A window into lysogeny: revealing temperate phage biology with transcriptomics.
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ABSTRACT:
SUBMITTER: Owen SV
PROVIDER: S-EPMC7067206 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The global transcriptional profile of novel T7-like Pseudomonas aeruginosa phage LUZ100 was obtained using the long read RNA sequencing technique ONT-cappable-seq. Using this approach we obtained a comprehensive genome-wide map of viral transcription start sites, terminators and transcription units and gained new insights in the molecular mechanisms of transcriptional regulation of T7-like temperate phages.
2023-02-08 | GSE211961 | GEO
Project description:Bacteriophages are the most abundant organisms on the planet and both lytic and temperate phages play key roles as shapers of ecosystems and drivers of bacterial evolution. Temperate phages can choose between (i) lysis: exploiting their bacterial hosts by producing multiple phage particles and releasing them by lysing the host cell, and (ii) lysogeny: establishing a potentially mutually beneficial relationship with the host by integrating their chromosome into the host cell's genome. Temperate phages exhibit lysogeny propensities in the curiously narrow range of 5-15%. For some temperate phages, the propensity is further regulated by the multiplicity of infection, such that single infections go predominantly lytic while multiple infections go predominantly lysogenic. We ask whether these observations can be explained by selection pressures in environments where multiple phage variants compete for the same host. Our models of pairwise competition, between phage variants that differ only in their propensity to lysogenize, predict the optimal lysogeny propensity to fall within the experimentally observed range. This prediction is robust to large variation in parameters such as the phage infection rate, burst size, decision rate, as well as bacterial growth rate, and initial phage to bacteria ratio. When we compete phage variants whose lysogeny strategies are allowed to depend upon multiplicity of infection, we find that the optimal strategy is one which switches from full lysis for single infections to full lysogeny for multiple infections. Previous attempts to explain lysogeny propensity have argued for bet-hedging that optimizes the response to fluctuating environmental conditions. Our results suggest that there is an additional selection pressure for lysogeny propensity within phage populations infecting a bacterial host, independent of environmental conditions.
| S-EPMC5526970 | biostudies-literature
Project description:Autographiviridae is a diverse yet distinct family of bacterial viruses marked by a strictly lytic lifestyle and a generally conserved genome organization. Here, we characterized Pseudomonas aeruginosa phage LUZ100, a distant relative of type phage T7. LUZ100 is a podovirus with a limited host range which likely uses lipopolysaccharide (LPS) as a phage receptor. Interestingly, infection dynamics of LUZ100 indicated moderate adsorption rates and low virulence, hinting at temperate characteristics. This hypothesis was supported by genomic analysis, which showed that LUZ100 shares the conventional T7-like genome organization yet carries key genes associated with a temperate lifestyle. To unravel the peculiar characteristics of LUZ100, ONT-cappable-seq transcriptomics analysis was performed. These data provided a bird's-eye view of the LUZ100 transcriptome and enabled the discovery of key regulatory elements, antisense RNA, and transcriptional unit structures. The transcriptional map of LUZ100 also allowed us to identify new RNA polymerase (RNAP)-promoter pairs that can form the basis for biotechnological parts and tools for new synthetic transcription regulation circuitry. The ONT-cappable-seq data revealed that the LUZ100 integrase and a MarR-like regulator (proposed to be involved in the lytic/lysogeny decision) are actively cotranscribed in an operon. In addition, the presence of a phage-specific promoter transcribing the phage-encoded RNA polymerase raises questions on the regulation of this polymerase and suggests that it is interwoven with the MarR-based regulation. This transcriptomics-driven characterization of LUZ100 supports recent evidence that T7-like phages should not automatically be assumed to have a strictly lytic life cycle. IMPORTANCE Bacteriophage T7, considered the "model phage" of the Autographiviridae family, is marked by a strictly lytic life cycle and conserved genome organization. Recently, novel phages within this clade have emerged which display characteristics associated with a temperate life cycle. Screening for temperate behavior is of utmost importance in fields like phage therapy, where strictly lytic phages are generally required for therapeutic applications. In this study, we applied an omics-driven approach to characterize the T7-like Pseudomonas aeruginosa phage LUZ100. These results led to the identification of actively transcribed lysogeny-associated genes in the phage genome, pointing out that temperate T7-like phages are emerging more frequent than initially thought. In short, the combination of genomics and transcriptomics allowed us to obtain a better understanding of the biology of nonmodel Autographiviridae phages, which can be used to optimize the implementation of phages and their regulatory elements in phage therapy and biotechnological applications, respectively.
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Project description:Bacteriophages are large and diverse components of the biosphere, and many phages are temperate. Upon infection, temperate phages can establish lysogeny in which a prophage is typically integrated into the bacterial chromosome. Here, we describe the phenomenon of tRNA-dependent lysogeny, a previously unrecognized behavior of some temperate phages. tRNA-dependent lysogeny is characterized by two unusual features. First, a phage-encoded tyrosine family integrase mediates site-specific recombination between a phage attP site and a bacterial attB site overlapping a host tRNA gene. However, attP and attB share only a short (~10 bp) common core such that a functional tRNA is not reconstructed upon integration. Second, the phage encodes a tRNA of the same isotype as the disrupted but essential host tRNA, complementing its loss, and consequently is required for the survival of lysogenic progeny. As expected, an integrase-defective phage mutant forms turbid plaques, and bacterial progeny are immune to superinfection, but they lack stability, and the prophage is rapidly lost. In contrast, a tRNA-defective phage mutant forms clear plaques and more closely resembles a repressor mutant, and lysogens are recovered only at very low frequency through the use of secondary attachment sites elsewhere in the host genome. Integration-proficient plasmids derived from these phages must also carry a cognate phage tRNA gene for efficient integration, and these may be useful tools for mycobacterial genetics. We show that tRNA-dependent lysogeny is used by phages within multiple different groups of related viruses and may be prevalent elsewhere in the broader phage community.IMPORTANCEBacteriophages are the most numerous biological entities in the biosphere, and a substantial proportion of phages are temperate, forming stable lysogens in which a prophage copy of the genome integrates into the bacterial chromosome. Many phages encode a variety of tRNA genes whose roles are poorly understood, although it has been proposed that they enhance translational efficiencies in lytic growth or that they counteract host defenses that degrade host tRNAs. Here, we show that phage-encoded tRNAs play key roles in the establishment of lysogeny of some temperate phages. They do so by compensating for the loss of tRNA function when phages integrate at an attB site overlapping a tRNA gene but fail to reconstruct the tRNA at the attachment junction. In this system of tRNA-dependent lysogeny, the phage-encoded tRNA is required for lysogeny, and deletion of the phage tRNA gives rise to a clear plaque phenotype and obligate lytic growth.
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Project description:Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.
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Project description:Vibrio cholerae uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin-2-ol (DPO) and receptor VqmA (VqmAVc), which together repress genes for virulence and biofilm formation. vqmA genes exist in Vibrio and in one vibriophage, VP882. Phage-encoded VqmA (VqmAPhage) binds to host-produced DPO, launching the phage lysis program via an antirepressor that inactivates the phage repressor by sequestration. The antirepressor interferes with repressors from related phages. Like phage VP882, these phages encode DNA-binding proteins and partner antirepressors, suggesting that they, too, integrate host-derived information into their lysis-lysogeny decisions. VqmAPhage activates the host VqmAVc regulon, whereas VqmAVc cannot induce phage-mediated lysis, suggesting an asymmetry whereby the phage influences host QS while enacting its own lytic-lysogeny program without interference. We reprogram phages to activate lysis in response to user-defined cues. Our work shows that a phage, causing bacterial infections, and V. cholerae, causing human infections, rely on the same signal molecule for pathogenesis.
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Project description:Ef11 is a temperate bacteriophage originally isolated by induction from a lysogenic Enterococcus faecalis strain recovered from an infected root canal, and the Ef11 prophage is widely disseminated among strains of E. faecalis. Because E. faecalis has emerged as a significant opportunistic human pathogen, we were interested in examining the genes and regulatory sequences predicted to be critical in the establishment/maintenance of lysogeny by Ef11 as a first step in the construction of the genome of a virulent, highly lytic phage that could be used in treating serious E. faecalis infections. Passage of Ef11 in E. faecalis JH2-2 yielded a variant that produced large, extensively spreading plaques in lawns of indicator cells, and elevated phage titres in broth cultures. Genetic analysis of the cloned virus producing the large plaques revealed that the variant was a recombinant between Ef11 and a defective FL1C-like prophage located in the E. faecalis JH2-2 chromosome. The recombinant possessed five ORFs of the defective FL1C-like prophage in place of six ORFs of the Ef11 genome. Deletion of the putative lysogeny gene module (ORFs 31-36) and replacement of the putative cro promoter from the recombinant phage genome with a nisin-inducible promoter resulted in no loss of virus infectivity. The genetic construct incorporating all the aforementioned Ef11 genomic modifications resulted in the generation of a variant that was incapable of lysogeny and insensitive to repressor, rendering it virulent and highly lytic, with a notably extended host range.
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Project description:Pneumococcal prophages are extremely frequent, but no role in pathogenesis has so far been attributed to them. We isolated a variant of phage MM1, named MM1-1998, from a serotype 24 strain of Streptococcus pneumoniae. We created three isogenic strain pairs (serotypes 3, 4, and 24) that differed only by the lysogenic presence of the MM1-1998 phage and did a phenotypic comparison. Lysogeny led to improved adherence to inert surfaces and pharyngeal cells compared to that with the cured variants of the strains. We found that lysogeny with MM1-1998 coincided with a more transparent phenotype and phage curing with more opaque colonies in all strain pairs, and we discovered that transparency was associated with more successful and stable lysogeny. Since transparency alone was possibly responsible for the adherence difference, we further compared the TIGR4 lysogen with an equally transparent variant of TIGR4 in order to reassess the role of phage or transparency separately. The results revealed that improved adherence was independently associated with lysogeny with the MM1-1998 phage. Other phenotypic differences such as faster growth, increased autolysis, and decreased intracellular hemolytic activity were more likely due to transparency. By improving the adherence of pneumococci, this prophage may contribute to their fitness and possibly to their persistence in humans.
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Project description:Recent environmental and metagenomic studies have considerably increased the repertoire of archaeal viruses and suggested that they play important roles in nutrient cycling in the biosphere. However, very little is known about how they regulate their life cycles and interact with their hosts. Here, we report that the life cycle of the temperate haloarchaeal virus SNJ1 is controlled by the product ORF4, a small protein belonging to the antitoxin MazE superfamily. We show that ORF4 controls the lysis-lysogeny switch of SNJ1 and mediates superinfection immunity by repression of genomic DNA replication of the superinfecting viruses. Bioinformatic analysis shows that ORF4 is highly conserved in two SNJ1-like proviruses, suggesting that the mechanisms for lysis-lysogeny switch and superinfection immunity are conserved in this group of viruses. As the lysis-lysogeny switch and superinfection immunity of archaeal viruses have been poorly studied, we suggest that SNJ1 could serve as a model system to study these processes.IMPORTANCE Archaeal viruses are important parts of the virosphere. Understanding how they regulate their life cycles and interact with host cells provide crucial insights into their biological functions and the evolutionary histories of viruses. However, mechanistic studies of the life cycle of archaeal viruses are scarce due to a lack of genetic tools and demanding cultivation conditions. Here, we discover that the temperate haloarchaeal virus SNJ1, which infects Natrinema sp. strain J7, employs a lysis-lysogeny switch and establishes superinfection immunity like bacteriophages. We show that its ORF4 is critical for both processes and acts as a repressor of the replication of SNJ1. These results establish ORF4 as a master regulator of SNJ1 life cycle and provides novel insights on the regulation of life cycles by temperate archaeal viruses and on their interactions with host cells.
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