Application of Distributive Conjugal DNA Transfer in Mycobacterium smegmatis To Establish a Genome-Wide Synthetic Genetic Array

Abstract

Genetic redundancy can obscure phenotypic effects of single-gene mutations. Two individual mutations may be viable separately but are lethal when combined, thus synthetically linking the two gene products in an essential process. Synthetic genetic arrays (SGA), in which defined mutations are combined, provide a powerful approach to identify novel genetic interactions and redundant pathways. A genome-scale SGA can offer an initial assignment of function to hypothetical genes by uncovering interactions with known genes or pathways. Here, we take advantage of the chromosomal conjugation system of to combine individual donor and recipient mutations on a genome-wide scale. We demonstrated the feasibility of a high-throughput mycobacterial SGA (mSGA) screen by using mutants of and as query genes, which were combined with an arrayed library of transposon mutants by conjugation. The mSGA identified interacting genes that we had predicted and, most importantly, identified novel interacting genes - encoding both proteins and a ncRNA. In combination with other molecular genetic approaches, the mSGA has great potential to both reduce the high number of conserved hypothetical protein annotations in mycobacterial genomes and further define mycobacterial pathways and gene interactions. is the model organism of choice for the study of mycobacterial pathogens because it is a fast growing, non-pathogenic species encoding many genes that are conserved throughout mycobacteria. In this work, we describe a synthetic genetic array (mSGA) approach for , which combines mutations on a genome-wide scale with high efficiency. Analysis of the double-mutant strains allows identification of interacting genes and pathways that are normally hidden by redundant biological pathways. The mSGA is a powerful genetic tool that allows functions to be assigned to the many conserved hypothetical genes found in all mycobacterial species.

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“…In addition, there is significant homology at the amino acid level for 60% of M. tb. genes that have M. smegmatis orthologs [111] . Nonetheless, M. tb.…”