Interesting phagy observations and questions for the...

Interesting phagyobservations and questions

for the curious:

XKCD LRRHood

Electron Microscopy

Janine Ilagan, Melissa Jurica

Capsid Width: 102 (3)Tail Length: 84 (9)

Capsid Width: 80 (3)Tail Length: 282 (13)

Values are in nanometers and are listed as: average (standard deviation)

Kim Davis, in the redwood forestwhere she isolated LRRHood(LiCle Red Riding Hood)

• Double stranded circular genome of 154,349 bp.

• Member of group C1, highly similar to Cali.

• Glimmer and Genemark were used to idenNfy221 potenNal protein coding genes

• Used trnascan and Aragorn(hCp://130.235.46.10/ARAGORN/) to idenNfy

• 32 predicted trna genes

• 1 predicted tmrna.

• The tmrna and 3 of the trnas were only found byAragorn

LRRHood Genome Sequence

• One tRNA with a CUA anN-‐codon

• Thus, it appears to recognizeUAG stop codons

• Homology to other bacterialtRNAs suggest it may chargedwith tryptophan

• 36 LRRHood genes end in UAG,and in several cases, readthroughof a UAG stop codon would jointwo annotated ORFs

Supressor tRNA?

• M. smegmaNs also has a tmRNA• Why does LRRHood have its own tmRNA if the host already has one?

•(and why does it carry extra tRNAs?)

What is the funcNon of LRRHoods tmRNA?

John Paul Donohue

The UCSC Genome Browser

genes

conservation

homology withgroup C1phages

John Paul Donohue

genes

conservation


highly variable


John Paul Donohue

genes

conservation


insertions


LRRHood has acquired a transcripNonalrepressor via a novel sequence inserNon


= 29 bp sequence flanking insertion inLRRHood and found at apparent insertionsite in group C1 phages (not to scale).

Does this repeat explain themechanism of the insertion?


LRRHood gp44:

• similar to transcriptional repressor protein found in grp A1, A2phages and the group F1 phage fruitloop

• 169/170 amino acids are identical to Bxb1 gp69

• gp69 DNA binding site is not present in LRRHood genome

• does repressor provide a growth advantage to LRRHood in amixed infection?

Phage Violet

Isolated by enrichment from a garden on UCSC campus

Julia Froud

Genome:-54,481 bp

-defined ends

-10bp 3’ overhangs

Annotation:93 proteinencoding genes

-no tRNAs

mycophageclass:

A1

other A phages

Violet belongs to the A1 cluster

tracks show conservation of other phage to Violet

Blastn shows Violet is most closely related to A1 clusterConstructed UCSC genome browser to examine relationships

Violet 59 MRTTREQLPRLSLEVIEALKATGETEADIARMYGVTPQAVSWHVHTYGGKLTARQVIRRE 118 MRTTREQLPRLSLEVIEALKATGETEADIARMYGVTPQAVSWHVHTYGGKLTARQVIRREBxb1 1 MRTTREQLPRLSLEVIEALKATGETEADIARMYGVTPQAVSWHVHTYGGKLTARQVIRRE 60

Violet 119 YPFKVPEPLSQCAPHKRLRDHGEYIATRGKGMKEYKLKRLRSFYRMLRENNWVVEFDPNI 178 YPFKVPEPLSQC PHKRLRDHGEYIATRGKGMKEYKLKRLRSFYRMLRENNWVVEFDPNIBxb1 61 YPFKVPEPLSQCTPHKRLRDHGEYIATRGKGMKEYKLKRLRSFYRMLRENNWVVEFDPNI 120

Violet 179 PPIPGVSKRGGWAYRERQESGEDLLIRVNEYTTLSEIGRHHIWRFPSVEP 228 PPIPGVSKRGGWAYRERQES EDLLIRVNEYTTLSEIGRHHIWRFPSVEPBxb1 121 PPIPGVSKRGGWAYRERQESDEDLLIRVNEYTTLSEIGRHHIWRFPSVEP 170

Helix Turn Helix

Violet Encodes a Protein Closely Related tothe Bxb1 Repressor

Violet has a sequence repeat that is identical to theBxb1 repressor binding site

Bxb1 Repressor Binding Motif

CTTGA CGTAACACT

AT

Sequence repeat in Violet

Submitted Violet genome to MEME, a motif finding program

http://meme.sdsc.edu

The repressor binding site is asymmetric.Does the repressor bind as a monomer?How does the repressor block transcription?

Violet has a sequence repeat that is identical to theBxb1 repressor binding site

location of sequence motifs

orientation: reverse forward

The Bxb1 repressor binding site also occurs and functionsin an orientation specific manor.

Violet forms lysogens and is in the sameimmunity group as Bxb1


L5 is a cluster Ax phage whose repressor andrepressor binding sites are different from Bxb1


Violet and Bxb1 are in the same immunity group

mycophageclass:

A1

other A phages A B C

Sequence insertions in the Violet genome

Inteins

Whole genomecomparison ofViolet to othergroup A phage.

http://exon.ucsc.edu

A ~1.5 kb segment of Violet appears unique in this group.

Different Blast programs give different resultsBlastn = no hits at NCBI. Woohoo! New sequence!

Nucleotide query

*

Different Blast programs give different resultsBlastn = no hits at NCBI. Woohoo! New sequence!Blastx = DNA methyltransfease edges, HNH nuclease inthe middle. Homing Intein? Intron?

Nucleotide query

**

Different Blast programs give different resultsBlastn = no hits at NCBI. Woohoo! New sequence!Blastx = DNA methyltransfease edges, HNH nuclease inthe middle. Homing Intein? Intron?

Blastp = DNA methyltransferase edges, No HNH nuclease?? Must be that two proteins are coded in overlapping frames.

Protein query

Nucleotide query

**

**

What is it?

TATAA element Shine-DalgarnoYellow and red ORF in the same reading frameBlue ORF in the +1 frame relative to red/yellow ORF

Sequence features near the junctions

The insertion doesn’t seem to be an intein

1. There isn’t enough yellow protein sequence on eitherside of the blue nuclease coding sequence to encode anintein.

2. Lacks conserved sequences seen in known inteins

The insertion doesn’t seem to be an intron

1. There aren’t convincing RNA structures that look like agroup I intron.

2. There aren’t convincing RNA structures that look like agroup II intron.

3. There aren’t convincing RNA structures that look like afolded RNA of any kind really.

Phages Optimus (cluster J) and SG4 (cluster F1)have similar regions as Violet (cluster A1)

*found at phagesdb by Blast! Thanks Pitt Crew!

1. Features found in Violet are same in Optimus and SG4.

2. Sequence differences between phage are greater in themethyltransferase parts than in the middle – many are silent mutationsor cause conservative amino acid substitutions.

Pattern may not be consistent with homing by gene conversion. Consistent with important protein coded in both frames. *

A new kind of mobile element? Hypotheses.

The yellow sequence is not excised as in an intein, but folds in such a way that itdoes not interrupt folding or function of the target protein.Structure prediction is 63% loop, weak predictions of helix and sheet.

Not a DNA transposon probably. These usually disrupt the target.Nuclease domain not like transposases. No direct repeats near edges.

Or maybe it isn’t mobile, and is a defective copy of an intein or an intron that has lostthose elements.Why the conserved pattern of nucleotide changes then?

Wet lab experiments will be required to test these ideas. *

Firecracker

Dotplot Analysis of the Firecracker Genome. Comparisonof the Fircracker genome to Corndog (left) and itself (right). Thisanalysis shows that Firecracker is closely related to Corndog and thatlike Corndog, it contains many short sequence repeats (visible as off-diagonal structures in the plots).

Predicted palindrome motif for Firecracker. Motif found byMEME sequence analysis of the firecracker genomic sequence. Themotif occurs more than 30 times in the Firecracker genome.

Repeats found in Firecracker. An unusual set ofrepeats occurred specifically in a large gap between predictedgenes 125 and 126. The image on the left shows these repeatsas depicted in a dotplot. BLAST searches indicate that thisregion is conserved in Corndog, but is otherwise unique.

Repeats found in Firecracker. An unusual set of repeats occurredspecifically in a large gap between predicted genes 125 and 126. The image onthe left shows these repeats as depicted in a dotplot. BLAST searches indicatethat this region is conserved in Corndog, but is otherwise unique.

Integration of phage Trouble in M. smeg. Disrupts the groEL gene

Integration of phage Ogopogo in M. smeg. Disrupts tRNALys

• Do the insertions of Trouble orOgopogo in lysogens preserve thefunction of groEL and tRNALys? If theydo, do the altered genes have exactlythe same function or altered functions?

Interesting phagy observations and questions for the...

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