SEA-PHAGES Logo

The official website of the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science program.

Welcome to the forums at seaphages.org. Please feel free to ask any questions related to the SEA-PHAGES program. Any logged-in user may post new topics and reply to existing topics. If you'd like to see a new forum created, please contact us using our form or email us at info@seaphages.org.

lysin A gene split with an intron?

| posted 23 Jun, 2020 06:27
Hi, I am QCing the cluster F phage Coco12, which has an HNH endonuclease inserted into lysin A, and am looking for guidance on annotating the lysin A bits.

The insertion seems to split the gene between domains. HHPred easily identifies the amidase domain in the downstream half, but it does not provide any evidence for a peptidase domain in the upstream half. BLAST does show that the upstream half has the same sequence as lysin As in Payne and Hatfull that contain the "N3" putative peptidase domain, as in Tweety gp30 (Payne and Hatfull, 2012).

Cluster F phage Frankie has nearly the same situation with an HNH endonuclease inserted in nearly the same position. To my surprise, the lysin A gene in Frankie is annotated as if it has an intron – in two sections that are connected together to make one lysin A polypeptide. The GenBank entry reads

gene     26909..28665
         /locus_tag="SEA_FRANKIE_33"
CDS      join(26909..27370,27946..28665)

I have attached phamerator screenshots of the Coco12 and Frankie lysin As aligned with intact lysin As.

Should Coco12 lysin A be annotated with an intron, as in Frankie? (I am thinking that bench evidence ought to support an annotation like this.)

Or, should each lysin A part be annotated with a domain function appended? We have good HHPred evidence supporting the amidase domain for the downstream section, but we lack HHPRed or CDD evidence for a peptidase function in the upstream bit. BLASTp shows that the upstream section is nearly identical to Tweety gp30, which is the canonical lysin A listed in Payne and Hatfull that carries the N3 hydrolase domain (M23B peptidase in phi29 gp13 tail protein).

Or is there another option that is the best in this case? Thanks! Kirk

#################################################

Addendum 6/26/20:
I said above that the first half of Coco12's lysin A did not hit anything on HHPRed. I have never fiddled with HHPred settings before, but I changed the MSA generation method from the default HHBlits=>UniRef30 to PSI-BLAST=>nr70 and this now hits a group of proteins that does include phi29 gp13. The region that matches phi29 gp13 is not to its m23 metallo-peptidase-like domain, but to its lysozyme domain. I've attached a screenshot and link to the HHPred output.
Edited 26 Jun, 2020 16:49
| posted 26 Jun, 2020 17:09
Hi Kirk,
Can you stitch the lysin back together in silico and get really good BLASTN matches to other lysins?
That would be the most compelling evidence for an intron, other than the HNH (the HNH in the gap is already pretty compelling).
| posted 26 Jun, 2020 18:47
Hi Welkin,
Yes, when I remove the inserted HNH and the 6 bp duplication that was created, a lysin A is made that is identical to others such as MilleniumForce lysin A. I've attached my notes and sequences.
Thanks, Kirk
 
Login to post a reply.