Indeed, EPI100 carrying pACYC184-recA also showed a clear growth advantage compared to the vector control when grown in LB broth. This finding verifies that RecA plays a significant role in bacterial growth in general and thus the GI colonisation promoting effect of recA is most likely due to a generally enhanced growth rate of the recA containing clone. Nevertheless, while the selection of RecA in the mouse
model is not a surprising finding it serves as a proof of principle, regarding the validity of the screening approach. The fact that pACYC184-galET was unable to ferment galactose in vitro was to be expected since EPI100 harbours deletions in galactokinase (GalK) this website and UTP-glucose-1-phosphate uridylyltransferase (GalU), both of which are necessary for growth on galactose [24–26]. Instead, we observed an intriguing decreased sensitivity to bile salts in vitro conferred by C3091-derived GalET. Further studies are needed to characterise the mechanism underlying this phenotype click here and its physiological implications. However, we speculate that incorporation of C3091 GalET-mediated sugar-residues into the bacterial membrane, i.e. as a part of LPS as previously described , may have an enhancing effect on the membrane stability, thus promoting decreased sensitivity
to bile salts and possibly other compounds such as antimicrobial peptides present in the mouse GI tract. In support of this, enterohaemorrhagic E. coli gal mutant strains have been shown to be 500-fold less able to colonise the GI tract of rabbits and 100-fold more
susceptible to antimicrobial peptides than the parent strain . Together with the sensor transmitter protein ArcB, ArcA constitutes a two-component ArcAB system which functions as a global regulator of genes involved in metabolism in response to oxygen availability, primarily favouring anaerobic growth . ArcA homologues have, moreover, been implicated in regulating the expression of virulence factors and proteins involved in serum resistance [28, 29]. To our knowledge, the EPI100 strain does not harbour mutations in ArcAB, thus indicating a cumulative effect of native and K. pneumoniae-derived ArcA activity promoting enhanced colonisation. To assess whether this effect was due to enhanced adaption to anaerobic growth in Buspirone HCl general, we tested EPI100 carrying pACYC184-arcA for its potential enhanced ability to grow under anaerobic conditions in LB broth in competition with the EPI100 vector control. We did not observe any significant differences in the growth rate between the two strains. Thus, although a growth promoting effect of ArcA in the intestinal environment cannot be excluded from these in vitro assays, the effect of ArcA on GI colonisation may instead be via the regulation of colonisation factors not related specifically to anaerobic growth. Notably, during screening of a K.