Lysobacter enzymogenes is a ubiquitous soil gammaproteobacterium that produces a broad-spectrum antifungal antibiotic, known as heat-stable antifungal factor (HSAF). To increase HSAF production for use against fungal crop diseases, it is important to understand how HSAF synthesis is regulated. To gain insights into transcriptional regulation of the HSAF synthesis gene cluster, we generated a library with deletion mutations in the genes predicted to encode response regulators of the two-component signaling systems in L. enzymogenes strain OH11. By quantifying HSAF production levels in the 45 constructed mutants, we identified two strains that produced significantly smaller amounts of HSAF. One of the mutations affected a gene encoding a conserved bacterial response regulator, PilR, which is commonly associated with type IV pilus synthesis. We determined that L. enzymogenes PilR regulates pilus synthesis and twitching motility via a traditional pathway, by binding to the pilA promoter and upregulating pilA expression. Regulation of HSAF production by PilR was found to be independent of pilus formation. We discovered that the pilR mutant contained significantly higher intracellular levels of the second messenger cyclic di-GMP (c-di-GMP) and that this was the inhibitory signal for HSAF production. Therefore, the type IV pilus regulator PilR in L. enzymogenes activates twitching motility while downregulating antibiotic HSAF production by increasing intracellular c-di-GMP levels. This study identifies a new role of a common pilus regulator in proteobacteria and provides guidance for increasing antifungal antibiotic production in L. enzymogenes.
Type IV pilus (T4P) is widespread in bacteria, yet its biogenesis mechanism and functionality is only partially elucidated in a limited number of bacterial species. Here, by using strain OH11 as the model organism, we reported the identification of 26 T4P structural or functional component (SFC) proteins in the Gram-negative Lysobacter enzymogenes, which is a biocontrol agent potentially exploiting T4P-mediated twitching motility for antifungal activity. Twenty such SFC coding genes were individually knocked-out in-frame to create a T4P SFC deletion library. By using combined phenotypic and genetic approaches, we found that 14 such SFCs, which were expressed from four operons, were essential for twitching motility. These SFCs included the minor pilins (PilE, PilX, PilV, and FimT), the anti-retraction protein PilY1, the platform protein PilC, the extension/extraction ATPases (PilB, PilT, and PilU), and the PilMNOPQ complex. Among these, mutation of pilT or pilU caused a hyper piliation, while the remaining 12 SFCs were indispensable for pilus formation. Ten (FimT, PilY1, PilB, PilT, PilU, and the PilMNOPQ complex) of the 14 SFC proteins, as well as PilA, were further shown to play a key role in L. enzymogenes biofilm formation. Overall, our results provide the first report to dissect the genetic basis of T4P biogenesis and its role in biofilm formation in L. enzymogenes in detail, which can serve as an alternative platform for studying T4P biogenesis and its antifungal function.
Given the existence of manufacturing defects and the accumulation of assembly errors, non-compliant assembly appears between components, especially for composite structure assembly. In the engineering application, the clamping force (CF) is often used to eliminate the clearance between mating components, but the improper CF may result in unwanted structure failure. Thus, on the premise of ensuring the safety of composite parts, this study proposes a procedure to systematically optimise the assembly CF. Firstly, the components mating surfaces were obtained by laser scanner, and the matching of actual surfaces was transformed and simplified based on ‘equivalent surface’ concept. Then, a mathematical optimisation model was established. The CF layout and magnitude were taken as variables, and the clearance elimination rate and the overall assembly force value were employed as objective functions. Finally, the improved genetic algorithm (GA) was used to solve this problem. A parametric finite element analysis (FEA) model was built, and model accuracy was verified by physical experiments. The finite element calculation and post-processing were carried out by Python script in ABAQUS®. Compared to the engineer’s traditional approach, the influence of form defects and part deformations were considered, which can help control the assembly stress well and ensure product performance.
A new mathematical modeling method, namely, the finite element method and the lumped mass method (LMM-FEM) mixed modeling, is applied to establish the overall multinode dynamic model of a four-stage helicopter main gearbox. The design of structural parameters of the shaft is the critical link in the four-stage gearbox; it affects the response of multiple input and output branches; however, only the meshing pairs were frequently shown in the dynamic model in previous research. Therefore, each shaft is also treated as a single node and the shaft parameters are coupled into the dynamic equations in this method, which is more accurate for the transmission chain. The differential equations of the system are solved by the Fourier series method, and the dynamic response of each meshing element is calculated. The sensitivity analysis method and parameter optimization method are applied to obtain the key shaft parameters corresponding to each meshing element. The results show that the magnitude of dynamic response in converging meshing pair and tail output pair is higher than that of other meshing pairs, and the wall thickness has great sensitivity to a rotor shaft. In addition, the sensitivity analysis method can be used to select the corresponding shaft node efficiently and choose parameters appropriately for reducing the system response.
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