Coenzyme A biosynthesis in Bacillus subtilis: discovery of a novel precursor metabolite for salvage and its uptake system.

The Gram-positive model bacterium Bacillus subtilis is used for many biotechnological applications, including the large-scale production of vitamins. For vitamin B5, a precursor for coenzyme A synthesis, there is so far no established fermentation process available, and the metabolic pathways that involve this vitamin are only partially understood. In this study, we have elucidated the complete pathways for the biosynthesis of pantothenate and coenzyme A in B. subtilis. Pantothenate can not only be synthesized but also be taken up from the medium. We have identified the enzymes and the transporter involved in the pantothenate biosynthesis and uptake. High-affinity vitamin B5 uptake in B. subtilis requires an ATP-driven energy coupling factor transporter with PanU (previously YhfU) as the substrate-specific subunit. Moreover, we have identified a salvage pathway for coenzyme A acquisition that acts on complex medium even in the absence of pantothenate synthesis. This pathway requires rewiring of sulfur metabolism resulting in the increased expression of a cysteine transporter. In the salvage pathway, the bacteria import cysteinopantetheine, a novel naturally occurring metabolite, using the cystine transport system TcyJKLMN. This work lays the foundation for the development of effective processes for vitamin B5 and coenzyme A production using B. subtilis. IMPORTANCE: Vitamins are essential components of the diet of animals and humans. Vitamins are thus important targets for biotechnological production. While efficient fermentation processes have been developed for several vitamins, this is not the case for vitamin B5 (pantothenate), the precursor of coenzyme A. We have elucidated the complete pathway for coenzyme A biosynthesis in the biotechnological workhorse Bacillus subtilis. Moreover, a salvage pathway for coenzyme A synthesis was found in this study. Normally, this pathway depends on pantetheine; however, we observed activity of the salvage pathway on complex medium in mutants lacking the pantothenate biosynthesis pathway even in the absence of supplemented pantetheine. This required rewiring of metabolism by expressing a cystine transporter due to acquisition of mutations affecting the regulation of cysteine metabolism. This shows how the hidden "underground metabolism" can give rise to the rapid formation of novel metabolic pathways.

Physiological Roles of an Acinetobacter-specific σ Factor.

The Gram-negative pathogen Acinetobacter baumannii is considered an "urgent threat" to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used by A. baumannii to mitigate cellular stresses may uncover new therapeutic targets. Many γ-proteobacteria use the extracytoplasmic function (ECF) σ factor, RpoE, to invoke envelope homeostasis networks in response to stress. Acinetobacter species contain the poorly characterized ECF "SigAb;" however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique to Acinetobacter species and distinct from RpoE. We combine promoter mutagenesis, motif scanning, and ChIP-seq to define the direct SigAb regulon, which consists of sigAb itself, the stringent response mediator, relA, and the uncharacterized small RNA, "sabS." However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown of sigAb or sabS resulted in increased copper sensitivity and excess copper induced SigAb-dependent transcription. Further, we found that two uncharacterized genes in the sigAb operon, "aabA" and "aabB", have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show that sigAb, aabA, and aabB are important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture of A. baumannii.

An energy coupling factor transporter of Streptococcus sanguinis impacts antibiotic susceptibility as well as metal and membrane homeostasis.

Streptococcus sanguinis is a prevalent member of human microbiome capable of acting as a causative agent of oral and respiratory infections. S. sanguinis competitive success within the infection niche is dependent on acquisition of metal ions and vitamins. Among the systems that bacteria use for micronutrient uptake is the energy coupling factor (ECF) transporter system EcfAAT. Here we describe physiological changes arising from EcfAAT transporter disruption. We found that EcfAAT contributes to S. sanguinis antibiotic sensitivity as well as metal and membrane homeostasis. Specifically, our work found that disruption of EcfAAT results in increased polymyxin susceptibility. We performed assessment of cell-associated metal content and found depletion of iron, magnesium, and manganese. Furthermore, membrane composition analysis revealed significant enrichment in unsaturated fatty acid species resulting in increased membrane fluidity. Our results demonstrate how disruption of a single EcfAAT transporter can have broad consequences on bacterial cell homeostasis. ECF transporters are of interest within the context of infection biology in bacterial species other than streptococci, hence work described here will further the understanding of how micronutrient uptake systems contribute to bacterial pathogenesis.

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.

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.

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.

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.

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.

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.

Fuzzy-based prediction of solar PV and wind power generation for microgrid modeling using particle swarm optimization.

Regardless of their nature of stochasticity and uncertain nature, wind and solar resources are the most abundant energy resources used in the development of microgrid systems. In microgrid systems and distribution networks, the uncertain nature of both solar and wind resources results in power quality and system stability issues. The randomization behavior of solar and wind energy resources is controlled through the precise development of a power prediction model. Fuzzy-based solar PV and wind prediction models may more efficiently manage this randomness and uncertain character. However, this method has several drawbacks, it has limited performance when the volumes of wind and solar resources historical data are huge in size and it has also many membership functions of the fuzzy input and output variables as well as multiple fuzzy rules available. The hybrid Fuzzy-PSO intelligent prediction approach improves the fuzzy system's limitations and hence increases the prediction model's performance. The Fuzzy-PSO hybrid forecast model is developed using MATLAB programming of the particle swarm optimization (PSO) algorithm with the help of the global optimization toolbox. In this paper, an error correction factor (ECF) is considered a new fuzzy input variable. It depends on the validation and forecasted data values of both wind and solar prediction models to improve the accuracy of the prediction model. The impact of ECF is observed in fuzzy, Fuzzy-PSO, and Fuzzy-GA wind and solar PV power forecasting models. The hybrid Fuzzy-PSO prediction model of wind and solar power generation has a high degree of accuracy compared to the Fuzzy and Fuzzy-GA forecasting models. The rest of this paper is organized as: Section II is about the analysis of solar and wind resources row data. The Fuzzy-PSO prediction model problem formulation is covered in Section III. Section IV, is about the results and discussion of the study. Section V contains the conclusion. The references and abbreviations are presented at the end of the paper.

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.

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.

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 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.

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.

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.

Novel switchable ECF sigma factor transcription system for improving thaxtomin A production in Streptomyces.

The application of the valuable natural product thaxtomin A, a potent bioherbicide from the potato scab pathogenic Streptomyces strains, has been greatly hindered by the low yields from its native producers. Here, we developed an orthogonal transcription system, leveraging extra-cytoplasmic function (ECF) sigma (σ) factor 17 (ECF17) and its cognate promoter Pecf17, to express the thaxtomin gene cluster and improve the production of thaxtomin A. The minimal Pecf17 promoter was determined, and a Pecf17 promoter library with a wide range of strengths was constructed. Furthermore, a cumate inducible system was developed for precise temporal control of the ECF17 transcription system in S. venezuelae ISP5230. Theoretically, the switchable ECF17 transcription system could reduce the unwanted influences from host and alleviate the burdens introduced by overexpression of heterologous genes. The yield of thaxtomin A was significantly improved to 202.1 ± 15.3  µ g/mL using the switchable ECF17 transcription system for heterologous expression of the thaxtomin gene cluster in S. venezuelae ISP5230. Besides, the applicability of this transcription system was also tested in Streptomyces albus J1074, and the titer of thaxtomin A was raised to as high as 239.3 ± 30.6 µg/mL. Therefore, the inducible ECF17 transcription system could serve as a complement of the generally used transcription systems based on strong native constitutive promoters and housekeeping σ factors for the heterologous expression of valuable products in diverse Streptomyces hosts.

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.

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.

Galactolipids from Arabidopsis thaliana can replace the function of gluco lipids in Bacillus subtilis.

Arabidopsis thaliana monogalactosyldiacylglycerol synthase 1 (AtMGD1) and digalactosyldiacylglycerol synthase 2 (AtDGD2) genes introduced into a Bacillus subtilis chromosome with disrupted galE, which encodes UDP-glucose 4-epi merase, enabled the mutant to produce monogalactosyldiacylglycerol. When galE mutant cells are cultivated in galactose containing medium they show ab normal morphology. This phenotype is correlated with a decrease in the amount of glucolipids. Nucleoids of the ugtP and galE mutants were stained by propidium iodide, which does not permeate intact cell membranes, whereas nucleoids of wild type and of a pssA mutant we examined were not stained. Expression of the AtMGD1 gene in a ugtP galE double mutant restored cell membrane integrity. Expression of galactolipid synthase genes from a multi-copy plasmid, pDGHisN4, allowed higher production of galactolipids. Activation of the extracytoplasmic function sigma factors SigM, SigV, and SigX, in the ugtP mutant was decreased by expression of AtMGD1, and SigX activity was strongly repressed when both AtMGD1 and AtDGD2 genes were expressed in the mutant. We conclude that the number of sugars that bind to diacylglycerol - rather than the exact sugar species - is important for glycolipid function in B. subtilis.