Bacterial Rhs proteins containing toxic domains are often secreted by type VI secretion systems (T6SSs) through unclear mechanisms. Here, we show that the T6SS Rhs-family effector TseI of Aeromonas dhakensis is subject to self-cleavage at both the N-and the Cterminus, releasing the middle Rhs core and two VgrG-interacting domains (which we name VIRN and VIRC). VIRC is an endonuclease, and the immunity protein TsiI protects against VIRC toxicity through direct interaction. Proteolytic release of VIRC and VIRN is mediated, respectively, by an internal aspartic protease activity and by two conserved glutamic residues in the Rhs core. Mutations abolishing self-cleavage do not block secretion, but reduce TseI toxicity. Deletion of VIRN or the Rhs core abolishes secretion. TseI homologs from Pseudomonas syringae, P. aeruginosa, and Vibrio parahaemolyticus are also self-cleaved. VIRN and VIRC interact with protein VgrG1, while the Rhs core interacts with protein TecI. We propose that VIRN and the Rhs core act as T6SS intramolecular chaperones to facilitate toxin secretion and function.

Highlights d Combinatorial effector inactivation delineates functions and physical contact d Tit-for-tat of P. aeruginosa responds to a specific effector TseL d Immunity-independent stress responses provide protection against TseL toxicity d Stress response is akin to microbial innate immunity for general protection

Tuberculosis (TB) is a major global health problem, and multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) are spreading throughout the world. However, conventional drug susceptibility test (DST) methods, which rely on the detection of the colony formation on a solid medium, require 1-2 months to the result. A rapid and accurate DST is necessary to identify patients with drug-resistant TB and treat them with appropriate drugs. Here, we used microscopic imaging of Mycobacterium tuberculosis (MTB) immobilized in an agarose matrix for a rapid DST. The agarose matrix, which was molded in a microfluidic chip, was inoculated with MTB, and TB drugs in liquid culture medium diffused throughout the agarose to reach the MTB immobilized in the agarose matrix. After the responses of MTB to drugs were tracked with an automated microscopic system, an image-processing program automatically determined the susceptibility and resistance of MTB to specific doses of TB drugs. The automatic DST system was able to assess the drug susceptibility of various drug-resistant clinical TB strains within 9 days with an accuracy comparable to that of conventional method. Our rapid DST method based on microscopic time-lapse imaging greatly reduces the time required for a DST and can be used to rapidly and accurately treat TB patients.

Although the unique nanostructure of bacterial cellulose (BC) imparts superior mechanochemical properties and thus allows for diverse applications, the high production cost of BC necessitates the development of more cost‐effective solutions, for example, those using lignocellulosic biomass as a substrate and relying on its pretreatment and saccharification to generate fermentable sugars. However, the various species (e.g., aliphatic acids, furans, and phenolics) produced during pretreatment may interfere with bacterial cell growth and BC production. Herein, we investigated the effects of aliphatic (acetic and formic) acids, furans (5‐hydroxymethylfurfural [5‐HMF] and furfural), and phenolics (syringaldehyde and p‐coumaric acid) on the production of BC. This production was enhanced at low aliphatic acid concentrations (1 g/L acetic acid and 0.5 g/L formic acid) but was suppressed by at least 90% in cases of 0.75 g/L formic acid, 0.4 g/L furfural, 4 g/L 5‐HMF, 2.5 g/L syringaldehyde, and 2.5 g/L p‐coumaric acid. BC production efficiencies of 97.86%, 76.66%, and 73.50% were observed for Miscanthus, barley straw, and pine tree hydrolysates, respectively, under optimal conditions. Therefore, these results provided the possibility to utilize the most abundant and sustainable lignocellulose on the planet for BC production.

Despite the benefits associated with the use of food waste (FW), there are mixed consumer perceptions regarding pork quality harvested from pigs fed FW. Twenty crossbred pigs were selected for the present study. Ten pigs were fed a conventional diet (control group), and the other 10 pigs were given a conventional diet and FW (FW group) during different growth stages. Meat quality in the FW group showed deteriorative qualities with higher lightness and yellowness synonymous to pale soft exudative meat. Drip loss in the experimental group was significantly higher than that in the control group (p<0.01). The contents of polyunsaturated fatty acids in the FW group were higher and those of saturated and monounsaturated fatty acids were lower than those in the control group. The contents of thiobarbituric acid were significantly different between the control and FW groups (p<0.05). There was also a significant difference between the control and FW groups in terms of off-flavor (p<0.05) after sensory evaluation. To conclude, the off-flavor noted, including other inferior pork quality traits, in the FW group implies that FW should not be used as swine feed.

The hypersensitive response (HR) is a robust immune response mediated by nucleotidebinding, leucine-rich repeat receptors (NLRs). However, the early molecular event that links activated NLRs to cell death is unclear.Here, we demonstrate that NLRs target plasma membrane H + -ATPases (PMAs) that generate electrochemical potential, an essential component of living cells, across the plasma membrane. CC A 309, an autoactive N-terminal domain of a coiled-coil NLR (CNL) in pepper, is associated with PMAs. Silencing or overexpression of PMAs reversibly affects cell death induced by CC A 309 in Nicotiana benthamiana.CC A 309-induced extracellular alkalization causes plasma membrane depolarization, followed by cell death. Coimmunoprecipitation analyses suggest that CC A 309 inhibits PMA activation by preoccupying the dephosphorylated penultimate threonine residue of PMA. Moreover, pharmacological experiments using fusicoccin, an irreversible PMA activator, showed that inhibition of PMAs contributes to CNL-type (but not Toll interleukin-1 receptor NLR-type) resistance protein-induced cell death.We suggest PMAs as primary targets of plasma membrane-associated CNLs leading to HRassociated cell death by disturbing the electrochemical gradient across the membrane. These results provide new insight into NLR-mediated cell death in plants, as well as innate immunity in higher eukaryotes.

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The Disc Agarose Channel (DAC) system utilizes microfluidics and imaging technologies and is fully automated and capable of tracking single cell growth to produce Mycobacterium tuberculosis (MTB) drug susceptibility testing (DST) results within 3~7 days. In particular, this system can be easily used to perform DSTs without the fastidious preparation of the inoculum of MTB cells. Inoculum effect is one of the major problems that causes DST errors. The DAC system was not influenced by the inoculum effect and produced reliable DST results. In this system, the minimum inhibitory concentration (MIC) values of the first-line drugs were consistent regardless of inoculum sizes ranging from ~103 to ~108 CFU/mL. The consistent MIC results enabled us to determine the critical concentrations for 12 anti-tuberculosis drugs. Based on the determined critical concentrations, further DSTs were performed with 254 MTB clinical isolates without measuring an inoculum size. There were high agreement rates (96.3%) between the DAC system and the absolute concentration method using Löwenstein-Jensen medium. According to these results, the DAC system is the first DST system that is not affected by the inoculum effect. It can thus increase reliability and convenience for DST of MTB. We expect that this system will be a potential substitute for conventional DST systems.