Investigation of the effects of abrb and cody deletions on the bacilysin overproducer B. subtilis HWA strain

Abstract

Bacillus subtilis is a gram-positive, rod-shaped bacterium and a highly studied model organism. It has a highly adaptable metabolism and diverse physiological states regulated according to environmental conditions. B. subtilis species go through sporulation to form endospores that can survive harsh conditions such as high temperatures, and UV radiation. They can also form biofilms, attach to plant roots or fungal hyphae, take up extracellular DNA by its natural competence, show surface motility, produce and secrete secondary metabolites. Approximately 4-5% of the B. subtilis genome codes for secondary metabolites, including antibiotic bacilysin. Bacilysin, the main focus of this study, is a non-ribosomal dipeptide by linking non-proteinogenic amino acid anticapsin and L-alanine. Bacilysin causes selective cell wall disruption against bacteria, fungi, and algae species, some of which are pathogenic. Bacilysin is a pleiotropic signaling molecule for B. subtilis cells as it affects diverse cellular functions such as sporulation, germination and outgrowth. Previous studies have shown that the absence of bacilysin can have negative effects on spore quality and germination. A bacilysin non-producer strain was more sensitive to heat, chemicals and lysozyme. Additionally, comparative transcriptome analysis of B. subtilis PY79 and a bacilysin non-producer strain revealed that some genes related to competence development and biofilm formation are also affected by bacilysin. In B. subtilis, bacilysin biosynthesis relies on the expression of the bacABCDEF operon and a monocistronic gene bacG. Bacilysin production is regulated according to both external and internal factors. It has been established that growth conditions such as medium contents, temperature, and pH affect bacilysin production level. At the transcriptional level, bacilysin biosynthesis is regulated mainly via two mechanisms: quorum sensing pathway and stringent response that occur through the direct-action of the positive transcriptional regulators, including ComA~P, Spo0A~P, and LutR as well as negative transcriptional regulators AbrB, CodY, and ScoC. Bacilysin has broad-range activity against bacteria, with heat stability up to 15 min at 100°C, and activity within the pH range of 1.4 to 12.0. These characteristics give bacilysin significant clinical importance and make it an effective alternative to traditional drugs and biocontrol agents. However, bacilysin is produced at low levels, cannot be extracted with organic solvents, and has a low isolation yield. In our group, the bacilysin production level was increased at 2.87- fold via editing the 5' untranslated region (5'UTR) of the bac operon using the CRISPR/Cas9 approach, thereby obtaining the bacilysin overproducing strain B. subtilis HWA. Subsequently, to further boost the bacilysin production level in the over-producing strain B. subtilis HWA, the aim of this study was to examine how production levels are affected by eliminating the global regulators AbrB and CodY. To achieve this, the mutant strains B. subtilis PY79-GT0A (ΔabrB::cat) and B. subtilis PY79-GT0C (unkU::spc ΔcodY) were constructed by transforming competent PY79 cells with chromosomal DNA from the abrB deleted mutant strain B. subtilis BAL 373 (trpC2 pheA1 ΔabrB::cat) and the codY deleted mutant strain B. subtilis TMH 307 (trpC2 unkU::spc ΔcodY), respectively. Similarly, the mutant strains B. subtilis HWA HWA-GTA (ΔabrB::cat) and B. subtilis HWA-GTC (unkU::spc ΔcodY) were constructed by transforming competent HWA cells. Furthermore, the codY-abrB double mutant strains B. subtilis PY79-GT0AC (ΔabrB::cat unkU::spc ΔcodY) and B. subtilis HWA HWA-GTAC (ΔabrB::cat unkU::spc ΔcodY) were constructed by transforming competent cells of GT0A and GTA with chromosomal DNA from B. subtilis TMH 307. Potential tryptophan and/or phenylalanine auxotrophic mutants were eliminated by restreaking on solid Spizizen Minimal Media. Bacilysin phenotypes of the selected mutants were first detected by transferring colonies via toothpicks onto bioassay plates using Staphylococcus aureus ATCC 9144. Subsequently, mutants were grown in PA media for 16-18 hours and the bacilysin levels in their culture fluids were detected via paper disk-diffusion bioassay. Results showed that abrB disruption in HWA and PY79 cells significantly increased bacilysin production, though each strain was affected differently based on its baseline bacilysin production level. The bacilysin level in HWA-GTA increased by 7.7% relative to parental HWA strain, while PY79-GT0A displayed a 21.8% bacilysin level relative to its parent PY79. Interestingly, while codY mutation alone did not significantly affect bacilysin activity in HWA or PY79, adding codY mutation to AbrB mutants of both strains caused further increases of 22.1% and 13.4% relative to HWA and HWA-GTA, respectively, and 25.7% and 3.2% relative to PY79 and PY79-GT0A, respectively. In a final attempt to further enhance bacilysin production, the scoC mutation as an additional negative regulator of the bac operon was combined with the abrB-codY double mutation strain. However, the constructed triple mutant strain could not grow in liquid media, demonstrating that simultaneous disruption of these three global regulators severely compromised growth abilities of B. subtilis cells. In summary, the findings of this thesis study are important to revealing that concurrent inactivation of AbrB and CodY, two key negative regulators of bacilysin biosynthesis, provides the potential for improving bacilysin production levels further, even in the high-producing strain HWA.