iTRAQ-based proteomic analysis of Bacillus subtilis strain NCD-2 regulated by PhoPR two-component system: A comparative analysis with transcriptomics revealed the regulation for fengycin production by branched chain amino acids.

The PhoPR two-component system (TCS) is a signal transduction pathway to regulate the phosphate starvation response in Bacillus subtilis and regulated fengycin production in strain NCD-2 under low phosphate condition. The purpose of this study was to characterize the proteome level responses in the phoP-null mutant (MP) and the phoR-null mutant (MR), and to integrate the proteomics with the transcriptomic data obtained previously. The metabolic pathway for fengycin was predicted based on omics analysis as well as molecular genetics assay. Results showed the proteins and genes associated with biosynthesis of branched chain amino acids (BCAAs) were regulated by PhoPR TCS, and liquid chromatography mass spectrometry (LC-MS) analysis also confirmed that the production of BCAAs was down-regulated in the MP and MR mutants, when compared to wild-type strain NCD-2. Protein network analysis showed that the BCAA metabolism was linked to the biosynthesis of lipopeptides. The MP and MR strains decreased the fengycin production when cultured in modified Landy medium supplied with 0.42 mM phosphate, however, the fengycin production could be restored when the glutamic acid was replaced with BCAAs that were added to modified Landy medium. The lpdV gene, which is responsible for the BCAA degradation process, was deleted in strain NCD-2. Compared with the wild-type strain, the lpdV mutant produced significantly less fengycin in the medium supplied with BCAAs. Considered together, the results of this study indicate that the PhoPR TCS regulates fengycin production by affecting BCAA biosynthesis.

Genome mining and UHPLC-QTOF-MS/MS to identify the potential antimicrobial compounds and determine the specificity of biosynthetic gene clusters in Bacillus subtilis NCD-2.

BACKGROUND: Bacillus subtilis strain NCD-2 is an excellent biocontrol agent against plant soil-borne diseases and shows broad-spectrum antifungal activities. This study aimed to explore some secondary metabolite biosynthetic gene clusters and related antimicrobial compounds in strain NCD-2. An integrative approach combining genome mining and structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was adopted to interpret the chemical origins of metabolites with significant biological activities. RESULTS: Genome mining revealed nine gene clusters encoding secondary metabolites with predicted functions, including fengycin, surfactin, bacillaene, subtilosin, bacillibactin, bacilysin and three unknown products. Fengycin, surfactin, bacillaene and bacillibactin were successfully detected from the fermentation broth of strain NCD-2 by UHPLC-QTOF-MS/MS. The biosynthetic gene clusters of bacillaene, subtilosin, bacillibactin, and bacilysin showed 100% amino acid sequence identities with those in B. velezensis strain FZB42, whereas the identities of the surfactin and fengycin gene clusters were only 83 and 92%, respectively. Further comparison revealed that strain NCD-2 had lost the fenC and fenD genes in the fengycin biosynthetic operon. The biosynthetic enzyme-related gene srfAB for surfactin was divided into two parts. Bioinformatics analysis suggested that FenE in strain NCD-2 had a similar function to FenE and FenC in strain FZB42, and that FenA in strain NCD-2 had a similar function to FenA and FenD in strain FZB42. Five different kinds of fengycins, with 26 homologs, and surfactin, with 4 homologs, were detected from strain NCD-2. To the best of our knowledge, this is the first report of a non-typical gene cluster related to fengycin synthesis. CONCLUSIONS: Our study revealed a number of gene clusters encoding antimicrobial compounds in the genome of strain NCD-2, including a fengycin synthetic gene cluster that might be unique by using genome mining and UHPLC-QTOF-MS/MS. The production of fengycin, surfactin, bacillaene and bacillibactin might explain the biological activities of strain NCD-2.

Spatial and temporal distribution, degradation, and metabolism of three neonicotinoid insecticides on different parts, especially pests' target feeding parts of apple tree.

BACKGROUND: Neonicotinoid insecticides (NIs) have been recently banned in some countries because of increased pest resistance and deleterious risks to non-target organisms. Recent studies considered all parts of crops as a whole part in plant protection. However, there are few reports focused on the distribution and metabolic trends of NIs on target feeding sites of different pests in apple orchards. RESULTS: The spatial and temporal distribution, absorption, degradation, and metabolism of three NIs, imidacloprid, acetamiprid, and thiamethoxam, on different parts of apple trees were studied under foliar spray and root irrigation treatments. In the spray treatment, the initial average concentration ratios (TCRs) were 31.6% for lower shoots, 23.3% for upper leaves, 23.2% for upper shoots, 21.0% for lower leaves, and 0.5% and 0.4% for upper and lower fruits, respectively. The average half-lives of the three NIs were 2.9 days for shoots, 7.4 days for leaves, and 10.8 days for fruits. The degradation rate of shoots was 2.5 times that of leaves, and 3.6 times that of fruits. Imidacloprid olefin and N-methyl acetamiprid were two of the main metabolites. In the root treatment, both roots and soils had high TCRs during the whole sampling period. Only imidacloprid was transmitted to above-ground parts of the plants, with TCRs of 0.38-50.94%. CONCLUSION: This study found significant differences in spatial and temporal distribution, degradation, metabolism, and trends of NIs on different pest target sites of apple trees. The data obtained may help promote scientific control of target pests and evaluation of safety for non-target species in orchards. © 2020 Society of Chemical Industry.

Brain-wide Organization of Neuronal Activity and Convergent Sensorimotor Transformations in Larval Zebrafish.

Simultaneous recordings of large populations of neurons in behaving animals allow detailed observation of high-dimensional, complex brain activity. However, experimental approaches often focus on singular behavioral paradigms or brain areas. Here, we recorded whole-brain neuronal activity of larval zebrafish presented with a battery of visual stimuli while recording fictive motor output. We identified neurons tuned to each stimulus type and motor output and discovered groups of neurons in the anterior hindbrain that respond to different stimuli eliciting similar behavioral responses. These convergent sensorimotor representations were only weakly correlated to instantaneous motor activity, suggesting that they critically inform, but do not directly generate, behavioral choices. To catalog brain-wide activity beyond explicit sensorimotor processing, we developed an unsupervised clustering technique that organizes neurons into functional groups. These analyses enabled a broad overview of the functional organization of the brain and revealed numerous brain nuclei whose neurons exhibit concerted activity patterns.

Navigational strategies underlying phototaxis in larval zebrafish.

Understanding how the brain transforms sensory input into complex behavior is a fundamental question in systems neuroscience. Using larval zebrafish, we study the temporal component of phototaxis, which is defined as orientation decisions based on comparisons of light intensity at successive moments in time. We developed a novel "Virtual Circle" assay where whole-field illumination is abruptly turned off when the fish swims out of a virtually defined circular border, and turned on again when it returns into the circle. The animal receives no direct spatial cues and experiences only whole-field temporal light changes. Remarkably, the fish spends most of its time within the invisible virtual border. Behavioral analyses of swim bouts in relation to light transitions were used to develop four discrete temporal algorithms that transform the binary visual input (uniform light/uniform darkness) into the observed spatial behavior. In these algorithms, the turning angle is dependent on the behavioral history immediately preceding individual turning events. Computer simulations show that the algorithms recapture most of the swim statistics of real fish. We discovered that turning properties in larval zebrafish are distinctly modulated by temporal step functions in light intensity in combination with the specific motor history preceding these turns. Several aspects of the behavior suggest memory usage of up to 10 swim bouts (~10 sec). Thus, we show that a complex behavior like spatial navigation can emerge from a small number of relatively simple behavioral algorithms.

The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins.

The hereditary hearing-vision loss disease, Usher syndrome I (USH1), is caused by defects in several proteins that can interact with each other in vitro. Defects in USH1 proteins are thought to be responsible for the developmental and functional impairments of sensory cells in the retina and inner ear. Harmonin/USH1C and Sans/USH1G are two of the USH1 proteins that interact with each other. Harmonin also binds to other USH1 proteins such as cadherin 23 (CDH23) and protocadherin 15 (PCDH15). However, the molecular basis governing the harmonin and Sans interaction is largely unknown. Here, we report an unexpected assembly mode between harmonin and Sans. We demonstrate that the N-terminal domain and the first PDZ domain of harmonin are tethered by a small-domain C-terminal to PDZ1 to form a structural and functional supramodule responsible for binding to Sans. We discover that the SAM domain of Sans, specifically, binds to the PDZ domain of harmonin, revealing previously unknown interaction modes for both PDZ and SAM domains. We further show that the synergistic PDZ1/SAM and PDZ1/carboxyl PDZ binding-motif interactions, between harmonin and Sans, lock the two scaffold proteins into a highly stable complex. Mutations in harmonin and Sans found in USH1 patients are shown to destabilize the complex formation of the two proteins.