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      Systematic overexpression of genes encoded by mycobacteriophage Waterfoul reveals novel inhibitors of mycobacterial growth

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          Abstract

          Bacteriophages represent an enormous reservoir of novel genes, many of which are unrelated to existing entries in public databases and cannot be assigned a predicted function. Characterization of these genes can provide important insights into the intricacies of phage–host interactions and may offer new strategies to manipulate bacterial growth and behavior. Overexpression is a useful tool in the study of gene-mediated effects, and we describe here the construction of a plasmid-based overexpression library of a complete set of genes for Waterfoul, a mycobacteriophage closely related to those infecting clinically important strains of Mycobacterium tuberculosis and/or Mycobacterium abscessus. The arrayed Waterfoul gene library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 32 Waterfoul gene products capable of inhibiting the growth of the host Mycobacterium smegmatis and providing a first look at the frequency and distribution of cytotoxic products encoded within a single mycobacteriophage genome. Several of these Waterfoul gene products were observed to confer potent anti-mycobacterial effects, making them interesting candidates for follow-up mechanistic studies.

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          Most cited references52

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          Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.

          We describe and validate a new membrane protein topology prediction method, TMHMM, based on a hidden Markov model. We present a detailed analysis of TMHMM's performance, and show that it correctly predicts 97-98 % of the transmembrane helices. Additionally, TMHMM can discriminate between soluble and membrane proteins with both specificity and sensitivity better than 99 %, although the accuracy drops when signal peptides are present. This high degree of accuracy allowed us to predict reliably integral membrane proteins in a large collection of genomes. Based on these predictions, we estimate that 20-30 % of all genes in most genomes encode membrane proteins, which is in agreement with previous estimates. We further discovered that proteins with N(in)-C(in) topologies are strongly preferred in all examined organisms, except Caenorhabditis elegans, where the large number of 7TM receptors increases the counts for N(out)-C(in) topologies. We discuss the possible relevance of this finding for our understanding of membrane protein assembly mechanisms. A TMHMM prediction service is available at http://www.cbs.dtu.dk/services/TMHMM/. Copyright 2001 Academic Press.
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            CDD/SPARCLE: the conserved domain database in 2020

            As NLM’s Conserved Domain Database (CDD) enters its 20th year of operations as a publicly available resource, CDD curation staff continues to develop hierarchical classifications of widely distributed protein domain families, and to record conserved sites associated with molecular function, so that they can be mapped onto user queries in support of hypothesis-driven biomolecular research. CDD offers both an archive of pre-computed domain annotations as well as live search services for both single protein or nucleotide queries and larger sets of protein query sequences. CDD staff has continued to characterize protein families via conserved domain architectures and has built up a significant corpus of curated domain architectures in support of naming bacterial proteins in RefSeq. These architecture definitions are available via SPARCLE, the Subfamily Protein Architecture Labeling Engine. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.
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              Engineered bacteriophages for treatment of a patient with a disseminated drug resistant Mycobacterium abscessus

              A 15-year-old cystic fibrosis patient with a disseminated Mycobacterium abscessus infection was treated with a three-phage cocktail following bilateral lung transplantation. Effective lytic phage derivatives that efficiently kill the infectious M. abscessus strain were developed by genome engineering and forward genetics. Intravenous phage treatment was well tolerated and associated with objective clinical improvement including sternal wound closure, improved liver function, and substantial resolution of infected skin nodules.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                G3 (Bethesda)
                Genetics
                g3journal
                G3: Genes|Genomes|Genetics
                Oxford University Press
                2160-1836
                August 2022
                21 June 2022
                21 June 2022
                : 12
                : 8
                : jkac140
                Affiliations
                Department of Science Education, Howard Hughes Medical Institute , Chevy Chase, MD 20185, USA
                Department of Science Education, Howard Hughes Medical Institute , Chevy Chase, MD 20185, USA
                Department of Biology, University of Maryland Baltimore County , Baltimore, MD 21250, USA
                Department of Biology, University of Maryland Baltimore County , Baltimore, MD 21250, USA
                Center for Molecular & Cellular Biosciences, University of Southern Mississippi , Hattiesburg, MS 39406, USA
                Department of Biology, Emmanuel College , Boston, MA 02115, USA
                Department of Science Education, Howard Hughes Medical Institute , Chevy Chase, MD 20185, USA
                Author notes
                Corresponding author: Department of Science Education, Howard Hughes Medical Institute, Chevy Chase, MD 20185, USA. Email: sivanathanv@ 123456hhmi.org
                [†]

                Danielle Heller and Isabel Amaya contributed equally to this work.

                Author information
                https://orcid.org/0000-0002-6581-1256
                https://orcid.org/0000-0002-6169-9711
                https://orcid.org/0000-0002-9644-2094
                https://orcid.org/0000-0002-2857-1206
                https://orcid.org/0000-0001-7163-4543
                Article
                jkac140
                10.1093/g3journal/jkac140
                9339283
                35727726
                dd11842d-4bf2-4e42-8a7b-e25b1b425187
                © The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 07 February 2022
                : 14 April 2022
                Page count
                Pages: 8
                Funding
                Funded by: HHMI-supported Science Education Alliance GENES (Gene function Exploration by a Network of Emerging Scientists);
                Categories
                Mutant Screen Report
                AcademicSubjects/SCI01180
                AcademicSubjects/SCI01140
                AcademicSubjects/SCI00010
                AcademicSubjects/SCI00960

                Genetics
                mycobacteriophage,mycobacterium smegmatis,cytotoxicity
                Genetics
                mycobacteriophage, mycobacterium smegmatis, cytotoxicity

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