Essential Gene Analysis in Acinetobacter baumannii by High-Density Transposon Mutagenesis and CRISPR Interference
| Autor: | Jinna Bai, Amy Y. Tang, Yunfei Dai, Andrew Farinha, Tim van Opijnen, Ralph R. Isberg, Germán Vargas-Cuebas, Edward Geisinger, Sapna Syal |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Acinetobacter baumannii
Transposable element Mutagenesis (molecular biology technique) Computational biology Microbiology 03 medical and health sciences Bacterial Proteins Gene silencing CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Molecular Biology Bacterial Capsules 030304 developmental biology 0303 health sciences CRISPR interference biology 030306 microbiology Gene Expression Regulation Bacterial biology.organism_classification Mutagenesis Essential gene Gene Knockdown Techniques DNA Transposable Elements Transposon mutagenesis Research Article |
| Zdroj: | J Bacteriol |
| ISSN: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.00565-20 |
| Popis: | Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Development of rationally designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii, and we developed a CRISPR interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii. IMPORTANCE New approaches are urgently needed to control A. baumannii, one of the most drug-resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|