Disruption of glucose homeostasis by bacterial infection orchestrates host innate immunity through NAD+/NADH balance.

Metabolic reprogramming is crucial for activating innate immunity in macrophages, and the accumulation of immunometabolites is essential for effective defense against infection. The NAD+/NADH (ratio of nicotinamide adenine dinucleotide and its reduced counterpart) redox couple serves as a critical node that integrates metabolic pathways and signaling events, but how this metabolite couple engages macrophage activation remains unclear. Here, we show that the NAD+/NADH ratio serves as a molecular signal that regulates proinflammatory responses and type I interferon (IFN) responses divergently. Salmonella Typhimurium infection leads to a decreased NAD+/NADH ratio by inducing the accumulation of NADH. Further investigation shows that an increased NAD+/NADH ratio correlates with attenuated proinflammatory responses and enhanced type I IFN responses. Conversely, a decreased NAD+/NADH ratio is linked to intensified proinflammatory responses and restrained type I IFN responses. These results show that the NAD+/NADH ratio is an essential cell-intrinsic factor that orchestrates innate immunity, which enhances our understanding of how metabolites fine-tune innate immunity.

A phosphodiesterase CpdB in Yersinia pseudotuberculosis degrades CDNs to inhibit innate immune response.

Yersinia pseudotuberculosis (Yptb) is a pathogenic gram-negative bacterium that can colonize the intestines of different animals. Its infection leads to the activation of the host's innate immunity. Both host and bacterial-derived cyclic dinucleotides (CDNs) could activate the innate immune response of host cells. In bacteria, CDNs like c-di-AMP, c-di-GMP, or 3'3'-cGAMP can be hydrolyzed by different hydrolases. Recent studies showed that the degradation of those second messengers helps the pathogen evade immune detection. In this study, we identified a hydrolase, YPK_3776, namely CpdB in Yptb. CpdB is predicted to bind bacterial-derived c-di-AMP, c-di-GMP, 3'3'-cGAMP and host-derived 2'3'-cGAMP. Surprisingly, by using high-performance liquid chromatography (HPLC), we found that CpdB could only degrade bacterial-derived CDNs but not host-derived 2'3'-cGAMP. In addition, CpdB has 2'3'-cNMP activity. Consistently, the Yptb mutant lacking the cpdB gene exhibited a higher level of intracellular c-di-GMP. Furthermore, the ∆cpdB mutant elicited stronger innate immune responses during Yptb infection in macrophages, suggesting CpdB enables Yptb to evade host immune surveillance. Furthermore, CpdB inhibited the Yptb-induced innate immune response in a STING-dependent manner. Finally, we showed the ∆cpdB infection in mice model exhibited in lower bacterial burden, as compared to wild-type strain infection, indicating CpdB is important for bacterial survival in the host. Together, we identified a cyclic dinucleotide hydrolase CpdB in Yptb that could degrade bacterial-derived CDNs which help the pathogen to evade immune detection via the STING pathway.

Comprehensive proteomic profiling of lung adenocarcinoma: development and validation of an innovative prognostic model.

Background: Lung adenocarcinoma (LUAD), a global leading cause of cancer deaths, remains inadequately addressed by current protein biomarkers. Our study focuses on developing a protein-based risk signature for improved prognosis of LUAD. Methods: We employed the least absolute shrinkage and selection operator (LASSO)-COX algorithm on The Cancer Genome Atlas database to construct a prognostic model incorporating six proteins (CD49B, UQCRC2, SMAD1, FOXM1, CD38, and KAP1). The model's performance was assessed using principal component, Kaplan-Meier (KM), and receiver operating characteristic (ROC) analysis, indicating strong predictive capability. The model stratifies LUAD patients into distinct risk groups, with further analysis revealing its potential as an independent prognostic factor. Additionally, we developed a predictive nomogram integrating clinicopathologic factors, aimed at assisting clinicians in survival prediction. Gene set enrichment analysis (GSEA) and examination of the tumor immune microenvironment were conducted, highlighting metabolic pathways in high-risk genes and immune-related pathways in low-risk genes, indicating varied immunotherapy sensitivity. Validation through immunohistochemistry from the Human Protein Atlas (HPA) database and immunofluorescence staining of clinical samples was performed, particularly focusing on CD38 expression. Results: Our six-protein model (CD49B, UQCRC2, SMAD1, FOXM1, CD38, KAP1) effectively categorized LUAD patients into high and low-risk groups, confirmed by principal component, KM, and ROC analyses. The model showed high predictive accuracy, with distinct survival differences between risk groups. Notably, CD38, traditionally seen as protective, was paradoxically associated with poor prognosis in LUAD, a finding supported by immunohistochemistry and immunofluorescence data. GSEA revealed that high-risk genes are enriched in metabolic pathways, while low-risk genes align with immune-related pathways, suggesting better immunotherapy response in the latter group. Conclusions: This study presented a novel prognostic protein model for LUAD, highlighting the CD38 expression paradox and enhancing our understanding of protein roles in lung cancer progression. It offered new clinical tools for prognosis prediction and provided assistance for future lung cancer pathogenesis research.

Salmonella T3SS-elicited inflammatory innate immune response inhibits type I IFN response in macrophages.

As a gram-negative intracellular bacterial pathogen, Salmonella enterica serovar Typhimurium (S. Typhimurium) invades different cell types including macrophages. Its infection in macrophages induces robust innate immune responses that are featured by proinflammatory and type I interferon (IFN) responses. The type III secretion systems (T3SSs) of S. Typhimurium play a crucial role in activating host inflammasome pathways. It has been recognized that the inflammasome pathways inhibit the type I IFN cascade. However, the potential role of T3SS in regulating the type I IFN response and the underlying mechanisms are largely unknown. In this study, we showed that S. Typhimurium infection activated strong proinflammatory, type I IFN and IFN-stimulated genes (ISGs) expression in macrophages. Furthermore, we showed that T3SS-defective S. Typhimurium mutant ΔinvC elicited attenuated inflammatory response but enhanced type I IFN and ISGs expression. Additionally, the inhibition of caspase-1 by a specific inhibitor VX-765 resulted in increased type I IFN response. Moreover, cell-permeable pan-caspase inhibitor Z-VAD-FMK also enhanced the type I IFN response upon S. Typhimurium infection. Intriguingly, compared with exponential phase S. Typhimurium infection, stationary phase bacteria triggered higher levels of type I IFN responses. Finally, the inhibition of caspase-1 by VX-765 substantially increased the intracellular S. Typhimurium burden. In conclusion, we demonstrated that the proinflammatory response induced by S. Typhimurium T3SS can inhibit the type I IFN response, which provides insight into the role of T3SS in orchestrating innate immunity during S. Typhimurium infection.

Rethreading Design of Ratiometric roGFP2 Mimetic Peptide for Hydrogen Peroxide Sensing.

Reconstruction of the miniaturized peptide to mimic the tailored functions of protein has been attractive but challenging. Herein, initialized from the crystal structure of redox-sensitive green fluorescent protein-2 (roGFP2), we propose a practical approach to construct the roGFP2 mimetic peptide by rethreading the aromatic residues adjacent to the chromophore fragment. By fine-tuning the residues of peptides, a mini tetrapeptide (Cys-Phe-Phe-His) was designed, which can act as a hydrogen peroxide sensor using its ratiometric fluorescence. The roGFP2 mimetic tetrapeptide is biocompatible and photostable and has competitive fluorescent properties with roGFP2 by the virtue of its assembly induced emissions. We expand the ratiometric tetrapeptide for sensing hydrogen peroxide in acidic chambers. The results provide a promising approach for the artificial design of miniaturized peptides with the desired function.

Quantitative Ratiometric Biosensors Based on Fluorescent Ferrocene-Modified Histidine Dipeptide Nanoassemblies.

Fluorescent proteins (FPs) provide a ratiometric readout for quantitative assessment of the destination of internalized biomolecules. FP-inspired peptide nanostructures that can compete with FPs in their capacity are the most preferred building blocks for the synthesis of fluorescent soft matter. However, realizing a ratiometric emission from a single peptide fluorophore remains exclusive since multicolor emission is a rare property in peptide nanostructures. Here, we describe a bioinspired peptidyl platform for ratiometric intracellular quantitation by employing a single ferrocene-modified histidine dipeptide. The intensiometric ratio of green to blue fluorescence correlates linearly with the concentration of the peptide by three orders of magnitude. The ratiometric fluorescence of the peptide is an assembly-induced emission originating from hydrogen bonds and aromatic interactions. Additionally, modular design enables ferrocene-modified histidine dipeptides to use as a general platform for the construction of intricate peptides that retain the ratiometric fluorescent properties. The ratiometric peptide technique promises flexibility in the design of a wide spectrum of stoichiometric biosensors for quantitatively understanding the trafficking and subcellular fate of biomolecules.

Pseudomonas aeruginosa H3-T6SS Combats H2O2 Stress by Diminishing the Amount of Intracellular Unincorporated Iron in a Dps-Dependent Manner and Inhibiting the Synthesis of PQS.

The type VI secretion system (T6SS), a protein translocation nanomachine, is widely distributed in Gram-negative bacteria and delivers effectors directly into target cells or the extracellular environment to help the bacteria gain a competitive fitness advantage and promote bacterial survival in harmful environments. In this study, we demonstrated that the synthesis of the Pseudomonas quinolone signal (PQS) in Pseudomonas aeruginosa PAO1 was inhibited by the H3-T6SS gene cluster under iron-rich conditions, and that this inhibition was relieved under iron starvation conditions. Conversely, PQS differentially regulated the expression of the H3-T6SS structural genes and the effector protein gene tseF. The expression of tseF was inhibited by PQS, while the expressions of the H3-T6SS structural genes were positively regulated by PQS. Further studies showed that the H3-T6SS was involved in the resistance of P. aeruginosa to oxidative stress caused by hydrogen peroxide (H2O2). Interestingly, H3-T6SS expression was neither induced by H2O2 stress nor regulated by OxyR (a global anti-oxidative transcriptional regulator) but was positively regulated by RpoS (a major transcription regulator of the stress response). In addition, we found that the clpV3 (a structural gene of H3-T6SS) mutation resulted in upregulation of two proteins related to PQS synthesis and many proteins related to oxidative stress resistance, while the expression of some iron storage proteins, especially Dps, were significantly downregulated. Furthermore, the clpV3 mutation led to an increase in the intracellular free Fe2+ content of P. aeruginosa. Further studies showed that both the PQS deficient mutation and overexpression of dps effectively restored the H2O2 sensitive phenotype of the H3-T6SS mutant. Finally, we proposed the following model of H3-T6SS-mediated resistance to H2O2 stress in P. aeruginosa. H3-T6SS not only reduces the intracellular free Fe2+ level by upregulating the expression of ferritin Dps, but also inhibits the synthesis of PQS to mediate the resistance of P. aeruginosa to H2O2 stress. This study highlights the important role of H3-T6SS in the ability of P. aeruginosa to combat H2O2 stress and provides a perspective for understanding the stress response mechanism of bacteria.

Salmonella Induces the cGAS-STING-Dependent Type I Interferon Response in Murine Macrophages by Triggering mtDNA Release.

Salmonella enterica serovar Typhimurium (S. Typhimurium) elicited strong innate immune responses in macrophages. To activate innate immunity, pattern recognition receptors (PRRs) in host cells can recognize highly conserved pathogen-associated molecular patterns (PAMPs). Here, we showed that S. Typhimurium induced a robust type I interferon (IFN) response in murine macrophages. Exposure of macrophages to S. Typhimurium activated a Toll-like receptor 4 (TLR4)-dependent type I IFN response. Next, we showed that type I IFN and IFN-stimulated genes (ISGs) were elicited in a TBK1-IFN-dependent manner. Furthermore, cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) and immune adaptor protein stimulator of interferon genes (STING) were also required for the induction of type I IFN response during infection. Intriguingly, S. Typhimurium infection triggered mitochondrial DNA (mtDNA) release into the cytosol to activate the type I IFN response. In addition, we also showed that bacterial DNA was enriched in cGAS during infection, which may contribute to cGAS activation. Finally, we showed that cGAS and STING deficient mice and cells were more susceptible to S. Typhimurium infection, signifying the critical role of the cGAS-STING pathway in host defense against S. Typhimurium infection. In conclusion, in addition to TLR4-dependent innate immune response, we demonstrated that S. Typhimurium induced the type I IFN response in a cGAS-STING-dependent manner and the S. Typhimurium-induced mtDNA release was important for the induction of type I IFN. This study elucidated a new mechanism by which bacterial pathogen activated the cGAS-STING pathway and also characterized the important role of cGAS-STING during S. Typhimurium infection. IMPORTANCE As one of the most common foodborne transmitted zoonotic pathogens, S. Typhimurium infection causes diarrheal disease in humans and animals. S. Typhimurium infection has been implicated as an inducer for the type I interferon (IFN) response in macrophages, but the mechanisms are not fully understood. In this study, we reported that in addition to TLR4-dependent response, the cytosolic surveillance pathway (CSP) cGAS-STING is also required for the activation of type I IFN response during S. Typhimurium infection. We further showed that the infection of S. Typhimurium triggered mtDNA release into the cytosol, which induces the type I IFN response. In addition, physical interactions between cGAS and S. Typhimurium DNA have been identified in the context of infection. Importantly, we also provided convincing in vivo and in vitro evidence that the cGAS-STING pathway was potently implicated in the host defense against S. Typhimurium infection. Together, we uncovered a mechanism by which type I IFN response is elicited during S. Typhimurium infection in murine macrophages in an mtDNA-cGAS-STING-dependent manner.

Aerobactin-Mediated Iron Acquisition Enhances Biofilm Formation, Oxidative Stress Resistance, and Virulence of Yersinia pseudotuberculosis.

Aerobactin is a citrate-hydroxamate siderophore that is critical for the virulence of pathogenic enteric bacteria. However, although the aerobactin-producing iucABCD-iutA operon is distributed widely in the genomes of Yersinia species, none of the pathogenic Yersinia spp. was found to produce aerobactin. Here, we showed that the iucABCD-iutA operon in the food-borne enteric pathogen Yersinia pseudotuberculosis YPIII is a functional siderophore system involved in iron acquisition. The expression of the operon was found to be directly repressed by the ferric uptake regulator (Fur) in an iron concentration-dependent manner. In addition, we demonstrated that the aerobactin-mediated iron acquisition contributes to bacterial growth under iron-limited conditions. Moreover, we provided evidence that aerobactin plays important roles in biofilm formation, resistance to oxidative stress, ROS removal, and virulence of Y. pseudotuberculosis. Overall, our study not only uncovered a novel strategy of iron acquisition in Y. pseudotuberculosis but also highlighted the importance of aerobactin in the pathogenesis of Y. pseudotuberculosis.

The transcriptional regulator Zur regulates the expression of ZnuABC and T6SS4 in response to stresses in Yersinia pseudotuberculosis.

Zinc homeostasis is crucial for the development and stress resistance of bacteria in the environment. Serial zinc sensing transcriptional regulators, zinc transporters and zinc binding proteins were found to maintain the zinc homeostasis in bacteria. Zur is a zinc uptake regulator that is widely distributed in species, and ZnuABC, as well as the Type VI Secretion System (T6SS4) function in zinc acquisition. Here, we report that the regulator Zur inhibits the expression of the ZnuABC which inhibition could be eliminated at low zinc level, and upregulates the T6SS4 operon in Yersinia pseudotuberculosis to facilitate Zn2+ uptake and oxidative stress resistance. Zur regulates the expression of ZnuABC and T6SS4 by directly binding to their promoter regions. Zur senses the Zn2+ concentration and represses ZnuABC in a Zn2+-containing environment. Zur works as an auxiliary regular activator of T6SS4, facilitating oxidative stress resistance. This study revealed the dual function of regulator Zur on ZnuABC and T6SS4, and enriched the knowledge of Zn2+ homeostasis maintenance in Y. pseudotuberculosis.

Identification of Robinia pseudoacacia target proteins responsive to Mesorhizobium amphore CCNWGS0123 effector protein NopT.

Nodulation outer proteins secreted via type 3 secretion systems are involved in the process of symbiosis between legume plants and rhizobia. To study the function of NopT in symbiosis, we mutated nopT in Mesorhizobium amphore CCNWGS0123 (GS0123), which can nodulate black locust (Robinia pseudoacacia). The nopT mutant induced higher levels of jasmonic acid, salicylic acid, and hydrogen peroxide accumulation in the roots of R. pseudoacacia compared with wild-type GS0123. The ΔnopT mutant induced higher disease-resistant gene expression 72 hours post-inoculation (hpi), whereas GS0123 induced higher disease-resistant gene expression earlier, at 36 hpi. Compared with the nopT mutant, GS0123 induced the up-regulation of most genes at 36 hpi and the down-regulation of most genes at 72 hpi. Proteolytically active NopT_GS0123 induced hypersensitive responses when expressed transiently in tobacco leaves (Nicotiana benthamiana). Two NopT_GS0123 targets in R. pseudoacacia were identified, ATP-citrate synthase alpha chain protein 2 and hypersensitive-induced response protein. Their interactions with NopT_GS0123 triggered resistance by the plant immune system. In conclusion, NopT_GS0123 inhibited the host plant immune system and had minimal effect on nodulation in R. pseudoacacia. Our results reveal the underlying molecular mechanism of NopT function in plant-symbiont interactions.

Siderophore-Mediated Iron Acquisition Enhances Resistance to Oxidative and Aromatic Compound Stress in Cupriavidus necator JMP134.

Many bacteria secrete siderophores to enhance iron uptake under iron-restricted conditions. In this study, we found that Cupriavidus necator JMP134, a well-known aromatic pollutant-degrading bacterium, produces an unknown carboxylate-type siderophore named cupriabactin to overcome iron limitation. Using genome mining, targeted mutagenesis, and biochemical analysis, we discovered an operon containing six open reading frames (cubA-F) in the C. necator JMP134 genome that encodes proteins required for the biosynthesis and uptake of cupriabactin. As the dominant siderophore of C. necator JMP134, cupriabactin promotes the growth of C. necator JMP134 under iron-limited conditions via enhanced ferric iron uptake. Furthermore, we demonstrated that the iron concentration-dependent expression of the cub operon is mediated by the ferric uptake regulator (Fur). Physiological analyses revealed that the cupriabactin-mediated iron acquisition system influences swimming motility, biofilm formation, and resistance to oxidative and aromatic compound stress in C. necator JMP134. In conclusion, we identified a carboxylate-type siderophore named cupriabactin, which plays important roles in iron scavenging, bacterial motility, biofilm formation, and stress resistance.IMPORTANCE Since siderophores have been widely exploited for agricultural, environmental, and medical applications, the identification and characterization of new siderophores from different habitats and organisms will have great beneficial applications. Here, we identified a novel siderophore-producing gene cluster in C. necator JMP134. This gene cluster produces a previously unknown carboxylate siderophore, cupriabactin. Physiological analyses revealed that the cupriabactin-mediated iron acquisition system influences swimming motility, biofilm formation, and oxidative stress resistance. Most notably, this system also plays important roles in increasing the resistance of C. necator JMP134 to stress caused by aromatic compounds, which provide a promising strategy to engineer more efficient approaches to degrade aromatic pollutants.

Manipulation of the silkworm immune system by a metalloprotease from the pathogenic bacterium Pseudomonas aeruginosa.

Antimicrobial peptide (AMP) production and melanization are two key humoral immune responses in insects. Induced synthesis of AMPs results from Toll and IMD signal transduction whereas melanization depends on prophenoloxidase (PPO) activation system. During invasion, pathogens produce toxins and other virulent factors to counteract host immune responses. Here we show that the pathways leading to PPO activation and AMP synthesis in the silkworm Bombyx mori are affected by a metalloprotease, named elastase B, secreted by Pseudomonas aeruginosa (PAO1). The metalloprotease gene (lasB) was expressed shortly after PAO1 cells had been injected into the larval silkworm hemocoel, leading to an increase of elastase activity. Injection of the purified PAO1 elastase B into silkworm hemolymph compromised PPO activation. In contrast, the protease caused a level increase of gloverin, an AMP in the hemolymph. To verify our results obtained using the purified elastase B, we infected B. mori with PAO1 ΔlasB mutant and found that PO activity in hemolymph of the PAO1 ΔlasB-infected larvae was significantly higher than that in the wild type-infected. The mutant-inhabited hemolymph had lower levels of gloverin and antimicrobial activity. PAO1 ΔlasB showed a decreased viability in the silkworm hemolymph whereas the host had a lower mortality. In addition, the effects caused by the ΔlasB mutant were restored by a complementary strain. These data collectively indicated that the elastase B produced by PAO1 is an important virulent factor that manipulates the silkworm immune system during infection.

Propioniciclava sinopodophylli sp. nov., isolated from leaves of Sinopodophyllum hexandrum (Royle) Ying.

A Gram-reaction-positive, facultatively anaerobic, rod-shaped and non-motile bacterial strain, designated TEYR-7T, was isolated from the leaves of Sinopodophyllum hexandrum collected from the Qinling Mountains in Shaanxi Province, northwest China. Growth of strain TEYR-7T occurred at 15-37 °C (optimum, 28-30 °C), at pH 6.0-9.0 (optimum, pH 7.0) and in the presence of 0-3 % (w/v) NaCl (optimum, 0-1 %). Propionate and acetate were produced from glucose fermentation. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TEYR-7T was a member of the phylum Actinobacteria, exhibiting the highest sequence similarity to Propioniciclava tarda DSM 22130T (94.3 %). The only respiratory quinone detected in strain TEYR-7T was menaquinone MK-9(H4) and the major cellular fatty acids (>10 %) were anteiso-C15 : 0 and C16 : 0. The polar lipid profile consisted of phosphatidylglycerol, diphosphatidylglycerol, two unidentified glycolipids, an unidentified phospholipid and three unidentified lipids. The genomic DNA G+C content was 71.2 mol%. meso-Diaminopimelic acid was detected in the peptidoglycan. On the basis of data from the present polyphasic taxonomic study, strain TEYR-7T is considered to represent a novel species of the genus Propioniciclava, for which the name Propioniciclava sinopodophylli sp. nov. is proposed. The type strain is TEYR-7T (=CCTCC AB 2015257T=KCTC 33808T).

A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition.

Iron sequestration by host proteins contributes to the defence against bacterial pathogens, which need iron for their metabolism and virulence. A Pseudomonas aeruginosa mutant lacking all three known iron acquisition systems retains the ability to grow in media containing iron chelators, suggesting the presence of additional pathways involved in iron uptake. Here we screen P. aeruginosa mutants defective in growth in iron-depleted media and find that gene PA2374, proximal to the type VI secretion system H3 (H3-T6SS), functions synergistically with known iron acquisition systems. PA2374 (which we have renamed TseF) appears to be secreted by H3-T6SS and is incorporated into outer membrane vesicles (OMVs) by directly interacting with the iron-binding Pseudomonas quinolone signal (PQS), a cell-cell signalling compound. TseF facilitates the delivery of OMV-associated iron to bacterial cells by engaging the Fe(III)-pyochelin receptor FptA and the porin OprF. Our results reveal links between type VI secretion, cell-cell signalling and classic siderophore receptors for iron acquisition in P. aeruginosa.

Graded Response of the Multifunctional 2-Cysteine Peroxiredoxin, CgPrx, to Increasing Levels of Hydrogen Peroxide in Corynebacterium glutamicum.

AIMS: Eukaryotic typical 2-cysteine (Cys) peroxiredoxins (Prxs) are multifunctional proteins subjected to complex regulation and play important roles in oxidative stress resistance, hydrogen peroxide (H2O2) signaling modulation, aging, and cancer, but the information on the biochemical functions and regulation mechanisms of prokaryotic atypical 2-Cys Prxs is largely lacking. RESULTS: In this study, we show that at low peroxide concentrations, the atypical 2-Cys Prx in Corynebacterium glutamicum (CgPrx) mainly exists as monomers and displays thioredoxin (Trx)-dependent peroxidase activity. Moderate oxidative stress causes reversible S-mycothiolation of the H2O2-sensing Cys63 residue, which keeps CgPrx exclusively in dimer form with neither peroxidase nor chaperone activity. Then, the increased levels of H2O2 could act as a messenger to oxidize the redox-sensitive regulator hydrogen peroxide-inducible gene activator, leading to activation of expression of the more efficient mycothiol peroxidase and catalase to eliminate excessive peroxide. If oxidative stress is too severe, the H2O2-sensing Cys63 becomes hyperoxidized to sulfonic acid, which irreversibly inactivates the peroxidase activity, and most of CgPrx will be converted to multimeric chaperones for salvage of damaged proteins. INNOVATION: We demonstrate for the first time that atypical 2-Cys CgPrx acts as both a Trx-dependent peroxidase and a molecular chaperone and plays a regulatory role in modulating the peroxide-mediated signaling cascades. CONCLUSION: These results reveal that CgPrx functions as a multifunctional protein crucial for adapting appropriate responses to different levels of oxidative challenge in C. glutamicum. Antioxid. Redox Signal. 26, 1-14.

Paenibacillus qinlingensis sp. nov., an indole-3-acetic acid-producing bacterium isolated from roots of Sinopodophyllum hexandrum (Royle) Ying.

A novel indole-3-acetic acid-producing bacterium, designated TEGT-2T, was isolated from the roots of Sinopodophyllum hexandrum collected from the Qinling Mountains in shaanxi province, northwestern China, and was subjected to a taxonomic study by using a polyphasic approach. Cells of strain TEGT-2T were Gram-stain-positive, strictly aerobic, endospore-forming rods and motile by means of peritrichous flagella. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TEGT-2T was a member of the genus Paenibacillus, exhibiting the highest sequence similarity to Paenibacillus pectinilyticus KCTC 13222T (97.9 %), Paenibacillus frigoriresistens CCTCC AB 2011150T (97.3 %), Paenibacillus ferrarius CCTCC AB 2013369T (96.9 %) and Paenibacillus alginolyticus NBRC 15375T (96.5 %). The only menaquinone detected was MK-7, and the major fatty acid was anteiso-C15 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phospholipids, an unidentified aminolipid and two unidentified lipids. meso-Diaminopimelic acid was detected in the peptidoglycan. The DNA G+C content was 46.6 mol%. DNA-DNA relatedness values for strain TEGT-2T with respect to its closest phylogenetic relatives Paenibacilluspectinilyticus KCTC 13222T and Paenibacillus. frigoriresistens CCTCC AB 2011150T were lower than 40 %. Based on the phenotypic, phylogenetic and genotypic data, strain TEGT-2T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus qinlingensis sp. nov. is proposed. The type strain is TEGT-2T (=CCTCC AB 2015258T=KCTC 33806T).

Functional characterization of a csoR-cueA divergon in Bradyrhizobium liaoningense CCNWSX0360, involved in copper, zinc and cadmium cotolerance.

Random mutagenesis in a symbiotic nitrogen-fixing Bradyrhizobium liaoningense CCNWSX0360 (Bln0360) using Tn5 identified five copper (Cu) resistance-related genes. They were functionally sorted into three groups: transmembrane transport (cueA and tolC); oxidation (copA); and protection of the membrane barrier (lptE and ctpA). The gene cueA, together with the upstream csoR (Cu-sensitive operon repressor), constituted a csoR-cueA divergon which plays a crucial role in Cu homeostasis. Deletion of cueA decreased the Cu tolerance of cells, and complementation of this mutant restored comparable Cu resistance to that of the wild-type. Transcriptional and fusion expression analysis demonstrated that csoR-cueA divergon was up-regulated by both the monovalent Cu+ and divalent Zn2+/Cd2+, and negatively regulated by transcriptional repressor CsoR, via a bidirectional promoter. Deletion of csoR renders the cell hyper-resistant to Cu, Zn and Cd. Although predicted to encode a Cu transporting P-type ATPase (CueA), cueA also conferred resistance to zinc and cadmium; two putative N-MBDs (N-terminal metal binding domains) of CueA were required for the Cu/Zn/Cd tolerance. Moreover, cueA is needed for nodulation competitiveness of B. liaoningense in Cu rich conditions. Together, the results demonstrated a crucial role for the csoR-cueA divergon as a component of the multiple-metal resistance machinery in B. liaoningense.

Overexpression of Mycothiol Disulfide Reductase Enhances Corynebacterium glutamicum Robustness by Modulating Cellular Redox Homeostasis and Antioxidant Proteins under Oxidative Stress.

Mycothiol (MSH) is the dominant low-molecular-weight thiol (LMWT) unique to high-(G+C)-content Gram-positive Actinobacteria, such as Corynebacterium glutamicum, and is oxidised into its disulfide form mycothiol disulfide (MSSM) under oxidative conditions. Mycothiol disulfide reductase (Mtr), an NADPH-dependent enzyme, reduces MSSM to MSH, thus maintaining intracellular redox homeostasis. In this study, a recombinant plasmid was constructed to overexpress Mtr in C. glutamicum using the expression vector pXMJ19-His6. Mtr-overexpressing C. glutamicum cells showed increased tolerance to ROS induced by oxidants, bactericidal antibiotics, alkylating agents, and heavy metals. The physiological roles of Mtr in resistance to oxidative stresses were corroborated by decreased ROS levels, reduced carbonylation damage, decreased loss of reduced protein thiols, and a massive increase in the levels of reversible protein thiols in Mtr-overexpressing cells exposed to stressful conditions. Moreover, overexpression of Mtr caused a marked increase in the ratio of reduced to oxidised mycothiol (MSH:MSSM), and significantly enhanced the activities of a variety of antioxidant enzymes, including mycothiol peroxidase (MPx), mycoredoxin 1 (Mrx1), thioredoxin 1 (Trx1), and methionine sulfoxide reductase A (MsrA). Taken together, these results indicate that the Mtr protein functions in C. glutamicum by protecting cells against oxidative stress.

Mycothiol protects Corynebacterium glutamicum against acid stress via maintaining intracellular pH homeostasis, scavenging ROS, and S-mycothiolating MetE.

Mycothiol (MSH) plays a major role in protecting cells against oxidative stress and detoxification from a broad range of exogenous toxic agents. In the present study, we reveal that intracellular MSH contributes significantly to the adaptation to acidic conditions in the model organism Corynebacterium glutamicum. We present evidence that MSH confers C. glutamicum with the ability to adapt to acidic conditions by maintaining pHi homeostasis, scavenging reactive oxygen species (ROS), and protecting methionine synthesis by the S-mycothiolation modification of methionine synthase (MetE). The role of MSH in acid adaptation was further confirmed by improving the acid tolerance of C. glutamicum by overexpressing the key MSH synthesis gene mshA. Hence, our work provides insights into a previously unknown, but important, aspect of the C. glutamicum cellular response to acid stress. The results reported here may help to understand acid tolerance mechanisms in acid sensitive bacteria and may open a new avenue for improving acid resistance in industry strains for the production of bio-based chemicals from renewable biomass.