ECF-Type ATP-Binding Cassette Transporters.

Energy-coupling factor (ECF)-type ATP-binding cassette (ABC) transporters catalyze membrane transport of micronutrients in prokaryotes. Crystal structures and biochemical characterization have revealed that ECF transporters are mechanistically distinct from other ABC transport systems. Notably, ECF transporters make use of small integral membrane subunits (S-components) that are predicted to topple over in the membrane when carrying the bound substrate from the extracellular side of the bilayer to the cytosol. Here, we review the phylogenetic diversity of ECF transporters as well as recent structural and biochemical advancements that have led to the postulation of conceptually different mechanistic models. These models can be described as power stroke and thermal ratchet. Structural data indicate that the lipid composition and bilayer structure are likely to have great impact on the transport function. We argue that study of ECF transporters could lead to generic insight into membrane protein structure, dynamics, and interaction.

Design of a tunable bacterial gene expression system using engineered σ factors.

Extracytoplasmic function (ECF) σ factors selectively upregulate expression of specific genes in bacteria. These σ factors, belonging to the σ70 family, are much smaller than the primary, housekeeping σ factor with two helical domains that interact with the Pribnow box and the -35 element of the promoter DNA. Structural studies reveal that promoter specificity in a σ factor is determined by the interactions between a loop (L3) and the Pribnow box element. Similarly, the efficiency of transcription initiation is governed by the polypeptide linker between the two promoter-binding domains. Both these polypeptide segments are dynamic and poorly conserved among ECF σ factor homologs. This feature hitherto limited insights from protein-DNA interactions to be correlated with transcription initiation efficiency. Here, we describe an approach to characterize these features that govern the dynamic range of gene expression using chimeric Escherichia coli σE. The L3 loop and linker polypeptides in these σE chimeras were replaced by the corresponding segments from 10 annotated and functional Mycobacterium tuberculosis ECF σ's. In vitro and in vivo measurements to determine the effect of these polypeptide replacements provided an experimentally validated σE chimera- gene expression level data set. We illustrate the utility of this chimeric σE library in improving the efficiency of a biosynthetic pathway in E. coli. In a two-enzyme step, unaffected by feedback inhibition and substrate concentration, we show an increase in desired product levels by altering the relative intracellular levels of the target enzymes using this library of σ factors. The chimeric σE library thus demonstrates the feasibility of engineering σ factors to achieve bespoke expression levels of target genes for diverse applications in synthetic microbiology. IMPORTANCE: The synthesis of organic compounds involves the action of multiple enzymes in a biosynthetic pathway. Incorporating such biosynthetic pathways into microbes often leads to substantial cellular and metabolic stress resulting in low titers of the target compound. This limitation can be offset, in part, by optimizing enzyme efficiency and cellular enzyme concentration. The former involves significant efforts to achieve improvements in catalytic efficiency with the caveat that the metabolic load on a microbial cell imposed by the overexpression of the exogenous enzyme could result in reduced cell fitness. Here, we demonstrate the feasibility of engineered σ factors to modulate gene expression levels without significant genetic engineering. We note that changing the sequence of two flexible polypeptide loops without any changes to the structural scaffold of the transcription initiation factor σE could modulate the expression levels of the target genes. This ability provides a route to improve the efficiency of a biosynthetic pathway without altering the overall genomic makeup. The σE chimera library thus provides an avenue for pre-determined conditional gene expression of specific genes in Escherichia coli.

Target repurposing unravels avermectins and derivatives as novel antibiotics inhibiting energy-coupling factor transporters (ECFTs).

Energy-coupling factor transporters (ECFTs) are membrane-bound ATP-binding cassette (ABC) transporters in prokaryotes that are found in pathogens against which novel antibiotics are urgently needed. To date, just 54 inhibitors of three molecular-structural classes with mostly weak inhibitory activity are known. Target repurposing is a strategy that transfers knowledge gained from a well-studied protein family to under-studied targets of phylogenetic relation. Forty-eight human ABC transporters are known that may harbor structural motifs similar to ECFTs to which particularly multitarget compounds may bind. We assessed 31 multitarget compounds which together target the entire druggable human ABC transporter proteome against ECFTs, of which nine showed inhibitory activity (hit rate 29.0%) and four demonstrated moderate to strong inhibition of an ECFT (IC50 values between 4.28 and 50.2 µM) as well as antibacterial activity against ECFT-expressing Streptococcus pneumoniae. Here, ivermectin was the most potent candidate (MIC95: 22.8 µM), and analysis of five ivermectin derivatives revealed moxidectin as one of the most potent ECFT-targeting antibacterial agents (IC50: 2.23 µM; MIC95: 2.91 µM). Distinct molecular-structural features of avermectins and derivatives as well as the differential biological response of the hit compounds in general provided first indications with respect to the structure-activity relationships and mode of action, respectively.

Regulation of the general stress response sigma factor σT by Lon-mediated proteolysis.

IMPORTANCE: Regulated protein degradation is a critical process in all cell types, which contributes to the precise regulation of protein amounts in response to internal and external cues. In bacteria, protein degradation is carried out by ATP-dependent proteases. Although past work revealed detailed insights into the operation principles of these proteases, there is limited knowledge about the substrate proteins that are degraded by distinct proteases and the regulatory role of proteolysis in cellular processes. This study reveals a direct role of the conserved protease Lon in regulating σT, a transcriptional regulator of the general stress response in α-proteobacteria. Our work is significant as it underscores the importance of regulated proteolysis in modulating the levels of key regulatory proteins under changing conditions.

Staphylococcus aureus SigS Induces Expression of a Regulatory Protein Pair That Modulates Its mRNA Stability.

SigS is the sole extracytoplasmic function sigma factor in Staphylococcus aureus and is necessary for virulence, immune evasion, and adaptation to toxic chemicals and environmental stressors. Despite the contribution of SigS to a myriad of critical phenotypes, the downstream effectors of SigS-dependent pathogenesis, immune evasion, and stress adaptation remain elusive. To address this knowledge gap, we analyzed the S. aureus transcriptome following transient overexpression of SigS. We identified a bicistronic transcript, upregulated 1,000-fold, containing two midsized genes, each containing single domains of unknown function (DUFs). We renamed these genes SigS-regulated orfA (sroA) and SigS-regulated orfB (sroB). We demonstrated that SigS regulation of the sroAB operon is direct by using in vitro transcription analysis. Using Northern blot analysis, we also demonstrated that SroA and SroB have opposing autoregulatory functions on the transcriptional architecture of the sigS locus, with SroA stimulating SigS mRNA levels and SroB stimulating s750 (SigS antisense) levels. We hypothesized that these opposing regulatory effects were due to a direct interaction. We subsequently demonstrated a direct interaction between SroA and SroB using an in vivo surrogate genetics approach via bacterial adenylate cyclase-based two-hybrid (BACTH) analysis. We demonstrated that the SroA effect on SigS is at the posttranscriptional level of mRNA stability, highlighting a mechanism likely used by S. aureus to tightly control SigS levels. Finally, we demonstrate that the sroAB locus promotes virulence in a murine pneumonia model of infection. IMPORTANCE SigS is necessary for S. aureus virulence, immune evasion, and adaptation to chemical and environmental stressors. These processes are critically important for the ability of S. aureus to cause disease. However, the SigS-dependent transcriptome has not been identified, hindering our ability to identify downstream effectors of SigS that contribute to these pathogenic and adaptive phenotypes. Here, we identify a regulatory protein pair that is a major direct target of SigS, known as SroA and SroB. SroA also acts to stimulate SigS expression at the posttranscriptional level of RNA turnover, providing insight into intrinsically low levels of SigS. The discovery of SroA and SroB increases our understanding of SigS and the S. aureus pathogenesis process.

Crystal structures of Streptomyces tsukubaensis sigma factor SigG1 and anti-sigma RsfG.

Transcription of specific genes in bacteria under environmental stress is frequently initiated by extracytoplasmic function (ECF) σ factors. ECFs σ factors harbour two conserved domains, σ2 and σ4, for transcription initiation by recognition of the promoter region and recruitment of RNA polymerase (RNAP). The crystal structure of Streptomyces tsukubaensis SigG1, an ECF56-family σ factor, was determined revealing σ2, σ4 and the additional carboxi-terminal domain SnoaL_2 tightly packed in a compact conformation. The structure of anti-sigma RsfG was also determined by X-ray crystallography and shows a rare ß-barrel fold. Analysis of the metal binding motifs inside the protein barrel are consistent with Fe(III) binding, which is in agreement with previous findings that the Streptomyces tsukubaensis ECF56 SigG1-RsfG system is involved in metal-ion homeostasis.

General Stress Signaling in the Alphaproteobacteria.

The Alphaproteobacteria uniquely integrate features of two-component signal transduction and alternative σ factor regulation to control transcription of genes that ensure growth and survival across a range of stress conditions. Research over the past decade has led to the discovery of the key molecular players of this general stress response (GSR) system, including the sigma factor σ(EcfG), its anti-σ factor NepR, and the anti-anti-σ factor PhyR. The central molecular event of GSR activation entails aspartyl phosphorylation of PhyR, which promotes its binding to NepR and thereby releases σ(EcfG) to associate with RNAP and direct transcription. Recent studies are providing a new understanding of complex, multilayered sensory networks that activate and repress this central protein partner switch. This review synthesizes our structural and functional understanding of the core GSR regulatory proteins and highlights emerging data that are defining the systems that regulate GSR transcription in a variety of species.

A structural and functional comparison between two recombinant human lubricin proteins: Recombinant human proteoglycan-4 (rhPRG4) vs ECF843.

Proteoglycan 4 (PRG4, lubricin) is a mucin-like glycoprotein present on the ocular surface that has both boundary lubricating and anti-inflammatory properties. Full-length recombinant human PRG4 (rhPRG4) has been shown to be clinically effective in improving signs and symptoms of dry eye disease (DED). In vitro, rhPRG4 has been shown to reduce inflammation-induced cytokine production and NFκB activity in corneal epithelial cells, as well as to bind to and inhibit MMP-9 activity. A different form of recombinant human lubricin (ECF843), produced from the same cell line as rhPRG4 but manufactured using a different process, was recently assessed in a DED clinical trial. However, ECF843 did not significantly improve signs or symptoms of DED compared to vehicle. Initial published characterization of ECF843 showed it had a smaller hydrodynamic diameter and was less negatively charged than native PRG4. Further examination of the structural and functional properties of ECF843 and rhPRG4 could contribute to the understanding of what led to their disparate clinical efficacy. Therefore, the objective of this study was to characterize and compare rhPRG4 and ECF843 in vitro, both biophysically and functionally. Hydrodynamic diameter and charge were measured by dynamic light scattering (DLS) and zeta potential, respectively. Size and molecular weight was determined for individual species by size exclusion chromatography (SEC) with in-line DLS and multi-angle light scattering (MALS). Bond structure was measured by Raman spectroscopy, and sedimentation properties were measured by analytical ultracentrifugation (AUC). Functionally, MMP-9 inhibition was measured using a commercial MMP-9 activity kit, coefficient of friction was measured using an established boundary lubrication test at a latex-glass interface, and collagen 1-binding ability was measured by quart crystal microbalance with dissipation (QCMD). Additionally, the ability of rhPRG4 and ECF843 to inhibit urate acid crystal formation and cell adhesion was assessed. ECF843 had a significantly smaller hydrodynamic diameter and was less negatively charged than rhPRG4, as assessed by DLS and zeta potential. Size was further explored with SEC-DLS-MALS, which indicated that while rhPRG4 had 3 main peaks, corresponding to monomer, dimer, and multimer as expected, ECF843 had 2 peaks that were similar in size and molecular weight compared to rhPRG4's monomer peak and a third peak that was significantly smaller in both size and molar mass than the corresponding peak of rhPRG4. Raman spectroscopy demonstrated that ECF843 had significantly more disulfide bonds, which are functionally determinant structures, relative to the carbon-carbon backbone compared to rhPRG4, and AUC indicated that ECF843 was more compact than rhPRG4. Functionally, ECF843 was significantly less effective at inhibiting MMP-9 activity and functioning as a boundary lubricant compared to rhPRG4, as well as being slower to bind to collagen 1. Additionally, ECF843 was significantly less effective at inhibiting urate acid crystal formation and at preventing cell adhesion. Collectively, these data demonstrate ECF843 and rhPRG4 are significantly different in both structure and function. Given that a protein's structure sets the foundation for its interactions with other molecules and tissues in vivo, which ultimately determine its function, these differences most likely contributed to the disparate DED clinical trial results.

High affinity iron uptake by pyoverdine in Pseudomonas aeruginosa involves multiple regulators besides Fur, PvdS, and FpvI.

Pseudomonas aeruginosa is a Gram-negative bacterium which can cause serious infections among immune-depressed people including cystic fibrosis patients where it can colonize the lungs causing chronic infections. Iron is essential for P. aeruginosa and can be provided via three sources under aerobic conditions: its own siderophores pyochelin (PCH) and pyoverdine (PVD), xenosiderophores, or heme, respectively. Pyoverdine is the high affinity siderophore and its synthesis and uptake involve more than 30 genes organized in different operons. Its synthesis and uptake are triggered by iron scarcity via the Fur regulator and involves two extra cytoplasmic sigma factors (ECF), PvdS for the biosynthesis of PVD and FpvI for the uptake via the TonB-dependent FpvA outer membrane transporter and other periplasmic and inner membrane proteins. It appeared recently that the regulation of PVD biosynthesis and uptake involves other regulators, including other ECF factors, and LysR regulators. This is the case especially for the genes coding for periplasmic and inner membrane proteins involved in the reduction of Fe3+ to Fe2+ and the transport of ferrous iron to the cytoplasm that appears to represent a crucial step in the uptake process.

The Global Regulator MftR Controls Virulence and Siderophore Production in Burkholderia thailandensis.

Burkholderia thailandensis is a member of the Burkholderia pseudomallei complex. It encodes the transcription factor MftR, which is conserved among the more pathogenic Burkholderia spp. and previously shown to be a global regulator of gene expression. We report here that a B. thailandensis strain in which the mftR gene is disrupted is more virulent in both Caenorhabditis elegans and onion. The ΔmftR strain exhibits a number of phenotypes associated with virulence. It is more proficient at forming biofilm, and the arcDABC gene cluster, which has been linked to anaerobic survival and fitness within a biofilm, is upregulated. Swimming and swarming motility are also elevated in ΔmftR cells. We further show that MftR is one of several transcription factors which control production of the siderophore malleobactin. MftR binds directly to the promoter driving expression of mbaS, which encodes the extracytoplasmic function sigma factor MbaS that is required for malleobactin production. Malleobactin is a primary siderophore in B. thailandensis as evidenced by reduced siderophore production in mbaS::Tc cells, in which mbaS is disrupted. Expression of mbaS is increased ~5-fold in ΔmftR cells, and siderophore production is elevated. Under iron-limiting conditions, mbaS expression is increased ~150-fold in both wild-type and ΔmftR cells, respectively, reflecting regulation by the ferric uptake regulator (Fur). The mbaS expression profiles also point to repression by a separate, ligand-responsive transcription factor, possibly ScmR. Taken together, these data indicate that MftR controls a number of phenotypes, all of which promote bacterial survival in a host environment. IMPORTANCE Bacterial pathogens face iron limitation in a host environment. To overcome this challenge, they produce siderophores, small iron-chelating molecules. Uptake of iron-siderophore complexes averts bacterial iron limitation. In Burkholderia spp., malleobactin or related compounds are the primary siderophores. We show here that genes encoding proteins required for malleobactin production in B. thailandensis are under the direct control of the global transcription factor MftR. Repression of gene expression by MftR is relieved when MftR binds xanthine, a purine metabolite present in host cells. Our work therefore identifies a mechanism by which siderophore production may be optimized in a host environment, thus contributing to bacterial fitness.

ß-Lactam Resistance in Azospirillum baldaniorum Sp245 Is Mediated by Lytic Transglycosylase and ß-Lactamase and Regulated by a Cascade of RpoE7→RpoH3 Sigma Factors.

Bacterial resistance to ß-lactam antibiotics is often mediated by ß-lactamases and lytic transglycosylases. Azospirillum baldaniorum Sp245 is a plant-growth-promoting rhizobacterium that shows high levels of resistance to ampicillin. Investigating the molecular basis of ampicillin resistance and its regulation in A. baldaniorum Sp245, we found that a gene encoding lytic transglycosylase (Ltg1) is organized divergently from a gene encoding an extracytoplasmic function (ECF) σ factor (RpoE7) in its genome. Inactivation of rpoE7 in A. baldaniorum Sp245 led to increased ability to form cell-cell aggregates and produce exopolysaccharides and biofilm, suggesting that rpoE7 might contribute to antibiotic resistance. Inactivation of ltg1 in A. baldaniorum Sp245, however, adversely affected its growth, indicating a requirement of Ltg1 for optimal growth. The expression of rpoE7, as well that of as ltg1, was positively regulated by RpoE7, and overexpression of RpoE7 conferred ampicillin sensitivity to both the rpoE7::km mutant and its parent. In addition, RpoE7 negatively regulated the expression of a gene encoding a ß-lactamase (bla1). Out of the 5 paralogs of RpoH encoded in the genome of A. baldaniorum Sp245, RpoH3 played major roles in conferring ampicillin sensitivity and in the downregulation of bla1. The expression of rpoH3 was positively regulated by RpoE7. Collectively, these observations reveal a novel regulatory cascade of RpoE7-RpoH3 σ factors that negatively regulates ampicillin resistance in A. baldaniorum Sp245 by controlling the expression of a ß-lactamase and a lytic transglycosylase. In the absence of a cognate anti-sigma factor, addressing how the activity of RpoE7 is regulated by ß-lactams will unravel new mechanisms of regulation of ß-lactam resistance in bacteria. IMPORTANCE Antimicrobial resistance is a global health problem that requires a better understanding of the mechanisms that bacteria use to resist antibiotics. Bacteria inhabiting the plant rhizosphere are a potential source of antibiotic resistance, but their mechanisms controlling antibiotic resistance are poorly understood. A. baldaniorum Sp245 is a rhizobacterium that is known for its characteristic resistance to ampicillin. Here, we show that an AmpC-type ß-lactamase and a lytic transglycosylase mediate resistance to ampicillin in A. baldaniorum Sp245. While the gene encoding lytic transglycosylase is positively regulated by an ECF σ-factor (RpoE7), a cascade of RpoE7 and RpoH3 σ factors negatively regulates the expression of ß-lactamase. This is the first evidence showing involvement of a regulatory cascade of σ factors in the regulation of ampicillin resistance in a rhizobacterium.

An Extracytoplasmic Function Sigma/Anti-Sigma Factor System Regulates ß-Glucanase Expression in Tannerella forsythia in Response to Fusobacterium nucleatum Sensing.

The periodontal pathogen Tannerella forsythia expresses a ß-glucanase (TfGlcA) whose expression is induced in response to Fusobacterium nucleatum, a bridge bacterium of the oral cavity. TfGlcA cleaves ß-glucans to release glucose, which can serve as a carbon source for F. nucleatum and other cohabiting organisms. A two-gene cluster encoding a putative extracytoplasmic function (ECF) sigma factor and a FecR-like anti-sigma factor has been recognized upstream of a TfGlcA operon. We characterized and analyzed the role of these putative ECF sigma and anti-sigma factors in the regulation of TfGlcA expression. For this purpose, deletion mutants were constructed and analyzed for ß-glucanase expression. In addition, an Escherichia coli-produced ECF sigma factor recombinant protein was evaluated for transcriptional and DNA binding activities. The results showed that the recombinant protein promoted transcription by the RNA polymerase core enzyme from the glcA promoter. Furthermore, in comparison to those in the parental strain, the ß-glucanase expression levels were significantly reduced in the ECF sigma-factor deletion mutant and increased significantly in the FecR anti-sigma factor deletion mutant. The levels did not change in the mutants following coincubation with the F. nucleatum whole cells or cell extracts. Finally, the levels of ß-glucanase produced by T. forsythia strains paralleled F. nucleatum biomass in cobiofilms. In conclusion, we identified a ß-glucanase operon regulatory system in T. forsythia comprising an ECF sigma factor (TfSigG) and a cognate FecR-like anti-sigma factor responsive to F. nucleatum and potentially other stimuli. IMPORTANCE Previous studies have shown that F. nucleatum forms robust biofilms with T. forsythia utilizing glucose from the hydrolysis of ß-glucans by T. forsythia ß-glucanase, induced by F. nucleatum. In this study, we showed that a regulatory system comprising of an ECF sigma factor, TfSigG, and a FecR-like anti-sigma factor, TfFecR, is responsible for the ß-glucanase induction in response to F. nucleatum, suggesting that this system plays roles in the mutualistic interactions of T. forsythia and F. nucleatum. The findings suggest the development and potential utility of small-molecule inhibitors targeting the ß-glucanase activity or the TfSigG/TfFecR system as therapeutic drugs against dental plaque formation and periodontitis.

High-Level Expression of Cell-Surface Signaling System Hxu Enhances Pseudomonas aeruginosa Bloodstream Infection.

Bloodstream infections (BSIs) caused by Pseudomonas aeruginosa are associated with a high mortality rate in the clinic. However, the fitness mechanisms responsible for the evolution of virulence factors that facilitate the dissemination of P. aeruginosa to the bloodstream are poorly understood. In this study, a transcriptomic analysis of the BSI-associated P. aeruginosa clinical isolates showed a high-level expression of cell-surface signaling (CSS) system Hxu. Whole-genome sequencing and comparative genomics of these isolates showed that a mutation in rnfE gene was responsible for the elevated expression of the Hxu-CSS pathway. Most importantly, deletion of the hxuIRA gene cluster in a laboratory strain PAO1 reduced its BSI capability while overexpression of the HxuIRA pathway promoted BSI in a murine sepsis model. We further demonstrated that multiple components in the blood plasma, including heme, hemoglobin, the heme-scavenging proteins haptoglobin, and hemopexin, as well as the iron-delivery protein transferrin, could activate the Hxu system. Together, these studies suggested that the Hxu-CSS system was an important signal transduction pathway contributing to the adaptive pathogenesis of P. aeruginosa in BSI.

Deciphering the role of the two conserved motifs of the ECF41 family σ factor in the autoregulation of its own promoter in Azospirillum brasilense Sp245.

In Azospirillum brasilense, an extra-cytoplasmic function σ factor (RpoE10) shows the characteristic 119 amino acid long C-terminal extension found in ECF41-type σ factors, which possesses three conserved motifs (WLPEP, DGGGR, and NPDKV), one in the linker region between the σ2 and σ4 , and the other two in the SnoaL_2 domain of the C-terminal extension. Here, we have described the role of the two conserved motifs in the SnoaL_2 domain of RpoE10 in the inhibition and activation of its activity, respectively. Truncation of the distal part of the C-terminal sequence of the RpoE10 (including NPDKV but excluding the DGGGR motif) results in its promoter's activation suggesting autoregulation. Further truncation of the C-terminal sequence up to its proximal part, including NPDKV and DGGGR motif, abolished promoter activation. Replacement of NPDKV motif with NAAAV in RpoE10 increased its ability to activate its promoter, whereas replacement of DGGGR motif led to reduced promoter activation. We have explored the dynamic modulation of σ2 -σ4 domains and the relevant molecular interactions mediated by the two conserved motifs of the SnoaL2 domain using molecular dynamics simulation. The analysis enabled us to explain that the NPDKV motif located distally in the C-terminus negatively impacts transcriptional activation. In contrast, the DGGGR motif found proximally of the C-terminal extension is required to activate RpoE10.

A Microfabricated Pistonless Syringe Pump Driven by Electro-Conjugate Fluid with Leakless On/Off Microvalves.

In contrast to microfluidic devices, bulky syringe pumps are widely used to deliver a small amount of solution with high accuracy. Miniaturizing the syringe pump is difficult due to the scale effect in the microscale where the friction of the piston-cylinder is dominant and there are few high-power microactuators. To solve these problems, an on-chip microsyringe pump without mechanical sliding parts and with high power sources is proposed. The microsyringe pump utilizes the interface between water and oil (electro-conjugate fluid, ECF) instead of a piston and an electrohydrodynamic (EHD) flow driven by ECF in place of a linear actuator. ECF as a functional fluid has two capabilities: a) making the water-oil interface in microchannels and b) generating an active ECF flow at an applied voltage to withdraw and infuse aqueous solution by the interface. To control the flow direction, ECF-driven leakless on/off microvalves are also integrated. It is demonstrated that the proposed ECF microsyringe pump synchronized with the ECF on/off microvalves can control the withdrawing and infusing of aqueous solution with high resolution and precision. The experiments prove the feasibility of the microsyringe pump to be embedded as a module for the precise and linear control of flow rates in microfluidic devices.

Development of a whole-cell biosensor for detection of antibiotics targeting bacterial cell envelope in Bacillus subtilis.

It is an urgent need to develop novel antibiotics to treat infections caused by multi-drug-resistant bacteria. One promising strategy could be the use of whole-cell biosensors, which have been extensively studied to monitor environmental pollutants and intracellular metabolites. Here, we used the σM-mediated regulatory system of Bacillus subtilis to construct a whole-cell biosensor for the detection of cell envelope-acting antibiotics. Using polymyxin B as the inducer for bacterial cell envelope stress and enhanced green fluorescent protein (EGFP) as the reporter, we found that the promoter of ypuA (PypuA) had the lowest background noise and the most significant changes in the fluorescence output. The whole-cell biosensor displayed dose-dependent and time-dependent responses in fluorescence signals. The detection range of this biosensor for polymyxin B was between 0.125 and 12 µg/mL. The response of the biosensor is specific to antibiotics that target the cell envelope. Besides determination in liquid cultures, the output signal of the biosensor can be easily determined on agar surfaces. Using this biosensor, we successfully detected polymyxins secreted by its producing strain and bacteria that produce cell envelope-acting antibiotics. KEY POINTS: • A whole-cell biosensor was constructed based on the σM-mediated regulatory system. • The response of the biosensor is specific to cell envelope-acting antibiotics. • The biosensor can be used to screen novel cell envelope-acting antibiotics.

Monitoring of Indoor Farming of Lettuce Leaves for 16 Hours Using Electrical Impedance Spectroscopy (EIS) and Double-Shell Model (DSM).

An electrical impedance spectroscopy (EIS) experiment was performed using a double-shell electrical model to investigate the feasibility of detecting physiological changes in lettuce leaves over 16 h. Four lettuce plants were used, and the impedance spectra of the leaves were measured five times per plant every hour at frequencies of 500 Hz and 300 kHz. Estimated R-C parameters were computed, and the results show that the lettuce leaves closely fit the double-shell model (DSM). The average resistance ratios of R1 = 10.66R4 and R1 = 3.34R2 show high resistance in the extracellular fluid (ECF). A rapid increase in resistance (R1, R2, and R4) and a decrease in capacitance (C3 and C5) during water uptake were observed. In contrast, a gradual decrease in resistance and an increase in capacitance were observed while the LED light was on. Comparative studies of leaf physiology and electrical value changes support the idea that EIS is a great technique for the early monitoring of plant growth for crop production.

Mechanisms of Action of Non-Canonical ECF Sigma Factors.

Extracytoplasmic function (ECF) sigma factors are subunits of the RNA polymerase specialized in activating the transcription of a subset of genes responding to a specific environmental condition. The signal-transduction pathways where they participate can be activated by diverse mechanisms. The most common mechanism involves the action of a membrane-bound anti-sigma factor, which sequesters the ECF sigma factor, and releases it after the stimulus is sensed. However, despite most of these systems following this canonical regulation, there are many ECF sigma factors exhibiting a non-canonical regulatory mechanism. In this review, we aim to provide an updated and comprehensive view of the different activation mechanisms known for non-canonical ECF sigma factors, detailing their inclusion to the different phylogenetic groups and describing the mechanisms of regulation of some of their representative members such as EcfG from Rhodobacter sphaeroides, showing a partner-switch mechanism; EcfP from Vibrio parahaemolyticus, with a phosphorylation-dependent mechanism; or CorE from Myxococcus xanthus, regulated by a metal-sensing C-terminal extension.

Use of a Novel Extremophilic Xylanase for an Environmentally Friendly Industrial Bleaching of Kraft Pulps.

Xylanases can boost pulp bleachability in Elemental Chlorine Free (ECF) processes, but their industrial implementation for producing bleached kraft pulps is not straightforward. It requires enzymes to be active and stable at the extreme conditions of alkalinity and high temperature typical of this industrial process; most commercial enzymes are unable to withstand these conditions. In this work, a novel highly thermo and alkaline-tolerant xylanase from Pseudothermotoga thermarum was overproduced in E. coli and tested as a bleaching booster of hardwood kraft pulps to save chlorine dioxide (ClO2) during ECF bleaching. The extremozyme-stage (EXZ) was carried out at 90 °C and pH 10.5 and optimised at lab scale on an industrial oxygen-delignified eucalyptus pulp, enabling us to save 15% ClO2 to reach the mill brightness, and with no detrimental effect on paper properties. Then, the EXZ-assisted bleaching sequence was validated at pilot scale under industrial conditions, achieving 25% ClO2 savings and reducing the generation of organochlorinated compounds (AOX) by 18%, while maintaining pulp quality and papermaking properties. Technology reproducibility was confirmed with another industrial kraft pulp from a mix of hardwoods. The new enzymatic technology constitutes a realistic step towards environmentally friendly production of kraft pulps through industrial integration of biotechnology.

An Extracytoplasmic Function Sigma/Anti-Sigma Factor System Regulates Hypochlorous Acid Resistance and Impacts Expression of the Type IV Secretion System in Brucella melitensis.

The intracellular bacterial pathogen Brucella causes persistent infections in various mammalian species. To survive and replicate within macrophages, these bacteria must be able to withstand oxidative stresses and express the type IV secretion system (T4SS) to evade host immune responses. The extracytoplasmic function (ECF) sigma factor system is a major signal transduction mechanism in bacteria that senses environmental cues and responds by regulating gene expression. In this study, we defined an ECF σ bcrS and its cognate anti-σ factor abcS in Brucella melitensis M28 by conserved domain analysis and a protein interaction assay. BcrS directly activates an adjacent operon, bcrXQP, that encodes a methionine-rich peptide and a putative methionine sulfoxide reductase system, whereas AbcS is a negative regulator of bcrS and bcrXQP. The bcrS-abcS and bcrXQP operons can be induced by hypochlorous acid and contribute to hypochlorous acid resistance in vitro. Next, RNA sequencing analysis and genome-wide recognition sequence search identified the regulons of BcrS and AbcS. Interestingly, we found that BcrS positively influences T4SS expression in an AbcS-dependent manner and that AbcS also affects T4SS expression independently of BcrS. Last, we demonstrate that abcS is required for the maintenance of persistent infection, while bcrS is dispensable in a mouse infection model. Collectively, we conclude that BcrS and AbcS influence expression of multiple genes responsible for Brucella virulence traits. IMPORTANCEBrucella is a notorious intracellular pathogen that induces chronic infections in animals and humans. To survive and replicate within macrophages, these bacteria require a capacity to withstand oxidative stresses and to express the type IV secretion system (T4SS) to combat host immune responses. In this study, we characterized an extracytoplasmic function sigma/anti-sigma factor system that regulates resistance to reactive chlorine species and T4SS expression, thereby establishing a potential link between two crucial virulence traits of Brucella. Furthermore, the anti-sigma factor AbcS contributes to Brucella persistent infection of mice. Thus, this work provides novel insights into Brucella virulence regulation as well as a potential drug target for fighting Brucella infections.