Biology subjects

Pollenz, R. S.

Publications and source records attributed to Pollenz, R. S..

3 recordsLinked to original sources

bioRxiv2026

Genetic dissection of Mycobacteriophage D29 host lysis reveals two lysis regulators and a novel lipoprotein that regulate the lysis event and are localized to distinct regions of the genome

Phage D29 infects Mycobacterium smegmatis mc2 155 and has a non-canonical lysis cassette that encodes two endolysin proteins (Lysin A and Lysin B) and a single two transmembrane domain (TMD) protein, LysA2a similar to F1 cluster phage LysF1a. A 1TMD LysF1b homolog, LysA2b, is encoded by a gene found downstream of the tape measure. Exogenous expression of both LysA2 proteins in tandem is a cytotoxic to M. smegmatis. Deletion of lysA2a produces phages that are lysis competent with a 10-minute triggering delay and 30% plaque size reduction. Deletion of lysA2b results in severe lysis defects manifest by 70% reduced plaque size, delayed lysis timing and reduced burst size. Deletion of both lysA2 genes results in phages that are viable and show lysis phenotypes like the lysF1b deletion. Genetic complementation of lysA2b deleted phage with the lysF1b gene fully complements the lysis phenotypes but alters the triggering time to that of an F1 cluster phage. Energy poisons trigger lysis prematurely in all phages with lysA2 gene deletions. Lysis recovery mutants (LRM) isolated from phages lacking the lysA2b genes generate wild type plaque size and have point mutations that map to TMD1 or the C-terminal region of the lysA2a gene. LRMs isolated from phages lacking both lysA2 genes show premature lysis and have mutations that all map to residue C31 of a novel lipoprotein (gene 64). Deletion of gene 64 does not change wild type D29 lysis phenotypes or rescue the lysis defects of any of the lysA2 mutants. A fitness/competition assay shows that loss of the lysA2 genes imposes a substantial competitive fitness cost. These finding support a lysis regulatory network model where the 2TMD protein is maintained in an inactive state until activated by its cognate 1TMD lysis regulator and the lipoprotein has accessory function that may enhance lysis efficiency.

microbiology↗

bioRxiv2025

Genetic Analysis of F1 Cluster Phages that Infect Mycobacterium smegmatis Identifies Two Distinct Holin-Like Proteins that Regulate the Host Lysis Event

Phages Girr and NormanBulbieJr (NBJ) infect Gram-positive Mycobacterium smegmatis mc2 155. Both phages contain conserved lysis cassettes that harbor two endolysin genes (lysin A and lysin B) and two genes encoding transmembrane domain (TMD) holin-like proteins. The first holin-like protein, termed LysF1a is 88 amino acids, has two TMDs and a predicted N-in-C-in membrane topology. The second, termed LysF1b, has a single N-terminal TMD and a predicted N-out-C-in topology making it distinct from the type III holins or spanins in size and membrane topology. Deletion of lysF1b results in severe lysis defect phenotypes manifest by reduced plaque size and changes to lysis timing in liquid culture. Deletion of both lysF1a and lysF1b genes results in phages that show the same lysis phenotypes as the single lysF1b deletion. Phages with only lysF1b are lysis competent and trigger lysis prematurely when exposed to energy poisons while phages with lysF1a or lysF1a/lysF1b deletions do not trigger prematurely. Deletion of genes upstream of the lysis cassette did not impact lysis phenotypes. Lysis recovery mutants were isolated from phages lacking the lysF1b gene and these mutants generated wild type plaque size but triggered lysis prematurely and showed [~]65% reductions in burst size. Genome sequencing identified different point mutations that mapped to TMD1 or the C-terminal region of the lysF1a gene. Infection of an M. smegmatis strain that does not produce lipomannan and lipoarabinomannan by either wild type phages or phages carrying the lysF1b deletion showed modest plaque size increases but did not fully complement the lysis defect of phages lacking the lysF1b gene. Collectively, the findings show that both LysF1a and LysF1b proteins are required for efficient bacterial lysis by these F1 cluster phages. LysF1a does not function as a pure antiholin but requires the expression of the LysF1b protein for efficient lysis functioning.

microbiology↗

bioRxiv2023

A genome-wide cytotoxicity screen of Cluster F1 mycobacteriophage Girr reveals novel inhibitors of Mycobacterium smegmatis growth

Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and functions cannot be assigned for >65% of the encoded proteins. One approach to begin experimentally elucidating the function of these uncharacterized gene products is genome-wide screening to identify phage genes that confer phenotypes of interest like inhibition of host growth. This study describes the results of a screen evaluating the effects of overexpressing each gene encoded by the temperate Cluster F1 mycobacteriophage Girr on the growth of the host bacterium Mycobacterium smegmatis. Overexpression of 29 of the 102 Girr genes ([~]28% of the genome) resulted in mild to severe cytotoxicity. Of the 29 toxic genes described, 12 have no known function (NKF) and are predominately small proteins of <125 amino acids. Overexpression of the majority of these 12 cytotoxic NKF proteins resulted in moderate to severe growth reduction and represent novel antimicrobial products. The remaining 17 toxic genes have predicted functions, encoding products involved in phage structure, DNA replication/modification, DNA binding/gene regulation, or other enzymatic activity. Comparison of this dataset with prior genome-wide cytotoxicity screens of mycobacteriophages Waterfoul and Hammy reveals some common functional themes, though several of the predicted Girr functions associated with cytotoxicity in our report, including genes involved in lysogeny, have not been described previously. This study, completed as part of the HHMI-supported SEA-GENES project, highlights the power of parallel, genome-wide overexpression screens to identify novel interactions between phages and their hosts.

microbiology↗