An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202 / Una bomba de eflujo en la isla genómica GI-M202a media la transferencia de resistencia a la polimixina B en Pandoraea pnomenusa M202

Background: Polymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront. Methods: Pandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling. Results: The MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter...(AU)

An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202.

BACKGROUND: Polymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront. METHODS: Pandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling. RESULTS: The MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites. CONCLUSIONS: These findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.

Novel mechanism of hydrogen peroxide for promoting efficient natamycin synthesis in Streptomyces.

The mechanism of regulation of natamycin biosynthesis by Streptomyces in response to oxidative stress is unclear. Here, we first show cholesterol oxidase SgnE, which catalyzes the formation of H2O2 from sterols, triggered a series of redox-dependent interactions to stimulate natamycin production in S. gilvosporeus. In response to reactive oxygen species, residues Cys212 and Cys221 of the H2O2-sensing consensus sequence of OxyR were oxidized, resulting in conformational changes in the protein: OxyR extended its DNA-binding domain to interact with four motifs of promoter p sgnM . This acted as a redox-dependent switch to turn on/off gene transcription of sgnM, which encodes a cluster-situated regulator, by controlling the affinity between OxyR and p sgnM , thus regulating the expression of 12 genes in the natamycin biosynthesis gene cluster. OxyR cooperates with SgnR, another cluster-situated regulator and an upstream regulatory factor of SgnM, synergistically modulated natamycin biosynthesis by masking/unmasking the -35 region of p sgnM depending on the redox state of OxyR in response to the intracellular H2O2 concentration. IMPORTANCE Cholesterol oxidase SgnE is an indispensable factor, with an unclear mechanism, for natamycin biosynthesis in Streptomyces. Oxidative stress has been attributed to the natamycin biosynthesis. Here, we show that SgnE catalyzes the formation of H2O2 from sterols and triggers a series of redox-dependent interactions to stimulate natamycin production in S. gilvosporeus. OxyR, which cooperates with SgnR, acted as a redox-dependent switch to turn on/off gene transcription of sgnM, which encodes a cluster-situated regulator, by masking/unmasking its -35 region, to control the natamycin biosynthesis gene cluster. This work provides a novel perspective on the crosstalk between intracellular ROS homeostasis and natamycin biosynthesis. Application of these findings will improve antibiotic yields via control of the intracellular redox pressure in Streptomyces.

TaERF87 and TaAKS1 synergistically regulate TaP5CS1/TaP5CR1-mediated proline biosynthesis to enhance drought tolerance in wheat.

Drought stress limits wheat production and threatens food security world-wide. While ethylene-responsive factors (ERFs) are known to regulate plant response to drought stress, the regulatory mechanisms responsible for a tolerant phenotype remain unclear. Here, we describe the positive regulatory role of TaERF87 in mediating wheat tolerance to drought stress. TaERF87 overexpression (OE) enhances drought tolerance, while silencing leads to drought sensitivity in wheat. RNA sequencing with biochemical assays revealed that TaERF87 activates the expression of the proline biosynthesis genes TaP5CS1 and TaP5CR1 via direct binding to GCC-box elements. Furthermore, proline accumulates to higher levels in TaERF87- and TaP5CS1-OE lines than that in wild-type plants under well-watered and drought stress conditions concomitantly with enhanced drought tolerance in these transgenic lines. Moreover, the interaction between TaERF87 and the bHLH transcription factor TaAKS1 synergistically enhances TaP5CS1 and TaP5CR1 transcriptional activation. TaAKS1 OE also increases wheat drought tolerance by promoting proline accumulation. Additionally, our findings verified that TaERF87 and TaAKS1 are targets of abscisic acid-responsive element binding factor 2 (TaABF2). Together, our study elucidates the mechanisms underlying a positive response to drought stress mediated by the TaABF2-TaERF87/TaAKS1-TaP5CS1/TaP5CR1 module, and identifies candidate genes for the development of elite drought-tolerant wheat varieties.

A gain-of-function allele of a DREB transcription factor gene ameliorates drought tolerance in wheat.

Drought is a major environmental factor limiting wheat production worldwide. However, the genetic components underlying wheat drought tolerance are largely unknown. Here, we identify a DREB transcription factor gene (TaDTG6-B) by genome-wide association study that is tightly associated with drought tolerance in wheat. Candidate gene association analysis revealed that a 26-bp deletion in the TaDTG6-B coding region induces a gain-of-function for TaDTG6-BDel574, which exhibits stronger transcriptional activation, protein interactions, and binding activity to dehydration-responsive elements (DRE)/CRT cis-elements than the TaDTG6-BIn574 encoded by the allele lacking the deletion, thus conferring greater drought tolerance in wheat seedlings harboring this variant. Knockdown of TaDTG6-BDel574 transcripts attenuated drought tolerance in transgenic wheat, whereas its overexpression resulted in enhanced drought tolerance without accompanying phenotypic abnormalities. Furthermore, the introgression of the TaDTG6-BDel574 elite allele into drought-sensitive cultivars improved their drought tolerance, thus providing a valuable genetic resource for wheat breeding. We also identified 268 putative target genes that are directly bound and transcriptionally regulated by TaDTG6-BDel574. Further analysis showed that TaDTG6-BDel574 positively regulates TaPIF1 transcription to enhance wheat drought tolerance. These results describe the genetic basis and accompanying mechanism driving phenotypic variation in wheat drought tolerance, and provide a novel genetic resource for crop breeding programs.

Variation in cis-regulation of a NAC transcription factor contributes to drought tolerance in wheat.

Drought is a major environmental factor limiting wheat production worldwide, and developing drought-tolerant cultivars is a central challenge for wheat breeders globally. Therefore, it is important to identify genetic components determining drought tolerance in wheat. In this study, we identified a wheat NAC gene (TaNAC071-A) that is tightly associated with drought tolerance by a genome-wide association study. Knockdown of TaNAC071-A in wheat attenuated plant drought tolerance, whereas its overexpression significantly enhanced drought tolerance through improved water-use efficiency and increased expression of stress-responsive genes. This heightened water-saving mechanism mitigated the yield loss caused by water deficit. Further candidate gene association analysis showed that a 108-bp insertion in the promoter of TaNAC071-A alters its expression level and contributes to variation in drought tolerance among wheat accessions. This insertion contains two MYB cis-regulatory elements (CREs) that can be directly bound by the MYB transcription activator, TaMYBL1, thereby leading to increased TaNAC071-A expression and plant drought tolerance. Importantly, introgression of this 108-bp insertion allele, TaNAC071-AIn-693, into drought-sensitive cultivars could improve their drought tolerance, demonstrating that it is a valuable genetic resource for wheat breeding. Taken together, our findings highlight a major breakthrough in determining the genetic basis underlying phenotypic variation in wheat drought tolerance and showcase the potential of exploiting CRE-containing indels for improving important agronomical traits.

Diamond Grinding Wheel Condition Monitoring Based on Acoustic Emission Signals.

Acoustic emission (AE) phenomenon has a direct relationship with the interaction of tool and material which makes AE the most sensitive one among various process variables. However, its prominent sensitivity also means the characteristics of random and board band. Feature representation is a difficult problem for AE-based monitoring and determines the accuracy of monitoring system. It is knottier for the situation of using diamond wheel grinding optical components, not only because of the complexity of grinding process but also the high requirement on surface and subsurface quality. This paper is dedicated to AE-based condition monitoring of diamond wheel during grinding brittle materials and feature representation is paid more attention. AE signal of brittle-regime grinding is modeled as a superposition of a series of burst-type AE events. Theory analysis manifested that original time waveform and frequency spectrum are all suitable for feature representation. Considering the convolution form of b-AE in time domain, a convolutional neural network with original time waveform of AE signals as the input is built for multi-class classification of wheel state. Detailed state division in a wheel's whole life cycle is realized and the accuracy is over 90%. Different from the overlapping in time domain, AE components of different crack mechanisms are probably separated in frequency domain. From this point of view, AE spectrums are more suitable for feature extraction than the original time waveform. In addition, the time sequence of AE samples is essential for the evaluation of wheel's life elapse and making use of sequential information is just the idea behind recurrent neural network (RNN). Therefore, long short-term memory (LSTM), a special kind of RNN, is used to build a regression prediction model of wheel state with AE spectrums as the model input and satisfactory prediction accuracy is acquired on the test set.

Characterization of wheat homeodomain-leucine zipper family genes and functional analysis of TaHDZ5-6A in drought tolerance in transgenic Arabidopsis.

BACKGROUND: Many studies in Arabidopsis and rice have demonstrated that HD-Zip transcription factors play important roles in plant development and responses to abiotic stresses. Although common wheat (Triticum aestivum L.) is one of the most widely cultivated and consumed food crops in the world, the function of the HD-Zip proteins in wheat is still largely unknown. RESULTS: To explore the potential biological functions of HD-Zip genes in wheat, we performed a bioinformatics and gene expression analysis of the HD-Zip family. We identified 113 HD-Zip members from wheat and classified them into four subfamilies (I-IV) based on phylogenic analysis against proteins from Arabidopsis, rice, and maize. Most HD-Zip genes are represented by two to three homeoalleles in wheat, which are named as TaHDZX_ZA, TaHDZX_ZB, or TaHDZX_ZD, where X denotes the gene number and Z the wheat chromosome on which it is located. TaHDZs in the same subfamily have similar protein motifs and intron/exon structures. The expression profiles of TaHDZ genes were analysed in different tissues, at different stages of vegetative growth, during seed development, and under drought stress. We found that most TaHDZ genes, especially those in subfamilies I and II, were induced by drought stress, suggesting the potential importance of subfamily I and II TaHDZ members in the responses to abiotic stress. Compared with wild-type (WT) plants, transgenic Arabidopsis plants overexpressing TaHDZ5-6A displayed enhanced drought tolerance, lower water loss rates, higher survival rates, and higher proline content under drought conditions. Additionally, the transcriptome analysis identified a number of differentially expressed genes between 35S::TaHDZ5-6A transgenic and wild-type plants, many of which are involved in stress response. CONCLUSIONS: Our results will facilitate further functional analysis of wheat HD-Zip genes, and also indicate that TaHDZ5-6A may participate in regulating the plant response to drought stress. Our experiments show that TaHDZ5-6A holds great potential for genetic improvement of abiotic stress tolerance in crops.

Regulatory changes in TaSNAC8-6A are associated with drought tolerance in wheat seedlings.

Wheat is a staple crop produced in arid and semi-arid areas worldwide, and its production is frequently compromised by water scarcity. Thus, increased drought tolerance is a priority objective for wheat breeding programmes, and among their targets, the NAC transcription factors have been demonstrated to contribute to plant drought response. However, natural sequence variations in these genes have been largely unexplored for their potential roles in drought tolerance. Here, we conducted a candidate gene association analysis of the stress-responsive NAC gene subfamily in a wheat panel consisting of 700 varieties collected worldwide. We identified a drought responsive gene, TaSNAC8-6A, that is tightly associated with drought tolerance in wheat seedlings. Further analysis found that a favourable allele TaSNAC8-6AIn-313 , carrying an insertion in the ABRE promoter motif, is targeted by TaABFs and confers enhanced drought-inducible expression of TaSNAC8-6A in drought-tolerant genotypes. Transgenic wheat and Arabidopsis TaSNAC8-6A overexpression lines exhibited enhanced drought tolerance through induction of auxin- and drought-response pathways, confirmed by transcriptomic analysis, that stimulated lateral root development, subsequently improving water-use efficiency. Taken together, our findings reveal that natural variation in TaSNAC8-6A and specifically the TaSNAC8-6AIn-313 allele strongly contribute to wheat drought tolerance and thus represent a valuable genetic resource for improvement of drought-tolerant germplasm for wheat production.