A Targeted Genome-scale Overexpression Platform for Proteobacteria.

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 overexpression face key limitations. Here, we develop a facile approach for genome-scale gene overexpression in bacteria we call, "CRISPRtOE" (CRISPR transposition and OverExpression). We create a platform for comprehensive gene targeting using CRISPR-associated transposition (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 Escherichia coli, recovering known antibiotic targets and genes with unexplored roles in antibiotic function. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria.

Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in Acinetobacter baumannii.

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 genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome.

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium Zymomonas mobilis has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rhodobacter nitrogen fixation (Rnf) respiratory complex are required for the anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts. IMPORTANCE The inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the alphaproteobacterium Zymomonas mobilis, have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use of Z. mobilis to create biofuels and bioproducts.

Tools for Genetic Engineering and Gene Expression Control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides.

Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts have the potential to form the backbone of the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans, Rhodobacter sphaeroides, and Zymomonas mobilis, show particular promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis, N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides. We screen a synthetic promoter library to identify inducible promoters with strong, regulated activity in both organisms. Combining Tn7 integration with promoters from our library, we establish CRISPR interference systems for N. aromaticivorans and R. sphaeroides that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these industrially important species and other Alphaproteobacteria.

Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in Acinetobacter baumannii.

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 (Tn-seq), 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 underpin 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.

Programmable Gene Knockdown in Diverse Bacteria Using Mobile-CRISPRi.

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.

A High-Efficacy CRISPR Interference System for Gene Function Discovery in Zymomonas mobilis.

Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes-including essential genes. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, are key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.IMPORTANCE Biofuels produced by microbial fermentation of plant feedstocks provide renewable and sustainable energy sources that have the potential to mitigate climate change and improve energy security. Engineered strains of the bacterium Z. mobilis can convert sugars extracted from plant feedstocks into next-generation biofuels like isobutanol; however, conversion by these strains remains inefficient due to key gaps in our knowledge about genes involved in metabolism and stress responses such as alcohol tolerance. Here, we develop CRISPRi as a tool to explore gene function in Z. mobilis We characterize genes that are essential for growth, required to ferment sugar to ethanol, and involved in resistance to isobutanol. Our Z. mobilis CRISPRi system makes it straightforward to define gene function and can be applied to improve strain engineering and increase biofuel yields.

Structural basis for transcription activation by Crl through tethering of σS and RNA polymerase.

In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti-σ-factors. In Escherichia coli, Salmonella enterica, and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σS-regulon by promoting the association of σS with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σS-RNAP in an open promoter complex with a σS-regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σS (σS2), the structure, along with p-benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP ß'-subunit that we call the ß'-clamp-toe (ß'CT). Deletion of the ß'CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-ß'CT interaction. We conclude that Crl activates σS-dependent transcription in part through stabilizing σS-RNAP by tethering σS2 and the ß'CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.

C-4 sterol demethylation enzymes distinguish bacterial and eukaryotic sterol synthesis.

Sterols are essential eukaryotic lipids that are required for a variety of physiological roles. The diagenetic products of sterol lipids, sterane hydrocarbons, are preserved in ancient sedimentary rocks and are utilized as geological biomarkers, indicating the presence of both eukaryotes and oxic environments throughout Earth's history. However, a few bacterial species are also known to produce sterols, bringing into question the significance of bacterial sterol synthesis for our interpretation of sterane biomarkers. Recent studies suggest that bacterial sterol synthesis may be distinct from what is observed in eukaryotes. In particular, phylogenomic analyses of sterol-producing bacteria have failed to identify homologs of several key eukaryotic sterol synthesis enzymes, most notably those required for demethylation at the C-4 position. In this study, we identified two genes of previously unknown function in the aerobic methanotrophic γ-Proteobacterium Methylococcus capsulatus that encode sterol demethylase proteins (Sdm). We show that a Rieske-type oxygenase (SdmA) and an NAD(P)-dependent reductase (SdmB) are responsible for converting 4,4-dimethylsterols to 4α-methylsterols. Identification of intermediate products synthesized during heterologous expression of SdmA-SdmB along with 13C-labeling studies support a sterol C-4 demethylation mechanism distinct from that of eukaryotes. SdmA-SdmB homologs were identified in several other sterol-producing bacterial genomes but not in any eukaryotic genomes, indicating that these proteins are unrelated to the eukaryotic C-4 sterol demethylase enzymes. These findings reveal a separate pathway for sterol synthesis exclusive to bacteria and show that demethylation of sterols evolved at least twice-once in bacteria and once in eukaryotes.

Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase.

Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

A distinct pathway for tetrahymanol synthesis in bacteria.

Tetrahymanol is a polycyclic triterpenoid lipid first discovered in the ciliate Tetrahymena pyriformis whose potential diagenetic product, gammacerane, is often used as a biomarker for water column stratification in ancient ecosystems. Bacteria are also a potential source of tetrahymanol, but neither the distribution of this lipid in extant bacteria nor the significance of bacterial tetrahymanol synthesis for interpreting gammacerane biosignatures is known. Here we couple comparative genomics with genetic and lipid analyses to link a protein of unknown function to tetrahymanol synthesis in bacteria. This tetrahymanol synthase (Ths) is found in a variety of bacterial genomes, including aerobic methanotrophs, nitrite-oxidizers, and sulfate-reducers, and in a subset of aquatic and terrestrial metagenomes. Thus, the potential to produce tetrahymanol is more widespread in the bacterial domain than previously thought. However, Ths is not encoded in any eukaryotic genomes, nor is it homologous to eukaryotic squalene-tetrahymanol cyclase, which catalyzes the cyclization of squalene directly to tetrahymanol. Rather, heterologous expression studies suggest that bacteria couple the cyclization of squalene to a hopene molecule by squalene-hopene cyclase with a subsequent Ths-dependent ring expansion to form tetrahymanol. Thus, bacteria and eukaryotes have evolved distinct biochemical mechanisms for producing tetrahymanol.

Structure of the RNA polymerase assembly factor Crl and identification of its interaction surface with sigma S.

Bacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica, Crl positively regulates the σ(S) regulon by binding to σ(S) to promote its association with core RNAP. We recently characterized the determinants in σ(S) responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-σ(S) interaction in E. coli. We show that the P. mirabilis, Vibrio harveyi, and E. coli Crl homologs function similarly in E. coli, indicating that Crl structure and function are likely conserved throughout gammaproteobacteria. We utilize phylogenetic conservation and bacterial two-hybrid analyses to predict residues in Crl important for the interaction with σ(S). The results of p-benzoylphenylalanine (BPA)-mediated UV cross-linking studies further support the model in which an evolutionarily conserved central cleft is the surface on Crl that binds to σ(S). Within this conserved binding surface, we identify a key residue in Crl that is critical for activation of Eσ(S)-dependent transcription in vivo and in vitro. Our study provides a physical basis for understanding the σ(S)-Crl interaction.

Spatial patterns of Aquificales in deep-sea vents along the Eastern Lau Spreading Center (SW Pacific).

The microbial diversity associated with actively venting deep-sea hydrothermal deposits is tightly connected to the geochemistry of the hydrothermal fluids. Although the dominant members of these deposits drive the structure of the microbial communities, it is less well understood whether the lower abundance groups are as closely connected to the geochemical milieu, or driven perhaps by biotic factors such as microbial community interactions. We used the natural geochemical gradients that exist in the back-arc basin, Eastern Lau Spreading Center and Valu-Fa Ridge (ELSC/VFR) in the Southwestern Pacific, to explore whether the chemolithotrophic Aquificales are influenced by geographical location, host-rock of the vent field or deposit type. Using a combination of cloning, DNA fingerprinting (DGGE) and enrichment culturing approaches, all genera of this order previously described at marine vents were detected, i.e., Desulfurobacterium, Thermovibrio, Aquifex, Hydrogenivirga, Persephonella and Hydrogenothermus. The comparison between clone libraries and DGGE showed similar patterns of distribution of different Aquificales whereas results differed for the enrichment cultures that were retrieved. However, the use of cultivation-based and -independent methods did provide complementary phylogenetic diversity overview of the Aquificales in these systems. Together, this survey revealed that the ELSC/VFR contains some of the largest diversity of Aquificales ever reported at a deep-sea vent area, that the diversity patterns are tied to the geography and geochemistry of the system, and that this geochemical diverse back-arc basin may harbor new members of the Aquificales.

Key features of σS required for specific recognition by Crl, a transcription factor promoting assembly of RNA polymerase holoenzyme.

Bacteria use multiple sigma factors to coordinate gene expression in response to environmental perturbations. In Escherichia coli and other γ-proteobacteria, the transcription factor Crl stimulates σ(S)-dependent transcription during times of cellular stress by promoting the association of σ(S) with core RNA polymerase. The molecular basis for specific recognition of σ(S) by Crl, rather than the homologous and more abundant primary sigma factor σ(70), is unknown. Here we use bacterial two-hybrid analysis in vivo and p-benzoyl-phenylalanine cross-linking in vitro to define the features in σ(S) responsible for specific recognition by Crl. We identify residues in σ(S) conserved domain 2 (σ(S)2) that are necessary and sufficient to allow recognition of σ(70) conserved domain 2 by Crl, one near the promoter-melting region and the other at the position where a large nonconserved region interrupts the sequence of σ(70). We then use luminescence resonance energy transfer to demonstrate directly that Crl promotes holoenzyme assembly using these specificity determinants on σ(S). Our results explain how Crl distinguishes between sigma factors that are largely homologous and activates discrete sets of promoters even though it does not bind to promoter DNA.

ppGpp and DksA likely regulate the activity of the extracytoplasmic stress factor sigmaE in Escherichia coli by both direct and indirect mechanisms.

One of the major signalling pathways responsible for intercompartmental communication between the cell envelope and cytoplasm in Escherichia coli is mediated by the alternative sigma factor, sigmaE. sigmaE has been studied primarily for its role in response to the misfolding of outer membrane porins. This response is essentially reactionary; cells are stressed, porin folding is disrupted, and the response is activated. sigmaE can also be activated following starvation for a variety of nutrients by the alarmone ppGpp. This response is proactive, as sigmaE is activated in the absence of any obvious damage to the cell envelope sensed by the stress signalling pathway. Here we examine the mechanism of regulation of sigmaE by ppGpp. ppGpp has been proposed to activate at least two alternative sigma factors, sigmaN and sigmaS, indirectly by altering the competition for core RNA polymerase between the alternative sigma factors and the housekeeping sigma factor, sigma70. In vivo experiments with sigmaE are consistent with this model. However, ppGpp and its cofactor DksA can also activate transcription by EsigmaEin vitro, suggesting that the effects of ppGpp on sigmaE activity are both direct and indirect.

A ubiquitous thermoacidophilic archaeon from deep-sea hydrothermal vents.

Deep-sea hydrothermal vents are important in global biogeochemical cycles, providing biological oases at the sea floor that are supported by the thermal and chemical flux from the Earth's interior. As hot, acidic and reduced hydrothermal fluids mix with cold, alkaline and oxygenated sea water, minerals precipitate to form porous sulphide-sulphate deposits. These structures provide microhabitats for a diversity of prokaryotes that exploit the geochemical and physical gradients in this dynamic ecosystem. It has been proposed that fluid pH in the actively venting sulphide structures is generally low (pH < 4.5), yet no extreme thermoacidophile has been isolated from vent deposits. Culture-independent surveys based on ribosomal RNA genes from deep-sea hydrothermal deposits have identified a widespread euryarchaeotal lineage, DHVE2 (deep-sea hydrothermal vent euryarchaeotic 2). Despite the ubiquity and apparent deep-sea endemism of DHVE2, cultivation of this group has been unsuccessful and thus its metabolism remains a mystery. Here we report the isolation and cultivation of a member of the DHVE2 group, which is an obligate thermoacidophilic sulphur- or iron-reducing heterotroph capable of growing from pH 3.3 to 5.8 and between 55 and 75 degrees C. In addition, we demonstrate that this isolate constitutes up to 15% of the archaeal population, providing evidence that thermoacidophiles may be key players in the sulphur and iron cycling at deep-sea vents.