Link to this post | posted 14 Aug, 2025 13:54

Pollenz

Repeated intergenic regulatory sequences have been identified in phages that infect most all of the SEA PHAGES hosts. These are recognized as short conserved regions that branch from one spot when two or more phages are loaded in Phamerator. Usually these are 25-50bp. FK phages have a HUGE region of >20 genes that have one of largest areas and longest repeats (See attachment). It is key to NOT change start calls for genes that are not the LORF in these regions and make sure that these sequences remain upstream of the start codon. RS Pollenz 4.53Mb

Link to this post | posted 14 Aug, 2025 13:22

Pollenz

The FK phages appear to have FOUR genes in the left arm that encode four distinct enzymes involved in cell wall metabolism. Example: Elver genes 8, 9, 10, 11. Gene 8 encodes the M23 peptidase activity associated with the endolysin (lysin A) and has high coverage to HHpred hits. Thus, a lysin A annotation is warranted here. Gene 9 encodes a hyaluronoglucosaminidase, often referred to as hyaluronidase an enzyme that degrades hyaluronic acid (HA). This activity can be found associated with minor tails in some phages. Best annotation call here is a hydrolase. Gene 10 encodes a polysaccharide deacetylase that is an esterase that catalyze the hydrolysis of ester bonds, in this case, acetyl groups attached to polysaccharides. Gene 11 encodes another peptidase, but does not show full coverage in HHpred and also does not have a defined lysin Alike domain activity (example M23, etc.), thus, best annotation is peptidase. Its likely that all of these work together to lyse the cell wall and more defined annotation of activities might be possible in later annotations, so always check Official functions list for updates on the lysin A annotation. RS Pollenz

Link to this post | posted 07 Aug, 2025 17:21

Pollenz

All Cluster A phages have the endolysin genes organized in the left arm of the phage. In some A clusters there is also a 2TMD protein associated with the lysin A and lysin B that MAY serve holin function. Clusters A1, A3, A5, A8, A10 and A19 do not have any TMD genes associated with the lysins. Instead, it appears that the putative TMD proteins (holins) are encoded by genes found downstream of the tape measure embedded within the minor tail genes (SEE POSTED ATTACHMENT). There can be 1, 2 or 3 TMD genes that do not hit to any minor tail proteins in HHpred but have been annotated as such but should be annotated as membrane proteins for the time being until wet lab data demonstrates that these are in fact the lysis proteins. Even cluster A phages with a 2TMD gene near the lysins, have TMD genes in this area. Note that many of these TMD proteins are in phams with members that CAN BE FOUND within the canonical lysis cassettes of phages in other clusters. Its always a good idea to look at other pham cluster members and map the location of the genes when there are mixed annotation functions across the pham. If the Phamerator map is toggled to show the blue TMD proteins in the phage, the presence of these are easily identified and since many minor tails of phage Bxb1 have now been resolved, Bxb1 serves as a good reference for not only the location of the minor tails, but the presence of the TMD genes in that region. RS Pollenz Edited 07 Aug, 2025 17:24 343Kb

Link to this post | posted 14 Jul, 2025 15:51

Pollenz

FK phages have two very large genes (>1400bp, >460 amino acids) found upstream of the tape measure (REF phage Elver genes 36 and 37). These proteins both have a very small HHpred hit (<50 amino acids) to PDB 2C3F_A: HYALURONIDASE, PHAGE ASSOCIATED; LYASE, HYALURONAN LYASE, PHAGE TAIL FIBRE from a streptococcal phage. There are no hits to Mycobacterial phage Bxb1 tail proteins. However, pham members for Elver 37 are found in AP1 phages in canonical locations downstream of the tape measure. Thus, a minor tail call appears supported. RS Pollenz

Link to this post | posted 14 Jul, 2025 15:20
Pollenz	FK phages appear to have two large (>800bp) genes upstream of the tape measure (Ref phage Elver genes 31 and 33) that are grouped to the same pham and are major tails found in numerous other phages. RS Pollenz Edited 14 Aug, 2025 13:07

Link to this post | posted 29 Apr, 2025 11:46

Pollenz

Hello Our lab at USF has been working on these cool TM genes now since 2022 (see: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0276603) and also have wet lab data supporting a lysis function for these 1TMD proteins in phages that infect M. smeg. In Arthrobacter phages, we also noted that the 1TMD appears to be upstream of the putative endolysins and that there are also 1-2 other TMD genes in the vicinity. We have also found up to 4 TMD encoding genes in phages that infect smeg and Gordonia as well as similar 1TMD encoded proteins that are NOT in the lysis cassette area with lysins. My opinion at the moment is that calling a LysZ function needs to wait until 1) the data is actually published and 2) that the function observed in the Corynebacterium is also confirmed in smeg and our other hosts. The 1TMD is clearly involved in lysis, but there are also other functions for very similar proteins described in the literature (see: https://journals.asm.org/doi/10.1128/mbio.00813-22?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed). RS Pollenz

Link to this post | posted 08 Mar, 2025 22:12

Pollenz

Hello A strategy to approach this "mess" is to pull up your phage and TWO FO annotated reference phages with decent identity. The BLAST of PrairieDogTown shows that JanetJ and Aoka are good ref phages that have good matches to PrairieDogTown in several genomic areas. Obviously if you have good nucleotide identity (PURPLE), you should have a similar genomic organization and can see what was done with the other annotated phages. Functional hits and # of pham members can be very helpful when deciding which genes are valid and which to delete. The guiding principles are also important here in regard to overlapping genes and transitions from REV to FOR. RS Pollenz

Link to this post | posted 11 Jun, 2023 13:58

Pollenz

The "immunity cassette" region of the FF phages is rather complex and not canonical as it contains several integrases and various DNA binding proteins. Although there are proteins from these phages grouped to pham 88787 (as of 6/11/23) that have a majority call of immunity repressor, a deeper look at the size of the FF cluster proteins in pham 88787 show that the FF phages all have proteins that are <90 amino acids. Thus, they all contain a clear N-terminal HTH domain that maps to both C1 and C2 repressors, but they all LACK the important C-terminal dimerization domain that is essential to the function of the canonical C1/C2 proteins from lambda and other phages. Note that the great majority of other proteins in the pham are from G1 phages that have 1) a much more clearly defined immunity cassette and 2) HTH proteins that have the C-terminal dimerization domain and can be better defined as immunity represors. So, a more conservative HTH DNA binding domain call for the FF phages is probably warranted at this time for these smaller proteins until clear wet lab data can identify the precise function of these smaller proteins and their exact role in lysogeny. RS Pollenz

Link to this post | posted 08 Jun, 2023 00:22
Pollenz	L5 is listed as GGGGGAA with two A's both in the paper and in the nice table. Its the same issue with using GGGAAA, that is formally listed as being GGGAAAA with 4 A's, not three. RS Pollenz

Link to this post | posted 07 Jun, 2023 20:26
Pollenz	Review of EA4 phages at the 2023 faculty meeting shows a sequence of CGGGGGAc that has been annotated many times as the slippery sequence. While this has the 5Gs, it does not match exactly to the sequences listed in the Guide as they have the sequence CGGGGGCG or GGGGGAA. While compelling, are we calling sequences that are "almost" identical? RS Pollenz