Detection and relative quantification of amine oxidase gene (yobN) in Bacillus subtilis: application of real-time quantitative PCR

Abstract

Degradation of undesirable biogenic amines (BAs) in foodstuffs by microorganisms is considered one of the most effective ways of eliminating their toxicity. In this study, we design two sets of primers for the detection and quantification of the amine oxidase gene (yobN) and endogenous (housekeeping) gene (gyrB) in Bacillus subtilis. Moreover, these sets can be used for relative quantification of yobN by real-time PCR (qPCR). We also tested the degradation of BAs by three bacterial strains (B. subtilis strains: IB1a, CCM 2216, CCM 2267) in a mineral medium over a two-day period. Their degradation abilities were verified by high performance liquid chromatography with UV detection (HPLC/UV). According to the results, two strains significantly (P \ 0.05) reduced histamine, tyramine, putrescine, and cadaverine. Moreover, our results indicate that the degradation ability of B. subtilis strains could be limited by sporulation because the gene encoding amine oxidase (yobN) is no longer expressed in the spores.

Isolation of Persister Cells of Bacillus subtilis and Determination of Their Susceptibility to Antimicrobial Peptides

“…Upregulated liaI indicated that increased cell envelop stress was associated to B. subtilis persister formation and/or the maintenance of this persistent physiological state. This result is consistent with earlier studies showing that cell envelope stress was strongly in- gyrase subunit B [39]), rrns (16s ribosomal RNA gene [40]), gapA (encoding GAPDH (Glyceraldehyde-3-phosphate dehydrogenase 1) [41]), rpoA (encoding DNA-directed RNA polymerase subunit alpha [42]), and hbsU (encoding DNA-binding protein HU1 [43]). After comparing the Ct values of each candidate gene, hbsU was selected as reference gene (Figure 5).…”

“…Due to the harsh stress condition of generating persisters, it was important to select a proper reference gene with approximately equal expression levels under different stresses. In this experiment, we selected five commonly used reference genes as candidates to choose a proper reference gene, including gyrB (encoding DNA gyrase subunit B [39]), rrns (16s ribosomal RNA gene [40]), gapA (encoding GAPDH (Glyceraldehyde-3-phosphate dehydrogenase 1) [41]), rpoA (encoding DNA-directed RNA polymerase subunit alpha [42]), and hbsU (encoding DNA-binding protein HU1 [43]). After comparing the Ct values of each candidate gene, hbsU was selected as reference gene (Figure 5).…”

“…Expression of 5 stress response-related genes was quantified relative to hbsU expression (Figure 6), including liaI (cell envelope stress [44]), skfA (Cannibalism sporulation related stress response [45]), recA (SOS response [46]), ctc (sig-B regulated general stress response [47]), and relA (stringent response [48]). gyrase subunit B [39]), rrns (16s ribosomal RNA gene [40]), gapA (encoding GAPDH (Glyceraldehyde-3-phosphate dehydrogenase 1) [41]), rpoA (encoding DNA-directed RNA polymerase subunit alpha [42]), and hbsU (encoding DNA-binding protein HU1 [43]). After comparing the Ct values of each candidate gene, hbsU was selected as reference gene (Figure 5).…”

A model industrial workhorse: Bacillus subtilis strain 168 and its genome after a quarter of a century

“…Finally, present in defective prophage 6 island, AoxN(YobN) is an amine oxidase with demonstrated activity on histamine, tyramine, putrescine and cadaverine. It co‐evolves with a variety of catalytic enzymes including N 6‐methyladenosine deaminase MadA and spore coat proteins, but it is not expressed in spores (Pištěková et al, 2022 ).…”