Analysis of plcR regulator gene expression in Bacillus spp. using GFP fusions and flow cytometry

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Vukojevic, Marina
McKillip, John L.
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Thesis (M.S.)
Department of Biology
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Carvacrol, the key extract of oregano oil, is known for its antimicrobial properties toward Bacillus cereus and other bacteria. As such, carvacrol may be considered beneficial in treatment of severe endophthalmitis (ocular infections) caused by B. cereus. However, in vitro, subinhibitory concentrations of carvacrol (SIC, 1 mM), have been shown to upregulate mRNA expression of B. cereus hblC and nheA enterotoxin genes by 5%. This is clinically relevant because B. cereus virulence may be exacerbated if endophthalmitis treatment involves incorrect/SIC levels of carvacrol. In Bacillus spp., hemolytic BL (HBL) and non-hemolytic (Nhe) toxin production is activated by at least one transcriptional regulator, a phospholipase C regulator (PlcR). This factor also regulates endotoxin production in the important insect pathogen, B. thuringiensis. Here we investigated whether plcR expression is upregulated by SIC of carvacrol in B. cereus ATTC14579 and B. thuringiensis subsp. israelensis as measured by flow cytometry. Green fluorescent protein (GFP)-expressing B. cereus ATTC14579 and B. thuringiensis subsp. israelensis were constructed in order to measure the effect of carvacrol on plcR using flow cytometry. The results show that SIC carvacrol treated transformed B. cereus ATTC14579 and B. thuringiensis subsp. israelensis increased plcR expression by 25% and 70% respectively, relative to untreated cultures. Our results reveal that plcR is upregulated to similar levels as nheA and hblC when subjected to SIC carvacrol. In other words, SIC carvacrol increases virulence of B. cereus and B. thuringiensis subsp. israelensis. Furthermore, results indicate that hblC and nheA are directly controlled by plcR regulator expression in response to the SIC carvacrol and do not apparently necessitate additional regulatory effector proteins. The implications of this study may further clarify the order of regulatory virulence events in B. cereus and B. thuringiensis.