173 research outputs found
Bacterially Speaking
Bacteria use a variety of means to communicate with one another and with their eukaryotic hosts. In some cases, social interactions allow bacteria to synchronize the behavior of all of the members of the group and thereby act like multicellular organisms. By contrast, some bacterial social engagements promote individuality among members within the group and thereby foster diversity. Here we explore the molecular mechanisms underpinning some recently discovered bacterial communication systems. These include long- and short-range chemical signaling channels; one-way, two-way, and multi-way communication; contact-mediated and contact-inhibited signaling; and the use and spread of misinformation or, more dramatically, even deadly information
Loss of PopZ At activity in Agrobacterium tumefaciens by Deletion or Depletion Leads to Multiple Growth Poles, Minicells, and Growth Defects.
Agrobacterium tumefaciens grows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing protein A. tumefaciens PopZ (PopZ At ), we created deletions of the five PopZ At domains and assayed their localization. In addition, we created a popZAt deletion strain (ΔpopZAt ) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZ At strain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZAt cells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZ At , to stabilize growth pole accumulation of PopZ AtIMPORTANCEAgrobacterium tumefaciens is a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZ At , a homologue of Caulobacter crescentus PopZ (PopZ Cc ), a protein essential in many aspects of pole identity in C. crescentus We report that the loss of PopZ At leads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth, understanding this process in A. tumefaciens may lead to the development of new strategies to prevent the proliferation of these pathogens. In addition, studies of A. tumefaciens will provide new insights into the evolution of the genetic networks that regulate bacterial polar growth and cell division
Biofilm streamers cause catastrophic disruption of flow with consequences for environmental and medical systems.
Biofilms are antibiotic-resistant, sessile bacterial communities that occupy most moist surfaces on Earth and cause chronic and medical device-associated infections. Despite their importance, basic information about biofilm dynamics in common ecological environments is lacking. Here, we demonstrate that flow through soil-like porous materials, industrial filters, and medical stents dramatically modifies the morphology of Pseudomonas aeruginosa biofilms to form 3D streamers, which, over time, bridge the spaces between obstacles and corners in nonuniform environments. We discovered that accumulation of surface-attached biofilm has little effect on flow through such environments, whereas biofilm streamers cause sudden and rapid clogging. We demonstrate that flow-induced shedding of extracellular matrix from surface-attached biofilms generates a sieve-like network that captures cells and other biomass, which add to the existing network, causing exponentially fast clogging independent of growth. These results suggest that biofilm streamers are ubiquitous in nature and strongly affect flow through porous materials in environmental, industrial, and medical systems
Information processing and signal integration in bacterial quorum sensing
Bacteria communicate using secreted chemical signaling molecules called
autoinducers in a process known as quorum sensing. The quorum-sensing network
of the marine bacterium {\it Vibrio harveyi} employs three autoinducers, each
known to encode distinct ecological information. Yet how cells integrate and
interpret the information contained within the three autoinducer signals
remains a mystery. Here, we develop a new framework for analyzing signal
integration based on Information Theory and use it to analyze quorum sensing in
{\it V. harveyi}. We quantify how much the cells can learn about individual
autoinducers and explain the experimentally observed input-output relation of
the {\it V. harveyi} quorum-sensing circuit. Our results suggest that the need
to limit interference between input signals places strong constraints on the
architecture of bacterial signal-integration networks, and that bacteria likely
have evolved active strategies for minimizing this interference. Here we
analyze two such strategies: manipulation of autoinducer production and
feedback on receptor number ratios.Comment: Supporting information is in appendi
Deducing Receptor Signaling Parameters from In Vivo Analysis: LuxN/AI-1 Quorum Sensing in Vibrio harveyi
SummaryQuorum sensing, a process of bacterial cell-cell communication, relies on production, detection, and response to autoinducer signaling molecules. LuxN, a nine-transmembrane domain protein from Vibrio harveyi, is the founding example of membrane-bound receptors for acyl-homoserine lactone (AHL) autoinducers. We used mutagenesis and suppressor analyses to identify the AHL-binding domain of LuxN and discovered LuxN mutants that confer both decreased and increased AHL sensitivity. Our analysis of dose-response curves of multiple LuxN mutants pins these inverse phenotypes on quantifiable opposing shifts in the free-energy bias of LuxN for occupying its kinase and phosphatase states. To understand receptor activation and to characterize the pathway signaling parameters, we exploited a strong LuxN antagonist, one of fifteen small-molecule antagonists we identified. We find that quorum-sensing-mediated communication can be manipulated positively and negatively to control bacterial behavior and, more broadly, that signaling parameters can be deduced from in vivo data
Individual contextual factors in the validation of the Bernese pain scale for neonates: protocol for a prospective observational study
Background: The Bernese Pain Scale for Neonates (BPSN) is a multidimensional pain assessment tool that is already
widely used in clinical settings in the German speaking areas of Europe. Recent findings indicate that pain
responses in preterm neonates are influenced by individual contextual factors, such as gestational age (GA), gender
and the number of painful procedures experienced. Currently, the BPSN does not consider individual contextual
factors. Therefore, the aim of this study is the validation of the BPSN using a large sample of neonates with
different GAs. Furthermore, the influence of individual contextual factors on the variability in pain reactions across
GA groups will be explored. The results will be used for a modification of the BPSN to account for individual
contextual factors in future clinical pain assessment in neonates.
Methods and design: This prospective multisite validation study with a repeated measures design will take place
in three university hospital neonatal intensive care units (NICUs) in Switzerland (Bern, Basel and Zurich). To examine
the impact of GA on pain responses and their variability, the infants will be stratified into six GA groups ranging
from 24 0/7 to 42 0/7. Among preterm infants, 2–5 routine capillary heel sticks within the first 14 days of life, and
among full-term infants, two heel sticks during the first days of life will be documented. For each heel stick,
measurements will be video recorded for each of three phases: baseline, heel stick, and recovery. The infants’ pain
responses will be rated according to the BPSN by five nurses who are blinded as to the number of each heel stick
and as to the measurement phases. Individual contextual factors of interest will be extracted from patient charts.
Discussion: Understanding and considering the influence of individual contextual factors on pain responses in a
revised version of the BPSN will help the clinical staff to more appropriately assess pain in neonates, particularly
preterm neonates hospitalized in NICUs. Pain assessment is a first step toward appropriate and efficient pain
management, which itself is an important factor in later motor and cognitive development in this vulnerable
patient population.
Trial registration: The study is registered in the database of Clinical Trial gov. Study ID-number: NCT 02749461.
Registration date: 12 April 2016.
Keywords: Pain assessment, Premature infants, Contextual factors, Diagnosti
Measurement of the copy number of the master quorum-sensing regulator of a bacterial cell
Quorum sensing is the mechanism by which bacteria communicate and synchronize
group behaviors. Quantitative information on parameters such as the copy number
of particular quorum-sensing proteins should contribute strongly to
understanding how the quorum-sensing network functions. Here we show that the
copy number of the master regulator protein LuxR in Vibrio harveyi, can be
determined in vivo by exploiting small-number fluctuations of the protein
distribution when cells undergo division. When a cell divides, both its volume
and LuxR protein copy number N are partitioned with slight asymmetries. We have
measured the distribution functions describing the partitioning of the protein
fluorescence and the cell volume. The fluorescence distribution is found to
narrow systematically as the LuxR population increases while the volume
partitioning is unchanged. Analyzing these changes statistically, we have
determined that N = 80-135 dimers at low cell density and 575 dimers at high
cell density. In addition, we have measured the static distribution of LuxR
over a large (3,000) clonal population. Combining the static and time-lapse
experiments, we determine the magnitude of the Fano factor of the distribution.
This technique has broad applicability as a general, in vivo technique for
measuring protein copy number and burst size.Comment: Main text 23 pages, 5 figures. Supporting material 19 pages, 7
figures. In new version, text revised, one figure reformatte
Non-uniform growth and surface friction determine bacterial biofilm morphology on soft substrates
During development, organisms acquire three-dimensional shapes with important
physiological consequences. While the basic mechanisms underlying morphogenesis
are known in eukaryotes, it is often difficult to manipulate them in vivo. To
circumvent this issue, here we present a study of developing Vibrio cholerae
biofilms grown on agar substrates in which the spatiotemporal morphological
patterns were altered by varying the agar concentration. Expanding biofilms are
initially flat, but later experience a mechanical instability and become
wrinkled. Whereas the peripheral region develops ordered radial stripes, the
central region acquires a zigzag herringbone-like wrinkle pattern. Depending on
the agar concentration, the wrinkles initially appear either in the peripheral
region and propagate inward (low agar concentration) or in the central region
and propagate outward (high agar concentration). To understand these
experimental observations, we developed a model that considers diffusion of
nutrients and their uptake by bacteria, bacterial growth/biofilm matrix
production, mechanical deformation of both the biofilm and the agar, and the
friction between them. Our model demonstrates that depletion of nutrients
beneath the central region of the biofilm results in radially-dependent growth
profiles, which in turn, produce anisotropic stresses that dictate the
morphology of wrinkles. Furthermore, we predict that increasing surface
friction (agar concentration) reduces stress anisotropy and shifts the location
of the maximum compressive stress, where the wrinkling instability first
occurs, toward the center of the biofilm, in agreement with our experimental
observations. Our results are broadly applicable to bacterial biofilms with
similar morphologies and also provide insight into how other bacterial biofilms
form distinct wrinkle patterns.Comment: 16 pages, 4 figures + supplementary information (36 pages, 14
figures
Quantifying the Integration of Quorum-Sensing Signals with Single-Cell Resolution
Cell-to-cell communication in bacteria is a process known as quorum sensing that relies on the production, detection, and response to the extracellular accumulation of signaling molecules called autoinducers. Often, bacteria use multiple autoinducers to obtain information about the vicinal cell density. However, how cells integrate and interpret the information contained within multiple autoinducers remains a mystery. Using single-cell fluorescence microscopy, we quantified the signaling responses to and analyzed the integration of multiple autoinducers by the model quorum-sensing bacterium Vibrio harveyi. Our results revealed that signals from two distinct autoinducers, AI-1 and AI-2, are combined strictly additively in a shared phosphorelay pathway, with each autoinducer contributing nearly equally to the total response. We found a coherent response across the population with little cell-to-cell variation, indicating that the entire population of cells can reliably distinguish several distinct conditions of external autoinducer concentration. We speculate that the use of multiple autoinducers allows a growing population of cells to synchronize gene expression during a series of distinct developmental stages
Gene dosage compensation calibrates four regulatory RNAs to control Vibrio cholerae quorum sensing
Quorum sensing is a mechanism of cell-to-cell communication that allows bacteria to coordinately regulate gene expression in response to changes in cell-population density. At the core of the Vibrio cholerae quorum-sensing signal transduction pathway reside four homologous small RNAs (sRNAs), named the quorum regulatory RNAs 1–4 (Qrr1–4). The four Qrr sRNAs are functionally redundant. That is, expression of any one of them is sufficient for wild-type quorum-sensing behaviour. Here, we show that the combined action of two feedback loops, one involving the sRNA-activator LuxO and one involving the sRNA-target HapR, promotes gene dosage compensation between the four qrr genes. Gene dosage compensation adjusts the total Qrr1–4 sRNA pool and provides the molecular mechanism underlying sRNA redundancy. The dosage compensation mechanism is exquisitely sensitive to small perturbations in Qrr levels. Precisely maintained Qrr levels are required to direct the proper timing and correct patterns of expression of quorum-sensing-regulated target genes
- …