The Gram-negative pathogen Acinetobacter baumannii is considered an "urgent threat" to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used by A. baumannii to mitigate cellular stresses may uncover new therapeutic targets. Many γ-proteobacteria use the extracytoplasmic function (ECF) σ factor, RpoE, to invoke envelope homeostasis networks in response to stress. Acinetobacter species contain the poorly characterized ECF "SigAb"; however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique to Acinetobacter species and distinct from RpoE-like ECFs. We combine promoter mutagenesis, motif scanning, and chromatin immunoprecipitation-sequencing (ChIP-seq) to define the direct SigAb regulon, which consists of genes encoding SigAb itself, the stringent response mediator, RelA, and the uncharacterized small RNA, "SabS." However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown of sigAb or sabS resulted in increased copper sensitivity and excess copper-induced SigAb-dependent transcription. Furthermore, we found that two uncharacterized genes in the sigAb operon, "aabA" and "aabB," have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show that sigAb, aabA, and aabB are important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture of A. baumannii. IMPORTANCE:Acinetobacter baumannii is a hospital-acquired pathogen, and many strains are resistant to multiple antibiotics. Understanding how A. baumannii senses and responds to stress may uncover novel routes to treat infections. Here, we examine how the Acinetobacter-specific transcription factor, SigAb, mitigates stress. We find that SigAb directly regulates only a small number of genes, but indirectly controls hundreds of genes that have substantial impacts on cell physiology. We show that SigAb is required for maximal growth, even during optimal conditions, and is acutely required during growth in the presence of elevated copper. Given that copper toxicity plays roles in pathogenesis and on copper-containing surfaces in hospitals, we speculate that SigAb function may be important in clinically relevant contexts.

Targeted, genome-scale gene perturbation screens using Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) and activation (CRISPRa) have revolutionized eukaryotic genetics, advancing medical, industrial, and basic research. Although CRISPRi knockdowns have been broadly applied in bacteria, options for genome-scale gene overexpression face key limitations. Here, we develop a facile approach for genome-scale overexpression in bacteria we call, "CRISPRtOE" (CRISPR transposition and OverExpression). We first create a platform for comprehensive gene targeting using CRISPR-associated transposons (CAST) and show that transposition occurs at a higher frequency in non-transcribed DNA. We then demonstrate that CRISPRtOE can upregulate gene expression in Proteobacteria with medical and industrial relevance by integrating synthetic promoters of varying strength upstream of target genes. Finally, we employ CRISPRtOE screening at the genome-scale in the model bacterium Escherichia coli and the non-model biofuel producer Zymomonas mobilis, recovering known and novel antibiotic and engineering targets. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria.

Gene expression systems that transcend species barriers are needed for cross-species analysis of gene function. In particular, expression systems that can be utilized in both model and pathogenic bacteria underpin comparative functional approaches that inform conserved and variable features of bacterial physiology. In this study, we develop replicative and integrative vectors alongside a novel, IPTG-inducible promoter that can be used in the model bacterium Escherichia coli K-12 as well as strains of the antibiotic-resistant pathogen, Acinetobacter baumannii. We generate modular vectors that transfer by conjugation at high efficiency and either replicate or integrate into the genome, depending on design. Embedded in these vectors, we also developed a synthetic, IPTG-inducible promoter, PabstBR, that induces to a high level but is less leaky than the commonly used trc promoter. We show that PabstBR is titratable at both the population and single-cell levels, regardless of species, highlighting the utility of our expression systems for cross-species functional studies. Finally, as a proof of principle, we use our integrating vector to develop a reporter for the E. coli envelope stress σ factor, RpoE, and deploy the reporter in E. coli and A. baumannii, finding that A. baumannii does not recognize RpoE-dependent promoters unless RpoE is heterologously expressed. We envision that these vector and promoter tools will be valuable for the community of researchers who study the fundamental biology of E. coli and A. baumannii.IMPORTANCEAcinetobacter baumannii is a multidrug-resistant, hospital-acquired pathogen with the ability to cause severe infections. Understanding the unique biology of this non-model bacterium may lead to the discovery of new weaknesses that can be targeted to treat antibiotic-resistant infections. In this study, we provide expression tools that can be used to study the gene function in A. baumannii, including in drug-resistant clinical isolates. These tools are also compatible with the model bacterium, Escherichia coli, enabling cross-species comparisons of gene function. We anticipate that the use of these tools by the scientific community will accelerate our understanding of Acinetobacter biology.

The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen, Acinetobacter baumannii, is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities. Although A. baumannii essential genes have been identified by transposon sequencing, they have not been prioritized by sensitivity to knockdown or antibiotics. Here, we take a systems biology approach to comprehensively characterize A. baumannii essential genes using CRISPR interference (CRISPRi). We show that certain essential genes and pathways are acutely sensitive to knockdown, providing a set of vulnerable targets for future therapeutic investigation. Screening our CRISPRi library against last-resort antibiotics uncovered genes and pathways that modulate beta-lactam sensitivity, an unexpected link between NADH dehydrogenase activity and growth inhibition by polymyxins, and anticorrelated phenotypes that may explain synergy between polymyxins and rifamycins. Our study demonstrates the power of systematic genetic approaches to identify vulnerabilities in Gram-negative pathogens and uncovers antibiotic-essential gene interactions that better inform combination therapies.IMPORTANCEAcinetobacter baumannii is a hospital-acquired pathogen that is resistant to many common antibiotic treatments. To combat resistant A. baumannii infections, we need to identify promising therapeutic targets and effective antibiotic combinations. In this study, we comprehensively characterize the genes and pathways that are critical for A. baumannii viability. We show that genes involved in aerobic metabolism are central to A. baumannii physiology and may represent appealing drug targets. We also find antibiotic-gene interactions that may impact the efficacy of carbapenems, rifamycins, and polymyxins, providing a new window into how these antibiotics function in mono- and combination therapies. Our studies offer a useful approach for characterizing interactions between drugs and essential genes in pathogens to inform future therapies.

The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities in relevant pathogens. The Gram-negative pathogen, Acinetobacter baumannii , poses an urgent threat by evading antibiotic treatment through both intrinsic and acquired mechanisms. Antibiotics kill bacteria by targeting essential gene products, but antibiotic-essential gene interactions have not been studied systematically in A. baumannii . Here, we use CRISPR interference (CRISPRi) to comprehensively phenotype A. baumannii essential genes. We show that certain essential genes are acutely sensitive to knockdown, providing a set of promising therapeutic targets. Screening our CRISPRi library against last-resort antibiotics revealed essential pathways that modulate beta-lactam resistance, an unexpected link between NADH dehydrogenase function and polymyxin killing, and the genetic basis for synergy between polymyxins and rifamycins. Our results demonstrate the power of systematic genetic approaches to identify weaknesses in Gram-negative pathogens and uncover antibiotic mechanisms that better inform combination therapies. Significance Acinetobacter baumannii is a hospital-acquired pathogen that is resistant to common antibiotic treatments. A. baumannii infections, we need to identify promising therapeutic targets and effective antibiotic combinations. Here, we characterize genes critical for A. baumannii viability and their interactions with antibiotics. We find that genes involved in proton gradient formation required for oxygen-dependent energy generation are central to A. baumannii physiology and represent appealing drug targets. We show that polymyxins interact with proton gradient genes, explaining how these antibiotics inhibit growth at sub-lethal concentrations and their efficacy in combination therapies. Our studies reveal antibiotic-gene interactions in A. baumannii that can inform future therapies.

Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene-knockdown tool that uses an RNA-protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile-CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile-CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile-CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile-CRISPRi vectors, Tn7 transfer of Mobile-CRISPRi to Gram-negative bacteria, and ICEBs1 transfer of Mobile-CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning of new sgRNA spacers into Mobile-CRISPRi vectors Basic Protocol 3: Tn7 transfer of Mobile-CRISPRi to Gram-negative bacteria Basic Protocol 4: ICEBs1 transfer of Mobile-CRISPRi to Bacillales Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3: Transformation of E. coli by electroporation Support Protocol 4: Transformation of CaCl2 -competent E. coli.

The causative agent of Lyme disease, Borrelia burgdorferi, requires shifts in gene expression to undergo its natural enzootic cycle between tick and vertebrate hosts. mRNA decay mechanisms play significant roles in governing gene expression in other bacteria, but are not yet characterized in B. burgdorferi. RNase III is an important enzyme in processing ribosomal RNA, but it also plays a role in mRNA decay in many bacteria. We compared RNA decay profiles and steady-state abundances of transcripts in wild-type Borrelia burgdorferi strain B31 and in an RNase III null (rnc(-)) mutant. Transcripts encoding RNA poly-merase subunits (rpoA and rpoS), ribosomal proteins (rpsD, rpsK, rpsM, rplQ, and rpsO), a nuclease (pnp), a flagellar protein (flaB), and a translational regulator (bpuR) decayed more rapidly in the wild-type strain than in the slow growing mt .- mutant indicating that RNA turnover is mediated by RNase III in the bacterium that causes Lyme disease. Additionally, in wild type bacteria, RNA decay rates of rpoS, rpoN, ospA, ospC, bpuR and dbpA transcripts are only modestly affected by changes in the osmolarity. (C) 2020 Elsevier Inc. All rights reserved.