Amanda M. Curry, Celia S. Fung, Michael A. Santana, Lindsey J. Stretz, Katie Mageeney (TA), Dr. Meg Kenna, Dr. Jutta Marzillier and Dr. Vassie WareDepartment of Biological Sciences, Lehigh University, Bethlehem, PA 18015

• Orphams: gp30, gp31, gp33 (no known mycobacteriophage family members); functions unknown

• Integrase: gp37(used as a Proof of Concept in experimental analyses)

• N-Specific gene: gp57

(found in Butters and Redi)

BRED and Butters

The research goal of the SEA- PHAGES project at Lehigh University is to isolate and characterize novel mycobacteriophages that infect the host, Mycobacterium smegmatis, thereby contributing to an understanding of mycobacteriophage genomic structural and functional diversity and contributing to the potential development and expansion of molecular tools to manipulate these phages and their hosts. Since 2009 students have isolated, purified, and characterized over 50 phages, including about a dozen phages whose genomes have been sequenced and annotated. During the 3rd year of the course, students discovered and further analyzed the temperate phage, Butters (N cluster phage). As one of the smallest known annotated mycobacteriophage genomes (41491bp with 66 genes proposed), at least 5% of the genome has no known phage genome counterpart (defining several orphams), as determined by BLAST analysis. Additionally, the latter half of the genome is interspersed with sequences common to several I1 and P cluster phages, thereby highlighting significant mosaicism in the Butters genome. Considering the large variation in genome size among the mycobacteriophages, Butters is an ideal candidate to explore questions about minimum size requirements for packaging the genome of a temperate mycobacteriophage. The Butters genome is also ideal to determine if an orpham is required for a productive lytic or lysogenic phage life cycle. As a first step to investigate these questions, 38 SEA-PHAGES students in the fall 2012 used the BRED technique to delete 5 Butters genes. Nine students continued the Butters project as independent researchers in the spring 2013. Deletions in the Butters integrase gene (gp37) were used as a “proof of concept” to demonstrate the efficiency of the technique and the proposed impact of integrase gene deletion on the life cycle of a temperate phage. Integrase deletions were constructed, analyzed by PCR, breakpoints determined by sequencing and mutant phages purified. Plaque morphologies from integrase mutants and wildtype phage were compared, showing a predicted change in plaque clarity and size for integrase mutant phages. Further, deletions in orphams gp30, gp31, gp33, and the N-specific gp57 gene were constructed and analyzed by PCR. We report on findings from the Butters deletion project here.

Summary of BRED Procedure (Hatfull Lab)

• What role(s) do Butters’ orphams play in the life cycle of one of the smallest known mycobacteriophages? Are these genes essential for a Butters infection in Mycobacterium smegmatis? – If not, do they impact the lysogenic cycle?

• What is the minimum genome size necessary for producing a Butters phage competent for infection?

• www.emc.maricopa.edu

• Enzyme responsible for site-specific insertion of phage genome into host genome, resulting in lysogenic life cycle

• Butters integrase is a member of the serine recombinase family

• As a proof of concept, we might predict that an integrase deletion would eliminate the lysogenic cycle and favor the lytic cycle. If so, a phenotypic change from turbid to clear plaques might be expected.

• Gene to be deleted

• 100 bp upstream • 100 bp downstream

• Forward primer

• Reverse primer

• Steps to design primers:1. Blast to confirm that gene flanking regions do not hybridize elsewhere in the Butters genome.

2. Forward Primer: 120 bp length comprised of 100 bp upstream of region to be deleted fused to 20 bp downstream of region to be deleted.

3. Reverse Primer: 100 bp length of reverse complement of 100 bp downstream of region to be deleted. Contains 20 bp overlap with forward primer.

4. PCR amplification to produce ~200 bp product to be used as a recombination substrate.

• duplex• region

Primary PCR

Picked Plaques

2% agarose, 1X TAE buffer 2% agarose, 1X TAE buffer

• M 1 2 3 4 5 6 7 8 9 16

676 -

350 -

222 -

# bp

Secondary PCR

Picked Plaques

Gene BRED Screening of Plaques

Plaque Phenotype

Sequence Data

Purified Phage

gp37 (Integrase)

gp30 (Orpham)

gp31 (Orpham)

gp33(Orpham)

gp57 (N-

specific)

• Founder: Lena Ma, Sea Class 2011-12• Characteristics:

– N cluster phage– 41,491 bp linear genome with defined physical ends

• 13 bp 3’ overhang• 66 annotated genes (GenBank accession no.

NC_021061)– Turbid plaques: (0.5-3mm after 24-72 hrs)– Temperate phage– Siphoviridae (1:3 head:tail ratio)

Hypotheses and Experimental Strategy

• Butters is a temperate phage, producing turbid plaques on M. smegmatis mc2155 lawns. The integrase gene is among the phage genes required for the Butters lysogenic life cycle. The role, if any, of Butters orphams (gp30, gp31, gp33) and N-specific gp57 in phage infection is unknown.

I. HYPOTHESIS: The integrase gene (gp37) is dispensable for a Butters lytic infection.

• EXPERIMENTAL PLAN• Use the Bacterial Recombineering Electroporated DNA (BRED) technique to make gp37 deletions to determine if integrase is required for phage lytic infection.• Determine if mutant phage can be recovered and if plaque phenotype is altered.

II. HYPOTHESIS: Selected orphams and N-specific gp57 are not required for a productive Butters infection in M. smegmatis.

• EXPERIMENTAL PLAN• Use BRED to make specific gene deletions to determine if mutant phage can be recovered and to determine if plaque phenotype is affected.

Mycobacterium phage Butters

N Cluster Phage Genome Comparisons in Phamerator

Questions of Interest from Genome Analysis

BRED Protocol

Primer Design for Recombination Substrate

Integrase (gp37)Genes of Interest

• WT Butters

• Plate with mixed plaques

WT plaques: turbid0.5mm after 24h

Plate with mixed plaques (post-BRED):Mixed plaque morphology:Clear (~2 mm) and turbid (0.5 mm)types after 24h

Plaque Phenotype Changes in Integrase Mutants

• Sequence data from Δgp37_4

Reverse primer

Forward primer

• Δgp37_4

• Δgp37_8

PCR analysis of integrase BRED plaques reveals smaller sized PCR products relative to wildtype

Expected WT PCR product: 1345 bp

Two integrase mutants: Δgp37_4 removes 1041 bp; Δgp37_8 removes 718 bp

Post-BRED plating reveals plaque size variation

PCR screening of BRED plaques shows smaller products compared to wildtype

Summary

• Integrase (gp37):– To date, two integrase mutants have been characterized by sequencing PCR products, showing a

complete gp37 deletion and a partial deletion of the 5’ end of gp37.– Butters integrase mutants form clear, larger plaques on M. smegmatis lawns, suggesting a lytic cycle

for mutant phages. – Integrase mutant phages have been purified and can be propagated in the absence of wildtype

Butters. – The largest integrase deletion results in a Butters genome of ~40,450 bp compared to wildtype

Butters of 41,491 bp.

• gp30:– Putative gp30 mutant plaques are significantly larger than wildtype Butters plaques. The factors

that contribute to the larger plaque size in this case (efficiency of adsorption of the phage to the host, length of the latent period, burst size of the phage) are unknown. Are gp30 mutants lysis-defective such that more progeny phage accumulate before lysis, allowing for a greater burst size? (Does gp30 affect lysis timing?)

– Putative gp30 plaques are turbid, suggesting that mutant phages are still competent to carry out the lysogenic cycle. Thus, gp30 would not be essential for the lysogenic cycle.

• gp57:– Putative gp57 mutant plaques are significantly larger bull-eye plaques, appearing with a

shorter incubation time than wildtype Butters plaques. – Putative gp57 plaques have a bull-eye appearance, suggesting they are still competent to

carry out the lysogenic cycle. Thus, gp57 would not be essential for the lysogenic cycle.

• Continuation of screening, sequence analysis for genomic characterization of deletions, and purification of mutants from each ORF to determine if each is necessary for a successful infection

• Purification of mutant phage for 3D reconstructions by cryo EM for comparison to wildtype Butters in collaboration with Dr. Susan Hafenstein at Penn State University College of Medicine

• Determination of ability of mutant phage to form lysogens (note wildtype Butters lysogen is available) and testing mutant lysogen characteristics

• Use of mutant Butters phage for BRED to determine if multiple deletions produce infectious particles or if phage genome packaging is sufficient for infectivity.

Work in Progress and Future Directions

ACKNOWLEDGMENTS: HHMI, Lehigh Univerisity Mountaintop Pilot Summer Projects, and Lehigh’s Department of Biological Sciences for financial support; HHMI SEA Staff for infrastructure assistance; Bekah Dedrick in the Hatfull Lab, Kevin Bradley, and Lu Barker for assistance with BRED protocols; The Hatfull lab including Welkin Pope for QC’ing the Butters genome; Maria Brace for SEA Program support; Lee Graham for laboratory support and EM imaging.

Introduction

• phagesdb.org protocols

gp33 gp57

gp31gp30

gp31gp30

WT

WT

gp57