Disulfide Bond Formation Involves a Quinhydrone-Type Charge–Transfer Complex

The chemistry of disulfide exchange in biological systems is well studied. However, the detailed mechanism of how oxidizing equivalents are derived to form disulfide bonds in proteins is not clear. In prokaryotic organisms, it is known that DsbB delivers oxidizing equivalents through DsbA to secreted proteins. DsbB becomes reoxidized by reducing quinones that are part of the membrane-bound electron-transfer chains. It is this quinone reductase activity that links disulfide bond formation to the electron transport system. We show here that purified DsbB contains the spectral signal of a quinhydrone, a charge-transfer complex consisting of a hydroquinone and a quinone in a stacked configuration. We conclude that disulfide bond formation involves a stacked hydroquinone-benzoquinone pair that can be trapped on DsbB as a quinhydrone charge-transfer complex. Quinhydrones are known to be redox-active and are commonly used as redox standards, but, to our knowledge, have never before been directly observed in biological systems. We also show kinetically that this quinhydrone-type charge-transfer complex undergoes redox reactions consistent with its being an intermediate in the reaction mechanism of DsbB. We propose a simple model for the action of DsbB where a quinhydrone-like complex plays a crucial role as a reaction intermediate

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes

Resipientunderdsøkelse av Begna, Randselva og Tyrifjorden i 2010 i forbindelse med utslipp fra Norske Skog Follum ASA og Huhtamaki Norway AS

Norske Skog Follum og Huhtamaki Norway AS har foretatt en resipientundersøkelse i henhold til vanndirektivet for å dokumentere effektene av sine utslipp til Begna, Randselva og Tyrifjorden. Virkningen av utslippene på vannkjemien (tot P, tot N, NO3, KOF, Mn, Al) var liten eller ikke påvisbar. Begroingsalgene indikerer god eller svært god tilstand på elvestasjonene, bunndyrene indikerer god eller svært god tilstand med unntak av moderat tilstand ved Hønefossen. Det biologiske mangfoldet varierte mye, med høyest mangfold på referansestasjonene. For Tyrifjoden var den økologiske tilstanden svært god der konsentrasjonene av fosfor og klorofyll a var de lavest som er målt siden 1978. Resultatene fra burforsøk med lokal ørret indikerer at aluminium i utslippet ved Follum Fabrikker foreligger på en lite giftig form og er lite gjelle-reaktivt.Norske Skog Follum AS

Investigation of the mechanism of the DsbB holoenzyme.

The DsbA-DsbB pathway is responsible for catalyzing de novo disulfide formation. DsbA is a thioredoxin-like protein that can oxidize a variety of substrate proteins. To function catalytically, DsbA must be maintained in an oxidized state in vivo. This is the role of the membrane protein DsbB. DsbB has the unique ability to generate a disulfide in DsbA by reducing quinone. A second distinct pathway is responsible for isomerizing non-native disulfides, the DsbC-DsbD pathway. The cell uses the disulfide isomerase DsbC to rearrange non-native disulfides, which allows mis-oxidized proteins to achieve their correct fold. To act efficiently DsbC must be maintained in a reduced state. This is the role of DsbD. This work describes 3 characteristics of DsbB that are vital to its in vivo role in disulfide formation. (1) The substrate specificity of DsbB, in part, is responsible for preventing a futile cycle between the DsbA-DsbB and DsbC-DsbD pathways. The DsbA-DsbB and DsbC-DsbD pathways have opposite redox requirements, and it is critical that they be kept isolated from one another in order to prevent a futile cycle. The substrate specificity of DsbB prevents cross-talk between these two pathways (Ch II). (2) During the reoxidation of DsbA, electrons pass through DsbB from the C104/C130 disulfide to the C41/C44 disulfide, and then finally on to quinone. The redox potentials of the two disulfides in DsbB indicate that neither is fit to be the lone reoxidant of DsbA. Thus, the reoxidation of DsbA by DsbB may be coupled to quinone reduction by DsbB (Ch III). (3) Purified DsbB has a brilliant purple color due to a bound cofactor. Based on spectral and kinetic analysis this cofactor appears to be a quinhydrone-like charge-transfer complex, which is involved in the catalytic activity of DsbB (Ch IV). Additionally, a model that DsbB and Ero1p might interact with their cofactors in a similar manner was investigated. Ero1p uses a conserved tryptophan (W200) and histidine (H231) to coordinate its flavin cofactor. The residues H91 and W145 of DsbB were identified as candidates that may be involved in the DsbB-quinone interaction, as W200 and H231 are involved in the Ero1p-flavin interaction. Based on analysis of mutations in H91 and W145, these residues do not appear to play a major role in the activity of DsbB (Ch V).Ph.D.Biological SciencesCellular biologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125204/2/3186742.pd

Turning a disulfide isomerase into an oxidase: DsbC mutants that imitate DsbA

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102154/1/emboj7593659.pd

Identified <i>L. monocytogenes</i> biofilm-formation genes.

<p><i>Putative functions were obtained from <a href="http://www.broadinstitute.org/annotation/genome/listeria_group/MultiHome.html" target="_blank">http://www.broadinstitute.org/annotation/genome/listeria_group/MultiHome.html</a></i>.</p><p><i>Based on DNA homologies with the L. monocytogenes 10403S genome database; lmrg refers to genetic loci within strain 10403S</i>.</p><p><i>% Compared to wild-type L. monocytogenes 10403S biofilm formation in two independent experiments</i>.</p><p>Identified <i>L. monocytogenes</i> biofilm-formation genes.</p

Biofilm formation by Δ<i>phoPR</i> and Δ<i>dltABCD L. monocytogenes</i>.

<p>Bacterial strains were inoculated into TSBYE media in 96-well plates and grown at 35°C for 24 hours. Cultures were then diluted 1:10 into fresh HTM media with 3% glucose and 0.1 mg/mL each cysteine and methionine in new 96-well PVC microtiter plates. Plates were incubated at 35°C for 96 hours, rinsed with dH₂O using a semi-automated cell washer, stained with crystal violet, rinsed with acetic acid and the OD₅₉₅ ±SD determined using a spectrophotometer. The data presented are representative of three independent experiments. *, p <0.05 (One-way ANOVA test).</p

CSLM analysis of <i>L. monocytogenes</i> biofilm production.

<p>Results presented are the means ±SD from two independent experiments performed in triplicate.</p><p>Student's <i>t-test</i> indicated a statistically significant difference between biofilm thickness formed by <i>L. monocytogenes</i> 10403S compared to mutant bacterial strains (p ≤ 0.05).</p><p>CSLM analysis of <i>L. monocytogenes</i> biofilm production.</p

Transmission and scanning electron microscopy analysis of <i>L. monocytogenes</i> EPS production.

<p><i>L. monocytogenes</i> 10403S bacteria in biofilms formed on dialysis tubing membranes (regenerated cellulose) (A) (bar = 100 nm) or planktonic bacteria grown in broth culture (B) (bar = 500 nm) were examined by TEM at 72 hours post-inoculation. (C) SEM of a <i>L. monocytogenes</i> biofilm developed on regenerated cellulose at 24 hours post-inoculation (bar = 10 µm). Arrows indicate EPS. For TEM, samples were fixed with 25% glutaraldehyde, rinsed with cacodylate buffer and stained with ruthenium red to visualize EPS material. For SEM, samples were rinsed with multiple dilutions of ethanol prior to visualization.</p

Scanning electron microscopy of a bean sprout inoculated with <i>L. monocytogenes</i>.

<p>Sterile bean sprouts were placed in HTM agar media with 3% glucose and inoculated with 10 µl of a 1:10 dilution of a 24-hour culture of 10403S. Following a 24 hour incubation, bean sprouts were processed for scanning electron microscopy (A) Bean sprout (bar = 1 mm) (B) magnified view of the white square from (A) (bar = 100 µm). (C) Bean sprout vegetative tissue colonized with <i>L. monocytogenes</i> (bar = 10 µm) (D) magnification of (C) (bar = 10 µm).</p