A Shift in Central Metabolism Accompanies Virulence Activation in Pseudomonas aeruginosa.

The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period.IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a further challenge to the development of therapeutics. Here, we identify that NADH activity has an important role in the induction of virulence in the pathogen P. aeruginosa This finding, coupled with the ubiquity of NADH in bacterial pathogens, opens up the possibility of targeting enzymes that process NADH as a potential broad antivirulence approach

The Use of Co-Culturing in Solid Substrate Cultivation and Possible Solutions to Scientific Challenges

This perspective systematically summarizes the use of solid substrate co‐cultures in agriculture, food, plant, and industrial biotechnology applications. The summarization is organized by organism, i.e. fungus, bacteria, yeast and then co‐cultivation of either two or three organisms. Generally, in solid substrate co‐culture, the organisms synergistically penetrate and degrade the solid substrate, thereby increasing product yield and productivity over a monoculture. Efforts to increase co‐culture performance include optimizing process parameters (pH, temperature, moisture, and oxygen demand) and defining the acceptable types of substrate. Scientific challenges exist in understanding the interactions between microbial stains, such as viability, suite of products, and bio‐transformations. The perspective details possible solutions to these challenges and highlights future research directions for co‐cultures using either solid or liquid fermentation

A Rapid Image-based Bacterial Virulence Assay Using Amoeba.

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa

Structural insights into the role of Bacillus subtilis YwfH (BacG) in tetrahydrotyrosine synthesis

The synthesis of the dipeptide antibiotic bacilysin involves the sequential action of multiple enzymes in the bac operon. YwfH (also referred to as BacG) catalyzes the stereoselective reduction of dihydro-hydroxyphenylpyruvate (H2HPP) to tetrahydro-hydroxyphenylpyruvate (H4HPP) in this biosynthetic pathway. YwfH is an NADPH-dependent reductase that facilitates the conjugate addition of a hydride at the C4 olefin terminus of H2HPP. Here, the structure of YwfH is described at three conformational steps: the apo form, an apo-like conformation and the NADPH complex. YwfH is structurally similar to other characterized short-chain dehydrogenase/reductases despite having marginal sequence similarity. The structures of YwfH in different conformational states provide a rationale for the ping-pong reaction mechanism. The identification and role of the residues in the catalytic tetrad (Lys113Tyr117Ser155Asn158) in proton transfer were examined by mutational analysis. Together, the structures and biochemical features revealed synchronized conformational changes that facilitate cofactor specificity and catalysis of H4HPP formation en route to tetrahydrotyrosine synthesis

Research Article Fault Tolerant Design for Magnetic Memories

Abstract: This study presents a Fault Tolerant memory cores based on the property of Component Reusability, a method for Fault Tolerance for content addressable memories. The memories used in the design are 256, 512, 1024 and 2048 bytes. The fault is injected into the circuitry operation by using Automatic Test Pattern Generators (ATPGs). The design has been implemented in Cadence 90 nm technology and tested with Fault Injection Circuits and ATPG effectiveness was found out to be 100% at a frequency of 500 MHZ

A Shift in Central Metabolism Accompanies Virulence Activation in Pseudomonas aeruginosa

The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a further challenge to the development of therapeutics. Here, we identify that NADH activity has an important role in the induction of virulence in the pathogen P. aeruginosa. This finding, coupled with the ubiquity of NADH in bacterial pathogens, opens up the possibility of targeting enzymes that process NADH as a potential broad antivirulence approach.The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa. We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period

A Shift in Central Metabolism Accompanies Virulence Activation in Pseudomonas aeruginosa.

The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period.IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a further challenge to the development of therapeutics. Here, we identify that NADH activity has an important role in the induction of virulence in the pathogen P. aeruginosa This finding, coupled with the ubiquity of NADH in bacterial pathogens, opens up the possibility of targeting enzymes that process NADH as a potential broad antivirulence approach

Nanopillared Surfaces Disrupt Pseudomonas aeruginosa Mechanoresponsive Upstream Motility.

Pseudomonas aeruginosa is an opportunistic, multidrug-resistant, human pathogen that forms biofilms in environments with fluid flow, such as the lungs of cystic fibrosis patients, industrial pipelines, and medical devices. P. aeruginosa twitches upstream on surfaces by the cyclic extension and retraction of its mechanoresponsive type IV pili motility appendages. The prevention of upstream motility, host invasion, and infectious biofilm formation in fluid flow systems remains an unmet challenge. Here, we describe the design and application of scalable nanopillared surface structures fabricated using nanoimprint lithography that reduce upstream motility and colonization by P. aeruginosa. We used flow channels to induce shear stress typically found in catheter tubes and microscopy analysis to investigate the impact of nanopillared surfaces with different packing fractions on upstream motility trajectory, displacement, velocity, and surface attachment. We found that densely packed, subcellular nanopillared surfaces, with pillar periodicities ranging from 200 to 600 nm and widths ranging from 70 to 215 nm, inhibit the mechanoresponsive upstream motility and surface attachment. This bacteria-nanostructured surface interface effect allows us to tailor surfaces with specific nanopillared geometries for disrupting cell motility and attachment in fluid flow systems