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1.
Nat Commun ; 12(1): 4174, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234105

RESUMO

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
2.
J Bacteriol ; 203(13): e0014921, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33875545

RESUMO

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.


Assuntos
Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Transferases/metabolismo , Animais , Antibacterianos/farmacologia , Ácido Aspártico Endopeptidases , Proteínas de Bactérias , Escherichia coli/genética , Feminino , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Camundongos , Peptidoglicano/metabolismo , Transferases/química , Transferases/genética , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/metabolismo
3.
Trends Microbiol ; 29(4): 334-345, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33036869

RESUMO

The outer membrane (OM) is a defining feature of Gram-negative bacteria that serves as a permeability barrier and provides rigidity to the cell. Critical to OM function is establishing and maintaining an asymmetrical bilayer structure with phospholipids in the inner leaflet and the complex glycolipid lipopolysaccharide (LPS) in the outer leaflet. Cells ensure this asymmetry by regulating the biogenesis of lipid A, the conserved and essential anchor of LPS. Here we review the consequences of disrupting the regulatory components that control lipid A biogenesis, focusing on the rate-limiting step performed by LpxC. Dissection of these processes provides critical insights into bacterial physiology and potential new targets for antibiotics able to overcome rapidly spreading resistance mechanisms.

4.
Nature ; 584(7821): 479-483, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32788728

RESUMO

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Assuntos
Membrana Celular/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/patogenicidade , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Estabilidade Enzimática , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Genes Essenciais , Hidrolases/química , Hidrolases/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/biossíntese , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , Virulência
5.
Elife ; 82019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31237236

RESUMO

Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.


Assuntos
Anticorpos Monoclonais/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Animais , Antibacterianos/farmacologia , Sítios de Ligação , Mapeamento de Epitopos , Epitopos/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , Ratos Sprague-Dawley , Relação Estrutura-Atividade
6.
J Bacteriol ; 201(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30322857

RESUMO

Integral ß-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the ß-barrel assembly machine (BAM). This essential complex, composed of a ß-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulate in vitro Thus, the probing of BAM function in living cells is critical to fully understand the mechanism of ß-barrel folding. We recently identified an anti-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function. Here, we show that the inhibitory effect of MAB1 is enhanced when BAM function is perturbed by either lowering the level of BamA or removing the nonessential BAM lipoproteins, BamB, BamC, or BamE. The disruption of BAM reduces BamA activity, increases outer membrane (OM) fluidity, and activates the σE stress response, suggesting the OM environment and BAM function are interconnected. Consistent with this idea, an increase in the membrane fluidity through changes in the growth environment or alterations to the lipopolysaccharide in the outer membrane is sufficient to provide resistance to MAB1 and enable the BAM to tolerate these perturbations. Amino acid substitutions in BamA at positions in the outer membrane spanning region or the periplasmic space remote from the extracellular MAB1 binding site also provide resistance to the inhibitory antibody. Our data highlight that the outer membrane environment is a critical determinant in the efficient and productive folding of ß-barrel membrane proteins by BamA.IMPORTANCE BamA is an essential component of the ß-barrel assembly machine (BAM) in the outer membranes of Gram-negative bacteria. We have used a recently described inhibitory anti-BamA antibody, MAB1, to identify the molecular requirements for BAM function. Resistance to this antibody can be achieved through changes to the outer membrane or by amino acid substitutions in BamA that allosterically affect the response to MAB1. Sensitivity to MAB1 is achieved by perturbing BAM function. By using MAB1 activity and functional assays as proxies for BAM function, we link outer membrane fluidity to BamA activity, demonstrating that an increase in membrane fluidity sensitizes the cells to BAM perturbations. Thus, the search for potential inhibitors of BamA function must consider the membrane environment in which ß-barrel folding occurs.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Lipoproteínas/metabolismo , Fluidez de Membrana , Dobramento de Proteína , Multimerização Proteica , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Análise Mutacional de DNA , Escherichia coli/genética , Proteínas de Escherichia coli/genética
7.
Sci Rep ; 8(1): 7136, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29740124

RESUMO

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP ß-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of ß-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/imunologia , Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Imunização , Conformação Proteica , Dobramento de Proteína , Transporte Proteico/genética , Transporte Proteico/imunologia , Vacinação
8.
Proc Natl Acad Sci U S A ; 115(14): 3692-3697, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29555747

RESUMO

The folding and insertion of integral ß-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ß-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli ß-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ß-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ß-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.


Assuntos
Antibacterianos/farmacologia , Anticorpos Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Fluidez de Membrana/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Escherichia coli/imunologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína
9.
Proc Natl Acad Sci U S A ; 112(44): E6038-47, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26483489

RESUMO

Five homologous noncoding small RNAs (sRNAs), called the Qrr1-5 sRNAs, function in the Vibrio harveyi quorum-sensing cascade to drive its operation. Qrr1-5 use four different regulatory mechanisms to control the expression of ∼ 20 mRNA targets. Little is known about the roles individual nucleotides play in mRNA target selection, in determining regulatory mechanism, or in defining Qrr potency and dynamics of target regulation. To identify the nucleotides vital for Qrr function, we developed a method we call RSort-Seq that combines saturating mutagenesis, fluorescence-activated cell sorting, high-throughput sequencing, and mutual information theory to explore the role that every nucleotide in Qrr4 plays in regulation of two mRNA targets, luxR and luxO. Companion biochemical assays allowed us to assign specific regulatory functions/underlying molecular mechanisms to each important base. This strategy yielded a regional map of nucleotides in Qrr4 vital for stability, Hfq interaction, stem-loop formation, and base pairing to both luxR and luxO, to luxR only, and to luxO only. In terms of nucleotides critical for sRNA function, the RSort-Seq analysis provided strikingly different results from those predicted by commonly used regulatory RNA-folding algorithms. This approach is applicable to any RNA-RNA interaction, including sRNAs in other bacteria and regulatory RNAs in higher organisms.


Assuntos
Escherichia coli/fisiologia , Nucleotídeos/fisiologia , Percepção de Quorum , RNA não Traduzido/fisiologia , Vibrio/fisiologia , Escherichia coli/genética , Vibrio/genética
10.
Cell ; 160(1-2): 228-40, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25579683

RESUMO

Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response.


Assuntos
Percepção de Quorum , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Vibrio/metabolismo , Sequência de Bases , Escherichia coli/genética , Sequências Repetidas Invertidas , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Vibrio/genética
11.
J Bacteriol ; 197(1): 73-80, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25313392

RESUMO

Bacteria use a chemical communication process called quorum sensing to monitor cell density and to alter behavior in response to fluctuations in population numbers. Previous studies with Vibrio harveyi have shown that LuxR, the master quorum-sensing regulator, activates and represses >600 genes. These include six genes that encode homologs of the Escherichia coli Bet and ProU systems for synthesis and transport, respectively, of glycine betaine, an osmoprotectant used during osmotic stress. Here we show that LuxR activates expression of the glycine betaine operon betIBA-proXWV, which enhances growth recovery under osmotic stress conditions. BetI, an autorepressor of the V. harveyi betIBA-proXWV operon, activates the expression of genes encoding regulatory small RNAs that control quorum-sensing transitions. Connecting quorum-sensing and glycine betaine pathways presumably enables V. harveyi to tune its execution of collective behaviors to its tolerance to stress.


Assuntos
Pressão Osmótica/fisiologia , Percepção de Quorum/fisiologia , Vibrio/fisiologia , Betaína/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Estresse Fisiológico , Transativadores/genética , Transativadores/metabolismo , Vibrio/genética
12.
EMBO J ; 32(15): 2158-71, 2013 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-23838640

RESUMO

Quorum sensing is a chemical communication process that bacteria use to control collective behaviours including bioluminescence, biofilm formation, and virulence factor production. In Vibrio harveyi, five homologous small RNAs (sRNAs) called Qrr1-5, control quorum-sensing transitions. Here, we identify 16 new targets of the Qrr sRNAs. Mutagenesis reveals that particular sequence differences among the Qrr sRNAs determine their target specificities. Modelling coupled with biochemical and genetic analyses show that all five of the Qrr sRNAs possess four stem-loops: the first stem-loop is crucial for base pairing with a subset of targets. This stem-loop also protects the Qrr sRNAs from RNase E-mediated degradation. The second stem-loop contains conserved sequences required for base pairing with the majority of the target mRNAs. The third stem-loop plays an accessory role in base pairing and stability. The fourth stem-loop functions as a rho-independent terminator. In the quorum-sensing regulon, Qrr sRNAs-controlled genes are the most rapid to respond to quorum-sensing autoinducers. The Qrr sRNAs are conserved throughout vibrios, thus insights from this work could apply generally to Vibrio quorum sensing.


Assuntos
Conformação de Ácido Nucleico , Percepção de Quorum/fisiologia , RNA Bacteriano/metabolismo , RNA não Traduzido/metabolismo , Regulon/fisiologia , Vibrio/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Bacteriano/genética , RNA não Traduzido/genética , Vibrio/genética
13.
J Bacteriol ; 195(3): 436-43, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23204455

RESUMO

Bacteria use a chemical communication process called quorum sensing to control transitions between individual and group behaviors. In the Vibrio harveyi quorum-sensing circuit, two master transcription factors, AphA and LuxR, coordinate the quorum-sensing response. Here we show that AphA regulates 167 genes, LuxR regulates 625 genes, and they coregulate 77 genes. LuxR strongly controls genes at both low cell density and high cell density, suggesting that it is the major quorum-sensing regulator. In contrast, AphA is absent at high cell density and acts to fine-tune quorum-sensing gene expression at low cell density. We examined two loci as case studies of coregulation by AphA and LuxR. First, AphA and LuxR directly regulate expression of the genes encoding the quorum-regulatory small RNAs Qrr2, Qrr3, and Qrr4, the consequence of which is a specifically timed transition between the individual and the group life-styles. Second, AphA and LuxR repress type III secretion system genes but at different times and to different extents. The consequence of this regulation is that type III secretion is restricted to a peak at mid-cell density. Thus, the asymmetric production of AphA and LuxR coupled with differences in their strengths and timing of target gene regulation generate a precise temporal pattern of gene expression.


Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Percepção de Quorum/fisiologia , Regulon/fisiologia , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Vibrio/metabolismo , Proliferação de Células , Escherichia coli/classificação , Escherichia coli/metabolismo , Mutação , Análise Serial de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Fatores de Tempo , Transativadores/genética , Fatores de Transcrição/fisiologia , Vibrio/genética
14.
Genes Dev ; 26(23): 2634-46, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23207918

RESUMO

Escherichia coli DksA is a transcription factor that binds to RNA polymerase (RNAP) without binding to DNA, destabilizing RNAP-promoter interactions, sensitizing RNAP to the global regulator ppGpp, and regulating transcription of several hundred target genes, including those encoding rRNA. Previously, we described promoter sequences and kinetic properties that account for DksA's promoter specificity, but how DksA exerts its effects on RNAP has remained unclear. To better understand DksA's mechanism of action, we incorporated benzoyl-phenylalanine at specific positions in DksA and mapped its cross-links to RNAP, constraining computational docking of the two proteins. The resulting evidence-based model of the DksA-RNAP complex as well as additional genetic and biochemical approaches confirmed that DksA binds to the RNAP secondary channel, defined the orientation of DksA in the channel, and predicted a network of DksA interactions with RNAP that includes the rim helices and the mobile trigger loop (TL) domain. Engineered cysteine substitutions in the TL and DksA coiled-coil tip generated a disulfide bond between them, and the interacting residues were absolutely required for DksA function. We suggest that DksA traps the TL in a conformation that destabilizes promoter complexes, an interaction explaining the requirement for the DksA tip and its effects on transcription.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-23125205

RESUMO

Quorum sensing is a process of cell-cell communication that allows bacteria to share information about cell density and adjust gene expression accordingly. This process enables bacteria to express energetically expensive processes as a collective only when the impact of those processes on the environment or on a host will be maximized. Among the many traits controlled by quorum sensing is the expression of virulence factors by pathogenic bacteria. Here we review the quorum-sensing circuits of Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, and Vibrio cholerae. We outline these canonical quorum-sensing mechanisms and how each uniquely controls virulence factor production. Additionally, we examine recent efforts to inhibit quorum sensing in these pathogens with the goal of designing novel antimicrobial therapeutics.


Assuntos
Bacillus cereus/fisiologia , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum , Staphylococcus aureus/fisiologia , Vibrio cholerae/fisiologia , Fatores de Virulência/biossíntese , Bacillus cereus/patogenicidade , Infecções Bacterianas/tratamento farmacológico , Regulação da Expressão Gênica , Humanos , Pseudomonas aeruginosa/patogenicidade , Staphylococcus aureus/patogenicidade , Vibrio cholerae/patogenicidade
16.
Genes Dev ; 25(4): 397-408, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21325136

RESUMO

Bacteria cycle between periods when they perform individual behaviors and periods when they perform group behaviors. These transitions are controlled by a cell-cell communication process called quorum sensing, in which extracellular signal molecules, called autoinducers (AIs), are released, accumulate, and are synchronously detected by a group of bacteria. AI detection results in community-wide changes in gene expression, enabling bacteria to collectively execute behaviors such as bioluminescence, biofilm formation, and virulence factor production. In this study, we show that the transcription factor AphA is a master regulator of quorum sensing that operates at low cell density (LCD) in Vibrio harveyi and Vibrio cholerae. In contrast, LuxR (V. harveyi)/HapR (V. cholerae) is the master regulator that operates at high cell density (HCD). At LCD, redundant small noncoding RNAs (sRNAs) activate production of AphA, and AphA and the sRNAs repress production of LuxR/HapR. Conversely, at HCD, LuxR/HapR represses aphA. This network architecture ensures maximal AphA production at LCD and maximal LuxR/HapR production at HCD. Microarray analyses reveal that 300 genes are regulated by AphA at LCD in V. harveyi, a subset of which is also controlled by LuxR. We propose that reciprocal gradients of AphA and LuxR/HapR establish the quorum-sensing LCD and HCD gene expression patterns, respectively.


Assuntos
Percepção de Quorum/genética , Proteínas Repressoras/fisiologia , Transativadores/fisiologia , Vibrio/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Sequência de Bases , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Análise em Microsséries , Modelos Biológicos , Dados de Sequência Molecular , Percepção de Quorum/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência do Ácido Nucleico , Transativadores/genética , Transativadores/metabolismo , Vibrio/genética , Vibrio/metabolismo
17.
Genes Dev ; 23(2): 236-48, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19171784

RESUMO

The Escherichia coli DksA protein inserts into the RNA polymerase (RNAP) secondary channel, modifying the transcription initiation complex so that promoters with specific kinetic characteristics are regulated by changes in the concentrations of ppGpp and NTPs. We used footprinting assays to determine the specific kinetic intermediate, RP(I), on which DksA acts. Genetic approaches identified substitutions in the RNAP switch regions, bridge helix, and trigger loop that mimicked, reduced, or enhanced DksA function on rRNA promoters. Our results indicate that DksA binding in the secondary channel of RP(I) disrupts interactions with promoter DNA at least 25 A away, between positions -6 and +6 (the transcription start site is +1). We propose a working model in which the trigger loop and bridge helix transmit effects of DksA to the switch region(s), allosterically affecting switch residues that control clamp opening/closing and/or that interact directly with promoter DNA. DksA thus inhibits the transition to RP(I). Our results illustrate in mechanistic terms how transcription factors can regulate initiation promoter-specifically without interacting directly with DNA.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas/genética , Regulação Alostérica , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Guanosina Tetrafosfato/metabolismo , Modelos Moleculares , Estrutura Terciária de Proteína , Transdução de Sinais , Supressão Genética
18.
J Mol Biol ; 383(2): 367-79, 2008 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-18760284

RESUMO

Sequence-based searches identified a new family of genes in proteobacteria, named rnk, which shares high sequence similarity with the C-terminal domains of the Gre factors (GreA and GreB) and the Thermus/Deinococcus anti-Gre factor Gfh1. We solved the X-ray crystal structure of Escherichia coli regulator of nucleoside kinase (Rnk) at 1.9 A resolution using the anomalous signal from the native protein. The Rnk structure strikingly resembles those of E. coli GreA and GreB and Thermus Gfh1, all of which are RNA polymerase (RNAP) secondary channel effectors and have a C-terminal domain belonging to the FKBP fold. Rnk, however, has a much shorter N-terminal coiled coil. Rnk does not stimulate transcript cleavage in vitro, nor does it reduce the lifetime of the complex formed by RNAP on promoters. We show that Rnk competes with the Gre factors and DksA (another RNAP secondary channel effector) for binding to RNAP in vitro, and although we found that the concentration of Rnk in vivo was much lower than that of DksA, it was similar to that of GreB, consistent with a potential regulatory role for Rnk as an anti-Gre factor.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , RNA Ribossômico/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
J Mol Biol ; 377(2): 551-64, 2008 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-18272182

RESUMO

Identification of the RNA polymerase (RNAP) binding site for ppGpp, a central regulator of bacterial transcription, is crucial for understanding its mechanism of action. A recent high-resolution X-ray structure defined a ppGpp binding site on Thermus thermophilus RNAP. We report here effects of ppGpp on 10 mutant Escherichia coli RNAPs with substitutions for the analogous residues within 3-4 A of the ppGpp binding site in the T. thermophilus cocrystal. None of the substitutions in E. coli RNAP significantly weakened its responses to ppGpp. This result differs from the originally reported finding of a substitution in E. coli RNAP eliminating ppGpp function. The E. coli RNAPs used in that study likely lacked stoichiometric amounts of omega, an RNAP subunit required for responses of RNAP to ppGpp, in part explaining the discrepancy. Furthermore, we found that ppGpp did not inhibit transcription initiation by T. thermophilus RNAP in vitro or shorten the lifetimes of promoter complexes containing T. thermophilus RNAP, in contrast to the conclusion in the original report. Our results suggest that the ppGpp binding pocket identified in the cocrystal is not the one responsible for regulation of E. coli ribosomal RNA transcription initiation and highlight the importance of inclusion of omega in bacterial RNAP preparations.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , RNA Ribossômico/genética , Transcrição Genética/genética , Sítios de Ligação , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/enzimologia , Modelos Moleculares , Mutação/genética , Regiões Promotoras Genéticas/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Nucleotídeos de Purina/química , Nucleotídeos de Purina/metabolismo , Homologia Estrutural de Proteína , Thermus thermophilus/enzimologia , Thermus thermophilus/genética
20.
J Mol Biol ; 366(4): 1243-57, 2007 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-17207814

RESUMO

Escherichia coli DksA, GreA, and GreB have similar structures and bind to the same location on RNA polymerase (RNAP), the secondary channel. We show that GreB can fulfil some roles of DksA in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rRNA promoters to respond to changes in the concentration of ppGpp and NTPs. However, unlike deletion of the dksA gene, deletion of greB had no effect on rRNA promoters in vivo. We show that the apparent affinities of DksA and GreB for RNAP are similar, but the cellular concentration of GreB is much lower than that of DksA. When over-expressed and in the absence of competing GreA, GreB almost completely complemented the loss of dksA in control of rRNA expression, indicating its inability to regulate rRNA transcription in vivo results primarily from its low concentration. In contrast to GreB, the apparent affinity of GreA for RNAP was weaker than that of DksA, GreA affected rRNA promoters only modestly in vitro and, even when over-expressed, GreA did not affect rRNA transcription in vivo. Thus, binding in the secondary channel is necessary but insufficient to explain the effect of DksA on rRNA transcription. Neither Gre factor was capable of fulfilling two other functions of DksA in transcription initiation: co-activation of amino acid biosynthetic gene promoters with ppGpp and compensation for the loss of the omega subunit of RNAP in the response of rRNA promoters to ppGpp. Our results provide important clues to the mechanisms of both negative and positive control of transcription initiation by DksA.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/genética , Transcrição Genética , Fatores de Elongação da Transcrição/genética , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/metabolismo , Regiões Promotoras Genéticas , RNA Ribossômico/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Fatores de Elongação da Transcrição/metabolismo
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