Link to this post | posted 15 Aug, 2017 18:29

kklyczek

Hi Veronique, This is the same pham I was investigating in cluster J phages, where it has been called as PAPS reductase. This pham (2324 currently) is found in A1, F1, F2, and J. It looks like it generally has not been assigned a function in the As and Fs. But as you say, there are good HHPred hits. For Klein_172 the top several hit are PAPS (phosphoadenosine phosphosulfate) reductase, https://toolkit.tuebingen.mpg.de/#/jobs/8893012, and I was leaning toward that call, though it is not yet on the approved function list. Phyre2 returned PAPS reductase with 100% confidence, but at 63% coverage: http://www.sbg.bio.ic.ac.uk/phyre2/phyre2_output/5a596153e1c66682/summary.html Adenine nucleotide alpha hydrolase would be a more general call - enzymes in this family share a ribonucleotide binding domain which is probably what is generating the various HHPred hits. But PAPS reductase seems like a common phage function - it's been called in several other clusters (in other phams, e.g. 2991 in cluster B3 Chandler_4) and is found in phages from other hosts. I am not sure what it's function is in phage multiplication, but it uses thioredoxin as an electron donor to reduce inorganic sulfate to form organic thiols (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064068/). I don't think ribonucleotide reductase is the best call. The example gene on the approved function list, RedRock_53, looks quite different from this pham, and hits several ribonucleotide reductases full length: https://toolkit.tuebingen.mpg.de/#/jobs/3357242. This enzyme reduces ribonucleotides to deoxyribonucleotides. Karen Edited 15 Aug, 2017 18:30

Link to this post | posted 10 Aug, 2017 14:37
kklyczek	David, Which phages have the second potential holins? The cluster J phages I've looked at have pham 5845 between the two lysins, which does not look like a holin via HHPred (attached) and does not seem to have a transmembrane region by TMHMM? Karen 129Kb

Link to this post | posted 03 Aug, 2017 15:56

kklyczek

Hi David, I agree - here are the HHPred results for LittleE_149 (pham 3133), using PDB, Pfam, and TIGRfam: https://toolkit.tuebingen.mpg.de/#/jobs/9976351 . The hits are to that central ATPase domain, and most of the descriptions are recombination/repair enzymes. Interestingly, the only sequence similarity between LittleE_149 and Klein_151 (pham 1260) is the N-terminal domain, not that central region (BLAST alignment attached). And to address Welkin's question - there is not another DNA helicase called in the genomes with pham 1260 (at least not that I have seen). So maybe DNA helicase for all of these? Karen Edited 03 Aug, 2017 17:15 34Kb

Link to this post | posted 21 Jul, 2017 12:56
kklyczek	I just noticed that several databases have been added, and the current choices include: PDB SCOPe95 SCOPe70 COG_KOG Pfam-A NCBI_conserved_domains SMART TIGRFAM PRK I think the annotation guide in the past has recommended using PDG, Pfam, and TIGRFAM. I am not familiar with some of the others but will do some exploring. Edited 21 Jul, 2017 12:56

Link to this post | posted 20 Jul, 2017 14:30

kklyczek

Pham 1260 in cluster J phages has been annotated as RecA, RepA, and DNA B helicase. All three versions appear in the maps in the cluster J paper (Pope et al. 2013, doi:10.1371/journal.pone.0069273) where the pham is 3492: Baka_149, Courthouse_142, Optimus_140. There are HHPred hits to all of these at >90% with 50–60% coverage, as well as to many other recombination/repair proteins. It looks like the hit is to an ATPase domain that all of these share? If further investigation is needed I’ll be happy to try that, but wanted to check with those of you with more cluster J experience first. HHPred results for Klein_151 are attached. Note that some J phages have a different pham (3133) at this location which has consistently (I think) been called DNA B helicase (e.g. LittleE_149). Edited 20 Jul, 2017 14:30 1.01Mb

Link to this post | posted 20 Jul, 2017 14:26

kklyczek

In addition to small and large terminase subunits, cluster J phages have a third gene with terminase homology. My question is about which gene to call small terminase. The cluster J paper (Pope et al. 2013, doi:10.1371/journal.pone.0069273) identifies the small terminase just upstream of the large terminase (e.g. Baka_5 and 6), and the additional gene (Baka_2) is also identified as a terminase. However, the annotations in Phamerator all have the Baka_2 homologue identified as small terminase, and Baka_5 is not labeled. HHPred results for Klein_2 and Klein_10 (Baka_5 homologue) are attached, showing that Klein_2 has more homology to large terminase and Klein_10 matches small terminase. 2.34Mb