Evolution of genetic switch complexity

Broussard, Gregory W.; Hatfull, Graham F.

doi:10.4161/bact.24186

Cited by 11 publications

(12 citation statements)

References 31 publications

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“…DS6A plaques are not evidently turbid on lawns of M. tuberculosis, suggesting that it is virulent in nature or that it is temperate but forms lysogens at relatively low frequencies, as reported for some other mycobacteriophages (41). DS6A genomic features reflect components characteristic of temperate phages, including a putative repressor gene (gp58), an integrase gene (gp56), and a putative attP site (gp50-gp51 intergenic region).…”

Section: Discussionmentioning

confidence: 99%

Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria

et al. 2016

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Mycobacteriophage DS6A is unique among the more than 8,000 isolated mycobacteriophages due to its ability to form plaques exclusively on mycobacteria belonging to the Mycobacterium tuberculosis complex (MTBC). Speculation surrounding this specificity has led to unsupported assertions in published studies and patents that nontuberculous mycobacteria (NTM) are wholly resistant to DS6A infection. In this study, we identified two independent nonessential regions in the DS6A genome and replaced them with an mVenus-expressing plasmid to generate fluorescent reporter phages ⌽ 2 GFP12 and ⌽ 2 GFP13. We show that even though DS6A is able to form plaques only on MTBC bacteria, infection of various NTM results in mVenus expression in transduced cells. The efficiency of DS6A in delivering DNA varied between NTM species. Additionally, we saw a striking difference in the efficiency of DNA delivery between the closely related members of the Mycobacterium abscessus complex, M. abscessus and Mycobacterium massiliense. We also demonstrated that TM4 and DS6A, two phages that do not form plaques on M. massiliense, differ in their ability to deliver DNA, suggesting that there is a phage-specific restriction between mycobacterial species. Phylogenetic analysis reveals that the DS6A genome has a characteristically mosaic structure but provided few insights into the basis for the specificity for MTBC hosts. This study demonstrates that the inability of the MTBC-specific phage DS6A to form plaques on NTM is more complex than previously thought. Moreover, the DS6A-derived fluorophages provide important new tools for the study of mycobacterial biology. IMPORTANCEThe coevolution of bacteria and their infecting phages involves a constant arms race for bacteria to prevent phage infection and phage to overcome these preventions. Although a diverse array of phage defense systems is well characterized in bacteria, very few phage restriction systems are known in mycobacteria. The DS6A mycobacteriophage is unique in the mycobacterial world in that it forms plaques only on members of the Mycobacterium tuberculosis complex. However, the novel DS6A reporter phages developed in this work demonstrate that DS6A can infect nontuberculous mycobacteria at various efficiencies. By comparing the abilities of DS6A and another phage, TM4, to infect and form plaques on various mycobacterial species, we can begin to discern new phage restriction systems employed within the genus. The standard treatment for a drug-susceptible Mycobacterium tuberculosis infection is four drugs (rifampin, isoniazid, pyrazinamide, and ethambutol) for 2 months, followed by 4 months of two drugs (rifampin and isoniazid). One of the key aspects of successfully treating tuberculosis is ensuring that the infecting strain is susceptible to each of the antibiotics given; therefore, quickly diagnosing antibiotic resistance is important for successful patient outcome as well as for lowering the incidence of the generation of drug-resistant mutants from improper antibiotic administra...

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Section: Discussionmentioning

confidence: 99%

Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria

et al. 2016

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“…The L5 repressor binds as a monomer at the asymmetric non-operator binding sites (referred to as ‘stoperators’) to block transcription elongation [ 11 ]. There are few examples other than phage lambda and its relatives where the molecular basis of the decision between lytic and lysogenic outcomes is well understood [ 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

An Unusual Phage Repressor Encoded by Mycobacteriophage BPs

2015

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Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection—lysogenic or lytic growth—as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33–34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.

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“…A novel system of immunity regulation has been described in several different types of mycobacteriophages, including BPs (Cluster G), Brujita (Cluster I), Charlie (Cluster N) and BigNuz (Cluster P) (164, 180). The characteristic feature—readily recognizable bioinformatically—is that the phage attachment site for integration ( attP ) is located within the coding sequence for the phage repressor (e.g., BPs gp33).…”

Section: Mycobacteriophage Gene Expression and Its Regulationmentioning

confidence: 99%

“…This feature is illustrated by the behavior of integration-proficient plasmid vectors (see below) that transform at unexpectedly low frequencies due to Int degradation; stabilization of Int leads to large increases in the transformation frequencies (164). The relative simplicity of these immunity systems with a few genes and DNA sites serving multiple functions suggests that these may have evolved relatively early in the development of phage immunity systems (180). …”

Section: Mycobacteriophage Gene Expression and Its Regulationmentioning

confidence: 99%

Molecular Genetics of Mycobacteriophages

Hatfull

2014

Microbiol Spectr

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Mycobacteriophages have provided numerous essential tools for mycobacterial genetics, including delivery systems for transposons, reporter genes, and allelic exchange substrates, and components for plasmid vectors and mutagenesis. Their genetically diverse genomes also reveal insights into the broader nature of the phage population and the evolutionary mechanisms that give rise to it. The substantial advances in our understanding of the biology of mycobacteriophages including a large collection of completely sequenced genomes indicates a rich potential for further contributions in tuberculosis genetics and beyond.

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Evolution of genetic switch complexity

Cited by 11 publications

References 31 publications

Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria

Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria

An Unusual Phage Repressor Encoded by Mycobacteriophage BPs

Molecular Genetics of Mycobacteriophages

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