment that spans the entire region from L to R, thus completing the sequence of this operon. This region contains all of the functions necessary for host cell lysis. Sequence analysis revealed five open reading frames, initially designated orf19 through orf23. All of the existing lysis mutants--ts60, am12, am76, and am218--were located in orf21, which must therefore correspond to gene K. The K gene product has extensive amino acid sequence similarity to the product of gene R of bacteriophage lambda, and its exhibits endolysin function. Site-directed mutagenesis and reverse genetics were used to create P2 amber mutants in each of the four other newly identified open reading frames. Both orf19 (gene X) and orf20 (gene Y) encode essential functions, whereas orf22 (lysA) and orf23 (lysB) are nonessential. Gene Y encodes a polypeptide with striking similarities to the family of holin proteins exemplified by gpS of phage lambda, and the Yam mutant displayed the expected properties of a holin mutant. The gene products of lysA and lysB, although nonessential, appear to play a role in the correct timing of lysis, since a lysA amber mutant caused slightly accelerated lysis and a lysB amber mutant slightly delayed lysis of nonpermissive strains. Gene X must encode a tail protein, since lysates from nonpermissive cells infected with the X amber mutant were complemented in vitro by similar lysates of cells infected with P2 head mutants but not with tail mutants.

SUBMITTER: Ziermann R

PROVIDER: S-EPMC196335 | biostudies-other | 1994 Aug

REPOSITORIES: biostudies-other

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Functions involved in bacteriophage P2-induced host cell lysis and identification of a new tail gene.

Ziermann R R Bartlett B B Calendar R R Christie G E GE

Journal of bacteriology 19940801 16

Successful completion of the bacteriophage P2 lytic cycle requires phage-induced lysis of its Escherichia coli host, a process that is poorly understood. Genetic analysis of lysis-deficient mutants defined a single locus, gene K, which lies within the largest late transcription unit of P2 and maps between head gene L and tail gene R. We determined and analyzed the DNA sequence of a ca. 2.1-kb EcoRV fragment that spans the entire region from L to R, thus completing the sequence of this operon. Th ...[more]

PMID: 8051010

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Project description:Rapid detection of bacterial pathogens is a critical unmet need for both food and environmental samples such as irrigation water. As a part of the Food safety Modernization Act (FSMA), The Produce Safety rule has established several requirements for testing for the presence of generic Escherichia coli in water, but the current method available for testing (EPA M1603) demands specified multiple colony verification and highly trained personnel to perform these tests. The purpose of the study was to assess a phage induced bacterial lysis using quantitative image analysis to achieve rapid detection of E. coli at low concentrations within 8 hours. This study aimed to develop a simple yet highly sensitive and specific approach to detect target bacteria in complex matrices. In the study, E. coli cells were first enriched in tryptic soy broth (TSB), followed by T7 phage induced lysis, concentration, staining and fluorescent imaging. Image analysis was conducted including image pre-processing, image segmentation and quantitatively analysis of cellular morphological features (area, eccentricity and full width at half maximum). Challenge experiments using realistic matrices, including simulated fresh produce wash water, coconut water and spinach wash water, demonstrated the method can be applied for use in situations that occur in food processing facilities. The results indicated E. coli cells that are lysed by T7 phages demonstrated significantly (P < 0.05) higher extracellular DNA release, altered cellular shape (from rod to circular) and diffused fluorescent signal intensity. Using this biosensing strategy, a sensitivity to detect Escherichia coli at 10 CFU/ml within 8 hours was achieved, both in laboratory medium and in complex matrices. The proposed phage based biosensing strategy enables rapid detection of bacteria and is applicable to analysis of food systems. Furthermore, the steps involved in this assay can be automated to enable detection of target bacteria in food facilities without extensive resources.

Project description:Bacterial viruses, or bacteriophages, are highly abundant in the biosphere and have a major impact on microbial populations. Many examples of phage interactions with their hosts, including establishment of dormant lysogenic and active lytic states, have been characterized at the level of the individual cell. However, much less is known about the dependence of these interactions on host metabolism and signal exchange within bacterial communities. In this report, we describe a lysogenic state of the enterobacterial phage T1, previously known as a classical lytic phage, and characterize the underlying regulatory circuitry. We show that the transition from lysogeny to lysis depends on bacterial population density, perceived via interspecies autoinducer 2. Lysis is further controlled by the metabolic state of the cell, mediated by the cyclic-3',5'-AMP (cAMP) receptor protein (CRP) of the host. We hypothesize that such combinations of cell density and metabolic sensing may be common in phage-host interactions.IMPORTANCE The dynamics of microbial communities are heavily shaped by bacterium-bacteriophage interactions. But despite the apparent importance of bacteriophages, our understanding of the mechanisms controlling phage dynamics in bacterial populations, and particularly of the differences between the decisions that are made in the dormant lysogenic and active lytic states, remains limited. In this report, we show that enterobacterial phage T1, previously described as a lytic phage, is able to undergo lysogeny. We further demonstrate that the lysogeny-to-lysis decision occurs in response to changes in the density of the bacterial population, mediated by interspecies quorum-sensing signal AI-2, and in the metabolic state of the cell, mediated by cAMP receptor protein. We hypothesize that this strategy enables the phage to maximize its chances of self-amplification and spreading in bacterial population upon induction of the lytic cycle and that it might be common in phage-host interactions.

Dataset Information

Functions involved in bacteriophage P2-induced host cell lysis and identification of a new tail gene.

Publications

Functions involved in bacteriophage P2-induced host cell lysis and identification of a new tail gene.

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