Discovery of the Gene Cluster in Bacillus velezensis AP183 Responsible for Bacillusin A Biosynthesis, Evaluation of the Use of B. velezensis AP183 in Preventing Staphylococcus aureus Infection, and Identification of Bacteriocins From a Soil Metagenome.
Type of DegreePhD Dissertation
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The spread of multidrug resistance among pathogenic bacteria is increasing worldwide. One of the multidrug resistance pathogens is methicillin-resistant S. aureus (MRSA) which poses a significant threat to health and causes severe infections in risk populations such as immunocompromised people. There is also alarming emergence of antibiotic resistant foodborne pathogens, however, there are a limited number of new antibiotics found in the last few decades. Therefore, it is essential to discover new antibiotics or antimicrobials to treat MRSA as well as other food borne pathogens. In this study both culture-dependent and culture-independent approaches were used to discover antibiotics or antimicrobials against methicillin-resistant clinical isolates of Staphylococcus aureus (MRSA) and other food borne pathogens. Rhizosphere-derived B. velezensis AP183 was previously discovered to produce bacillusin A, a novel and potent macrodiolide antibiotic capable of inhibiting methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus faecium. Genome sequencing of B. velezensis AP183 followed by predicting biosynthetic gene clusters (BGCs) revealed an approximately 70-Kb type I polyketide synthase (PKS) cluster. This type 1 PKS BGC was predicted to synthesize bacillusin A in B. velezensis AP183 because bacillusin A is a macrocyclic polyene compound generally synthesized by type 1 PKS. This study characterized the bacillusin A BGC in B. velezensis AP183. An in-frame gene knockout method was used to delete the 44 Kb region, predicted to encode the type I modular PKS responsible for bacillusin A biosynthesis. The mutant did not produce bacillusin A as revealed by a loss of activity as well as lost the anti-MRSA activity and by LC-MS. A CRISPR/Cas9 based method was used to capture and integrate the 70 kb predicted BGC into B. subtilis 168 and successful expression of bacillusin A was observed by LC-MS analysis as well as anti-MRSA activity. A previous study found that B. velezensis AP183 showed potent inhibition of S. aureus proliferation and bioluminescence in a mouse cutaneous wound (P = 0.0198). This study conducted the experiments to evaluate the ability of B. velezensis AP183 to prevent S. aureus biofilm formation on a tracheostomy tube substrate. B. velezensis AP183 could form a biofilm on a tracheostomy tube inner cannula substrate, and that this biofilm was antagonistic to S. aureus colonization. B. velezensis AP183 was also observed to inhibit the growth of S. aureus isolates originated from bovine mastitis cases. To evaluate the inflammatory response of mammary tissue to intramammary inoculation with B. velezensis AP183, we used high dose and low dose inocula in dairy cows. At the high dose, a significant increase in somatic cell count (SCC) and clinical mastitis was observed at all post-inoculation time points (P < 0.01), which resolved quickly compared to S. aureus-induced mastitis; in contrast, the lower dose of B. velezensis AP183 resulted in a slight increase of SCC and no clinical mastitis. In groups treated with B. velezensis AP183, SCC and abundance of S. aureus decreased with significant reductions in S. aureus after three days post-inoculation with AP183 (P = 0.04). A milk microbiome analysis revealed significant reductions in S. aureus relative abundance in the AP183-treated group by eight days post-inoculation (P = 0.02). These data indicate that B. velezensis AP183 can inhibit S. aureus biofilm formation and its proliferation in murine and bovine disease models. A metagenomic approach was used to discover novel bacteriocins encoded by soil microorganisms sampled from a long-term agricultural rotation (Cullars Rotation) at Auburn University. A previous study from our lab generated a soil metagenomic library containing 19,200 clones in a bacterial artificial chromosome vector with an average insert size of 110kb. The contig sequences from this library were used to screen bacteriocins using BAGEL3/4 and antiSMASH bioinformatics pipelines. In silico screening of soil metagenome identified a total of 136 different types of bacteriocins that contained class I, class II and class III bacteriocins. Few classes I and class III bacteriocins were selected for further study. For the expression of class III bacteriocin, the selected clones were identified from the cryopreserved metagenomic library, amplified by PCR and subcloned and expressed using the Expresso Rhamnose SUMO Cloning and Expression system. For class I bacteriocin expression, the structural gene were amplified first and subcloned in the Expresso Rhamnose SUMO Cloning and Expression system. Later the plasmid construct was transformed into corresponding metagenomic clones containing the whole BGC and expressed. The expression of bacteriocin was confirmed by SDS-PAGE analysis. The expressed proteins of class III bacteriocin were partly soluble as we observed significant amount of protein in the pellet on SDS PAGE gel, it could be due to the aggregation of the expressed protein. No antimicrobial activity was observed against the indicator food borne organisms used in this study. For both class of bacteriocin, we observed successful expression of the bacteriocin but did not observe the expected modification in the lanthipeptides which accompanied by loss of molecular weight due to dehydration step in post translational modification. We did not observe any antimicrobial activity of any of the putative expressed bacteriocin against the selected food borne pathogens.