Link to this post | posted 02 Mar, 2023 23:27

kklyczek

Hi Amaya, Sorry to take so long to reply. We have discussed this offline, but I wanted to post that discussion here. Yes, this protein has very good, full-length HHPred alignments with thiamine-binding domains in a few proteins. Some notes are in these slides. However, a couple of reasons to be cautious about calling it thiamine-binding protein include: - This binding domain is in the ACT domain family, which is found in a lot of different proteins with various functions, binding various ligands (E. coli 3-phosphoglycerate dehydrogenase is the model ACT domain protein). There are equally good alignments in HHPred with several proteins of unknown function. The London gp47 domain contains some but not all of the key residues described for thiamine binding in this paper. Could it be binding something else instead? - Both of the thiamine-binding proteins matched by London gp47 in HHPred are part of a cluster of genes involved in thiamine transport (Bacillus) or oxidative stress responses (Thermotoga). What function would this protein have in the absence of the other proteins in these clusters? Its location in the phage genome is just upstream of the integrase gene, on the opposite strand by itself. Nick Edgington and his students ran Elezi gp47 through AlphaFold and saw very similar folding to the Thermatoga protein TM0486 (see last slide in attachment). Is this sufficient evidence to make the call, or are the other issues still pertinent? Karen Edited 02 Mar, 2023 23:29

Link to this post | posted 17 Aug, 2022 17:29

kklyczek

Rick, Debbie, Kristen, Not all members of this pham have the RecT-like gene immediately downstream. Should those also be called RecE-like anyway? There are (at least) two patterns: 1. For cluster P2 phage Tortellini gp55, the next gene is not RecT, but has weak hits to a similar ssDNA binding protein (see attached phamerator map and HHPred results for Tortellini gp56) The rest of the cluster P phages do have the RecE-RecT combination described for ShaboiShabazz. 2. In some cluster AY phages (Persistance gp62, Seahorse gp72), there is a ssDNA binding protein 3-4 genes downstream (Persistance gp65, Seahorse gp76). a) Is this close enough to be identified as a RecE-RecT-like? b) HHPred hits for the ssDNA binding protein (Persistance_65) do not specifically mention RecT but rather M.smeg SSBb and related proteins. It is challenging to distinguish between RecE-like exonuclease, RecB exonuclease domain, and Cas4 family exonuclease based just on alignments, since they are so similar. I was investigating cluster P1 phage Langerak, which has a gene in this pham (gp46). HHPred alignments to all three types of hits are in the attachment. They all include the same motifs that Rick identified for RecE in ShaboiShabazz gp42 and many of the specific residues. One could argue that there is better alignment to RecE (there are larger gaps in alignments to RecB-like and Cas4) – is this be sufficient to call RecE in the absence of RecT? Does it come down to specific residues? Or is a the more general exonuclease call the better option in the absence of RecT? Thanks Karen 2.21Mb

Link to this post | posted 17 Aug, 2022 17:23

kklyczek

Hi Kathleen, After looking into this a bit more, I think I need to change my recommendation to RecE-like exonuclease for Langerak_46, based on the RecT-like ssDNA binding protein gene immediately downstream (Langerak_47). Please see this forum thread https://seaphages.org/forums/topic/5186/. I missed this as I was focused on the Cas4 vs RecB question but, as Debbie pointed out, context is important! The genes mentioned in that post, Che9c_60 and ShaboiShabazz_42, are in the same pham as Langerak_46. This is a large pham including several clusters. Most (but not all) of the genes have a RecT-like ssDNA binding protein immediately downstream, and if they do they should be called RecE-like based on this analysis. RecE-like exonucleases, Cas4 family exonucleases, and the exonuclease domain of RecB-like helicase/exonucleases all have very similar folds and motifs (all are part of the PD-(D/E)XK nuclease superfamily). All will likely have HHPred hits to crystal structures for RecE (e.g. 3H4R_A) and Cas4 (e.g. 4R5Q_A). Based on the information in the official function list and the forum thread linked above, the following guidelines might be useful for calling proteins with these HHPred hits: a. If the protein also has a helicase domain, call RecB-like helicase/exonuclease b. If the gene immediately downstream is a RecT-like ssDNA binding protein, call RecE-like exonuclease c. If neither a nor b are true, call Cas4-family exonuclease I have not yet found an example of a phage RecB-like helicase/exonuclease. Here is the HHPred result for a bacterial RecB-like protein that has both domains, as an example. https://toolkit.tuebingen.mpg.de/jobs/5897366_4 Karen

Link to this post | posted 04 Aug, 2022 19:08

kklyczek

Hi Kathleen, The exonucleases are confusing! You are correct that Langerak_46 has HHPred hits to nuclease/helicases as well as the Cas4-family exonucleases. However, the nuclease/helicase hits are only aligning to the C-terminal parts of those proteins (see attached screenshots). My interpretation is that Langerak_46 is aligning to the exonuclease domains of the RecB-like proteins, but it does not contain the helicase domain. So I think the best call given the information in the official function list is Cas4-family exonuclease. The prototype gene product in the official function list for RecB-like exonuclease/helicase is RedRock_72, but the HHPred results for that protein show only alignment to the exonuclease (https://toolkit.tuebingen.mpg.de/jobs/9540395_8), and in fact that gene is currently annotated as Cas4 exonuclease. I will try to find an example of a RecB-like protein so you can see how to identify the two domains. I am also not entirely sure how to distinguish Cas4-family exonuclease from other members of the PD-(D/E)XK superfamily that show up as HHPred hits, but am looking in to that further. Karen Edited 04 Aug, 2022 19:13 654Kb

Link to this post | posted 20 Jul, 2022 23:35

kklyczek

Hi Fred, I recently investigated a similar gene in clusters AZ and EH; Eraser_30 is an example. Your Skinny gene product has similar HHPred results, with many strong hits with >95% probability and >90% query coverage. Looking at the PDB hits, the query sequence aligns to almost the entire crystal sequence, so these are good matches (see attached file). The hits include descriptions like homing endonuclease, LADLIDADG domain, hydrolase, some of which are describing different levels of specificity (LADLIDADG is a type of homing endonucleases, which are all hydrolases). The LADLIDADG endonucleases are one family of homing endonucleases (others are HNH endonucleases and G-I-Y Y-I-G endonucleases). The name refers to the consensus sequence of a motif in the DNA binding site. The conserved features of LADLIDADG endonucleases include: - Approximately 100 amino acids with Alpha-beta-beta-alpha-beta-beta-alpha secondary structure - LADLIGADG motif starting in the first alpha helix - Aromatic amino acid right before the motif The Skinny gene product meets these criteria, and has good alignment to the target sequence throughout, so I would recommend calling it LAGLIDADG endonuclease. Other approved functions that are accurate include endonuclease or nuclease or hydrolase, but I think there is enough evidence for the more specific call. I note that HNH endonuclease has been called for some of these genes, but those endonucleases have different structural features, and there is no evidence for HNH endonuclease here. It is also interesting to note that Skinny is the only cluster M phage genome that has this pham! Karen Edited 20 Jul, 2022 23:36 522Kb

Link to this post | posted 13 Nov, 2021 16:31

kklyczek

Hi All, I just wanted to make sure that everyone knows that Kocuria kristinae NRRL B-14843 is actually Microbacterium paraoxydans (by 16S sequence). K. kristinae NRRL B-14835 has been verified as K. kristinae. We have had good success isolating phage on the M. paraoxydans but no luck so far with bona fide K. kristinae. It grows well and makes a nice lawn, but no phages isolated from about two dozen samples attempted. I stongly recommend verifying any potential new hosts by 16S sequence, especially if obtaining them directly from NRRL. They archive samples based on what the submitters tell them and do not verify submissions. We learned that lesson the hard way. Karen Edited 13 Nov, 2021 16:32

Link to this post | posted 04 Feb, 2019 01:13

kklyczek

I just reviewed the annotation for Gordonia cluster CS3 phage Lahirium. Its portal gene appears to contain the same intein found in Woes. (Most of the CS3 phages have this.) It looks like Woes was recently modified so the intein is mentioned in a note rather than in the function, i.e. the GenBank entry now includes a note "contains intein" and the function is just "portal protein" - it was "portal protein with intein". I just wanted to confirm that this is the approved way to document the intein. Thanks! Karen Edited 04 Feb, 2019 02:40

Link to this post | posted 14 Sep, 2018 13:20

kklyczek

Hi Nikki, In addition to the excellent suggestions by Debbie and Dustin, the observation that the TA was not solidifying in the bottle made me wonder if the agar was thoroughly mixed when the TA came out of the autoclave? We have seen that the agar can settle to the bottom, especially if it is a large flask and if the agar was not completely dissolved before autoclaving. If it is not mixed when it comes out of the autoclave, the first few bottles may not have enough agar to solidify. That could also explain why it worked for some students and not others? Karen

Link to this post | posted 01 Jul, 2018 14:53
kklyczek	Hi Chris, We encountered something very similar in the Rhodococcus cluster CA phages and decided to call those tRNA-other and not specify the anti-codon (this is allowed in GenBank). If it looks like a legitimate tRNA otherwise, that seems like a reasonable option here. Karen

Link to this post | posted 20 Jun, 2018 12:32
kklyczek	That slippery sequence is only in 2 of the 6 EC cluster phages - see the attached alignment of the 3'end of the putative G gene (KaiHaiDragon_33, pham 45474). Karen 152Kb