Biology subjects

Heller, D. M.

Publications and source records attributed to Heller, D. M..

3 recordsLinked to original sources

bioRxiv2026

Identification of novel inhibitors of Mycobacterium smegmatis growth through genome-wide overexpression of Cluster P3 mycobacteriophage Xavia genes

We examined whether genes encoded by the mycobacteriophage Xavia disrupt growth of Mycobacterium smegmatis, a widely used mycobacterial model. Seventy-one Xavia genes were individually expressed using an inducible plasmid system and assessed for effects on colony formation. Two genes were lethal even without induction, indicating toxicity under basal expression. Induction of sixteen additional genes reduced bacterial growth, spanning structural proteins, lysogeny regulators, DNA-associated enzymes, a lysis protein, and several genes with no known function. These findings expand functional insights into mycobacteriophage gene repertoires and identify candidates for future mechanistic studies. AbstractBacteriophage genomes encode large numbers of genes with no known function, and many of these genes affect essential host processes when expressed in a heterologous system. For mycobacteriophages, genome-wide overexpression in Mycobacterium smegmatis provides a direct way to identify proteins that impair growth and to determine which mycobacterial pathways are sensitive to phage gene products. To evaluate the cytotoxic potential of the Cluster P3 phage Xavia, a lineage that has not undergone functional screening, we constructed an arrayed pExTra library containing 71 predicted Xavia genes under control of the anhydrotetracycline inducible promoter pTet. All constructs were sequence-verified and transformed into M. smegmatis, and induction allowed measurement of gene-specific effects on growth. Two genes prevented recovery of transformants, suggesting toxicity under basal promoter leakiness. Inducible expression of 16 additional genes impaired growth, and these inhibitory proteins include structural components, regulators of lysogeny, enzymes of DNA metabolism, a lysis factor, and several proteins with no known function. Four of the strongest inhibitors were genes with no known function. These results extend functional screening into the previously untested P3 branch of Actinobacteriophages and identify new proteins that require mechanistic analysis.

microbiology↗

bioRxiv2025

Functional Characterization of 123 Genes from Mycobacteriophage LeBron Uncovers Cytotoxic Gene Families in Cluster L1

Bacteriophages represent a vast reservoir of genetic diversity however, functional annotation remains a major challenge, as most predicted gene products lack detectable similarity to characterized gene families. Experimental approaches such as systematic overexpression screens provide an avenue for identifying phage genes that influence bacterial physiology. Here, we report an overexpression screen of all 123 predicted protein-coding genes from Cluster L1 mycobacteriophage LeBron, the first representative of this cluster to undergo genome-wide functional analysis. Expression assays in Mycobacterium smegmatis revealed that 39 genes (32%) impaired host growth, with nineteen of these toxic genes (49%) assigned no known function. The proportion of cytotoxic genes observed in LeBron is comparable to findings from Clusters K and F, despite minimal sequence conservation across clusters. Interestingly, a subset of LeBrons toxic genes appear to be functionally analogous to previously identified toxic genes in other clusters, suggesting conserved biological activities carried out by non-homologous proteins. Additionally, this analysis uncovered several novel gene families that elicit strong cytotoxic effects, expanding the known catalog of phage-derived bacterial growth inhibitors. These results provide new insights into phage gene functions and demonstrate the value of genome-wide expression screening for uncovering conserved and cluster-specific interactions between bacteriophages and their hosts.

microbiology↗

bioRxiv2024

Genome-wide screen overexpressing mycobacteriophage Amelie genes identifies multiple inhibitors of mycobacterial growth

The genome sequences of thousands of bacteriophages have been determined and functions for many of the encoded genes have been assigned based on homology to characterized sequences. However, functions have not been assigned to more than two-thirds of the identified phage genes as they have no recognizable sequence features. Recent genome-wide overexpression screens have begun to identify bacteriophage genes that encode proteins that reduce or inhibit bacterial growth. This study describes the construction of a plasmid-based overexpression library of 76 genes encoded by Cluster K1 mycobacteriophage Amelie, which is genetically similar to Cluster K phages Waterfoul and Hammy recently described in similar screens and closely related to phages that infect clinically important mycobacteria. 26 out of the 76 genes evaluated in our screen, encompassing 34% of the genome, reduced growth of the host bacterium Mycobacterium smegmatis to various degrees. More than one-third of these 26 toxic genes have no known function, and 10 of the 26 genes almost completely abolished host growth upon overexpression. Notably, while several of the toxic genes identified in Amelie shared homologs with other Cluster K phages recently screened, this study uncovered eight previously unknown gene families that exhibit cytotoxic properties, thereby broadening the repertoire of known phage-encoded growth inhibitors. This work, carried out under the HHMI-supported SEA-GENES project (Science Education Alliance Gene-function Exploration by a Network of Emerging Scientists), underscores the importance of comprehensive overexpression screens in elucidating genome-wide patterns of phage gene function and novel interactions between phages and their hosts.

microbiology↗