Redox-Based Regulation of Bacterial Development and Behavior.

Severe changes in the environmental redox potential, and resulting alterations in the oxidation states of intracellular metabolites and enzymes, have historically been considered negative stressors, requiring responses that are strictly defensive. However, recent work in diverse organisms has revealed that more subtle changes in the intracellular redox state can act as signals, eliciting responses with benefits beyond defense and detoxification. Changes in redox state have been shown to influence or trigger chromosome segregation, sporulation, aerotaxis, and social behaviors, including luminescence as well as biofilm establishment and dispersal. Connections between redox state and complex behavior allow bacteria to link developmental choices with metabolic state and coordinate appropriate responses. Promising future directions for this area of study include metabolomic analysis of species- and condition-dependent changes in metabolite oxidation states and elucidation of the mechanisms whereby the redox state influences circadian regulation.

Energy Transfer Between i-Motif DNA Encapsulated Silver Nanoclusters and Fluorescein Amidite Efficiently Visualizes the Redox State of Live Cells.

The redox regulation, maintaining a balance between oxidation and reduction in living cells, is vital for cellular homeostasis, intricate signaling networks, and appropriate responses to physiological and environmental cues. Here, a novel redox sensor, based on DNA-encapsulated silver nanoclusters (DNA/AgNCs) and well-defined chemical fluorophores, effectively illustrating cellular redox states in live cells is introduced. Among various i-motif DNAs, the photophysical property of poly-cytosines (C20)-encapsulated AgNCs that sense reactive oxygen species (ROS) is adopted. However, the sensitivity of C20/AgNCs is insufficient for evaluating ROS levels in live cells. To overcome this drawback, the ROS sensing mechanism of C20/AgNCs through gel electrophoresis, mass spectrometry, and small-angle X-ray scattering is primarily defined. Then, by tethering fluorescein amidite (FAM) and Cyanine 5 (Cy5) dyes to each end of the C20/AgNCs sensor, an Energy Transfer (ET) between AgNCs and FAM is achieved, resulting in intensified green fluorescence upon ROS detection. Taken together, the FAM-C20/AgNCs-Cy5 redox sensor enables dynamic visualization of intracellular redox states, yielding insights into oxidative stress-related processes in live cells.

Redox properties and PAS domain structure of the Escherichia coli energy sensor Aer indicate a multistate sensing mechanism.

The Per-Arnt-Sim (PAS; named for the representative proteins: Period, Aryl hydrocarbon receptor nuclear translocator protein and Single-minded) domain of the dimeric Escherichia coli aerotaxis receptor Aer monitors cellular respiration through a redox-sensitive flavin adenine dinucleotide (FAD) cofactor. Conformational shifts in the PAS domain instigated by the oxidized FAD (FADOX)/FAD anionic semiquinone (FADASQ) redox couple traverse the HAMP (histidine kinases, adenylate cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and kinase control domains of the Aer dimer to regulate CheA kinase activity. The PAS domain of Aer is unstable and has not been previously purified. Here, residue substitutions that rescue FAD binding in an FAD binding-deficient full-length Aer variant were used in combination to stabilize the Aer PAS domain. We solved the 2.4 Å resolution crystal structure of this variant, Aer-PAS-GVV, and revealed a PAS fold that contains distinct features associated with FAD-based redox sensing, such as a close contact between the Arg115 side chain and N5 of the isoalloxazine ring and interactions of the flavin with the side chains of His53 and Asn85 that are poised to convey conformational signals from the cofactor to the protein surface. In addition, we determined the FADox/FADASQ formal potentials of Aer-PAS-GVV and full-length Aer reconstituted into nanodiscs. The Aer redox couple is remarkably low at -289.6 ± 0.4 mV. In conclusion, we propose a model for Aer energy sensing based on the low potential of Aer-PAS-FADox/FADASQ couple and the inability of Aer-PAS to bind to the fully reduced FAD hydroquinone.

ToxT Regulon Is Nonessential for Vibrio cholerae Colonization in Adult Mice.

Vibrio cholerae is the causative agent of cholera, a life-threatening diarrheal disease in humans. The ability of V. cholerae to colonize the intestine of different animals is a key factor for its fitness and transmissibility between hosts. Many virulence factors, including the ToxT regulon, have been identified to be the major components allowing V. cholerae to colonize the small intestine of suckling mice; however, the mechanism of V. cholerae colonization in the adult mammalian intestine is unclear. In this study, using the streptomycin-treated adult mouse animal model, we characterized the role of the ToxT regulon in V. cholerae colonization in adult mammalian intestine. We first found that the activity of TcpP regulating ToxT regulon expression was attenuated by intestinal reactive oxygen species (ROS). We then found that V. cholerae containing a deletion of the ToxT regulon showed a competition advantage in colonizing adult mice; however, a mutant containing a constitutively active ToxT regulon showed a significant defect in colonizing adult mice. Constitutively producing the virulence factors in the ToxT regulon causes a V. cholerae competition defect in nutrient-limiting conditions. The results of this study demonstrate that modulating the activity of the ToxT regulon through ROS sensed by TcpP is critical for V. cholerae to enhance its colonization in the intestine of adult mice. IMPORTANCE Vibrio cholerae can inhabit both marine and freshwater ecosystems and can also enter and proliferate in the intestine of different animals which consume contaminated food or water. To successfully colonize the intestines of different hosts, V. cholerae coordinates its gene expression in response to different environments. Here, we describe how V. cholerae modulates the activity of the ToxT regulon by TcpP sensing ROS signals in the intestine of adult mice to better survive in this environment. We found that the constitutively active ToxT regulon causes V. cholerae growth retardation and colonization defect in adult mice. Our work highlights the distinctive role that regulating the activity of the ToxT regulon plays for V. cholerae to achieve full survival fitness in the adult mammalian intestine.

Extracellular Cysteines Are Critical to Form Functional Cx46 Hemichannels.

Connexin (Cxs) hemichannels participate in several physiological and pathological processes, but the molecular mechanisms that control their gating remain elusive. We aimed at determining the role of extracellular cysteines (Cys) in the gating and function of Cx46 hemichannels. We studied Cx46 and mutated all of its extracellular Cys to alanine (Ala) (one at a time) and studied the effects of the Cys mutations on Cx46 expression, localization, and hemichannel activity. Wild-type Cx46 and Cys mutants were expressed at comparable levels, with similar cellular localization. However, functional experiments showed that hemichannels formed by the Cys mutants did not open either in response to membrane depolarization or removal of extracellular divalent cations. Molecular-dynamics simulations showed that Cys mutants may show a possible alteration in the electrostatic potential of the hemichannel pore and an altered disposition of important residues that could contribute to the selectivity and voltage dependency in the hemichannels. Replacement of extracellular Cys resulted in "permanently closed hemichannels", which is congruent with the inhibition of the Cx46 hemichannel by lipid peroxides, through the oxidation of extracellular Cys. These results point to the modification of extracellular Cys as potential targets for the treatment of Cx46-hemichannel associated pathologies, such as cataracts and cancer, and may shed light into the gating mechanisms of other Cx hemichannels.

Multistep Signaling in Nature: A Close-Up of Geobacter Chemotaxis Sensing.

Environmental changes trigger the continuous adaptation of bacteria to ensure their survival. This is possible through a variety of signal transduction pathways involving chemoreceptors known as methyl-accepting chemotaxis proteins (MCP) that allow the microorganisms to redirect their mobility towards favorable environments. MCP are two-component regulatory (or signal transduction) systems (TCS) formed by a sensor and a response regulator domain. These domains synchronize transient protein phosphorylation and dephosphorylation events to convert the stimuli into an appropriate cellular response. In this review, the variability of TCS domains and the most common signaling mechanisms are highlighted. This is followed by the description of the overall cellular topology, classification and mechanisms of MCP. Finally, the structural and functional properties of a new family of MCP found in Geobacter sulfurreducens are revisited. This bacterium has a diverse repertoire of chemosensory systems, which represents a striking example of a survival mechanism in challenging environments. Two G. sulfurreducens MCP-GSU0582 and GSU0935-are members of a new family of chemotaxis sensor proteins containing a periplasmic PAS-like sensor domain with a c-type heme. Interestingly, the cellular location of this domain opens new routes to the understanding of the redox potential sensing signaling transduction pathways.

The redox-sensing transcriptional repressor Rex is important for regulating the products distribution in Thermoanaerobacterium aotearoense SCUT27.

The redox-sensing transcriptional repressor Rex (Rex) displayed diverse functions in different microbial species. Nowadays, only part function of rex has been verified in vitro and alcohol dehydrogenase gene (adhE) as the target of Rex has been widely reported. In this study, rex was knocked out in Thermoanaerobacterium aotearoense SCUT27 (GDMCC 60765) and the carbon metabolic distribution analysis was performed. Results showed that the ethanol yield (mol product/mol carbon) of SCUT27(Δrex) had increased by 75.00-90.91%, cell growth improved by 27.27-36.36%, and acetic acid and lactic acid decreased by 58.33-61.54% accompanied with the yield of hydrogen decreased by 46.15-58.35% within different carbon sources. The ability of sugar consumption of SCUT27(Δrex) had improved about 74.19-130.55% with the improvement of total ATP concentration and the cofactors NADH and NAD+ concentrations. In addition, the specific activities of alcohol dehydrogenase of SCUT27(Δrex) with NADH and NADPH as cofactors were improved by 119.26-140.28% and 35.66-47.69%, respectively. After ldh was further knocked out in SCUT27(Δrex), SCUT27(ΔldhΔrex) showed higher ethanol production and yield when various carbon resources were used as substrates (including glucose, xylose, glucose/xylose mixture and three kinds of lignocellulosic hydrolysates). This study confirms that Rex is an important regulator for determining products distribution in SCUT27 and deletion of rex and ldh is a promising strategy for enhanced ethanol production.

Zinc Differentially Modulates the Assembly of Soluble and Polymerized Vimentin.

The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress. We previously showed that vimentin interacts with zinc, which affects its assembly and redox sensing. Here, we used vimentin wt and C328S, an oxidation-resistant mutant showing improved NaCl-induced polymerization, to assess the impact of zinc on soluble and polymerized vimentin by light scattering and electron microscopy. Zinc acts as a switch, reversibly inducing the formation of vimentin oligomeric species. High zinc concentrations elicit optically-detectable vimentin structures with a characteristic morphology depending on the support. These effects also occur in vimentin C328S, but are not mimicked by magnesium. Treatment of vimentin with micromolar ZnCl2 induces fibril-like particles that do not assemble into filaments, but form aggregates upon subsequent addition of NaCl. In contrast, when added to NaCl-polymerized vimentin, zinc increases the diameter or induces lateral association of vimentin wt filaments. Remarkably, these effects are absent or attenuated in vimentin C328S filaments. Therefore, the zinc-vimentin interaction depends on the chemical environment and on the assembly state of the protein, leading to atypical polymerization of soluble vimentin, likely through electrostatic interactions, or to broadening and lateral association of preformed filaments through mechanisms requiring the cysteine residue. Thus, the impact of zinc on vimentin assembly and redox regulation is envisaged.

Modulation of Pseudomonas aeruginosa Quorum Sensing by Glutathione.

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of a battery of secreted products. At least some of these products are shared among the population and serve as public goods. When P. aeruginosa is grown on casein as the sole carbon and energy source, the QS-induced extracellular protease elastase is required for growth. We isolated a P. aeruginosa variant, which showed increased production of QS-induced factors after repeated transfers in casein broth. This variant, P. aeruginosa QS*, had a mutation in the glutathione synthesis gene gshA We describe several experiments that show a gshA coding variant and glutathione affect the QS response. The P. aeruginosa QS transcription factor LasR has a redox-sensitive cysteine (C79). We report that GshA variant cells with a LasR C79S substitution show a similar QS response to that of wild-type P. aeruginosa Surprisingly, it is not LasR but the QS transcription factor RhlR that is more active in bacteria containing the variant gshA Our results demonstrate that QS integrates information about cell density and the cellular redox state via glutathione levels.IMPORTANCEPseudomonas aeruginosa and other bacteria coordinate group behaviors using a chemical communication system called quorum sensing (QS). The QS system of P. aeruginosa is complex, with several regulators and signals. We show that decreased levels of glutathione lead to increased gene activation in P. aeruginosa, which did not occur in a strain carrying the redox-insensitive variant of a transcription factor. The ability of P. aeruginosa QS transcription factors to integrate information about cell density and cellular redox state shows these transcription factors can fine-tune levels of the gene products they control in response to at least two types of signals or cues.

Staphylococcus aureus adaptation to aerobic low-redox-potential environments: implications for an intracellular lifestyle.

Methicillin-resistant Staphylococcus aureus is a 'superbug' that is responsible for extensive death and morbidity. Chronic S. aureus infections are associated with the presence of intracellular bacteria and the host cytosol is an aerobic low-redox-potential (Eh) environment. How S. aureus adapts to aerobic low-Eh environments is understudied. A low external Eh, imposed by the non-metabolizable reductant dithiothreitol, resulted in transcriptional reprogramming mediated by the redox-responsive transcription factors AgrA, Rex and SrrBA, resulting in a shift towards fermentative metabolism. Accordingly, in the presence of the host cytoplasmic reductant glutathione, the aerobic respiration of S. aureus was impaired, the intracellular NADH:NAD+ ratio increased, lactate dehydrogenase was induced, resistance to the aminoglycoside antibiotic gentamicin was enhanced and greater numbers of small-colony variants (SCVs) were detected. These observations suggest that entry of S. aureus into the aerobic low-Eh environment of the host cytosol could result in adaptive responses that promote the formation of SCVs.

Characterization of Frex as an NADH sensor for in vivo applications in the presence of NAD+ and at various pH values.

The fluorescent biosensor Frex, recently introduced as a sensitive tool to quantify the NADH concentration in living cells, was characterized by time-integrated and time-resolved fluorescence spectroscopy regarding its applicability for in vivo measurements. Based on the purified sensor protein, it is shown that the NADH dependence of Frex fluorescence can be described by a Hill function with a concentration of half-maximal sensor response of K D ≈ 4 µM and a Hill coefficient of n ≈ 2. Increasing concentrations of NADH have moderate effects on the fluorescence lifetime of Frex, which changes by a factor of two from about 500 ps in the absence of NADH to 1 ns under fluorescence-saturating NADH concentrations. Therefore, the observed sevenfold rise of the fluorescence intensity is primarily ascribed to amplitude changes. Notably, the dynamic range of Frex sensitivity towards NADH highly depends on the NAD+ concentration, while the apparent K D for NADH is only slightly affected. We found that NAD+ has a strong inhibitory effect on the fluorescence response of Frex during NADH sensing, with an apparent NAD+ dissociation constant of K I ≈ 400 µM. This finding was supported by fluorescence lifetime measurements, which showed that the addition of NAD+ hardly affects the fluorescence lifetime, but rather reduces the number of Frex molecules that are able to bind NADH. Furthermore, the fluorescence responses of Frex to NADH and NAD+ depend critically on pH and temperature. Thus, for in vivo applications of Frex, temperature and pH need to be strictly controlled or considered during data acquisition and analysis. If all these constraints are properly met, Frex fluorescence intensity measurements can be employed to estimate the minimum NADH concentration present within the cell at sufficiently low NAD+ concentrations below 100 µM.

Redox sensing molecular mechanism of an iron metabolism regulatory protein FBXL5.

FBXL5 is a subunit of the SCFFBXL5 ubiquitin ligase complex that targets the proteasomal degradation of iron regulatory protein IRP2, which is an important regulator in iron metabolism. The degradation of FBXL5 itself is regulated in an iron- and oxygen-responsive manner through its diiron center containing Hr-like domain. Although the crystal structure of the Hr-like domain of FBXL5 and its degradation based on iron/oxygen sensing has been reported, the redox sensing molecular mechanism is still not clear. Herein the redox properties of FBXL5 were investigated via EPR, direct electrochemistry, SRCD, fluorescence emission spectroscopy, and redox kinetics. The results indicated that the conformation and function of FBXL5 are tuned by the redox states of the diiron center. The redox reactions of the diiron center are accompanied with conformational changes and iron release, which are associated with FBXL5 stability and degradation. These results provide insights into the redox sensing mechanism by which FBXL5 can serve as an iron metabolism regulator within mammalian cells.

VDAC3 gating is activated by suppression of disulfide-bond formation between the N-terminal region and the bottom of the pore.

The voltage-dependent anion channels (VDACs), VDAC1, VDAC2, and VDAC3, are pore-forming proteins that control metabolite flux between mitochondria and cytoplasm. VDAC1 and VDAC2 have voltage-dependent gating activity, whereas VDAC3 is thought to have weak activity. The aim of this study was to analyze the channel properties of all three human VDAC isoforms and to clarify the channel function of VDAC3. Bacterially expressed recombinant human VDAC proteins were reconstituted into artificial planar lipid bilayers and their gating activities were evaluated. VDAC1 and VDAC2 had typical voltage-dependent gating activity, whereas the gating of VDAC3 was weak, as reported. However, gating of VDAC3 was evoked by dithiothreitol (DTT) and S-nitrosoglutathione (GSNO), which are thought to suppress disulfide-bond formation. Several cysteine mutants of VDAC3 also exhibited typical voltage-gating. Our results indicate that channel gating was induced by reduction of a disulfide-bond linking the N-terminal region to the bottom of the pore. Thus, channel gating of VDAC3 might be controlled by redox sensing under physiological conditions.

Lactobacillus oligofermentans glucose, ribose and xylose transcriptomes show higher similarity between glucose and xylose catabolism-induced responses in the early exponential growth phase.

BACKGROUND: Lactobacillus oligofermentans has been mostly isolated from cold-stored packaged meat products in connection with their spoilage, but its precise role in meat spoilage is unknown. It belongs to the L. vaccinostercus group of obligate heterofermentative lactobacilli that generally ferment pentoses (e.g. xylose and ribose) more efficiently than hexoses (e.g. glucose). However, more efficient hexose utilization can be induced. The regulation mechanisms of the carbohydrate catabolism in such bacteria have been scarcely studied. To address this question, we provided the complete genome sequence of L. oligofermentans LMG 22743(T) and generated time course transcriptomes during its growth on glucose, ribose and xylose. RESULTS: The genome was manually annotated and its main functional features were examined. L. oligofermentans was confirmed to be able to efficiently utilize several hexoses and maltose, which is, presumably, induced by its repeated cultivation with glucose in vitro. Unexpectedly, in the beginning of the exponential growth phase, glucose- and xylose-induced transcriptome responses were more similar, whereas toward the end of the growth phase xylose and ribose transcriptomes became more alike. The promoter regions of genes simultaneously upregulated both on glucose and xylose in comparison with ribose (particularly, hexose and xylose utilization genes) were found to be enriched in the CcpA- binding site. Transcriptionally, no glucose-induced carbon catabolite repression was detected. The catabolism of glucose, which requires initial oxidation, led to significant overexpression of the NAD(P)H re-oxidation genes, the upstream regions of which were found to contain a motif, which was highly similar to a Rex repressor binding site. CONCLUSIONS: This paper presents the second complete genome and the first study of carbohydrate catabolism-dependent transcriptome response for a member of the L. vaccinostercus group. The transcriptomic changes detected in L. oligofermentans for growth with different carbohydrates differ significantly from those of facultative heterofermentative lactobacilli. The mechanism of CcpA regulation, putatively contributing to the observed similarities between glucose- and xylose-induced transcriptome responses and the absence of stringent carbon catabolite control, requires further studies. Finally, the cell redox balance maintenance, in terms of the NAD(P)+/NAD(P)H ratio, was predicted to be regulated by the Rex transcriptional regulator, supporting the previously made inference of Rex-regulons for members of the Lactobacillaceae family.

Cytochrome b 6 f function and localization, phosphorylation state of thylakoid membrane proteins and consequences on cyclic electron flow.

Both the structure and the protein composition of thylakoid membranes have an impact on light harvesting and electron transfer in the photosynthetic chain. Thylakoid membranes form stacks and lamellae where photosystem II and photosystem I localize, respectively. Light-harvesting complexes II can be associated to either PSII or PSI depending on the redox state of the plastoquinone pool, and their distribution is governed by state transitions. Upon state transitions, the thylakoid ultrastructure and lateral distribution of proteins along the membrane are subject to significant rearrangements. In addition, quinone diffusion is limited to membrane microdomains and the cytochrome b 6 f complex localizes either to PSII-containing grana stacks or PSI-containing stroma lamellae. Here, we discuss possible similarities or differences between green algae and C3 plants on the functional consequences of such heterogeneities in the photosynthetic electron transport chain and propose a model in which quinones, accepting electrons either from PSII (linear flow) or NDH/PGR pathways (cyclic flow), represent a crucial control point. Our aim is to give an integrated description of these processes and discuss their potential roles in the balance between linear and cyclic electron flows.

Nitrosopersulfide (SSNO-) targets the Keap-1/Nrf2 redox system.

Nitric oxide (NO), hydrogen sulfide and polysulfides have been proposed to contribute to redox signaling by activating the Keap-1/Nrf2 stress response system. Nitrosopersulfide (SSNO-) recently emerged as a bioactive product of the chemical interaction of NO or nitrosothiols with sulfide; upon decomposition it generates polysulfides and free NO, triggering the activation of soluble guanylate cyclase, inducing blood vessel relaxation in vitro and lowering blood pressure in vivo. Whether SSNO- itself interacts with the Keap-1/Nrf2 system is unknown. We therefore sought to investigate the ability of SSNO- to activate Nrf2-dependent processes in human vascular endothelial cells, and to compare the pharmacological effects of SSNO- with those of its precursors NO and sulfide at multiple levels of target engagement. We here demonstrate that SSNO- strongly increases nuclear levels, binding activity and transactivation activity of Nrf2, thereby increasing mRNA expression of Hmox-1, the gene encoding for heme oxygenase 1, without adversely affecting cell viability. Under all conditions, SSNO- appeared to be more potent than its parent compounds, NO and sulfide. SSNO--induced Nrf2 transactivation activity was abrogated by either NO scavenging with cPTIO or inhibition of thiol sulfuration by high concentrations of cysteine, implying a role for both persulfides/polysulfides and NO in SSNO- mediated Nrf2 activation. Taken together, our studies demonstrate that the Keap-1/Nrf2 redox system is a biological target of SSNO-, enriching the portfolio of bioactivity of this vasoactive molecule to also engage in the regulation of redox signaling processes. The latter suggests a possible role as messenger and/or mediator in cellular sensing and adaptations processes.

Significance of redox-active cysteines in human FAD synthase isoform 2.

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399-C402, C303-C312, both in the PAPS domain), and two unstable disulfides (C39-C50; C440-C464). Whereas the C39-C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.

Oxygen Microenvironments in E. coli Biofilm Nutrient Transport Channels: Insights from Complementary Sensing Approaches.

Chemical gradients and the emergence of distinct microenvironments in biofilms are vital to the stratification, maturation and overall function of microbial communities. These gradients have been well characterised throughout the biofilm mass but the microenvironment of recently discovered nutrient transporting channels in Escherichia coli biofilms remains unexplored. This study employs three different oxygen sensing approaches to provide a robust quantitative overview of the oxygen gradients and microenvironments throughout the biofilm transport channel networks formed by E. coli macrocolony biofilms. Oxygen nanosensing combined with confocal laser scanning microscopy established that the oxygen concentration changes along the length of biofilm transport channels. Electrochemical sensing provided precise quantification of the oxygen profile in the transport channels, showing similar anoxic profiles compared with the adjacent cells. Anoxic biosensing corroborated these approaches, providing an overview of the oxygen utilisation throughout the biomass. The discovery that transport channels maintain oxygen gradients contradicts the previous literature that channels are completely open to the environment along the apical surface of the biofilm. We provide a potential mechanism for the sustenance of channel microenvironments via orthogonal visualisations of biofilm thin sections showing thin layers of actively growing cells. This complete overview of the oxygen environment in biofilm transport channels primes future studies aiming to exploit these emergent structures for new bioremediation approaches.

Oxygen affinities of DosT and DosS sensor kinases with implications for hypoxia adaptation in Mycobacterium tuberculosis.

DosT and DosS are heme-based kinases involved in sensing and signaling O2 tension in the microenvironment of Mycobacterium tuberculosis (Mtb). Under conditions of low O2, they activate >50 dormancy-related genes and play a pivotal role in the induction of dormancy and associated drug resistance during tuberculosis infection. In this work, we reexamine the O2 binding affinities of DosT and DosS to show that their equilibrium dissociation constants are 3.3±1 µM and 0.46±0.08 µM respectively, which are six to eight-fold stronger than what has been widely referred to in literature. Furthermore, stopped-flow kinetic studies reveal association and dissociation rate constants of 0.84 µM-1s-1 and 2.8 s-1, respectively for DosT, and 7.2 µM-1s-1 and 3.3 s-1, respectively for DosS. Remarkably, these tighter O2 binding constants correlate with distinct stages of hypoxia-induced non-replicating persistence in the Wayne model of Mtb. This knowledge opens doors to deconvoluting the intricate interplay between hypoxia adaptation stages and the signal transduction capabilities of these important heme-based O2 sensors.

Mitochondrial ribosome biogenesis and redox sensing.

Mitoribosome biogenesis is a complex process involving RNA elements encoded in the mitochondrial genome and mitoribosomal proteins typically encoded in the nuclear genome. This process is orchestrated by extra-ribosomal proteins, nucleus-encoded assembly factors, which play roles across all assembly stages to coordinate ribosomal RNA processing and maturation with the sequential association of ribosomal proteins. Both biochemical studies and recent cryo-EM structures of mammalian mitoribosomes have provided insights into their assembly process. In this article, we will briefly outline the current understanding of mammalian mitoribosome biogenesis pathways and the factors involved. Special attention is devoted to the recent identification of iron-sulfur clusters as structural components of the mitoribosome and a small subunit assembly factor, the existence of redox-sensitive cysteines in mitoribosome proteins and assembly factors, and the role they may play as redox sensor units to regulate mitochondrial translation under stress.