Lipopolysaccharide transport regulates bacterial sensitivity to a cell wall-degrading intermicrobial toxin.

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the functional basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli (Eco) genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, lipopolysaccharide, that modulate Tae1 toxicity in vivo. Disruption of genes in early lipopolysaccharide biosynthesis provided Eco with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study reveals the complex functional underpinnings of susceptibility to Tae1 and T6SS which regulate the impact of toxin-substrate interactions in vivo.

Three Bacterial DedA Subfamilies with Distinct Functions and Phylogenetic Distribution.

Recent studies in bacteria have suggested that the broadly conserved but enigmatic DedA proteins function as undecaprenyl-phosphate (UndP) flippases, recycling this essential lipid carrier. To determine whether all DedA proteins have UndP flippase activity, we performed a phylogenetic analysis and correlated our findings to previously published experimental results and predicted structures. We uncovered three major DedA subfamilies: one contains UndP flippases, the second contains putative phospholipid flippases and is associated with aerobic metabolism, and the third is found only in specific Gram-negative phyla. IMPORTANCE DedA family proteins are highly conserved and nearly ubiquitous integral membrane proteins found in archaea, bacteria, and eukaryotes. Recent work revealed that eukaryotic DedA proteins are phospholipid scramblases and that some bacterial DedA proteins are undecaprenyl phosphate flippases. We performed a phylogenetic analysis of this protein family in bacteria that revealed 3 DedA subfamilies with distinct phylogenetic distributions, genomic contexts, and putative functions. Our bioinformatic analysis lays the groundwork for future experimental studies on the role of DedA proteins in maintaining and modifying the membrane.

Lipopolysaccharide integrity primes bacterial sensitivity to a cell wall-degrading intermicrobial toxin.

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the molecular basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa , Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, surface lipopolysaccharide, that modulate Tae1 toxicity in vivo . Disruption of lipopolysaccharide synthesis provided Escherichia coli (Eco) with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study highlights the consequences of co-regulating essential pathways on recipient fitness during interbacterial competition, and how antibacterial toxins leverage cellular vulnerabilities that are both direct and indirect to their specific targets in vivo .

Three bacterial DedA subfamilies with distinct functions and phylogenetic distribution.

Recent studies in bacteria suggested that the broadly conserved but enigmatic DedA proteins function as undecaprenyl-phosphate (UndP) flippases, recycling this essential lipid carrier. To determine whether all DedA proteins have UndP flippase activity, we performed a phylogenetic analysis and correlated it to previously published experimental results and predicted structures. We uncovered three major DedA subfamilies: one contains UndP flippases, the second contains putative phospholipid flippases and is associated with aerobic metabolism, and the third is found only in specific Gram-negative phyla. IMPORTANCE: DedA-family proteins are highly conserved and nearly ubiquitous integral membrane proteins found in Archaea, Bacteria, and Eukaryotes. Recent work revealed that eukaryotic DedA proteins are phospholipid scramblases and some bacterial DedA proteins are undecaprenyl phosphate flippases. We perform a phylogenetic analysis of this protein family in Bacteria revealing 3 DedA subfamilies with distinct phylogenetic distributions, genomic contexts, and putative functions. Our analysis lays the groundwork for a deeper understanding of DedA proteins and their role in maintaining and modifying the membrane.

Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli.

CRISPR interference (CRISPRi) has facilitated the study of essential genes in diverse organisms using both high-throughput and targeted approaches. Despite the promise of this technique, no comprehensive arrayed CRISPRi library targeting essential genes exists for the model bacterium Escherichia coli, or for any Gram-negative species. Here, we built and characterized such a library. Each of the ∼500 strains in our E. coli library contains an inducible, chromosomally integrated single guide RNA (sgRNA) targeting an essential (or selected nonessential) gene and can be mated with a pseudo-Hfr donor strain carrying a dcas9 cassette to create a CRISPRi knockdown strain. Using this system, we built an arrayed library of CRISPRi strains and performed population and single-cell growth and morphology measurements as well as targeted follow-up experiments. These studies found that inhibiting translation causes an extended lag phase, identified new modulators of cell morphology, and revealed that the morphogene mreB is subject to transcriptional feedback regulation, which is critical for the maintenance of morphology. Our findings highlight canonical and noncanonical roles for essential genes in numerous aspects of cellular homeostasis. IMPORTANCE Essential genes make up only ∼5 to 10% of the genetic complement in most organisms but occupy much of their protein synthesis and account for almost all antibiotic targets. Despite the importance of essential genes, their intractability has, until recently, hampered efforts to study them. CRISPRi has facilitated the study of essential genes by allowing inducible and titratable depletion. However, all large-scale CRISPRi studies in Gram-negative bacteria thus far have used plasmids to express CRISPRi components and have been constructed in pools, limiting their utility for targeted assays and complicating the determination of antibiotic effects. Here, we use a modular method to construct an arrayed library of chromosomally integrated CRISPRi strains targeting the essential genes of the model bacterium Escherichia coli. This library enables targeted studies of essential gene depletions and high-throughput determination of antibiotic targets and facilitates studies targeting the outer membrane, an essential component that serves as the major barrier to antibiotics.

Computational pipeline for designing guide RNAs for mismatch-CRISPRi.

CRISPR interference is an increasingly popular method for perturbing gene expression. Guided by single-guide RNAs (sgRNAs), nuclease-deficient Cas9 proteins bind to specific DNA sequences and hinder transcription. Specificity is achieved through complementarity of the sgRNAs to the DNA. Changing complementarity by introducing single-nucleotide mismatches can be exploited to tune knockdown. Here, we present a computational pipeline to identify sgRNAs targeting specific genes in a bacterial genome, filter them, and titrate their activity by introducing mismatches. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2020).

Bacterial CRISPR screens for gene function.

In this review we describe the application of CRISPR tools for functional genomics screens in bacteria, with a focus on the use of interference (CRISPRi) approaches. We review recent developments in CRISPRi titration, which has enabled essential gene functional screens, and genome-scale pooled CRISPRi screens. We summarize progress toward enabling CRISPRi screens in non-model and pathogenic bacteria, including the development of new dCas9 variants. Taking into account the current state of the field, we provide a forward-looking analysis of CRISPRi strategies for determining gene function in bacteria.

Rewiring the specificity of extracytoplasmic function sigma factors.

Bacterial 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 extracytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their putative regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the autoregulatory 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 footprinting pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that some ECF σs are conserved global regulators controlling many genes throughout the genome, which are important under many conditions, while others are local regulators, controlling a few closely linked genes in response to specific stimuli in select species. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.

Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis.

Essential genes are the hubs of cellular networks, but lack of high-throughput methods for titrating gene expression has limited our understanding of the fitness landscapes against which their expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate defined levels of CRISPRi activity and demonstrated its broad applicability. Using libraries of mismatched sgRNAs predicted to span the full range of knockdown levels, we characterized the expression-fitness relationships of most essential genes in Escherichia coli and Bacillus subtilis. We find that these relationships vary widely from linear to bimodal but are similar within pathways. Notably, despite ∼2 billion years of evolutionary separation between E. coli and B. subtilis, most essential homologs have similar expression-fitness relationships with rare but informative differences. Thus, the expression levels of essential genes may reflect homeostatic or evolutionary constraints shared between the two organisms.

Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.

A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.

Growth-Phase-Specific Modulation of Cell Morphology and Gene Expression by an Archaeal Histone Protein.

UNLABELLED: In all three domains of life, organisms use nonspecific DNA-binding proteins to compact and organize the genome as well as to regulate transcription on a global scale. Histone is the primary eukaryotic nucleoprotein, and its evolutionary roots can be traced to the archaea. However, not all archaea use this protein as the primary DNA-packaging component, raising questions regarding the role of histones in archaeal chromatin function. Here, quantitative phenotyping, transcriptomic, and proteomic assays were performed on deletion and overexpression mutants of the sole histone protein of the hypersaline-adapted haloarchaeal model organism Halobacterium salinarum. This protein is highly conserved among all sequenced haloarchaeal species and maintains hallmark residues required for eukaryotic histone functions. Surprisingly, despite this conservation at the sequence level, unlike in other archaea or eukaryotes, H. salinarum histone is required to regulate cell shape but is not necessary for survival. Genome-wide expression changes in histone deletion strains were global, significant but subtle in terms of fold change, bidirectional, and growth phase dependent. Mass spectrometric proteomic identification of proteins from chromatin enrichments yielded levels of histone and putative nucleoid-associated proteins similar to those of transcription factors, consistent with an open and transcriptionally active genome. Taken together, these data suggest that histone in H. salinarum plays a minor role in DNA compaction but important roles in growth-phase-dependent gene expression and regulation of cell shape. Histone function in haloarchaea more closely resembles a regulator of gene expression than a chromatin-organizing protein like canonical eukaryotic histone. IMPORTANCE: Histones comprise the major protein component of eukaryotic chromatin and are required for both genome packaging and global regulation of expression. The current paradigm maintains that archaea whose genes encode histone also use these proteins to package DNA. In contrast, here we demonstrate that the sole histone encoded in the genome of the salt-adapted archaeon Halobacterium salinarum is both unessential and unlikely to be involved in DNA compaction despite conservation of residues important for eukaryotic histones. Rather, H. salinarum histone is required for global regulation of gene expression and cell shape. These data are consistent with the hypothesis that H. salinarum histone, strongly conserved across all other known salt-adapted archaea, serves a novel role in gene regulation and cell shape maintenance. Given that archaea possess the ancestral form of eukaryotic histone, this study has important implications for understanding the evolution of histone function.

Dynamic Metabolite Profiling in an Archaeon Connects Transcriptional Regulation to Metabolic Consequences.

Previous work demonstrated that the TrmB transcription factor is responsible for regulating the expression of many enzyme-coding genes in the hypersaline-adapted archaeon Halobacterium salinarum via a direct interaction with a cis-regulatory sequence in their promoters. This interaction is abolished in the presence of glucose. Although much is known about the effects of TrmB at the transcriptional level, it remains unclear whether and to what extent changes in mRNA levels directly affect metabolite levels. In order to address this question, here we performed a high-resolution metabolite profiling time course during a change in nutrients using a combination of targeted and untargeted methods in wild-type and ΔtrmB strain backgrounds. We found that TrmB-mediated transcriptional changes resulted in widespread and significant changes to metabolite levels across the metabolic network. Additionally, the pattern of growth complementation using various purines suggests that the mis-regulation of gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low phosphoribosylpyrophosphate (PRPP) levels. We confirmed these low PRPP levels using a quantitative mass spectrometric technique and found that they are associated with a metabolic block in de novo purine synthesis, which is partially responsible for the growth defect of the ΔtrmB mutant strain in the absence of glucose. In conclusion, we show how transcriptional regulation of metabolism affects metabolite levels and ultimately, phenotypes.

A transcription factor links growth rate and metabolism in the hypersaline adapted archaeon Halobacterium salinarum.

Co-ordinating metabolism and growth is a key challenge for all organisms. Despite fluctuating environments, cells must produce the same metabolic outputs to thrive. The mechanisms underlying this 'growth homeostasis' are known in bacteria and eukaryotes, but remain unexplored in archaea. In the model archaeon Halobacterium salinarum, the transcription factor TrmB regulates enzyme-coding genes in diverse metabolic pathways in response to glucose. However, H. salinarum is thought not to catabolize glucose. To resolve this discrepancy, we demonstrate that TrmB regulates the gluconeogenic production of sugars incorporated into the cell surface S-layer glycoprotein. Additionally, we show that TrmB-DNA binding correlates with instantaneous growth rate, likely because S-layer glycosylation is proportional to growth. This suggests that TrmB transduces a growth rate signal to co-regulated metabolic pathways including amino acid, purine, and cobalamin biosynthesis. Remarkably, the topology and function of this growth homeostatic network appear conserved across domains despite extensive alterations in protein components.

Hsp104 overexpression cures Saccharomyces cerevisiae [PSI+] by causing dissolution of the prion seeds.

The [PSI(+)] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI(+)], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI(+)]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI(+)] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI(+)] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.

Protein-DNA binding dynamics predict transcriptional response to nutrients in archaea.

Organisms across all three domains of life use gene regulatory networks (GRNs) to integrate varied stimuli into coherent transcriptional responses to environmental pressures. However, inferring GRN topology and regulatory causality remains a central challenge in systems biology. Previous work characterized TrmB as a global metabolic transcription factor in archaeal extremophiles. However, it remains unclear how TrmB dynamically regulates its ∼100 metabolic enzyme-coding gene targets. Using a dynamic perturbation approach, we elucidate the topology of the TrmB metabolic GRN in the model archaeon Halobacterium salinarum. Clustering of dynamic gene expression patterns reveals that TrmB functions alone to regulate central metabolic enzyme-coding genes but cooperates with various regulators to control peripheral metabolic pathways. Using a dynamical model, we predict gene expression patterns for some TrmB-dependent promoters and infer secondary regulators for others. Our data suggest feed-forward gene regulatory topology for cobalamin biosynthesis. In contrast, purine biosynthesis appears to require TrmB-independent regulators. We conclude that TrmB is an important component for mediating metabolic modularity, integrating nutrient status and regulating gene expression dynamics alone and in concert with secondary regulators.

Sequestration of Sup35 by aggregates of huntingtin fragments causes toxicity of [PSI+] yeast.

Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.

Inhibition of protein-tyrosine phosphatases by mild oxidative stresses is dependent on S-nitrosylation.

Previous studies have shown that a Ca(2+)-dependent nitric-oxide synthase (NOS) is activated as part of a cellular response to low doses of ionizing radiation. Genetic and pharmacological inhibitor studies linked this NO signaling to the radiation-induced activation of ERK1/2. Herein, a mechanism for the radiation-induced activation of Tyr phosphorylation-dependent pathways (e.g. ERK1/2) involving the inhibition of protein-Tyr phosphatases (PTPs) by S-nitrosylation is tested. The basis for this mechanism resides in the redox-sensitive active site Cys in PTPs. These studies also examined oxidative stress induced by low concentrations of H(2)O(2). S-Nitrosylation of total cellular PTP and immunopurified SHP-1 and SHP-2 was detected as protection of PTP enzymatic activity from alkylation by N-ethylmaleimide and reversal by ascorbate. Both radiation and H(2)O(2) protected PTP activity from alkylation by a mechanism reversible by ascorbate and inhibited by NOS inhibitors or expression of a dominant negative mutant of NOS-1. Radiation and H(2)O(2) stimulated a transient increase in cytoplasmic free [Ca(2+)]. Radiation, H(2)O(2), and the Ca(2+) ionophore, ionomycin, also stimulated NOS activity, and this was associated with an enhanced S-nitrosylation of the active site Cys(453) determined by isolation of S-nitrosylated wild type but not active site Cys(453) --> Ser SHP-1 mutant by the "biotin-switch" method. Thus, one consequence of oxidative stimulation of NO generation is S-nitrosylation and inhibition of PTPs critical in cellular signal transduction pathways. These results support the conclusion that a mild oxidative signal is converted to a nitrosative one due to the better redox signaling properties of NO.