Biology subjects

Osadnik, H.

Publications and source records attributed to Osadnik, H..

3 recordsLinked to original sources

bioRxiv2020

Rewiring the specificity of extra-cytoplasmic function sigma factors

SUMMARYBacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators’ DNA binding and precise determination of biologically significant binding sites. Here, we address these challenges by solving the DNA-specificity code of extra-cytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the auto-regulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid – nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic foot-printing pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that ECF σs play varied roles: some are global regulators controlling many genes throughout the genome that are important under many conditions, while others are local regulators, controlling few closely linked genes in response to specific stimuli. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2020

Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs

The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated the inner workings of RNAP, and mutations in genes encoding RNAP drive adaptation of Escherichia coli to many health- and industry-relevant environments, yet a paucity of systematic analyses has hampered our understanding of the fitness benefits and trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and illuminate a connection between transcription and cell division. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

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↗