Biology subjects

Harden, M. M.

Publications and source records attributed to Harden, M. M..

3 recordsLinked to original sources

bioRxiv2021

A CRISPR interference screen reveals a role for cell wall teichoic acids in conjugation in Bacillus subtilis

Conjugative elements are widespread in bacteria and include plasmids and integrative and conjugative elements (ICEs). They transfer from donor to recipient cells via an element-encoded type IV secretion system. These elements interact with and utilize host functions for their lifecycles. We sought to identify essential host genes involved in the lifecycle of the integrative and conjugative element ICEBs1 of Bacillus subtilis. We constructed a library of strains for inducible knockdown of essential B. subtilis genes using CRISPR interference. Each strain expressed one guide RNA in ICEBs1. We induced partial interference of essential genes and identified those that caused an acute defect in acquisition of ICEBs1 by recipient cells. This screen revealed that reducing expression of genes needed for synthesis of cell wall teichoic acids caused a decrease in conjugation. Using three different ways to reduce their synthesis, we found that wall teichoic acids were necessary in both donors and recipients for efficient conjugative transfer of ICEBs1. Further, we found that depletion of wall teichoic acids caused cells involved in ICEBs1 conjugation to die, most likely from damage to the cell envelope. Our results indicate that wall teichoic acids help protect against envelope stress caused by an active conjugation machinery.

microbiology↗

bioRxiv2020

An integrative and conjugative element encodes an abortive infection system to protect host cells from predation by a bacteriophage

Bacteriophages and integrative and conjugative elements (ICEs, a.k.a. conjugative transposons) are mobile genetic elements that can move from one bacterial cell to another and often coexist in a host genome. Many bacterial species contain at least one temperate (lysogenic) phage and an integrative and conjugative element. Most strains of Bacillus subtilis are lysogenic for the phage SP{beta} and also contain the integrative and conjugative element ICEBs1. We found that the presence of ICEBs1 in cells inhibited production of SP{beta}, during both activation of a lysogen and following de novo infection. The ICEBs1 gene yddK (renamed spbK for SP{beta} killing) was both necessary and sufficient for the anti-SP{beta} activity. Co-expression of spbK and yonE, in the absence of other ICEBs1 and SP{beta} genes, resulted in inhibition of cell growth and loss of cell viability. These results indicate that together spbK and yonE affected a host process needed for normal growth and that spbK constitutes an abortive infection system. We found that this anti-SP{beta} phenotype protected populations of B. subtilis from predation by SP{beta}, likely providing selective pressure for the maintenance of ICEBs1 in B. subtilis populations. spbK encodes a TIR (Toll-interleukin-1 receptor)-domain protein with similarity to both plant antiviral proteins and animal innate immune signaling proteins. We postulate that selective many uncharacterized cargo genes in ICEs may confer selective advantage to cells by protecting against other mobile elements.

microbiology↗

bioRxiv2018

Mobile-CRISPRi: Enabling Genetic Analysis of Diverse Bacteria

Introductory paragraphThe vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. CRISPRi, a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs (sgRNAs), has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to human. However, the difficulty of establishing effective CRISPRi systems in non-model bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish \"Mobile-CRISPRi\", a suite of CRISPRi systems that combine modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in Proteobacteria and Firmicutes at the individual gene scale by examining drug-gene synergies and at the library scale by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microbe interactions.

microbiology↗