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Functional requirements for bacteriophage growth: gene essentiality and expression in mycobacteriophage Giles.

ABSTRACT: Bacteriophages represent a majority of all life forms, and the vast, dynamic population with early origins is reflected in their enormous genetic diversity. A large number of bacteriophage genomes have been sequenced. They are replete with novel genes without known relatives. We know little about their functions, which genes are required for lytic growth, and how they are expressed. Furthermore, the diversity is such that even genes with required functions - such as virion proteins and repressors - cannot always be recognized. Here we describe a functional genomic dissection of mycobacteriophage Giles, in which the virion proteins are identified, genes required for lytic growth are determined, the repressor is identified, and the transcription patterns determined. We find that although all of the predicted phage genes are expressed either in lysogeny or in lytic growth, 45% of the predicted genes are non-essential for lytic growth. We also describe genes required for DNA replication, show that recombination is required for lytic growth, and that Giles encodes a novel repressor. RNAseq analysis reveals abundant expression of a small non-coding RNA in a lysogen and in late lytic growth, although it is non-essential for lytic growth and does not alter lysogeny.

SUBMITTER: Dedrick RM

PROVIDER: S-EPMC3641587 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Functional requirements for bacteriophage growth: gene essentiality and expression in mycobacteriophage Giles.

Dedrick Rebekah M RM Marinelli Laura J LJ Newton Gerald L GL Pogliano Kit K Pogliano Joseph J Hatfull Graham F GF

Molecular microbiology 20130408 3

Bacteriophages represent a majority of all life forms, and the vast, dynamic population with early origins is reflected in their enormous genetic diversity. A large number of bacteriophage genomes have been sequenced. They are replete with novel genes without known relatives. We know little about their functions, which genes are required for lytic growth, and how they are expressed. Furthermore, the diversity is such that even genes with required functions - such as virion proteins and repressor ...[more]

PMID: 23560716

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Project description:UnlabelledMycobacteriophages are viruses that infect mycobacterial hosts and are prevalent in the environment. Nearly 700 mycobacteriophage genomes have been completely sequenced, revealing considerable diversity and genetic novelty. Here, we have determined the protein complement of mycobacteriophage Giles by mass spectrometry and mapped its genome-wide protein interactome to help elucidate the roles of its 77 predicted proteins, 50% of which have no known function. About 22,000 individual yeast two-hybrid (Y2H) tests with four different Y2H vectors, followed by filtering and retest screens, resulted in 324 reproducible protein-protein interactions, including 171 (136 nonredundant) high-confidence interactions. The complete set of high-confidence interactions among Giles proteins reveals new mechanistic details and predicts functions for unknown proteins. The Giles interactome is the first for any mycobacteriophage and one of just five known phage interactomes so far. Our results will help in understanding mycobacteriophage biology and aid in development of new genetic and therapeutic tools to understand Mycobacterium tuberculosis.ImportanceMycobacterium tuberculosis causes over 9 million new cases of tuberculosis each year. Mycobacteriophages, viruses of mycobacterial hosts, hold considerable potential to understand phage diversity, evolution, and mycobacterial biology, aiding in the development of therapeutic tools to control mycobacterial infections. The mycobacteriophage Giles protein-protein interaction network allows us to predict functions for unknown proteins and shed light on major biological processes in phage biology. For example, Giles gp76, a protein of unknown function, is found to associate with phage packaging and maturation. The functions of mycobacteriophage-derived proteins may suggest novel therapeutic approaches for tuberculosis. Our ORFeome clone set of Giles proteins and the interactome data will be useful resources for phage interactomics.

Project description:UnlabelledMycobacterium species such as M. smegmatis and M. tuberculosis encode at least two translesion synthesis (TLS) polymerases, DinB1 and DinB2, respectively. Although predicted to be linked to DNA repair, their role in vivo remains enigmatic. M. smegmatis mc(2)155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes for DinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class II ribonucleotide reductase in M. smegmatis ?DRKIN, a strain derived from mc(2)155 in which one copy of the duplication is lost, resulted in DNA replication defects and growth inhibition. The inhibitory effect could be linked to the deficiency of dTTP that resulted under these circumstances. The selective inhibition observed in the ?DRKIN strain was found to be due solely to a reduced dosage of dinB2 in this strain. Mycobacterium bovis, which is closely related to M. tuberculosis, the tuberculosis pathogen, was found to be highly susceptible to gene 50 overexpression. Incidentally, these slow-growing pathogens harbor one copy of dinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with the effect being maximum in cells deficient in DinB2.ImportanceMycobacterium species, such as M. tuberculosis, the tuberculosis pathogen, are known to encode several Y family DNA polymerases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this protein has been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo function remains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid mycobacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymerase DinB2 for the first time, this study also paves the way for the development of drugs that can kill mycobacteria by inducing a dTTP-deficient state.

Project description:BackgroundIdentifying factors affecting gene expression variation is a challenging problem in genetics. Previous studies have shown that the presence of TATA box, the number of cis-regulatory elements, gene essentiality, and protein interactions significantly affect gene expression variation. Nonetheless, the need to obtain a more complete understanding of such factors and how their interactions influence gene expression variation remains a challenge. The growth rates of yeast cells under several DNA-damaging conditions have been studied and a gene's toxicity degree is defined as the number of such conditions that the growth rate of the yeast deletion strain is significantly affected. Since toxicity degree reflects a gene's importance to cell survival under DNA-damaging conditions, we expect that it is negatively associated with gene expression variation. Mutations in both cis-regulatory elements and transcription factors (TF) regulating a gene affect the gene's expression and thus we study the relationship between gene expression variation and the number of TFs regulating a gene. Most importantly we study how these factors interact with each other influencing gene expression variation.ResultsUsing yeast as a model system, we evaluated the effects of four separate factors and their interactions on gene expression variation: protein interaction degree, toxicity degree, number of TFs, and the presence of TATA box. Results showed that 1) gene expression variation is negatively correlated with the protein interaction degree in the protein interaction network, 2) essential genes tend to have less expression variation than non-essential genes and gene expression variation decreases with toxicity degree, and 3) the number of TFs regulating a gene is the most important factor influencing gene expression variation (R2 = 8-14%). In addition, the number of TFs regulating a gene was found to be an important factor influencing gene expression variation for both TATA-containing and non-TATA-containing genes, but with different association strength. Moreover, gene expression variation was significantly negatively correlated with toxicity degree only for TATA-containing genes.ConclusionThe finding that distinct mechanisms may influence gene expression variation in TATA-containing and non-TATA-containing genes, provides new insights into the mechanisms that underlie the evolution of gene expression.

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Functional requirements for bacteriophage growth: gene essentiality and expression in mycobacteriophage Giles.

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Functional requirements for bacteriophage growth: gene essentiality and expression in mycobacteriophage Giles.

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