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Link to this post \| posted 15 Feb, 2019 12:53
scaruso	We would like to add the gene function “DNA delivery” (or equivalent wording to that effect) to the official functions list. This is the function assigned to the Tectivirus Enterobacteria phage PRD1 p32 and p34. It is also a function commonly assigned in Tectiviridae. Please see: Jalasvuori M, Koskinen K. 2018. Extending the hosts of Tectiviridae into four additional genera of Gram-positive bacteria and more diverse Bacillus species. Virology 518:136–142. PMID: 29481984 We identified this function in Streptomyces phage Forthebois gp22 and gp28 by HHPred to PRD1 p32 and p34. The functions of PRD1 p32 and p34 are described here: Grahn AM, Daugelavicius R, Bamford DH. 2002. The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry. J Virol 76:4866–72. PMID: 11967303 “The amino acid sequence of P32 shows characteristic elements for integral membrane proteins. According to the orientation of the predicted ɑ-helix, the topology of protein P32 would be such that the N-terminal part spans the membrane, leaving the bulk of the protein (C terminus) in the cytoplasmic side of the CM. If our model of a clathrin-like engulfment of the phage membrane from the host cytoplasmic membrane during particle assembly is valid, the C terminus of P32 would face outward on the virus membrane surface… Sequence analysis revealed another interesting feature, a close similarity of P32 to another small phage membrane protein, P34 (Fig. 5). This is a second example of a high level of sequence similarity between two PRD1 gene products, suggesting a single gene origin for the two elements.” Saren A-M, Ravantti JJ, Benson SD, Burnett RM, Paulin L, Bamford DH, Bamford JKH. 2005. A snapshot of viral evolution from genome analysis of the tectiviridae family. J Mol Biol 350:427–40. PMID: 15946683 “The known PRD1 genes encode replication (P1, P, capsid (P3, P30), vertex (P2, P5, P16, P31), DNA packaging (P6, P9,P20, P22), DNA delivery (P11, P18, P32, P34), host cell lysis (P7, P14, P15, P35) and assembly (P10, P17) proteins. The genome is packaged into a preformed empty capsid through a unique portal vertex and injected into the host cell through a tubular structure derived from the viral membrane.” “High conservation occurs in the DNA polymerase encoding sequence (gene I), except close to the gene end. This is also the case with the region encoding the carboxyl terminal end of the packaging ATPase (gene IX), the regions coding for the small membrane protein linking the packaging vertex to the membrane (gene XXII), the coat protein (gene III), and the small membrane proteins important for DNA delivery (genes XVIII, XXXII, XXXIV).” >PRD1_p32 MGEFGKTLITIVTAIIGVAIIAVIVSQRSNTAGVIQSATSGFSNILKSALAPII >PRD1_p34 MNDFVGPIVTVLTAIIGVAILAVLVSRNSNTAGVIKAGSGGFSSMLGTALSPVTGGTGFAMTNNYSGF >Forthebois_gp22 MNGDKVFNVLGAIVTVALVTTIVSRPTSAQVIKAMGDAFSGSIRAALGKZ >Forthebois_gp28 MKGDDVIKVLMGIIAVALVTTIVMRPNSATVIKAAGSAFSGSLRAAMGKZ Forthebois gp22 Forthebois gp28

Link to this post | posted 15 Feb, 2019 12:53

We would like to add the gene function “DNA delivery” (or equivalent wording to that effect) to the official functions list. This is the function assigned to the Tectivirus Enterobacteria phage PRD1 p32 and p34. It is also a function commonly assigned in Tectiviridae. Please see:

Jalasvuori M, Koskinen K. 2018. Extending the hosts of Tectiviridae into four additional genera of Gram-positive bacteria and more diverse Bacillus species. Virology 518:136–142. PMID: 29481984

We identified this function in Streptomyces phage Forthebois gp22 and gp28 by HHPred to PRD1 p32 and p34. The functions of PRD1 p32 and p34 are described here:

Grahn AM, Daugelavicius R, Bamford DH. 2002. The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry. J Virol 76:4866–72. PMID: 11967303

“The amino acid sequence of P32 shows characteristic elements for integral membrane proteins. According to the orientation of the predicted ɑ-helix, the topology of protein P32 would be such that the N-terminal part spans the membrane, leaving the bulk of the protein (C terminus) in the cytoplasmic side of the CM. If our model of a clathrin-like engulfment of the phage membrane from the host cytoplasmic membrane during particle assembly is valid, the C terminus of P32 would face outward on the virus membrane surface…

Sequence analysis revealed another interesting feature, a close similarity of P32 to another small phage membrane protein, P34 (Fig. 5). This is a second example of a high level of sequence similarity between two PRD1 gene products, suggesting a single gene origin for the two elements.”

Saren A-M, Ravantti JJ, Benson SD, Burnett RM, Paulin L, Bamford DH, Bamford JKH. 2005. A snapshot of viral evolution from genome analysis of the tectiviridae family. J Mol Biol 350:427–40. PMID: 15946683

“The known PRD1 genes encode replication (P1, P smile

, capsid (P3, P30), vertex (P2, P5, P16, P31), DNA packaging (P6, P9,P20, P22), DNA delivery (P11, P18, P32, P34), host cell lysis (P7, P14, P15, P35) and assembly (P10, P17) proteins. The genome is packaged into a preformed empty capsid through a unique portal
vertex and injected into the host cell through a tubular structure derived from the viral membrane.”

“High conservation occurs in the DNA polymerase encoding sequence (gene I), except close to the gene end. This is also the case with the region encoding the carboxyl terminal end of the packaging ATPase (gene IX), the regions coding for the small membrane protein linking the packaging vertex to the membrane (gene XXII), the coat protein (gene III), and the small membrane proteins important for DNA delivery (genes XVIII, XXXII, XXXIV).”

>PRD1_p32
MGEFGKTLITIVTAIIGVAIIAVIVSQRSNTAGVIQSATSGFSNILKSALAPII

>PRD1_p34
MNDFVGPIVTVLTAIIGVAILAVLVSRNSNTAGVIKAGSGGFSSMLGTALSPVTGGTGFAMTNNYSGF

>Forthebois_gp22
MNGDKVFNVLGAIVTVALVTTIVSRPTSAQVIKAMGDAFSGSIRAALGKZ

>Forthebois_gp28
MKGDDVIKVLMGIIAVALVTTIVMRPNSATVIKAAGSAFSGSLRAAMGKZ

Forthebois gp22

Forthebois gp28

Posted in: Request a new function on the SEA-PHAGES official list → DNA delivery

Link to this post \| posted 27 Nov, 2018 17:47
scaruso	Thanks, Debbie. I appreciate you looking at it. Steve

Posted in: Functional Annotation → A3 Holin?

Link to this post \| posted 27 Nov, 2018 01:33
scaruso	jparker Steven Caruso I see that the web phamerator can make maps, and understand that other features are coming. Is there a way to have it access alternate databases that I don't see (like Bacillus phages), of is that one of the not yet ready for prime time features? Thanks! steve We went out on a limb this quarter and tried to isolate bacillus phages. It worked, woohoo! However, I didn't think it through, and am just realizing that there isn't phamerator set up for bacillus. Is there a work around, or is this in the works? Thanks! Jordan Not that we are aware of yet. We are still using the old fashioned one for Bacillus phages for now, though we eagerly anticipate a web-based version for the students. There is a benefit to having access to the old version, though, if you want to be able to pull pham data for analysis, though, for either family. Steve

Link to this post | posted 27 Nov, 2018 01:33

scaruso

jparker
Steven Caruso
I see that the web phamerator can make maps, and understand that other features are coming. Is there a way to have it access alternate databases that I don't see (like Bacillus phages), of is that one of the not yet ready for prime time features?

Thanks!

steve

We went out on a limb this quarter and tried to isolate bacillus phages. It worked, woohoo! However, I didn't think it through, and am just realizing that there isn't phamerator set up for bacillus. Is there a work around, or is this in the works?
Thanks!
Jordan

Not that we are aware of yet. We are still using the old fashioned one for Bacillus phages for now, though we eagerly anticipate a web-based version for the students. There is a benefit to having access to the old version, though, if you want to be able to pull pham data for analysis, though, for either family.

Steve

Posted in: Web Phamerator → Are we there yet?

Link to this post \| posted 24 Nov, 2018 15:22
scaruso	I was thinking Synteny might be compelling, but I ran the gene with the other databases in HHPred, here's what I find: TIGR01673 holin_LLH; phage holin, LL-H family. This model represents a putative phage holin from a number of phage and prophage regions of Gram-positive bacteria. Probability: 91.2 E-value: 2.9 Score: 27.85 Aligned Cols: 83 Identities: 14% Similarity: 0.192 The second gene's (24921) best hit is to an uncharacterized membrane protein in the host seen by mass spec. NP_217999.1 membrane protein [Mycobacterium tuberculosis H37Rv] Probability: 99.07 E-value: 1.1E-12 Score: 92.87 Aligned Cols: 77 Identities: 30% Similarity: 0.671 So, seems like a reasonable call, but I can understand if you want to let it go. As for the other gene I mentioned above, I can't find any reason to call () an excise or DNA binding protein other than that it was called that by someone. So I'm going to call it NKF, though if you have info I would be grateful for it. Otherwise, it's all done. You can see the whole thing in PECAAN (or on a DNAM) if you like for reference. Thanks, Steve Edited 25 Nov, 2018 18:07

Link to this post | posted 24 Nov, 2018 15:22

scaruso

I was thinking Synteny might be compelling, but I ran the gene with the other databases in HHPred, here's what I find:

TIGR01673 holin_LLH; phage holin, LL-H family. This model represents a putative phage holin from a number of phage and prophage regions of Gram-positive bacteria.
Probability: 91.2 E-value: 2.9 Score: 27.85 Aligned Cols: 83 Identities: 14% Similarity: 0.192

The second gene's (24921) best hit is to an uncharacterized membrane protein in the host seen by mass spec.

NP_217999.1 membrane protein [Mycobacterium tuberculosis H37Rv]
Probability: 99.07 E-value: 1.1E-12 Score: 92.87 Aligned Cols: 77 Identities: 30% Similarity: 0.671

So, seems like a reasonable call, but I can understand if you want to let it go.

As for the other gene I mentioned above, I can't find any reason to call () an excise or DNA binding protein other than that it was called that by someone. So I'm going to call it NKF, though if you have info I would be grateful for it.

Otherwise, it's all done. You can see the whole thing in PECAAN (or on a DNAM) if you like for reference.

Thanks,

Steve

Edited 25 Nov, 2018 18:07

Posted in: Functional Annotation → A3 Holin?

Link to this post \| posted 23 Nov, 2018 18:36
scaruso	Thank you. I see it in the text, now, though the table at the end shows "F." Thanks, again. Steve

Posted in: Functional Annotation → Portal/Head-t-o-tail Case Study Question

Link to this post \| posted 23 Nov, 2018 15:58
scaruso	There are no genes in the left (forward) part of the genome with transmembrane domains that haven't been assigned functions other than the one above. On the right side after all the forward genes: The first (stop = 24921) gene has a TMD according to PECAAN's TmHmm. It also has a poor HHPred hit to: PF16935.5 ; Hol_Tox ; Putative Holin-like Toxin (Hol-Tox) Probability: 25.57, E-value: 420.0, Score: 17.93, Aligned Cols: 34, Identities: 9%, Similarity: 0.036, PECAAN HHPred is a little different with a prob of 32.3. coverage is only 35.1 The next, oddball forward gene has four TM domains. It has been identified as a DNA binding protein and, curiously as Excise in another phage. I'm stuck on NKF without proof beyond those calls, though. So it could also be looked at. No HHPreds to anything like Holin, though, or anything else. The last has stop=39165, has 1 TMD, but is only 105 bp long. No significant HHPred hits on PECAAN or external. The only issue is, I used the TmHmm program in PECANN to screen all 84 genes. It caught the three above, but I don't know if it would catch every possibility. I don't know what others would be found with SOSUI. Edited 24 Nov, 2018 15:12

Link to this post | posted 23 Nov, 2018 15:58

scaruso

There are no genes in the left (forward) part of the genome with transmembrane domains that haven't been assigned functions other than the one above.

On the right side after all the forward genes:

The first (stop = 24921) gene has a TMD according to PECAAN's TmHmm. It also has a poor HHPred hit to:
PF16935.5 ; Hol_Tox ; Putative Holin-like Toxin (Hol-Tox)
Probability: 25.57, E-value: 420.0, Score: 17.93, Aligned Cols: 34, Identities: 9%, Similarity: 0.036,
PECAAN HHPred is a little different with a prob of 32.3. coverage is only 35.1

The next, oddball forward gene has four TM domains. It has been identified as a DNA binding protein and, curiously as Excise in another phage.
I'm stuck on NKF without proof beyond those calls, though. So it could also be looked at.
No HHPreds to anything like Holin, though, or anything else.

The last has stop=39165, has 1 TMD, but is only 105 bp long. No significant HHPred hits on PECAAN or external.

The only issue is, I used the TmHmm program in PECANN to screen all 84 genes.
It caught the three above, but I don't know if it would catch every possibility.
I don't know what others would be found with SOSUI.

Edited 24 Nov, 2018 15:12

Posted in: Functional Annotation → A3 Holin?

Link to this post \| posted 22 Nov, 2018 18:01
scaruso	I'm following up on Zetzy, an A3, that we used in the Hack-a-thon, and I have a quick question. I was able to call two genes NKF (that had been labeled otherwise in other phages), and another as head-to-tail adapter based on the Case Study and the table provided at end, that outlines the PDB hit requirements (5A21_D). But the next gene's PDB his is to 5A21_E, which is not listed. The only 'E' version of the entry contains SPP1 proteins 15, 16, and 17. Any thoughts? 'G/17' is downstream, so could this be 'F/16"? HHPred: https://toolkit.tuebingen.mpg.de/#/jobs/Zet_13195 Protein: MSLLDGGPQYEDILVFPEEAVTDEDGNTKTRPSATGIPAKARFQVQGQSGTSARRAEQDNEGFESEKVYRMRFPRSWDAEHGVLGAQSEIEWRGKRWALFGDVNFYNSSRRTARIDYTVKRYZ

Link to this post | posted 22 Nov, 2018 18:01

scaruso

I'm following up on Zetzy, an A3, that we used in the Hack-a-thon, and I have a quick question.

I was able to call two genes NKF (that had been labeled otherwise in other phages), and another as head-to-tail adapter based on the Case Study and the table provided at end, that outlines the PDB hit requirements (5A21_D). But the next gene's PDB his is to 5A21_E, which is not listed. The only 'E' version of the entry contains SPP1 proteins 15, 16, and 17.

Any thoughts? 'G/17' is downstream, so could this be 'F/16"?

HHPred: https://toolkit.tuebingen.mpg.de/#/jobs/Zet_13195

Protein: MSLLDGGPQYEDILVFPEEAVTDEDGNTKTRPSATGIPAKARFQVQGQSGTSARRAEQDNEGFESEKVYRMRFPRSWDAEHGVLGAQSEIEWRGKRWALFGDVNFYNSSRRTARIDYTVKRYZ

Posted in: Functional Annotation → Portal/Head-t-o-tail Case Study Question

Link to this post \| posted 21 Nov, 2018 12:18
scaruso	Not in that area or its environs, there are a lot of identified genes on the left side, but I'll need to get back to you about the rest of the genome. The HHPred search was completed using only the normal DBs suggested by the manual. You might note, though, that it's common to get different results. I check all interesting ones by hand, still. See this example, where the PDB hit from HHPred on the web and on PECAAN had the same match, but very different probabilities (88.55 vs 55.6). https://toolkit.tuebingen.mpg.de/#/jobs/Zet_13556 I'll be working on it over the next week and will update. Steve Edited 22 Nov, 2018 18:05

Link to this post | posted 21 Nov, 2018 12:18

scaruso

Not in that area or its environs, there are a lot of identified genes on the left side, but I'll need to get back to you about the rest of the genome.

The HHPred search was completed using only the normal DBs suggested by the manual. You might note, though, that it's common to get different results. I check all interesting ones by hand, still.

See this example, where the PDB hit from HHPred on the web and on PECAAN had the same match, but very different probabilities (88.55 vs 55.6). https://toolkit.tuebingen.mpg.de/#/jobs/Zet_13556

I'll be working on it over the next week and will update.

Steve

Edited 22 Nov, 2018 18:05

Posted in: Functional Annotation → A3 Holin?

Link to this post \| posted 20 Nov, 2018 20:55
scaruso	I have what I believe is a decent candidate for the holin in the A3 subcluster, in Zetzy and would appreciate some feedback. It is immediately downstream of some tRNAs, and upstream of Lysin A and B. Such that, if correct, it would be: Terminase, small subunit Minor tail protein Minor tail protein NKF (probably a minor tail protein) tRNAs (x3) Holin Lysin A Lysin B Terminase, large subunit Portal Capsid maturation Scaffolding Major capsid protein The described synteny shows the Holin between Lysin A and Lysin B, but this seems like a reasonable variation, right in front of Lysin A and B. A link to the HHPred results can be found here: https://toolkit.tuebingen.mpg.de/#/jobs/Zet_3980. I have attached a PDF of the result as well. SOSUI shows that it has a single transmembrane region near the N-terminus: LATIAKLIAQALLPIIAKQIAEE, and it is 255 aa long. The HHPred suggests it is a class VI holin, which has a single transmembrane region near the N-terminus, but it is also in line with the T4 (class III) family, which is closer in size. The gene product: MLATIAKLIAQALLPIIAKQIAEEFGKHVEPLTKALVTAVTEAAATGAERGADKLTDYIPGKLDDQIIDPIVKRGLEIFRDLTRZ Thanks! Steve Edited 21 Nov, 2018 12:15 3.44Mb

Link to this post | posted 20 Nov, 2018 20:55

scaruso

I have what I believe is a decent candidate for the holin in the A3 subcluster, in Zetzy and would appreciate some feedback.

It is immediately downstream of some tRNAs, and upstream of Lysin A and B. Such that, if correct, it would be:

Terminase, small subunit
Minor tail protein
Minor tail protein
NKF (probably a minor tail protein)
tRNAs (x3)
Holin
Lysin A
Lysin B
Terminase, large subunit
Portal
Capsid maturation
Scaffolding
Major capsid protein

The described synteny shows the Holin between Lysin A and Lysin B, but this seems like a reasonable variation, right in front of Lysin A and B.

A link to the HHPred results can be found here: https://toolkit.tuebingen.mpg.de/#/jobs/Zet_3980. I have attached a PDF of the result as well.

SOSUI shows that it has a single transmembrane region near the N-terminus: LATIAKLIAQALLPIIAKQIAEE, and it is 255 aa long. The HHPred suggests it is a class VI holin, which has a single transmembrane region near the N-terminus, but it is also in line with the T4 (class III) family, which is closer in size.

The gene product: MLATIAKLIAQALLPIIAKQIAEEFGKHVEPLTKALVTAVTEAAATGAERGADKLTDYIPGKLDDQIIDPIVKRGLEIFRDLTRZ

Thanks!

Steve

Edited 21 Nov, 2018 12:15

Posted in: Functional Annotation → A3 Holin?

Link to this post \| posted 20 Aug, 2018 17:22
scaruso	David Bollivar This is a tougher one. Again, just my two cents. The evidence points to this having a LysM domain, but that can be a little misleading because the LysM domain essentially is a peptidoglycan binding domain. Your CDD hit is just to that part of the predicted protein. Unfortunately, there is not an approved function with just the LysM domain, so the question is do you have enough evidence for the endolysin part. There are suggestions (the LD transpeptidase hit for example) but I would suggest it is not strong enough to make this call. I personally would leave this NKF. I can buy that. Thank you for your second look. Steve

Link to this post | posted 20 Aug, 2018 17:22

scaruso

David Bollivar
This is a tougher one. Again, just my two cents. The evidence points to this having a LysM domain, but that can be a little misleading because the LysM domain essentially is a peptidoglycan binding domain. Your CDD hit is just to that part of the predicted protein. Unfortunately, there is not an approved function with just the LysM domain, so the question is do you have enough evidence for the endolysin part. There are suggestions (the LD transpeptidase hit for example) but I would suggest it is not strong enough to make this call. I personally would leave this NKF.

I can buy that. Thank you for your second look.
Steve

Posted in: Functional Annotation → WheeHeim LysM

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