Link to this post | posted 07 May, 2021 15:56

kbutela

I'd like some community input on how everyone is thinking about the call of Cas4 family exonuclease vs. RecE-like exonuclease. According to the approved functions list, we call RecB-like exonuclease/helicase if we can confidently identify both a helicase and an exonuclease domain present in the protein. Cas4 family exonuclease is called if we see alignments to the crystal structure 3H4R_A and to the PD-(D/E)XK nuclease superfamily (PF12705.7, among others) according to the functions list. It's a little more unclear on calling the RecE-like exonuclease. Presumably we should be looking for alignments to RecE crystal structures. I've been using this as a guide for my own annotations. Using Che9c_60 as a model for RecE-like exonuclease (from approved list), HHPred reveals matches to 3H4R_A and PF12705.7. Che9c_61 is called as a RecT-like ssDNA binding protein. There are no alignments to anything labeled as RecE. My question is: should we be calling RecE-like exonuclease for a gene that has the exonuclease only domains and is immediately upstream of a RecT-like ssDNA binding protein (regardless of hits to 3H4E_A and PD-(D/E)XK nuclease superfamilies), and reserve Cas4 family exonuclease for genes that are not immediately upstream or very close to a RecT-like ssDNA binding protein? My immediate thoughts are to use the presence of RecT as a guide to call RecE-like exonuclease, but I want to hear what you all think.

Link to this post | posted 04 May, 2021 20:35

kbutela

Hi Beckie- The HHPred hits support your observations of an acetyltransferase domain linked via a series of transmembrane domains to the SGNH domains. I found an article (https://mbio.asm.org/content/11/4/e01364-20)"Acetylation of Surface Carbohydrates in Bacterial Pathogens Requires Coordinated Action of a Two-Domain Membrane-Bound Acyltransferase" describing AT3, and one class seems to fit what you described here: "Among bacterial AT3 carbohydrate acetyltransferases, there are two known domain architectures, proteins consisting of an AT3 domain only (AT3-only) and an N-terminal AT3 domain linked to an extracytoplasmic domain, commonly an SGNH domain (AT3-SGNH fused). The SGNH domain is fused through addition of an 11th TMH and linking region." There is a conserved motif (GG-F/Y-XGV-D/P/V) found in a diverse set of these proteins from Gram pos and neg bacteria within the AT3 domain. I downloaded the same sequences that contained both domains from the paper and repeated their T-coffee alignment (attached. LonelyBoi does contain this motif, but the first letter is A not G. Otherwise it is identical to the rest of the group. Other conserved residues based on OafA (WP_000639473.1) are S112, N113, and Y122. LonelyBoi does contain these conserved residues based on the alignment to OafA. In the SGNH domain, the following residues are conserved using OafA as the reference: G409, D 410, S411. LonelyBoi contains these residues in the same area in the alignment. Essential catalytic residues experimentally determined in OatA (WP_000379821) G502, T503, and N504 (not conserved across the entire group of sequences) are found in LonelyBoi at the corresponding alignment area with the exception of LonelyBoi having S corresponding to the G502 residue. I used Figure S3 as a reference to map out how LonelyBoi compares to other sequences in terms of catalytic residues, and it appears that LonelyBoi has all of the highly conserved residues but not on the less conserved residues. Based on this information, I propose a new function: Membrane bound acyltransferase-3 (AT3) domain-containing protein. This is a more generic family level call, but I don't think we have enough wet bench evidence to further narrow down the function as to what this particular enzyme is doing. A quick article search suggests that phages carry this gene to modify cell surface peptidoglycans and lipopolysaccharides. 25Kb

Link to this post | posted 14 Apr, 2021 16:47

kbutela

Gene 59 in Gordonia phage Madi (Cluster DG) has a nearly identical alignment (99.7% probability with 99% query coverage) in HHPred to the crystal structure of UDP-galactopyranose mutase from Mycobacterium smegmatis https://www.rcsb.org/structure/5ER9. UDP-galactopyranose mutase converts galactopyranose to galactofuranose, which is needed for cell wall biosynthesis in bacteria. Not sure why a phage would have it but cool to consider. https://pubmed.ncbi.nlm.nih.gov/24096172/. Metamorphoo gp87 is called in the official functions list as UDP-glucose dehydrogenase, which is involved in the biosynthetic pathway for hexuronic acid-containing bacterial surface glycostructures. Other phages in this cluster use the more generic term "oxioreductase." There are numerous hits for oxioreductases in the HHPred results, mostly from other proteins that match with probability greater than 90% but around 70-80% query coverage. Would we be able to make a more specific functional call for this gene? HHPred results are attached. 3.02Mb

Link to this post | posted 08 Feb, 2021 23:38

kbutela

We are hiring two full-time lab instructors (8-month) in the Department of Biological Sciences at the University of Pittsburgh with an anticipated start date of August 2021. Instructors will teach sections of Foundations of Biology lab (all research-based introductory labs including SEA-PHAGES phage discovery and bioinformatics, Duckweed Survivor, Flower Microbiome, Neural Defects, DNA Regulation and Disease), Introductory to Microbiology Lab, Physiology Lab, or some combination of these courses. Positions are outside of the tenure stream subject to annual review and there are three promotion levels at the instructor level (instructor I, instructor II, and Senior Instructor. Positions require an MS or PhD in biology. The link below has more information. Applicants can apply online at: http://join.pitt.edu/. https://cfopitt.taleo.net/careersection/pitt_faculty_external/jobdetail.ftl?job=21000771&tz=GMT-05%3A00&tzname=America%2FNew_York I am happy to answer any questions interested applicants may have! Applications will be reviewed starting March 1st. Edited 08 Feb, 2021 23:39

Link to this post | posted 04 Dec, 2020 17:55

kbutela

Hi all- I recently discovered that DNA Master (version 5.23.6 updated 8 Nov 2020) is adding in an extra column into the basic Genome Profile called "Arm." It shows up in my files as Column E, in between Strand and Feature_Start. If you are trying to run Starterator 1.2 on a Whole Unphamerated Phage for QC purposes and do not delete this "Arm" column from the .csv Genome Profile, running Starterator 1.2 on the SEA VM fails and dumps out a blank report. Deleting the "Arm" column from the Genome Profile will enable you to successfully run a Starterator 1.2 report on a Whole Unphamerated Phage. I do not see a checkbox option on DNA Master to remove the "Arm" column from the Genome Profile output, so you will have to manually edit the csv output file.

Link to this post | posted 16 Jul, 2020 18:59
kbutela	Some Cluster A phages (see Backyardigan gp25 for an example https://phagesdb.org/genes/Backyardigan_CDS_25/) have a large overlap for the start of the tape measure protein with the previous gene. The overlapping start site is conserved and includes all coding potential. Some older annotations call the shorter start, but per SMART the large overlap start is the best call.

Link to this post | posted 17 Jun, 2020 16:36

kbutela

Hi all! My team is hiring a few Foundations of Biology lab instructors (full time and part time) for this upcoming fall. We are anticipating some form of in-person instruction, although we are pushing very hard for an all online lab option for phage genome annotation. All of our courses are authentic-research based. The position requires at least a master's degree in a biology-related field and some teaching experience is preferred. I'm happy to answer any questions you may have! Please forward this to anyone who you think may be looking for a position. https://cfopitt.taleo.net/careersection/pitt_faculty_external/jobdetail.ftl?job=20002782&tz=GMT-04%3A00&tzname=America%2FNew_York

Link to this post | posted 30 May, 2019 15:53
kbutela	For now (per SMART discussion 5-30-19), Welkin recommends just calling this as "terminase" for the functional call. In the notes section, you can add "has intein or HNH domain"

Link to this post | posted 30 May, 2019 15:52
kbutela	For now (per SMART discussion 5-30-19), Welkin recommends just calling this as "terminase" for the functional call. In the notes section, you can add "has intein or HNH domain"

Link to this post | posted 09 May, 2019 19:11

kbutela

I am reviewing Verity and Zipp, two Cluster DE phages. Gp2 is rather long (2646 bp), and in both phages has strong HHPred matches to terminases on the C-terminal side of the protein and strong HHPred matches to a variety of HNH endonucleases and intein domains on the N-terminal side of the protein (see attached). On the approved functions list, there is an option for "terminase, large subunit (nuclease domain)" for Cluster AY genomes, but I don't think that's an appropriate match here because running the example of Auxillum gp3 in HHPred gives full length hits to various terminase, nuclease domains. Would it be appropriate to call this function as "terminase with intein domain?" 2.49Mb