Aspergillus niger is a cell factory widely used in industries to produce proteases, organic acids, drugs, and other substances. The hyphal morphology of A. niger is a complex differentiated elongated tubular structure, which limits its basic research and application. In this study, the mpkA , bck1 , steC , and Tpk2 genes were successfully deleted using a quick way to knock out genes based on the RNP (Ribonucleoprotein) complex. The study showed that the knockout of mpkA and bck1 kinase gene strains resulted in smaller, denser colonies, short rod-shaped hypha, and a significant increase in glucoamylase secretion. The mechanism of short hypha formation and high protein production for A. niger is the cell wall integrity signaling (CWIS) pathway. The CWIS pathway passed through the bck1 - mkkA - mpkA tertiary kinase to deliver phosphorylation signals to the rlmA transcription factor, which regulated the expression of the cell wall synthesis gene agsA , thus regulating hyphal morphology. The mpkA kinase regulated the expression of the transcription factor amyR , which affected the expression of the genes glaA and amyA , thus enhancing the expression of proteins in A. niger . This study provides a strategy for the regulation of hyphal morphology and promotes the application of A. niger in industrial production.
Shiga toxin-producing Escherichia coli (STEC) is known as a kind of foodborne pathogens that can produce Shiga toxin type1, type 2, or both, encoded by stx1 and stx2 genes, respectively. Rapid and accurate detection of STEC and stx1 / stx2 subtyping assay in food and environment is essential for foodborne outbreak investigation. In this study, a method for the accurate and rapid subtyping of STEC single colony using asymmetric polymerase chain reaction (asPCR) combined with lateral flow immunoassay (LFIA) was established. AsPCR was conducted using biotinylated stx1 and digoxin-labeled stx2 primers to obtain several single stranded DNA amplicons labeled with biotin or/and digoxin. Subsequently, these amplicons were tagged with specific probes labeled with carboxylate-modified red polystyrene microspheres using sequential hybridization. Finally, STEC with different Shiga toxin gene types could be distinguished depending on the appearance of different test lines on an LFIA strip. Thus, the STEC single colony could be identified and subtyped accurately and conveniently using this method. This method showed high specificity. Twenty-four strains of STEC in milk were detected and subtyped using our method, which combines asPCR with LFIA.
Abstract Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that seriously threats the public and animal health. The major virulence of STEC is Shiga toxin (Stx) which is encoded by Stx prophage. Stx prophage induction leads to the production of Stx. Our recent research has shown that dietary functional sugars can differently regulate Shiga toxin type 2 (Stx2) prophage induction. However, high-throughput assays are required to find more dietary inhibitors for Stx prophage induction. This study aims to establish the recA dual-luciferase reporter system for rapidly screening the functional sugars that can inhibit Stx prophage induction. Results showed that the recA dual-luciferase reporter plasmid was successfully constructed as confirmed by DNA sequencing. Established recA dual-luciferase reporter system showed similar screening results when compared with traditional temperate phage determination method (i.e., qPCR) and suggested that L-arabinose is a dietary functional Stx prophage induction inhibitor. Supplementation with L-arabinose in culture medium reduces Stx production in STEC isolated from fecal samples of diarrhea piglets. Collectively, the recA dual-luciferase reporter system is a promising tool for convenient and rapid screening of dietary Stx prophage induction inhibitor, especially suitable for large-scale screening experiments.
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