AKSF1 Isolated From the Rice-Virulent Strain Acidovorax avenae K1 Is a Novel Effector That Suppresses PAMP-Triggered Immunity in Rice

Microbial pathogens deliver effectors into plant cells to suppress plant immune responses and modulate host metabolism in order to support infection processes. We sought to determine if the Acidovorax avenae rice-virulent K1 strain can suppress pathogen-associated molecular pattern–triggered immunity (PTI) induced by flagellin isolated from the rice-avirulent N1141 strain. The flagellin-triggered PTI, including H2O2 generation, callose deposition, and expression of several immune-related genes were strongly suppressed in K1 preinoculated cultured rice cells in a type III secretion system (T3SS)-dependent manner. By screening 4,562 transposon-tagged mutants based on their suppression ability, we found that 156 transposon-tagged K1 mutants lost the ability to suppress PTI induction. Mutant sequence analysis, comprehensive expression analysis using RNA sequencing, and the prediction of secretion through T3SS showed that a protein named A. avenae K1 suppression factor 1 (AKSF1) suppresses flagellin-triggered PTI in rice. Translocation of AKSF1 protein into rice cells is dependent on the T3SS during infection, an AKSF1-disruption mutant lost the ability to suppress PTI responses, and expression of AKSF1 in the AKSF1-disruption mutant complemented the suppression activity. When AKSF1-disruption mutants were inoculated into the host rice plant, reduction of the disease symptoms and suppression of bacterial growth were observed. Taken together, our results demonstrate that AKSF1 is a novel effector that can suppress the PTI in a host rice plant. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CCO “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

A Novel CreA-Mediated Regulation Mechanism of Cellulase Expression in the Thermophilic Fungus Humicola insolens

The thermophilic fungus Humicola insolens produces cellulolytic enzymes that are of great scientific and commercial interest; however, few reports have focused on its cellulase expression regulation mechanism. In this study, we constructed a creA gene (carbon catabolite repressor gene) disruption mutant strain of H. insolens that exhibited a reduced radial growth rate and stouter hyphae compared to the wild-type (WT) strain. The creA disruption mutant also expressed elevated pNPCase (cellobiohydrolase activities), pNPGase (β-glucosidase activities), and xylanase levels in non-inducing fermentation with glucose. Unlike other fungi, the H. insolens creA disruption mutant displayed lower FPase (filter paper activity), CMCase (carboxymethyl cellulose activity), pNPCase, and pNPGase activity than observed in the WT strain when fermentation was induced using Avicel, whereas its xylanase activity was higher than that of the parental strain. These results indicate that CreA acts as a crucial regulator of hyphal growth and is part of a unique cellulase expression regulation mechanism in H. insolens. These findings provide a new perspective to improve the understanding of carbon catabolite repression regulation mechanisms in cellulase expression, and enrich the knowledge of metabolism diversity and molecular regulation of carbon metabolism in thermophilic fungi.

Glucosinolate Distribution in the Aerial Parts of sel1-10, a Disruption Mutant of the Sulfate Transporter SULTR1;2, in Mature Arabidopsis thaliana Plants

Plants take up sulfur (S), an essential element for all organisms, as sulfate, which is mainly attributed to the function of SULTR1;2 in Arabidopsis. A disruption mutant of SULTR1;2, sel1-10, has been characterized with phenotypes similar to plants grown under sulfur deficiency (−S). Although the effects of −S on S metabolism were well investigated in seedlings, no studies have been performed on mature Arabidopsis plants. To study further the effects of −S on S metabolism, we analyzed the accumulation and distribution of S-containing compounds in different parts of mature sel1-10 and of the wild-type (WT) plants grown under long-day conditions. While the levels of sulfate, cysteine, and glutathione were almost similar between sel1-10 and WT, levels of glucosinolates (GSLs) differed between them depending on the parts of the plant. GSLs levels in the leaves and stems were generally lower in sel1-10 than those in WT. However, sel1-10 seeds maintained similar levels of aliphatic GSLs to those in WT plants. GSL accumulation in reproductive tissues is likely to be prioritized even when sulfate supply is limited in sel1-10 for its role in S storage and plant defense.

The disruption of verM activates the production of gliocladiosin A and B in Clonostachys rogersoniana

Two dipeptides (Gliocladiosin A and B) conjugated with a macrolide were identified in the verM disruption mutant of Clonostachys rogersoniana.

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum

ABSTRACTCrude glycerol, the major by-product of biodiesel production, is an attractive bioprocessing feedstock owing to its abundance, low cost, and high degree of reduction. In line with the advent of the biodiesel industry,Clostridium pasteurianumhas gained prominence as a result of its unique capacity to convert waste glycerol inton-butanol, a high-energy biofuel. However, no efforts have been directed at abolishing the production of 1,3-propanediol (1,3-PDO), the chief competing product ofC. pasteurianumglycerol fermentation. Here, we report rational metabolic engineering ofC. pasteurianumfor enhancedn-butanol production through inactivation of the gene encoding 1,3-PDO dehydrogenase (dhaT). In spite of current models of anaerobic glycerol dissimilation, culture growth and glycerol utilization were unaffected in thedhaTdisruption mutant (dhaT::Ll.LtrB). Metabolite characterization of thedhaT::Ll.LtrB mutant revealed an 83% decrease in 1,3-PDO production, encompassing the lowestC. pasteurianum1,3-PDO titer reported to date (0.58 g liter−1). With 1,3-PDO formation nearly abolished, glycerol was converted almost exclusively ton-butanol (8.6 g liter−1), yielding a highn-butanol selectivity of 0.83 gn-butanol g−1of solvents compared to 0.51 gn-butanol g−1of solvents for the wild-type strain. Unexpectedly, high-performance liquid chromatography (HPLC) analysis ofdhaT::Ll.LtrB mutant culture supernatants identified a metabolite peak consistent with 1,2-propanediol (1,2-PDO), which was confirmed by nuclear magnetic resonance (NMR). Based on these findings, we propose a new model for glycerol dissimilation byC. pasteurianum, whereby the production of 1,3-PDO by the wild-type strain and low levels of both 1,3-PDO and 1,2-PDO by the engineered mutant balance the reducing equivalents generated during cell mass synthesis from glycerol.IMPORTANCEOrganisms from the genusClostridiumare perhaps the most notable native cellular factories, owing to their vast substrate utilization range and equally diverse variety of metabolites produced. The ability ofC. pasteurianumto sustain redox balance and glycerol fermentation despite inactivation of the 1,3-PDO pathway is a testament to the exceptional metabolic flexibility exhibited by clostridia. Moreover, identification of a previously unknown 1,2-PDO-formation pathway, as detailed herein, provides a deeper understanding of fermentative glycerol utilization in clostridia and will inform future metabolic engineering endeavors involvingC. pasteurianum. To our knowledge, theC. pasteurianum dhaTdisruption mutant derived in this study is the only organism that produces both 1,2- and 1,3-PDOs. Most importantly, the engineered strain provides an excellent platform for highly selective production ofn-butanol from waste glycerol.

Induction of Virulence Gene Expression in Staphylococcus aureus by Pulmonary Surfactant

ABSTRACTWe performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression inStaphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of thesigBgene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of theessC,psiA, andhlgBgenes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest thatS. aureusresists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.