RESUMO
The three-dimensional structures of chromosomes are increasingly being recognized as playing a major role in cellular regulatory states. The efficiency and promiscuity of phage Mu transposition was exploited to directly measure in vivo interactions between genomic loci in E. coli. Two global organizing principles have emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation.
Assuntos
Bacteriófago mu/genética , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/química , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Conformação de Ácido Nucleico , Transposases/genética , Transposases/metabolismoRESUMO
Efflux is a common mechanism of resistance to antibiotics. We show that efflux itself promotes accumulation of antibiotic-resistance mutations (ARMs). This phenomenon was initially discovered in a bacterial swarm where the linked phenotypes of high efflux and high mutation frequencies spatially segregated to the edge, driven there by motility. We have uncovered and validated a global regulatory network connecting high efflux to downregulation of specific DNA-repair pathways even in non-swarming states. The efflux-DNA repair link was corroborated in a clinical "resistome" database: genomes with mutations that increase efflux exhibit a significant increase in ARMs. Accordingly, efflux inhibitors decreased evolvability to antibiotic resistance. Swarms also revealed how bacterial populations serve as a reservoir of ARMs even in the absence of antibiotic selection pressure. High efflux at the edge births mutants that, despite compromised fitness, survive there because of reduced competition. This finding is relevant to biofilms where efflux activity is high.
Assuntos
Antibacterianos , Bactérias , Resistência Microbiana a Medicamentos , Transporte Biológico , Antibacterianos/farmacologia , Bactérias/genéticaRESUMO
The importance of memory in bacterial decision-making is relatively unexplored. We show here that a prior experience of swarming is remembered when Escherichia coli encounters a new surface, improving its future swarming efficiency. We conducted >10,000 single-cell swarm assays to discover that cells store memory in the form of cellular iron levels. This "iron" memory preexists in planktonic cells, but the act of swarming reinforces it. A cell with low iron initiates swarming early and is a better swarmer, while the opposite is true for a cell with high iron. The swarming potential of a mother cell, which tracks with its iron memory, is passed down to its fourth-generation daughter cells. This memory is naturally lost by the seventh generation, but artificially manipulating iron levels allows it to persist much longer. A mathematical model with a time-delay component faithfully recreates the observed dynamic interconversions between different swarming potentials. We demonstrate that cellular iron levels also track with biofilm formation and antibiotic tolerance, suggesting that iron memory may impact other physiologies.
Assuntos
Escherichia coli , Ferro , Escherichia coli/genética , AntibacterianosRESUMO
FliL is a bacterial flagellar protein demonstrated to associate with, and regulate ion flow through, the stator complex in a diverse array of bacterial species. FliL is also implicated in additional functions such as stabilizing the flagellar rod, modulating rotor bias, sensing the surface, and regulating gene expression. How can one protein do so many things? Its location is paramount to understanding its numerous functions. This review will look at the evidence, attempt to resolve some conflicting findings, and offer new thoughts on FliL.
RESUMO
FliL is an essential component of the flagellar machinery in some bacteria, but a conditional one in others. The conditional role is for optimal swarming in some bacteria. During swarming, physical forces associated with movement on a surface are expected to exert a higher load on the flagellum, requiring more motor torque to move. FliL was reported to enhance motor output in several bacteria and observed to assemble as a ring around ion-conducting stators that power the motor. In this study we identify a common new function for FliL in diverse bacteria-Escherichia coli, Bacillus subtilis, and Proteus mirabilis. During swarming, all these bacteria show increased cell speed and a skewed motor bias that suppresses cell tumbling. We demonstrate that these altered motor parameters, or "motor remodeling," require FliL. Both swarming and motor remodeling can be restored in an E. coli fliL mutant by complementation with fliL genes from P. mirabilis and B. subtilis, showing conservation of a swarming-associated FliL function across phyla. In addition, we demonstrate that the strong interaction we reported earlier between FliL and the flagellar MS-ring protein FliF is confined to the RBM-3 domain of FliF that links the periplasmic rod to the cytoplasmic C-ring. This interaction may explain several phenotypes associated with the absence of FliL.
Assuntos
Proteínas de Bactérias , Proteínas de Membrana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Movimento , Flagelos/metabolismoRESUMO
The Gam protein of transposable phage Mu is an ortholog of eukaryotic and bacterial Ku proteins, which carry out nonhomologous DNA end joining (NHEJ) with the help of dedicated ATP-dependent ligases. Many bacteria carry Gam homologs associated with either complete or defective Mu-like prophages, but the role of Gam in the life cycle of Mu or in bacteria is unknown. Here, we show that MuGam is part of a two-component bacterial NHEJ DNA repair system. Ensemble and single-molecule experiments reveal that MuGam binds to DNA ends, slows the progress of RecBCD exonuclease, promotes binding of NAD+-dependent Escherichia coli ligase A, and stimulates ligation. In vivo, Gam equally promotes both precise and imprecise joining of restriction enzyme-digested linear plasmid DNA, as well as of a double-strand break (DSB) at an engineered I-SceI site in the chromosome. Cell survival after the induced DSB is specific to the stationary phase. In long-term growth competition experiments, particularly upon treatment with a clastogen, the presence of gam in a Mu lysogen confers a distinct fitness advantage. We also show that the role of Gam in the life of phage Mu is related not to transposition but to protection of genomic Mu copies from RecBCD when viral DNA packaging begins. Taken together, our data show that MuGam provides bacteria with an NHEJ system and suggest that the resulting fitness advantage is a reason that bacteria continue to retain the gam gene in the absence of an intact prophage.
Assuntos
Bacteriófago mu/metabolismo , Reparo do DNA por Junção de Extremidades/fisiologia , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Virais/metabolismo , Bacteriófago mu/genética , Bacteriófago mu/crescimento & desenvolvimento , DNA Ligases/química , Empacotamento do DNA/fisiologia , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Exodesoxirribonuclease V/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Estrutura Quaternária de Proteína , Homologia Estrutural de Proteína , Proteínas Virais/químicaRESUMO
In Escherichia coli and Salmonella, the c-di-GMP effector YcgR inhibits flagellar motility by interacting directly with the motor to alter both its bias and speed. Here, we demonstrate that in both of these bacteria, YcgR acts sequentially, altering motor bias first and then decreasing motor speed. We show that when c-di-GMP levels are high, deletion of ycgR restores wild-type motor behavior in E. coli, indicating that YcgR is the only motor effector in this bacterium. Yet, motility and chemotaxis in soft agar do not return to normal, suggesting that there is a second mechanism that inhibits motility under these conditions. In Salmonella, c-di-GMP-induced synthesis of extracellular cellulose has been reported to entrap flagella and to be responsible for the YcgR-independent motility defect. We found that this is not the case in E. coli Instead, we found through reversion analysis that deletion of rssB, which codes for a response regulator/adaptor protein that normally directs ClpXP protease to target σS for degradation, restored wild-type motility in the ycgR mutant. Our data suggest that high c-di-GMP levels may promote altered interactions between these proteins to downregulate flagellar gene expression.IMPORTANCE Flagellum-driven motility has been studied in E. coli and Salmonella for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in E. coli, we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σS regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated.
Assuntos
GMP Cíclico/análogos & derivados , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/genética , Flagelos/fisiologia , Regulon/fisiologia , Fatores de Transcrição/fisiologia , GMP Cíclico/fisiologia , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão GênicaRESUMO
We describe a mechanism of flagellar motor control by the bacterial signaling molecule c-di-GMP, which regulates several cellular behaviors. E. coli and Salmonella have multiple c-di-GMP cyclases and phosphodiesterases, yet absence of a specific phosphodiesterase YhjH impairs motility in both bacteria. yhjH mutants have elevated c-di-GMP levels and require YcgR, a c-di-GMP-binding protein, for motility inhibition. We demonstrate that YcgR interacts with the flagellar switch-complex proteins FliG and FliM, most strongly in the presence of c-di-GMP. This interaction reduces the efficiency of torque generation and induces CCW motor bias. We present a "backstop brake" model showing how both effects can result from disrupting the organization of the FliG C-terminal domain, which interacts with the stator protein MotA to generate torque. Inhibition of motility and chemotaxis may represent a strategy to prepare for sedentary existence by disfavoring migration away from a substrate on which a biofilm is to be formed.
Assuntos
Quimiotaxia/fisiologia , Proteínas de Escherichia coli/metabolismo , Flagelos/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Modelos Moleculares , Proteínas Motores Moleculares/genética , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TorqueRESUMO
The genome of transposable phage Mu is packaged as a linear segment, flanked by several hundred base pairs of non-Mu DNA. The linear ends are held together and protected from nucleases by the phage N protein. After transposition into the Escherichia coli chromosome, the flanking DNA (FD) is degraded, and the 5-bp gaps left in the target are repaired to generate a simple Mu insertion. Our study provides insights into this repair pathway. The data suggest that the first event in repair is removal of the FD by the RecBCD exonuclease, whose entry past the N-protein block is licensed by the transpososome. In vitro experiments reveal that, when RecBCD is allowed entry into the FD, it degrades this DNA until it arrives at the transpososome, which presents a barrier for further RecBCD movement. RecBCD action is required for stimulating endonucleolytic cleavage within the transpososome-protected DNA, leaving 4-nt flanks outside both Mu ends. This end product of collaboration between the transpososome and RecBCD resembles the intermediate products of Tn7 and retroviral and retrotransposon transposition, and may hint at a common gap-repair mechanism in these diverse transposons.
Assuntos
Bacteriófago mu/genética , Bacteriófago mu/metabolismo , Elementos de DNA Transponíveis/genética , Exodesoxirribonuclease V/metabolismo , Substituição de Aminoácidos , Reparo do DNA , DNA Viral/genética , DNA Viral/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Escherichia coli K12/virologia , Células HEK293 , Humanos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Transposases/química , Transposases/genética , Transposases/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
We report a new cellular interaction between the infecting transposable phage Mu and the host Escherichia coli replication machinery during repair of Mu insertions, which involves filling-in of short target gaps on either side of the insertion, concomitant with degradation of extraneous long flanking DNA (FD) linked to Mu. Using the FD as a marker to follow repair, we find that after transposition into the chromosome, the unrepaired Mu is indefinitely stable until the replication fork arrives at the insertion site, whereupon the FD is rapidly degraded. When the fork runs into a Mu target gap, a double strand end (DSE) will result; we demonstrate fork-dependent DSEs proximal to Mu. These findings suggest that Pol III stalled at the transpososome is exploited for co-ordinated repair of both target gaps flanking Mu without replicating the intervening 37 kb of Mu, disassembling the stable transpososome in the process. This work is relevant to all transposable elements, including retroviral elements like HIV-1, which share with Mu the common problem of repair of their flanking target gaps.
Assuntos
Bacteriófago mu/genética , Reparo do DNA , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Transposases/genética , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Replicação do DNA , DNA Viral/genética , Escherichia coli/metabolismo , Recombinação Genética , Transposases/metabolismoRESUMO
The E. coli chromosome is compacted by segregation into 400-500 supercoiled domains by both active and passive mechanisms, for example, transcription and DNA-protein association. We find that prophage Mu is organized as a stable domain bounded by the proximal location of Mu termini L and R, which are 37 kbp apart on the Mu genome. Formation/maintenance of the Mu 'domain' configuration, reported by Cre-loxP recombination and 3C (chromosome conformation capture), is dependent on a strong gyrase site (SGS) at the center of Mu, the Mu L end and MuB protein, and the E. coli nucleoid proteins IHF, Fis and HU. The Mu domain was observed at two different chromosomal locations tested. By contrast, prophage λ does not form an independent domain. The establishment/maintenance of the Mu domain was promoted by low-level transcription from two phage promoters, one of which was domain dependent. We propose that the domain confers transposition readiness to Mu by fostering topological requirements of the reaction and the proximity of Mu ends. The potential benefits to the host cell from a subset of proteins expressed by the prophage may in turn help its long-term stability.
Assuntos
Bacteriófago mu/genética , Cromossomos Bacterianos/genética , Cromossomos/genética , Escherichia coli/genética , Bacteriófago lambda/genética , DNA Girase/genética , Replicação do DNA/genética , DNA Topoisomerases Tipo II/genética , Lisogenia , Replicação Viral/genéticaRESUMO
Phage Mu is unique among transposable elements in employing a transposition enhancer. The enhancer DNA segment is the site where the transposase MuA binds and makes bridging interactions with the two Mu ends, interwrapping the ends with the enhancer in a complex topology essential for assembling a catalytically active transpososome. The enhancer is also the site at which regulatory proteins control divergent transcription of genes that determine the phage lysis-lysogeny decision. Here we report a third function for the enhancer - that of regulating degradation of extraneous DNA attached to both ends of infecting Mu. This DNA is protected from nucleases by a phage protein until Mu integrates into the host chromosome, after which it is rapidly degraded. We find that leftward transcription at the enhancer, expected to disrupt its topology within the transpososome, blocks degradation of this DNA. Disruption of the enhancer would lead to the loss or dislocation of two non-catalytic MuA subunits positioned in the transpososome by the enhancer. We provide several lines of support for this inference, and conclude that these subunits are important for activating degradation of the flanking DNA. This work also reveals a role for enhancer topology in phage development.
Assuntos
Bacteriófago mu/enzimologia , Bacteriófago mu/genética , Elementos de DNA Transponíveis , DNA/metabolismo , Sítios de Ligação , Evolução Molecular , Hidrólise , Ligação Proteica , Recombinação Genética , Proteínas Virais/metabolismoRESUMO
Chemotaxis enhances the fitness of Salmonella enterica serotype Typhimurium (S. Typhimurium) during colitis. However, the chemotaxis receptors conferring this fitness advantage and their cognate signals generated during inflammation remain unknown. Here we identify respiratory electron acceptors that are generated in the intestinal lumen as by-products of the host inflammatory response as in vivo signals for methyl-accepting chemotaxis proteins (MCPs). Three MCPs, including Trg, Tsr and Aer, enhanced the fitness of S. Typhimurium in a mouse colitis model. Aer mediated chemotaxis towards electron acceptors (energy taxis) in vitro and required tetrathionate respiration to confer a fitness advantage in vivo. Tsr mediated energy taxis towards nitrate but not towards tetrathionate in vitro and required nitrate respiration to confer a fitness advantage in vivo. These data suggest that the energy taxis receptors Tsr and Aer respond to distinct in vivo signals to confer a fitness advantage upon S. Typhimurium during inflammation by enabling this facultative anaerobic pathogen to seek out favorable spatial niches containing host-derived electron acceptors that boost its luminal growth.
Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia , Colite/microbiologia , Metabolismo Energético , Proteínas de Membrana/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Transporte/metabolismo , Colite/imunologia , Transporte de Elétrons , Feminino , Inflamação , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Proteínas Quimiotáticas Aceptoras de Metil , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Neutrófilos/imunologia , Nitratos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/metabolismo , Salmonelose Animal/imunologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/fisiologia , Ácido Tetratiônico/metabolismoRESUMO
Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5' flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli-the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps.
Assuntos
Bacteriófago mu , Reparo do DNA , Elementos de DNA Transponíveis/genética , Escherichia coli , Recombinação Homóloga/genética , Bacteriófago mu/genética , Bacteriófago mu/crescimento & desenvolvimento , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma , Mutagênese Insercional , Mutação , Transposases/metabolismoRESUMO
Flagella are highly complex rotary molecular machines that enable bacteria to not only migrate to optimal environments but to also promote range expansion, competitiveness, virulence, and antibiotic survival. Flagellar motility is an energy-demanding process, where the sum of its production (biosynthesis) and operation (rotation) costs has been estimated to total ~10% of the entire energy budget of an E. coli cell. The acquisition of such a costly adaptation process is expected to secure short-term benefits by increasing competitiveness and survival, as well as long-term evolutionary fitness gains. While the role of flagellar motility in bacterial survival has been widely reported, its direct influence on the rate of evolution remains unclear. We show here that both production and operation costs contribute to elevated mutation frequencies. Our findings suggest that flagellar movement may be an important player in tuning the rate of bacterial evolution.
RESUMO
Many approaches for measuring three-dimensional chromosomal conformations rely upon formaldehyde crosslinking followed by subsequent proximity ligation, a family of methods exemplified by 3C, Hi-C, etc. Here we provide an alternative crosslinking-free procedure for high-throughput identification of long-range contacts in the chromosomes of enterobacteria, making use of contact-dependent transposition of phage Mu to identify distant loci in close contact. The procedure described here will suffice to provide a comprehensive map of transposition frequencies between tens of thousands of loci in a bacterial genome, with the resolution limited by the diversity of the insertion site library used and the sequencing depth applied.
Assuntos
Mapeamento Cromossômico , Cromossomos Bacterianos , Escherichia coli , Escherichia coli/genética , Cromossomos Bacterianos/genética , Mapeamento Cromossômico/métodos , Bacteriófago mu/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Elementos de DNA Transponíveis/genéticaRESUMO
We show in this study that Salmonella cells, which do not upregulate flagellar gene expression during swarming, also do not increase flagellar numbers per µm of cell length as determined by systematic counting of both flagellar filaments and hooks. Instead, doubling of the average length of a swarmer cell by suppression of cell division effectively doubles the number of flagella per cell. The highest agar concentration at which Salmonella cells swarmed increased from the normal 0.5% to 1%, either when flagella were overproduced or when expression of the FliL protein was enhanced in conjunction with stator proteins MotAB. We surmise that bacteria use the resulting increase in motor power to overcome the higher friction associated with harder agar. Higher flagellar numbers also suppress the swarming defect of mutants with changes in the chemotaxis pathway that were previously shown to be defective in hydrating their colonies. Here we show that the swarming defect of these mutants can also be suppressed by application of osmolytes to the surface of swarm agar. The "dry" colony morphology displayed by che mutants was also observed with other mutants that do not actively rotate their flagella. The flagellum/motor thus participates in two functions critical for swarming, enabling hydration and overriding surface friction. We consider some ideas for how the flagellum might help attract water to the agar surface, where there is no free water.
Assuntos
Flagelos/fisiologia , Salmonella enterica/genética , Salmonella enterica/fisiologia , Ágar , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular , Fatores Quimiotáticos/genética , Quimiotaxia/genética , Quimiotaxia/fisiologia , DNA Bacteriano/genética , Flagelos/genética , Flagelos/metabolismo , Fricção , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Transdução de Sinais/genéticaRESUMO
Movement over an agar surface via swarming motility is subject to formidable challenges not encountered during swimming. Bacteria display a great deal of flexibility in coping with these challenges, which include attracting water to the surface, overcoming frictional forces, and reducing surface tension. Bacteria that swarm on "hard" agar surfaces (robust swarmers) display a hyperflagellated and hyperelongated morphology. Bacteria requiring a "softer" agar surface (temperate swarmers) do not exhibit such a dramatic morphology. For polarly flagellated robust swarmers, there is good evidence that restriction of flagellar rotation somehow signals the induction of a large number of lateral flagella, but this scenario is apparently not relevant to temperate swarmers. Swarming bacteria can be further subdivided by their requirement for multiple stators (Mot proteins) or a stator-associated protein (FliL), secretion of essential polysaccharides, cell density-dependent gene regulation including surfactant synthesis, a functional chemotaxis signaling pathway, appropriate cyclic (c)-di-GMP levels, induction of virulence determinants, and various nutritional requirements such as iron limitation or nitrate availability. Swarming strategies are as diverse as the bacteria that utilize them. The strength of these numerous designs stems from the vantage point they offer for understanding mechanisms for effective colonization of surface niches, acquisition of pathogenic potential, and identification of environmental signals that regulate swarming. The signature swirling and streaming motion within a swarm is an interesting phenomenon in and of itself, an emergent behavior with properties similar to flocking behavior in diverse systems, including birds and fish, providing a convenient new avenue for modeling such behavior.
Assuntos
Fenômenos Fisiológicos Bacterianos , Flagelos/fisiologia , Ágar , Bactérias/genética , Bactérias/patogenicidade , Proteínas de Bactérias/metabolismo , Quimiotaxia , Meios de Cultura , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Fricção , Proteínas de Membrana/metabolismo , Percepção de Quorum , Tensão Superficial , VirulênciaRESUMO
flhE belongs to the flhBAE flagellar operon in Enterobacteria, whose first two members function in Type III secretion (T3S). In Salmonella enterica, absence of FlhE affects swarming, but not swimming, motility. Based on a chance observation of a 'green' colony phenotype of flhE mutants on pH indicator plates containing glucose, we have established that this phenotype is associated with lysis of flagellated cells in an acidic environment created by glucose metabolism. The flhE mutant phenotype of Escherichia coli is similar overall to that of S. enterica but is seen in the absence of glucose and, unlike in S. enterica, causes a substantial growth defect. flhE mutants have a lowered cytoplasmic pH in both bacteria, indicative of a proton leak. GFP reporter assays indicate that the leak is dependent on the flagellar system, is present before the T3S system switches to secretion of late substrates, and gets worse after the switch and upon filament assembly, leading to cell lysis. We show that FlhE is a periplasmic protein that co-purifies with flagellar basal bodies. FlhE may act as a plug or a chaperone to regulate proton flow through the flagellar T3S system.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Escherichia coli/metabolismo , Flagelos/metabolismo , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Escherichia coli/química , Escherichia coli/genética , Flagelos/química , Flagelos/genética , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Prótons , Salmonella typhimurium/química , Salmonella typhimurium/genética , Deleção de SequênciaRESUMO
Chemoreceptors McpB and McpC in Salmonella enterica have been reported to promote chemotaxis in LB motility-plate assays. Of the chemicals tested as potential effectors of these receptors, the only response was towards L-cysteine and its oxidized form, L-cystine. Although enhanced radial migration in plates suggested positive chemotaxis to both amino acids, capillary assays failed to show an attractant response to either, in cells expressing only these two chemoreceptors. In vivo fluorescence resonance energy transfer (FRET) measurements of kinase activity revealed that in wild-type bacteria, cysteine and cystine are chemoeffectors of opposing sign, the reduced form being a chemoattractant and the oxidized form a repellent. The attractant response to cysteine was mediated primarily by Tsr, as reported earlier for Escherichia coli. The repellent response to cystine was mediated by McpB/C. Adaptive recovery upon cystine exposure required the methyl-transferase/-esterase pair, CheR/CheB, but restoration of kinase activity was never complete (i.e. imperfect adaptation). We provide a plausible explanation for the attractant-like responses to both cystine and cysteine in motility plates, and speculate that the opposing signs of response to this redox pair might afford Salmonella a mechanism to gauge and avoid oxidative environments.