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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 (SGAs), 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 Mycobacterium smegmatis 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 esx3, fxbA, and recA 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 noncoding RNA (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.IMPORTANCE Mycobacterium smegmatis is the model organism of choice for the study of mycobacterial pathogens, because it is a fast-growing nonpathogenic species harboring many genes that are conserved throughout mycobacteria. In this work, we describe a synthetic genetic array (mSGA) approach for M. smegmatis, which combines mutations on a genome-wide scale with high efficiency. Analysis of the double mutant strains enables the identification of interacting genes and pathways that are normally hidden by redundant biological pathways. The mSGA is a powerful genetic tool that enables functions to be assigned to the many conserved hypothetical genes found in all mycobacterial species.

SUBMITTER: Judd J

PROVIDER: S-EPMC5637176 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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

Judd Julius J Boucher Nathalie N Van Roey Erik E Gray Todd A TA Derbyshire Keith M KM

Journal of bacteriology 20170919 20

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 (SGAs), 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 intera ...[more]

PMID: 28784812

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Dataset Information

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

Publications

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

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