Figure 1 shows a diagram illustrating the malX-malI intergenic region with the transcription GSK3235025 datasheet start sites for the malX and malI promoters, the corresponding −10 elements, and the DNA site for CRP that is located at position −41.5 with respect to the malX transcription start and position −43.5 with respect to the malI transcription start. Figure 1 also shows the locations of two 16 base pair elements, suggested to be the operator targets for the MalI repressor. The aim of the work described here was to investigate this suggestion and to determine the functional operator(s) for each promoter. In a previous
work, Lloyd et al. (2008) described how the malX promoter could be assayed by cloning the malX100 fragment into the lac expression vector plasmid, pRW50. Measurements of β-galactosidase expression in M182 or its Δcrp derivative showed the malX promoter to be a typical Class II CRP-dependent promoter, which is consistent with the location of the DNA site for CRP (West et al., 1993). Lloyd et al. (2008) also reported that expression of the malX promoter∷lac fusion carried by pRW50 is unaffected by the introduction of a multicopy plasmid carrying the malX-malI intergenic region, suggesting that the level of chromosomally
encoded MalI is insufficient to repress the malX promoter significantly. Thus, to set up a system to measure MalI-dependent repression of the malX promoter, we cloned the malI gene into plasmid pACYC184 to generate pACYC-malI. Reverse transcriptase Measurements of β-galactosidase expression in M182 cells carrying buy Stem Cell Compound Library pRW50 with the malX100 promoter show that the presence of pACYC-malI causes an ∼30-fold reduction in expression, compared with the control with the empty pACYC-ΔHN plasmid (Table 1, upper panel). The experiment was then repeated with M182 cells carrying pRW50 with the malX400 promoter fragment, in which the malX promoter sequence upstream of the DNA site for CRP had been removed (illustrated in Fig. 1). The data in Table 1 (upper panel) show that neither malX promoter
activity nor repression by MalI is substantially affected by the deletion, and thus sequences upstream of the DNA site for CRP must play little or no role. On MacConkey lactose indicator plates, colonies of M182 carrying pRW50 with either the malX100 or malX400 promoter fragments, together with pACYC-malI, appear as white Lac− colonies. In contrast, if pACYC-malI is replaced with pACYC-ΔHN, colonies have a bright red, clear Lac+ appearance. Thus, to pinpoint the operator sequences essential for repression of the malX promoter by MalI, we used error-prone PCR to generate a library of random mutations in the malX400 promoter fragment and screened for mutations that resulted in pink or red colonies of cells containing pACYC-malI. We reasoned that such colonies would contain pRW50 carrying the malX400 fragment with mutations that interfered with MalI binding.