SEA-PHAGES | All posts created by mcclory

Link to this post \| posted 19 Nov, 2024 00:44
mcclory	I just wanted to give a bump to this comment, as it was very helpful for our students working with G. rubripertincta phages this semester. The G. rub phages were consistently producing tiny, cloudy plaques that were barely perceptible to students when we were using standard PYCa top agar (we often only knew plaques were present from a spot test). We diluted our 25 mL PYCa top agar aliquots with 10 mL of PYCa media (for 0.29% agar) + 0.5 mL 30% glycerol (0.43%, I think this was a calculation error, but it worked so we continued with this after). I did a side-by-side comparison using a lysate one student was working with and got noticeably larger plaques. After that we had the whole class switch and all G. rub phages produced much more apparent plaques. Thanks for this tip.

Link to this post | posted 19 Nov, 2024 00:44

I just wanted to give a bump to this comment, as it was very helpful for our students working with G. rubripertincta phages this semester. The G. rub phages were consistently producing tiny, cloudy plaques that were barely perceptible to students when we were using standard PYCa top agar (we often only knew plaques were present from a spot test).

We diluted our 25 mL PYCa top agar aliquots with 10 mL of PYCa media (for 0.29% agar) + 0.5 mL 30% glycerol (0.43%, I think this was a calculation error, but it worked so we continued with this after). I did a side-by-side comparison using a lysate one student was working with and got noticeably larger plaques. After that we had the whole class switch and all G. rub phages produced much more apparent plaques.

Thanks for this tip.

Posted in: Phage Discovery/Isolation → Webbed plate hack for low-titer stocks and small plaques

Link to this post \| posted 03 Jun, 2023 19:03
mcclory	Cluster EC phages have annotated an ERF family ssDNA binding protein or ERF family DNA pairing protein (E.G. Kowalski_48 or Megan_47). Make sure to check the official functions list for the current function name. ERF protein of phage P22 plays a role in circularizing the linear genome upon entry into the cell (1-3). Iyer, Koonin and Aravind 2002 (4) did a phylogenetic analysis of these DNA binding proteins that has been used to define the conserved domain (pfam04404). In order to call a gene with this function, make sure it includes the defining elements below in addition to supporting evidence from HHPred, BLAST and synteny. These proteins should contain the following sequence elements. 1) A conserved motif: GuXXoYhp+YXhXXhh (where G is glycine, Y-tyrosine, u is a tiny residue, h-hydrophobic, p is a polar residue, o is an alcohol residue, + is a basic residue, and X is any residue). a. Example, Megan_47 contains the sequence GSAITYARRYALTAAT that matches this motif (attached figure). 2) Shortly downstream of 1, a DXD motif. a. Example, Megan_47 contains the sequence DND 8 residues downstream of the motif above (attached figure). The attached figure contains a multiple sequence alignment of genes from this cluster with the above elements highlighted. These proteins are also predicted to have α + β fold secondary structure composed of 5 helices and 3-4 sheets. If you run secondary structure prediction of the full-length protein, compare the prediction with the figure in reference 4 (Figure 3). This note was developed in collaboration with Christine Byrum during the 2023 SEA Faculty meeting. References: 1) Botstein D, Matz MJ: A recombination function essential to the growth of bacteriophage P22. J Mol Biol. 1970, 54: 417-440. 2) Weaver S, Levine M: Recombinational circularization of Salmonella phage P22 DNA. Virology. 1977, 76: 29-38. 3) Poteete AR: Location and sequence of the erf gene of phage P22. Virology. 1982, 119: 422-429. 4) Iyer LM, Koonin EV, Aravind L. Classification and evolutionary history of the single-strand annealing proteins, RecT, Redbeta, ERF and RAD52. BMC Genomics. 2002 Mar 21;3:8. doi: 10.1186/1471-2164-3-8. Epub 2002 Mar 21. PMID: 11914131; PMCID: PMC101383. https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-3-8 Edited 03 Jun, 2023 19:55 1.47Mb

Link to this post | posted 03 Jun, 2023 19:03

mcclory

Cluster EC phages have annotated an ERF family ssDNA binding protein or ERF family DNA pairing protein (E.G. Kowalski_48 or Megan_47). Make sure to check the official functions list for the current function name. ERF protein of phage P22 plays a role in circularizing the linear genome upon entry into the cell (1-3).

Iyer, Koonin and Aravind 2002 (4) did a phylogenetic analysis of these DNA binding proteins that has been used to define the conserved domain (pfam04404). In order to call a gene with this function, make sure it includes the defining elements below in addition to supporting evidence from HHPred, BLAST and synteny.

These proteins should contain the following sequence elements.
1) A conserved motif: GuXXoYhp+YXhXXhh (where G is glycine, Y-tyrosine, u is a tiny residue, h-hydrophobic, p is a polar residue, o is an alcohol residue, + is a basic residue, and X is any residue).
a. Example, Megan_47 contains the sequence GSAITYARRYALTAAT that matches this motif (attached figure).
2) Shortly downstream of 1, a DXD motif.
a. Example, Megan_47 contains the sequence DND 8 residues downstream of the motif above (attached figure).

The attached figure contains a multiple sequence alignment of genes from this cluster with the above elements highlighted.

These proteins are also predicted to have α + β fold secondary structure composed of 5 helices and 3-4 sheets. If you run secondary structure prediction of the full-length protein, compare the prediction with the figure in reference 4 (Figure 3).

This note was developed in collaboration with Christine Byrum during the 2023 SEA Faculty meeting.

References:
1) Botstein D, Matz MJ: A recombination function essential to the growth of bacteriophage P22. J Mol Biol. 1970, 54: 417-440.
2) Weaver S, Levine M: Recombinational circularization of Salmonella phage P22 DNA. Virology. 1977, 76: 29-38.
3) Poteete AR: Location and sequence of the erf gene of phage P22. Virology. 1982, 119: 422-429.
4) Iyer LM, Koonin EV, Aravind L. Classification and evolutionary history of the single-strand annealing proteins, RecT, Redbeta, ERF and RAD52. BMC Genomics. 2002 Mar 21;3:8. doi: 10.1186/1471-2164-3-8. Epub 2002 Mar 21. PMID: 11914131; PMCID: PMC101383. https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-3-8

Edited 03 Jun, 2023 19:55

Posted in: Cluster EC Annotation Tips → ERF family proteins: necessary elements to call this function

Link to this post \| posted 16 Sep, 2022 21:18
mcclory	On 9/16/22 SEA-PHAGES hosted a Zoom meeting about phage hosts. Our breakout group was composed of SEA faculty from institutions that used Microbacterium foliorum as a host. I took brief notes from the breakout session. I'll relay things I found interesting, but it would be great for anyone who attended (or who wasn unable to make it) to expand on them. Participants: Sean McClory, Carlos Partida, Hui-Min Chung, Karen Klyczek, Louise Temple, Alejandra Mussi, Alexandra Jerby, Dustin Edwards, Elivara Eivazova, Emily Savage, Iain Duffy, Maria Elena Baez Flores, Ricardo Parra, Sean Coleman, Shallee Page, Yesmi Ahumada, Eric Engstrom Alejandra mentioned that she had selected M. foliorum because that host had the best success with direct isolations her students collected. I found this interesting, because we started working with M. foliorum when, after a dissapointing semester with our original host M. smeg, I collected a half-dozen samples from around our small campus (where most student samples came from) and did a direct isolation with each of the hosts we had in the freezer. M. foliorum was the winner and our next semester we had signficiantly better student success. Several participants mentioned isolating wild Microbacterium species and using them as hosts. Hui-Min in particular had an interesting project. Her students at University of Western Florida were interested in marine phages, so they collected a few wild bacteria isolates from the local beach. One is a Microbacterium species. Her students have had success isolating phages from marine samples (using the wild isolate hosts???). One of these phages does infect M. foliorum. Hui-Min also mentioned that clam extracts were a potentially great source of phages - I didn't catch all the details about this, but I'd love to hear more. Louise Temple mentioned that many Microbacterium species have a high number of prophages. We began a brief discussion of this, but ran out of time. If anyone has tips for detecting prophages in wild Mircobacterium spp. I think there are several people interested. After the breakout room, Vic shared a pro-tip before we left: grass clippings, particularly partially decaying grass clippings, may be an excellent source of M. foliorum phages. I'm looking forward to trying this. Good to see everyone! Good luck phage hunting!

Link to this post | posted 16 Sep, 2022 21:18

mcclory

On 9/16/22 SEA-PHAGES hosted a Zoom meeting about phage hosts. Our breakout group was composed of SEA faculty from institutions that used Microbacterium foliorum as a host.

I took brief notes from the breakout session. I'll relay things I found interesting, but it would be great for anyone who attended (or who wasn unable to make it) to expand on them.

Participants: Sean McClory, Carlos Partida, Hui-Min Chung, Karen Klyczek, Louise Temple, Alejandra Mussi, Alexandra Jerby, Dustin Edwards, Elivara Eivazova, Emily Savage, Iain Duffy, Maria Elena Baez Flores, Ricardo Parra, Sean Coleman, Shallee Page, Yesmi Ahumada, Eric Engstrom

Alejandra mentioned that she had selected M. foliorum because that host had the best success with direct isolations her students collected. I found this interesting, because we started working with M. foliorum when, after a dissapointing semester with our original host M. smeg, I collected a half-dozen samples from around our small campus (where most student samples came from) and did a direct isolation with each of the hosts we had in the freezer. M. foliorum was the winner and our next semester we had signficiantly better student success.

Several participants mentioned isolating wild Microbacterium species and using them as hosts. Hui-Min in particular had an interesting project. Her students at University of Western Florida were interested in marine phages, so they collected a few wild bacteria isolates from the local beach. One is a Microbacterium species. Her students have had success isolating phages from marine samples (using the wild isolate hosts???). One of these phages does infect M. foliorum. Hui-Min also mentioned that clam extracts were a potentially great source of phages - I didn't catch all the details about this, but I'd love to hear more.

Louise Temple mentioned that many Microbacterium species have a high number of prophages. We began a brief discussion of this, but ran out of time. If anyone has tips for detecting prophages in wild Mircobacterium spp. I think there are several people interested.

After the breakout room, Vic shared a pro-tip before we left: grass clippings, particularly partially decaying grass clippings, may be an excellent source of M. foliorum phages. I'm looking forward to trying this.

Good to see everyone! Good luck phage hunting!

Posted in: Microbacterium → Notes from September 2022 Faculty Meeting

Link to this post \| posted 26 Aug, 2020 23:31
mcclory	I was brainstorming about the possibility of introducing phage students to genetics beyond phages using some of the same tools they've mastered in the course. Has anyone used DNA Master to annotate or explore non-phage genomes?

Posted in: DNA Master → Using DNA Master beyond phages

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