Genomic Analysis and Comparative Proteomics of Temperate

Mycobacteriophages,

By: Carmen A AguirreThe College of St. Scholastica

Background • Bacteriophages are viruses that infect and

replicate within bacteria

• Mycobacteriophages specifically target mycobacteria (e.g. M. smegmatis, M.tuberculosis)

• There are two types of Phages:

Lytic: obligate lysis of host

Temperate: Lytic or Lysogenic cycle

Applications:

• Alternatives to antibiotics for resistant infections

• Biocontrol agents in agriculture

• Valuable research tool

• Untapped reservoir of genetic diversity

Overview• Introduction to SEA-Phages program • Methods of Isolation• General Overview of Annotated Mycobacteriophages• Overview of comparative genomics and stoperator

sequences• General Overview of Mass Spec Techniques• Mass Spectrometry Protein Identification • Future Directions

SEA-PHAGES ProgramSEA Phage Hunters Advancing Genomics and Evolutionary Science• National experiment in bacteriophage genomics• Students isolate, name, sequence, and analyze

newly-discovered bacteriophages isolated on M. smegmatis and other hosts

• Research-based curricula• Advance science education on a national scale• Authentic scientific discovery

Collect soil sample

Enrichment

Direct plating

Streak method≥ 3 times

Serial dilution/Titer

Spot test

MTL Harvestand titer Web Plate

Empirical test

10-plate infection

Isolate DNA

ARCHIVE

HTL harvestand titer

HTL

Electron Microscopy

HTL

HTL

Restriction Digest

Quality Control Gel SEQUENCING

CENTER

APPROVED Genomic

DNAIN

SILICO

Genome Sequence

Methods

CSS Phage StatsTotal Phage Isolated during 3 years= 34

5 sequenced and annotated4 in GenBankClusters are groups of phages that have nucleotide sequence similarity 14 Cluster A 1 Cluster E 2 Cluster B 1 Cluster CFamilies are groups of phages sharing similar electron microscopy morphologyMyoviridae: double-stranded DNA genomes with contractile tailsSiphoviridae: double-stranded DNA genomes and long, flexible, non-contractile tailsPodoviridae: double-stranded DNA genomes and short, stubby, non-contractile tails

QuinNkiroGeneral

InformationCluster: A3Family: SiphoviridaeType: TemperatePlaque Morphology: Large , Turbid plaques

GenomicsGenome: 50,066bpProtein Coding Regions: 88tRNA: 2Stoperator: 18

HetaeriaGeneral Information

Cluster: A4

Family: Siphoviridae

Type: Lytic?

Plaque Morphology: Small, Clear plaques

Genomics

Genome: 51,374bp

Protein Coding Regions: 87

Stoperator: 17

Stoperator Sequences

• DNA sequences that bind repressors thus prevent RNA elongation (transcription)

• Often found before genes that promote lysis

fDNAfRepressor bound to

Stoperator

Stoperator Sequences

• In many phages, multiple stoperator sequences are found in the genome, with most located in the second half of the genome

• Stoperator sequences have polarity (directionality) that correlates with direction of transcription of target gene

• Usually found in intergenic region near target gene

Stoperator polarity and location in mycobacteriophage L5 Brown et al. The EMBO Journal Vol.16 No.19 pp.5914–5921

Stoperators in our PhagesHetaeria

• Consensus sequence generated using 3 matches and 25 pseudo matches to original search sequence

• 27/28 sequences found within relevant genes at the tail end

QuinnKiro • 18 putative sequences• Mostly at the end of genome• Core TCAAG mutated in the three matches within Lysin

genes

Unique Quinnkiro stoperator results• NCBI:

Sequence GTGCGATGTCAAG found 11 times

• DNAMaster-Additional pseudo matches found around Lysin A and B

genes-Multiple stoperator sites found within Lysin A and B-Core TCAAG sequence mutated in one case-Sequence polarity reversed in one matchWithin Lysin A (4490 - 6052): Within and after Lysin B (6052 -

7017):

Comparative Genomics

• Looked for our consensus sequence in other clusters

• Recorded how many exact and pseudo matches

were found

• Investigated the gene functions of the likely target

genes

Phage Sequence Hits Gene Function

QuinnKiro (A) GTGCGATGTCAAG 18 Lysin A, B

Caelakin (A) GTGCGATGTCAAG 17 Lysin B, DNA Pol.

Hetaeria (B) GTGCGATGTCAAG 28 Helicase, Terminase

ZygoTaiga (C) GTGCGATGCCGAG 14 Nucleotide Binding Protein, Hydrolase

Hawkeye (D) GCGCGATGTCAAG 3 DNA Pol. III

Bruin (E) GCGCGATGTGGAC 2 DNAB-like Helicase

Drago (F) GTGCGATGCCAAC 2 NKF

Avrafan (G) GTGCGAGGTCGAG 1 Lysin B

Damien (H) GTGCGATGTCCCG 3 NKF

Babsiella (I) GCGCGATGTCAAC 1 HiCa antitoxin

• Stoperator sequence of GTGCGATGTCAAG found in QuinnKiro, Caelakin, and Hetaeria.

• Sequence found in nearly all clusters of phages, including non-temperate

• Stoperator sequence near LysA and B genes or areas that promote lysis/assembly

• most sequences in the second half of genome near genes with no known function

QuinnKiro• exhibited some unusual stoperator sequences associated with the lysin genes:

• Multiple sequences embedded in coding region• Apparent reversal of polarity

Speculation:• What is the purpose of stoperator sequences in lytic phages?

• An outcome of genetic mosaicism? • Common mycophage ancestor temperate?

Stoperator Genomics Conclusions

Phage protein identification through mass spectrometry

Goals: -Detection of phage proteins -verify annotation

-How does host (smeg) protein expression respond to phage infection/prophage presence?

Methods:

-First attempt: Harvest raw lysate or PEG-precipitated phage sample

-Second attempt: Infected Cell Pellet

-Analyze: digested proteins through triple TCF mass spec

-Analyze the peptides with scaffold program

Peg Precipitation Protocol summary

• Able to identify proteins that are abundant and unique

• Several phage structural proteins were identified

• Sample concentration may need to be increased to achieve better detection

• Many orphan peptides remain unidentified (likely smeg protein)

Infected Cell Pellet ProtocolGrow Smeg Cultures of Varying concentrations~12hrs

Run Samples and Find one of OD=0.4

Infect Culture with Lysate at MOI=10 ~3-3.5hrs

Detected Peptides Analyzed with Sccafold Viewer

Subject to triple TOF mass spec

Sample Peptides with >1000 counts/sec fragmented

Pellet Cells

Infected Cell Pellet analysis-Hetaeria

• Able to identify proteins that are abundant and unique• Proteins functioning in: Assembly, Structure, Genome

Replication, and Other.• Able to detect over half predicted proteins• Able to cover a significant percentage of sequence• Identify amino acid modifications

Identified Peptides with Functions

Amino Acid Modifications

Peptide Fragments

Summary of Hetaeria Mass Spec Data

• Over half of in silico predicted proteins detected

• Over a quarter of start sites verified• Proteins of varying functions identified• Ability to locate amino acid modifications• Ability to identify size of proteins and variety• Increased sequence coverage• Increasing understanding of the Lytic Phage

Currently• Waiting on Mass spec data on infected cell pellet for

Quinnkiro (A3) and Brusacoram (P)-Temperate phages?• Interesting data on proteins involved in switching from

lysogeny• Compare identified proteins between lytic and temperate

phages• Preparing Caelakin clear plaque mutant with increase phage

protein expression. • Examine how growth time and conditions affect phage and

host protein expression • Immunity Repressors?

Acknowledgements• Dr. Daniel Westholm-Professor• The College of St. Scholastica SEA-PHAGES Research Students• Sequencing

• Virginia Commonwealth University (Hetaeria, Severus, ZygoTaiga)• North Carolina State University (QuinnKiro, Caelakin)

• SEA-PHAGES• Howard Hughes Medical Institution• University of Pittsburg-Hatful Lab

• Mass Spectrometry• Mayo Clinic Proteomic Core Lab

• Electron Microscopy• Mayo Clinic Microscopy Lab