Here, we transcriptionally and phenotypically characterized the clpB gene from Enterococcus faecalis. Northern blot analysis identified a monocistronic mRNA strongly induced at 48 and 50 6C. In silico analysis identified that the clpB gene encodes a protein of 868 aa with a predicted molecular mass of approximately 98 kDa, presenting two conserved ATP-binding domains. Sequence analysis also identified a CtsR-binding box upstream of the putative "10 sequence, and inactivation of the ctsR gene resulted in an approximately 2-log increase in clpB mRNA expression, confirming ClpB as a member of the CtsR regulon. While expression of clpB was induced by heat stress, a DclpB strain grew relatively well under many different stressful conditions, including elevated temperatures. However, expression of ClpB appears to play a major role in induced thermotolerance and in pathogenesis, as assessed by using the Galleria mellonella virulence model.

Meticillin-resistant Staphylococcus aureus isolates were classified into three mupirocin susceptibility groups by the disc diffusion method using 5 and 200 μg mupirocin discs. The zone diameter observed for a 5 μg disc distinguished MupS from the resistant strains (either MupRL or MupRH). On the other hand, a 200 μg disc distinguished the high-resistance MupRH strains from the other two (MupS or MupRL). Thus, the concomitant use of 5 and 200 μg mupirocin discs allowed the clear distinction among the three mupirocin susceptibility groups, MupS, MupRL or MupRH.

The intrinsic ruggedness of Enterococcus faecalis is responsible for its widespread distribution in nature and is often viewed as an important virulence determinant. Previously, we showed that the ClpB ATPase is negatively regulated by CtsR and is required for thermotolerance and virulence in a Galleria mellonella invertebrate model. Here, we used in silico, Northern blot and quantitative real-time PCR analyses to identify additional members of the CtsR regulon, namely the clpP peptidase and the clpC and clpE ATPases. When compared to the parent strain, virulence of the ΔctsR strain in G. mellonella was significantly attenuated.

Taken together, the results confirmed that aureocin A53 has potential to be used as a food preservative, representing an alternative to the use of nisin in biopreservation of dairy products.