Many bacteria use extracellular signaling molecules to coordinate group behavior, a process referred to as quorum sensing (QS). However, some QS molecules are hydrophobic in character and are ...probably unable to diffuse across the bacterial cell envelope. How these molecules are disseminated between bacterial cells within a population is not yet fully understood. Here we show that the marine pathogen
packages the hydrophobic QS molecule CAI-1, a long-chain amino ketone, into outer membrane vesicles. Electron micrographs indicate that outer membrane vesicles of variable size are predominantly produced and released into the surroundings during stationary phase of
, which correlates with the timing of CAI-1-dependent signaling. The large vesicles (diameter < 55 nm) can trigger a QS phenotype in CAI-1 non-producing
and
cells. Packaging of CAI-1 into outer membrane vesicles might stabilize the molecule in aqueous environments and facilitate its distribution over distances.
Formation of membrane vesicles is ubiquitous among bacteria. These vesicles are involved in protein and DNA transfer and offer new approaches for vaccination. Gram-negative bacteria use among others hydrophobic signaling molecules for cell-cell communication, however due to their hydrophobic character it is unclear how these molecules are disseminated between bacterial cells. Here we show that the marine
packages one of its quorum sensing molecules, the long-chain ketone CAI-1, into outer membrane vesicles (OMVs). Isolated CAI-1-containing vesicles trigger a quorum sensing phenotype in CAI-1 non-producing
and also in
cells. Packaging of CAI-1 into OMVs not only solubilize, stabilize and concentrate this class of molecules, but facilitate their distribution between bacteria that live in aqueous environments.
Quorum sensing is a typical communication system among Gram-negative bacteria used to control group-coordinated behavior via small diffusible molecules dependent on cell number. The key components of ...a quorum sensing system are a LuxI-type synthase, producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor that detects AHLs to control expression of specific target genes. Six conserved amino acids are present in the signal-binding domain of AHL-sensing LuxR-type proteins, which are important for ligand-binding and -specificity as well as shaping the ligand-binding pocket. However, many proteobacteria possess LuxR-type regulators without a cognate LuxI synthase, referred to as LuxR solos. The two LuxR solos PluR and PauR from Photorhabdus luminescens and Photorhabdus asymbiotica, respectively, do not sense AHLs. Instead PluR and PauR sense alpha-pyrones and dialkylresorcinols, respectively, and are part of cell-cell communication systems contributing to the overall virulence of these Photorhabdus species. However, PluR and PauR both harbor substitutions in the conserved amino acid motif compared to that in AHL sensors, which appeared to be important for binding the corresponding signaling molecules. Here we analyze the role of the conserved amino acids in the signal-binding domain of these two non-AHL LuxR-type receptors for their role in signal perception. Our studies reveal that the conserved amino acid motif alone is essential but not solely responsible for ligand-binding.
The acid stress response is an important factor influencing the transmission of intestinal microbes such as the enterobacterium Escherichia coli. E. coli activates three inducible acid resistance ...systems - the glutamate decarboxylase, arginine decarboxylase, and lysine decarboxylase systems to counteract acid stress. Each system relies on the activity of a proton-consuming reaction catalyzed by a specific amino acid decarboxylase and a corresponding antiporter. Activation of these three systems is tightly regulated by a sophisticated interplay of membrane-integrated and soluble regulators. Using a fluorescent triple reporter strain, we quantitatively illuminated the cellular individuality during activation of each of the three acid resistance (AR) systems under consecutively increasing acid stress. Our studies highlight the advantages of E. coli in possessing three AR systems that enable division of labor in the population, which ensures survival over a wide range of low pH values.
Dialkylresorcinols as bacterial signaling molecules Brameyer, Sophie; Kresovic, Darko; Bode, Helge B. ...
Proceedings of the National Academy of Sciences,
01/2015, Letnik:
112, Številka:
2
Journal Article
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It is well recognized that bacteria communicate via small diffusible molecules, a process termed quorum sensing. The best understood quorum sensing systems are those that use acylated homoserine ...lactones (AHLs) for communication. The prototype of those systems consists of a LuxI-like AHL synthase and a cognate LuxR receptor that detects the signal. However, many proteobacteria possess LuxR receptors, yet lack any LuxI-type synthase, and thus these receptors are referred to as LuxR orphans or solos. In addition to the well-known AHLs, little is known about the signaling molecules that are sensed by LuxR solos. Here, we describe a novel cell–cell communication system in the insect and human pathogen Photorhabdus asymbiotica . We identified the LuxR homolog PauR to sense dialkylresorcinols (DARs) and cyclohexanediones (CHDs) instead of AHLs as signals. The DarABC synthesis pathway produces the molecules, and the entire system emerged as important for virulence. Moreover, we have analyzed more than 90 different Photorhabdus strains by HPLC/MS and showed that these DARs and CHDs are specific to the human pathogen P. asymbiotica . On the basis of genomic evidence, 116 other bacterial species are putative DAR producers, among them many human pathogens. Therefore, we discuss the possibility of DARs as novel and widespread bacterial signaling molecules and show that bacterial cell–cell communication goes far beyond AHL signaling in nature.
Significance Bacteria can communicate with each other by small diffusible molecules, a process termed quorum sensing. Many bacteria use acylated homoserine lactones (AHLs) as signals, which are sensed by so-called LuxR-type receptors. With the photopyrones from the insect pathogenic bacterium Photorhabdus luminescens , we recently identified the first quorum sensing molecules different from AHLs that are sensed by a LuxR-type receptor. Here we describe the second novel quorum sensing molecule sensed by a LuxR-type receptor of Photorhabdus species, PauR of the human pathogen Photorhabdus asymbiotica . We demonstrate that P. asymbiotica communicates via dialkylresorcinols (DARs) and cyclohexanediones (CHDs). As the synthesis pathway is widespread, and often present in human pathogens, we discuss DARs and CHDs as novel and widespread signaling molecules.
Membrane proteins account for about one third of the cellular proteome, but it is still unclear how dynamic they are and how they establish functional contacts with cytoplasmic interaction partners. ...Here, we consider a membrane-integrated one-component receptor that also acts as a transcriptional activator, and analyze how it kinetically locates its specific binding site on the genome. We focus on the case of CadC, the pH receptor of the acid stress response Cad system in E. coli. CadC is a prime example of a one-component signaling protein that directly binds to its cognate target site on the chromosome to regulate transcription. We combined fluorescence microscopy experiments, mathematical analysis, and kinetic Monte Carlo simulations to probe this target search process. Using fluorescently labeled CadC, we measured the time from activation of the receptor until successful binding to the DNA in single cells, exploiting that stable receptor-DNA complexes are visible as fluorescent spots. Our experimental data indicate that CadC is highly mobile in the membrane and finds its target by a 2D diffusion and capture mechanism. DNA mobility is constrained due to the overall chromosome organization, but a labeled DNA locus in the vicinity of the target site appears sufficiently mobile to randomly come close to the membrane. Relocation of the DNA target site to a distant position on the chromosome had almost no effect on the mean search time, which was between four and five minutes in either case. However, a mutant strain with two binding sites displayed a mean search time that was reduced by about a factor of two. This behavior is consistent with simulations of a coarse-grained lattice model for the coupled dynamics of DNA within a cell volume and proteins on its surface. The model also rationalizes the experimentally determined distribution of search times. Overall our findings reveal that DNA target search does not present a much bigger kinetic challenge for membrane-integrated proteins than for cytoplasmic proteins. More generally, diffusion and capture mechanisms may be sufficient for bacterial membrane proteins to establish functional contacts with cytoplasmic targets.
Dedifferentiation is a critical response to tissue damage, yet is not well understood, even at a basic phenomenological level. Developing
cells undergo highly efficient dedifferentiation, completed ...by most cells within 24 hr. We use this rapid response to investigate the control features of dedifferentiation, combining single cell imaging with high temporal resolution transcriptomics. Gene expression during dedifferentiation was predominantly a simple reversal of developmental changes, with expression changes not following this pattern primarily associated with ribosome biogenesis. Mutation of genes induced early in dedifferentiation did not strongly perturb the reversal of development. This apparent robustness may arise from adaptability of cells: the relative temporal ordering of cell and molecular events was not absolute, suggesting cell programmes reach the same end using different mechanisms. In addition, although cells start from different fates, they rapidly converged on a single expression trajectory. These regulatory features may contribute to dedifferentiation responses during regeneration.
Bacterial histidine kinase/response regulator systems operate at the interface between environmental cues and physiological states. Escherichia coli contains two LytS/LytTR-type histidine ...kinase/response regulator systems, BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB, which have been identified as pyruvate-responsive two-component systems. Since they exhibit remarkable similarity, we analyzed their phylogenetic distribution within the γ-proteobacteria, and experimentally characterized them in a set of representative species. We found that BtsS/BtsR is the predominant LytS/LytTR-type two-component system among γ-proteobacteria, whereas YpdA/YpdB primarily appears in a supplementary role. Based on our observations in E. coli, we used the highly conserved DNA-binding motifs to test the in vivo functionality of both systems in various genera, including Salmonella, Enterobacter, Citrobacter, Xenorhabdus, Yersinia, Aeromonas and Vibrio. The results suggest that, in all cases tested, BtsS/BtsR and YpdA/YpdB respond to different levels of pyruvate in the environment.
Fluctuating environments and individual physiological diversity force bacteria to constantly adapt and optimize the uptake of substrates. We focus here on two very similar two-component systems ...(TCSs) of
belonging to the LytS/LytTR family: BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB. Both TCSs respond to extracellular pyruvate, albeit with different affinities, typically during postexponential growth, and each system regulates expression of a single transporter gene,
and
, respectively. To obtain insights into the biological significance of these TCSs, we analyzed the activation of the target promoters at the single-cell level. We found unimodal cell-to-cell variability; however, the degree of variance was strongly influenced by the available nutrients and differed between the two TCSs. We hypothesized that activation of either of the TCSs helps individual cells to replenish carbon resources. To test this hypothesis, we compared wild-type cells with the
mutant under two metabolically modulated conditions: protein overproduction and persister formation. Although all wild-type cells were able to overproduce green fluorescent protein (GFP), about half of the
population was unable to overexpress GFP. Moreover, the percentage of persister cells, which tolerate antibiotic stress, was significantly lower in the wild-type cells than in the
population. Hence, we suggest that the BtsS/BtsR and YpdA/YpdB network contributes to a balancing of the physiological state of all cells within a population.
Histidine kinase/response regulator (HK/RR) systems enable bacteria to respond to environmental and physiological fluctuations.
and other members of the
possess two similar LytS/LytTR-type HK/RRs, BtsS/BtsR (formerly YehU/YehT) and YpdA/YpdB, which form a functional network. Both systems are activated in response to external pyruvate, typically when cells face overflow metabolism during post-exponential growth. Single-cell analysis of the activation of their respective target genes
and
revealed cell-to-cell variability, and the range of variation was strongly influenced by externally available nutrients. Based on the phenotypic characterization of a
mutant compared to the parental strain, we suggest that this TCS network supports an optimization of the physiological state of the individuals within the population.
All living cells have a large number of proteins that are anchored with one transmembrane helix in the cytoplasmic membrane. Almost nothing is known about their spatiotemporal organization in whole ...cells. Here we report on the localization and dynamics of one representative, the pH sensor and transcriptional regulator CadC in
. Fluorophore-tagged CadC was detectable as distinct cluster only when the receptor was activated by external stress, which results in DNA-binding. Clusters immediately disappeared under non-stress conditions. CadC variants that mimic the active state of CadC independent of environmental stimuli corroborated the correlation between CadC clustering and binding to the DNA, as did altering the number or location of the DNA-binding site(s) in whole cells. These studies reveal a novel diffusion-and-capture mechanism to organize a membrane-integrated receptor dependent on the DNA in a rod-shaped bacterium.
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