Relative amount of CII was measured after regular intervals (0, 5, 10, 15, 20 minutes) by western blotting followed by quantification using densitometric analysis. Corresponding western blots showing the stability of CII in different host strains are shown in the right panel. These results pose an intriguing selleck products question. Why does the deletion of an inhibitor of CII A 769662 proteolysis promote lysogeny? One can think of the following possibilities:

(i) A proper assembly of HflB that is necessary for its activity against cytosolic substrates, may require HflKC; or (ii) In the absence of HflKC, HflB is guided towards its membrane-associated substrates [26], indirectly stabilizing the cytosolic substrate CII. However, from in vivo proteolysis experiments we found that in AK990 cells (ΔhflKC), exogenous CII was not stabilized (Figure 1), confirming that HflB was active against CII even in the absence of hflKC. This result rules out both the possibilities mentioned above. It may be noted that similar results were

also obtained by Kihara et al [26]. Therefore, an increase in lambda lysogeny upon overexpression of host HflKC [26] is not at all surprising, since HflKC inhibits Selleckchem SAHA HDAC the proteolysis of CII. Effect of increasing concentrations of HflKC on the proteolysis of CII in vitro The paradoxical effect of an increase in the lysogenic frequency of λ upon deletion as Olopatadine well as overexpression of hflKC has been reported [26]. A possible reason behind this paradox could

be that a critical molar ratio between HflB and HflKC, believed to be 1:1 in wild type cells [35], is necessary for a proper proteolysis of CII by HflB. Both the increase or decrease of HflKC would offset this critical ratio and could lead to a stabilization of CII, promoting lysogeny. To examine this possibility, we carried out the proteolysis of CII by HflB in vitro, in the presence of three different concentrations of HflKC (Figure 2). In the first case, when HflKC was absent (mimicking the deletion of HflKC), CII (8 μM) was rapidly cleaved by HflB. The rate of proteolysis was much slower when HflKC was added in a molar ratio of HflKC:HflB = 1:1. The proteolysis was inhibited further when HflKC was added in excess (HflKC:HflB = 2:1). If the above hypothesis was true, proteolysis of CII should have been maximum at a molar ratio of 1:1. Therefore we conclude that HflKC acts as a simple inhibitor of CII proteolysis and the stabilization of CII in the absence of HflKC may involve other factors. Figure 2 Effect of varying concentrations of HflKC on in vitro proteolysis of CII. CII (8 μM) was treated with GST-HflB (1 μM), in the presence of His-HflKC in various concentrations: 0 (open circles), 1 μM (squares) and 2 μM (triangles). Samples were taken out at various time points, run on a 15% SDS-PAGE, and the CII bands were quantitated by densitometry.