Tackling against spreading of antimicrobial resistance, which is an intimidating
global problem, must focus on undiscovered systems, that may have a potential to
discover new antimicrobial compounds. Rhizosphere has enormous potential and
can be described as nutrient-rich environment that offers optimal growth conditions
for soil-dwelling bacteria such as PGPR (Plant Growth Promoting Rhizobacteria), a
specialised group of bacteria that promote plant growth and yield. Bacillus subtilis,
an improtant member of the PGPR group, is used in our master's thesis, where we
constructed B. subtilis mutants with deletions in specific genes involved in the
bacillaene or surfactin biosynthesis. According to the findings, secondary
metabolism is energy consuming, and master's thesis focused mainly on the
assumption that the deletion of one gene in surfactin or bacillaene biosynthesis
affects the production of the other. Therefore, srfAA or pksC gene promoter was
fused with a gene encoding for yellow fluorescent protein (yfp), which enabled us
to measure the yellow fluorescence intensity on spectrofluorimeter or observe cells
under confocal laser scanning microscope. We hypothesised that deletion of pks or
srfAA gene reduces energetic burden, resulting in higher levels of pksC promoter
expression after deleting surfactin (srfAA) pathway as well as in higher levels of
srfAA promoter expression, when deleting bacillaene pathway (pks). Based on
yellow fluorescence intensity measurements our hypothesis was confirmed. In
addition, surfactin dropplet test was performed to assess the amount of surfactin
produced by B. subtilis wild type strain or bacillaene mutant. We found that the
bacillaene mutant produces quantitatively more surfactin compared to the wild type
and therefore confirmed our third hypothesis, where we inffered on positive
correlation between surfactin transcriptional and translational level. The expression
of the pksC promoter in the population is heterogenous, confirming our second
hypothesis. We have demonstrated by confocal laser scanning microscopy that only
a specialised B. subtilis sub-population is responsible for higher levels of pksC
promoter expression.
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