ABSTRACT
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.
Subject(s)
Lipopolysaccharides , Type VI Secretion Systems , Lipopolysaccharides/metabolism , Bacterial Proteins/metabolism , Type VI Secretion Systems/metabolism , Escherichia coli/metabolism , Cell Wall/metabolism , Pseudomonas aeruginosa/metabolismABSTRACT
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 .
ABSTRACT
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 â¼2 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.
Subject(s)
Bacillus subtilis/genetics , Escherichia coli/genetics , Genes, Essential/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , CRISPR-Cas Systems , Escherichia coli/metabolism , Gene Expression/genetics , Gene Expression Regulation, Bacterial/genetics , Genes, Essential/physiology , Genetic Fitness/geneticsABSTRACT
The Gram-positive bacterium Bacillus subtilis uses serine not only as a building block for proteins but also as an important precursor in many anabolic reactions. Moreover, a lack of serine results in the initiation of biofilm formation. However, excess serine inhibits the growth of B. subtilis. To unravel the underlying mechanisms, we isolated suppressor mutants that can tolerate toxic serine concentrations by three targeted and non-targeted genome-wide screens. All screens as well as genetic complementation in Escherichia coli identified the so far uncharacterized permease YbeC as the major serine transporter of B. subtilis. In addition to YbeC, the threonine transporters BcaP and YbxG make minor contributions to serine uptake. A strain lacking these three transporters was able to tolerate 100 mM serine whereas the wild type strain was already inhibited by 1 mM of the amino acid. The screen for serine-resistant mutants also identified mutations that result in increased serine degradation and in increased expression of threonine biosynthetic enzymes suggesting that serine toxicity results from interference with threonine biosynthesis.
Subject(s)
Amino Acid Transport Systems/metabolism , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Metabolic Networks and Pathways , Serine/metabolism , Threonine/metabolism , Amino Acid Transport Systems/genetics , Bacillus subtilis/drug effects , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Mutation , Serine/pharmacology , Threonine/geneticsABSTRACT
DNA topoisomerases play essential roles in chromosome organization and replication. Most bacteria possess multiple topoisomerases which have specialized functions in the control of DNA supercoiling or in DNA catenation/decatenation during recombination and chromosome segregation. DNA topoisomerase I is required for the relaxation of negatively supercoiled DNA behind the transcribing RNA polymerase. Conflicting results have been reported on the essentiality of the topA gene encoding topoisomerase I in the model bacterium Bacillus subtilis. In this work, we have studied the requirement for topoisomerase I in B. subtilis. All stable topA mutants carried different chromosomal amplifications of the genomic region encompassing the parEC operon encoding topoisomerase IV. Using a fluorescent amplification reporter system we observed that each individual topA mutant had acquired such an amplification. Eventually, the amplifications were replaced by a point mutation in the parEC promoter region which resulted in a fivefold increase of parEC expression. In this strain both type I topoisomerases, encoded by topA and topB, were dispensable. Our results demonstrate that topoisomerase IV at increased expression is necessary and sufficient to take over the function of type 1A topoisomerases.
Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/genetics , DNA Topoisomerase IV/metabolism , DNA Topoisomerases, Type I/metabolism , Bacterial Proteins/metabolism , Chromosomes, Bacterial , DNA Replication , DNA, Bacterial/genetics , DNA, Superhelical/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/metabolism , Genome, Bacterial , Mutation , Phenotype , Point Mutation , Promoter Regions, GeneticABSTRACT
The 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. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans1-6. However, the difficulty of establishing effective CRISPRi systems across 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 combines 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 gammaproteobacteria and Bacillales 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-microorganism interactions.
Subject(s)
Bacteria/genetics , Bacterial Proteins/genetics , Bacteriological Techniques/methods , CRISPR-Cas Systems , Genetic Techniques , Anti-Bacterial Agents/pharmacology , Bacteria/classification , Bacteria/drug effects , Bacterial Proteins/metabolism , Conjugation, Genetic , Drug Resistance, Microbial/genetics , Gene Library , Gene Regulatory Networks , Gene Targeting , Genes, Essential/genetics , Genome, Bacterial/geneticsABSTRACT
The recent explosion of research on the microbiota has highlighted the important interplay between commensal microorganisms and the health of their cognate hosts. Metabolites isolated from commensal bacteria have been demonstrated to possess a range of antimicrobial activities, and it is widely believed that some of these metabolites modulate host behavior, affecting predisposition to disease and pathogen invasion. Our access to the local marine mammal stranding network and previous successes in mining the fish microbiota poised us to test the hypothesis that the marine mammal microbiota is a novel source of commensal bacteria-produced bioactive metabolites. Examination of intestinal contents from five marine mammals led to the identification of a Micromonospora strain with potent and selective activity against a panel of Gram-positive pathogens and no discernible human cytotoxicity. Compound isolation afforded a new complex glycosylated polyketide, phocoenamicin, with potent activity against the intestinal pathogen Clostridium difficile, an organism challenging to treat in hospital settings. Use of our activity-profiling platform, BioMAP, clustered this metabolite with other known ionophore antibiotics. Fluorescence imaging and flow cytometry confirmed that phocoenamicin is capable of shifting membrane potential without damaging membrane integrity. Thus, exploration of gut microbiota in hosts from diverse environments can serve as a powerful strategy for the discovery of novel antibiotics against human pathogens.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/pharmacology , Clostridioides difficile/drug effects , Gastrointestinal Microbiome , Mammals , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Biological Products/chemistry , Biological Products/isolation & purification , Biological Products/metabolism , Biological Products/pharmacology , Drug Discovery/methods , Gram-Positive Bacteria/drug effects , Molecular Structure , Structure-Activity Relationship , WorkflowABSTRACT
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.
Subject(s)
Bacillus subtilis/genetics , High-Throughput Screening Assays/methods , Amino Acids , Gene Deletion , Gene Library , Genomic Library , Genomics , Sequence Deletion/genetics , Spores, Bacterial/geneticsABSTRACT
The cell wall of most Gram-positive bacteria contains equal amounts of peptidoglycan and the phosphate-rich glycopolymer wall teichoic acid (WTA). During phosphate-limited growth of the Gram-positive model organism Bacillus subtilis 168, WTA is lost from the cell wall in a response mediated by the PhoPR two-component system, which regulates genes involved in phosphate conservation and acquisition. It has been thought that WTA provides a phosphate source to sustain growth during starvation conditions; however, WTA degradative pathways have not been described for this or any condition of bacterial growth. Here, we uncover roles for the Bacillus subtilis PhoP regulon genes glpQ and phoD as encoding secreted phosphodiesterases that function in WTA metabolism during phosphate starvation. Unlike the parent 168 strain, ΔglpQ or ΔphoD mutants retained WTA and ceased growth upon phosphate limitation. Characterization of GlpQ and PhoD enzymatic activities, in addition to X-ray crystal structures of GlpQ, revealed distinct mechanisms of WTA depolymerization for the two enzymes; GlpQ catalyzes exolytic cleavage of individual monomer units, and PhoD catalyzes endo-hydrolysis at nonspecific sites throughout the polymer. The combination of these activities appears requisite for the utilization of WTA as a phosphate reserve. Phenotypic characterization of the ΔglpQ and ΔphoD mutants revealed altered cell morphologies and effects on autolytic activity and antibiotic susceptibilities that, unexpectedly, also occurred in phosphate-replete conditions. Our findings offer novel insight into the B. subtilis phosphate starvation response and implicate WTA hydrolase activity as a determinant of functional properties of the Gram-positive cell envelope.
Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins , Cell Wall/enzymology , Phosphoric Diester Hydrolases , Bacillus subtilis/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Wall/genetics , Crystallography, X-Ray , Hydrolysis , Mutation , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Regulon/physiologyABSTRACT
Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.
Subject(s)
Bacillus subtilis/genetics , Genes, Bacterial , Genes, Essential , CRISPR-Cas Systems , Gene Knockdown Techniques , Gene Library , Gene Regulatory Networks , Molecular Targeted TherapyABSTRACT
High-throughput functional genomic technologies are accelerating progress in understanding the diversity of bacterial life and in developing a systems-level understanding of model bacterial organisms. Here we highlight progress in deep-sequencing-based functional genomics, show how whole genome sequencing is enabling phenotyping in organisms recalcitrant to genetic approaches, recount the rapid proliferation of functional genomic approaches to non-growth phenotypes, and discuss how advances are enabling genome-scale resource libraries for many different bacteria.
Subject(s)
Bacteria/genetics , Genome, Bacterial , High-Throughput Nucleotide Sequencing , DNA Barcoding, Taxonomic/methods , Genetic Variation , Genomics , High-Throughput Nucleotide Sequencing/methods , PhenotypeABSTRACT
Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. Transport of the cell wall precursor lipid II in Escherichia coli requires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show that Bacillus subtilis cells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized gene amj (alternate to MurJ; ydaH) and B. subtilis MurJ (murJBs; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJBs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJBs in E. coli supports lipid II flipping and viability in the absence of E. coli MurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σ(M) and cells lacking MurJBs increase amj transcription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition.
Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/growth & development , Bacterial Proteins/metabolism , Cell Wall/physiology , Gene Expression Regulation, Bacterial/physiology , Morphogenesis/physiology , Phospholipid Transfer Proteins/metabolism , Chromatography, High Pressure Liquid , Microscopy, Fluorescence , Phylogeny , Plasmids/genetics , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Uridine Diphosphate N-Acetylmuramic Acid/metabolismABSTRACT
Cells often mount transcriptional responses and activate specific sets of genes in response to stress-inducing signals such as heat or reactive oxygen species. Transcription factors in the RpoH family of bacterial alternative σ factors usually control gene expression during a heat shock response. Interestingly, several α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. We investigated the target promoters of Rhodobacter sphaeroides RpoH(I) and RpoH(II) using genome-scale data derived from gene expression profiling and the direct interactions of each protein with DNA in vivo. We found that the RpoH(I) and RpoH(II) regulons have both distinct and overlapping gene sets. We predicted DNA sequence elements that dictate promoter recognition specificity by each RpoH paralog. We found that several bases in the highly conserved TTG in the -35 element are important for activity with both RpoH homologs; that the T-9 position, which is over-represented in the RpoH(I) promoter sequence logo, is critical for RpoH(I)-dependent transcription; and that several bases in the predicted -10 element were important for activity with either RpoH(II) or both RpoH homologs. Genes that are transcribed by both RpoH(I) and RpoH(II) are predicted to encode for functions involved in general cell maintenance. The functions specific to the RpoH(I) regulon are associated with a classic heat shock response, while those specific to RpoH(II) are associated with the response to the reactive oxygen species, singlet oxygen. We propose that a gene duplication event followed by changes in promoter recognition by RpoH(I) and RpoH(II) allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria.
Subject(s)
DNA-Binding Proteins , Heat-Shock Proteins , Heat-Shock Response/genetics , Oxidative Stress/genetics , Sigma Factor , Singlet Oxygen/metabolism , Base Sequence , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Evolution, Molecular , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Heat-Shock Proteins/physiology , Hot Temperature , Promoter Regions, Genetic , Reactive Oxygen Species/metabolism , Rhodobacter sphaeroides/genetics , Rhodobacter sphaeroides/physiology , Sigma Factor/genetics , Sigma Factor/metabolism , Sigma Factor/physiology , Transcription, GeneticABSTRACT
Vibrio vulnificus is an opportunistic human pathogen that causes severe infections in susceptible individuals. While the components of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) have been shown to regulate numerous targets, little such information is available for the V. vulnificus PTS. Here we show that enzyme IIA(Glc) of the PTS regulates the peptidase activity of a mammalian insulysin homolog in V. vulnificus. While interaction of IIA(Glc) with the insulysin homolog is independent of the phosphorylation state of IIA(Glc), only unphosphorylated IIA(Glc) activates the insulysin homolog. Taken together, our results suggest that the V. vulnificus insulysin-IIA(Glc) complex plays a role in survival in the host by sensing glucose.
Subject(s)
Glucose/metabolism , Insulysin/chemistry , Insulysin/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Sequence Homology, Amino Acid , Vibrio vulnificus/enzymology , Animals , Biological Transport , Female , Humans , Mice , Mice, Inbred ICR , Phosphorylation , Survival Analysis , Vibrio vulnificus/metabolism , Vibrio vulnificus/pathogenicityABSTRACT
In bacteria, multiple sigmas direct RNA polymerase to distinct sets of promoters. Housekeeping sigmas direct transcription from thousands of promoters, whereas most alternative sigmas are more selective, recognizing more highly conserved promoter motifs. For sigma(32) and sigma(28), two Escherichia coli Group 3 sigmas, altering a few residues in Region 2.3, the portion of sigma implicated in promoter melting, to those universally conserved in housekeeping sigmas relaxed their stringent promoter requirements and significantly enhanced melting of suboptimal promoters. All Group 3 sigmas and the more divergent Group 4 sigmas have nonconserved amino acids at these positions and rarely transcribe >100 promoters. We suggest that the balance of "melting" and "recognition" functions of sigmas is critical to setting the stringency of promoter recognition. Divergent sigmas may generally use a nonoptimal Region 2.3 to increase promoter stringency, enabling them to mount a focused response to altered conditions.
Subject(s)
Bacterial Proteins/metabolism , Nucleic Acid Denaturation , Promoter Regions, Genetic/genetics , Sigma Factor/metabolism , Amino Acid Sequence , Bacteria/genetics , Bacteria/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Molecular Sequence Data , Phylogeny , Sequence AlignmentABSTRACT
Sigma28 controls the expression of flagella-related genes and is the most widely distributed alternative sigma factor, present in motile Gram-positive and Gram-negative bacteria. The distinguishing feature of sigma28 promoters is a long -10 region (GCCGATAA). Despite the fact that the upstream GC is highly conserved, previous studies have not indicated a functional role for this motif. Here we examine the functional relevance of the GCCG motif and determine which residues in sigma28 participate in its recognition. We find that the GCCG motif is a functionally important composite element. The upstream GC constitutes an extended -10 motif and is recognized by R91, a residue in Domain 3 of sigma28. The downstream CG is the upstream edge of -10 region of the promoter; two residues in Region 2.4, D81 and R84, participate in its recognition. Consistent with their role in base-specific recognition of the promoter, R91, D81 and D84 are universally conserved in sigma28 orthologues. Sigma28 is the second Group 3 sigma shown to use an extended -10 region in promoter recognition, raising the possibility that other Group 3 sigmas will do so as well.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Promoter Regions, Genetic , Sigma Factor/metabolism , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Mutagenesis , Plasmids , Sigma Factor/genetics , Transcription, GeneticABSTRACT
Sigma32 controls expression of heat shock genes in Escherichia coli and is widely distributed in proteobacteria. The distinguishing feature of sigma32 promoters is a long -10 region (CCCCATNT) whose tetra-C motif is important for promoter activity. Using alanine-scanning mutagenesis of sigma32 and in vivo and in vitro assays, we identified promoter recognition determinants of this motif. The most downstream C (-13) is part of the -10 motif; our work confirms and extends recognition determinants of -13C. Most importantly, our work suggests that the two upstream Cs (-16, -15) constitute an 'extended -10' recognition motif that is recognized by K130, a residue universally conserved in beta- and gamma-proteobacteria. This residue is located in the alpha-helix of sigmaDomain 3 that mediates recognition of the extended -10 promoter motif in other sigmas. K130 is not conserved in alpha- and delta-/epsilon-proteobacteria and we found that sigma32 from the alpha-proteobacterium Caulobacter crescentus does not need the extended -10 motif for high promoter activity. This result supports the idea that K130 mediates extended -10 recognition. Sigma32 is the first Group 3 sigma shown to use the 'extended -10' recognition motif.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Heat-Shock Proteins/metabolism , Promoter Regions, Genetic , Sigma Factor/metabolism , Amino Acid Substitution , Base Sequence , Caulobacter crescentus/genetics , Caulobacter crescentus/metabolism , Conserved Sequence , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Heat-Shock Proteins/genetics , Molecular Sequence Data , Mutagenesis , Plasmids , Sigma Factor/genetics , Transcription, GeneticABSTRACT
While the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (carbohydrate PTS) have been shown to regulate numerous targets, little such information is available for the nitrogen-metabolic phosphotransferase system (nitrogen-metabolic PTS). To elucidate the physiological role of the nitrogen-metabolic PTS, we carried out phenotype microarray (PM) analysis with Escherichia coli K-12 strain MG1655 deleted for the ptsP gene encoding the first enzyme of the nitrogen-metabolic PTS. Together with the PM data, growth studies revealed that a ptsN (encoding enzyme IIA(Ntr)) mutant became extremely sensitive to leucine-containing peptides (LCPs), while both ptsP (encoding enzyme I(Ntr)) and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity of LCPs was found to be due to leucine and the dephospho-form of enzyme IIA(Ntr) was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho-form of enzyme IIA(Ntr) is required for derepression of the ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step common to the biosynthesis of the branched-chain amino acids.
Subject(s)
Acetolactate Synthase/genetics , Acetolactate Synthase/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Gene Deletion , Gene Fusion , Genes, Reporter , Leucine , Oligonucleotide Array Sequence Analysis , Operon , RNA, Bacterial/analysis , RNA, Messenger/analysis , beta-Galactosidase/geneticsABSTRACT
The bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes as well as effecting sugar transport. The crr gene product (enzyme IIA(Glc) (IIA(Glc))) mediates some of these regulatory phenomena. In this report, we characterize a novel IIA(Glc)-binding protein from Escherichia coli extracts, discovered using ligand-fishing with surface plasmon resonance spectroscopy. This protein, which we named FrsA (fermentation/respiration switch protein), is the 47-kDa product of the yafA gene, previously denoted as "function unknown." FrsA forms a 1:1 complex specifically with the unphosphorylated form of IIA(Glc), with the highest affinity of any protein thus far shown to interact with IIA(Glc). Orthologs of FrsA have been found to exist only in facultative anaerobes belonging to the gamma-proteobacterial group. Disruption of frsA increased cellular respiration on several sugars including glucose, while increased FrsA expression resulted in an increased fermentation rate on these sugars with the concomitant accumulation of mixed-acid fermentation products. These results suggest that IIA(Glc) regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with FrsA.