Lcn972 is a non pore-forming bacteriocin that inhibits the synthesis of peptidoglycan at the septum in Lactococcus MK5108 concentration lactis. Moreover, the response of a number of Gram-positive bacterial species towards cell wall active antibiotics has been studied
recently by using genome-wide transcription analysis [19, 23–27]. Essentially, these reports describe a very complex system involving the concerted action of extracellular sigma factors and two-component systems (TCSs) . LiaRS, the B. subtilis homologue of CesSR, was unable to activate liaI expression in B. subtilis in response to AS-48 treatment. Therefore, the effect of AS-48 on bacterial gene expression clearly differs from the mechanisms described earlier for B. subtilis . The precise way in which Givinostat in vivo BC4206 responds to the presence of AS-48 needs to be deciphered by further experimental work, including determining the target genes of BC4206 and the PFT�� research buy exact signal sensed by this PadR-type regulator. The structure and function of the BC4207 membrane protein and its role in the resistance mechanism against AS-48 is also particularly intriguing and target of our future research. Conclusion B. cereus cells, when
treated with bacteriocin AS-48, increase the expression of the BC4207 gene coding for a putative membrane protein. Targeted inactivation of the BC4207 protein might be useful to increase the effect of AS-48 on food poisoning B. cereus cells. Methods Bacterial strains, growth conditions and preparation of cells for RNA isolation
Bacillus cereus ATCC 14579 and B. subtilis 168 strains from glycerol stocks were grown overnight on TY broth at 30°C, with shaking at 225 rpm. Cultures were diluted to a final OD600 of 0.15 in fresh TY medium. B. cereus ATCC14579 and B. subtilis 168 strains containing pATK33 or pLM5 were grown in the Suplatast tosilate presence of 50 and 10 μg/ml of kanamycin, respectively. Growth of B. cereus and B. subtilis in the presence of various concentration of bacteriocin was monitored every 15 minutes using a TECAN GENios Absorbance Reader (TECAN). When cultures reached an OD600 of 0.3, purified enterocin AS-48 was added to the cultures at a concentration of 0.5 μg/ml, which was the maximal concentration not inhibiting growth, cells were harvested after 15 or 30 min by centrifugation and cell pellets were immediately frozen in liquid nitrogen and stored at -80°C until RNA isolation. Six independent biological replicates were used for microarray analysis. For quantitative RT-PCR, cells were treated with nisin and bacitracin at a subinhibitory concentration of 2 μg/ml and 25 μg/ml, respectively. Purification of AS-48 Enterocin AS-48 was purified to homogeneity by reversed-phase high-performance chromatography as described elsewhere .