Figure 3.

Derepression of bio operon transcription by expression of biotin accepting proteins. In host strain CY1740 which carries the φ(bioFC-lacZ)501 fusion, the lacZ and lacY genes are fused to the rightward bio promoter (that of bioBFCD) resulting a lactose-positive phenotype when bio operon transcription is derepressed and a lactose-negative phenotype when the operon is repressed. This strain also carries a deletion of the chromosomal lactose operon and ia a biotin auxotroph due to the insertion of the lactose utilization genes into bioF. (A). The host strain was transformed with either the empty vector plasmid or plasmids encoding the Pep-ME, Pep-85 or AccB-87 fusion proteins. Also included was a strain CY1740 derivative that carried a plasmid encoding a mutant AccB-87 fusion protein (K122M) in which the lysine targeted for biotinylation had been replaced with methionine. Four independent transformants from each transformation were patched onto an LB-ampicillin plate that was incubated at 37°C for 8 h. The cells were then transferred by replica plating to a plate of MacConkey agar supplemented with 100 nM biotin (ca. 115 nM total) and ampicillin. After overnight incubation at 37°C derepression of bio operon transcription results in bright red colonies due to lactose utilization whereas repressed colonies remain colorless due to their inability to metabolize lactose. (B) Expression at various biotin concentrations of derivatives of strain CY1740 carrying plasmids encoding maltose binding protein fusions to biotin accepting peptides 85 (■) or ME (●). The maltose binding protein fusion to AccB-87 (▲) and the vector plasmid encoding a maltose binding protein that lacked a fusion partner (◆) are also shown. β-Galactosidase activities were assayed 4 h after induction of the maltose binding protein fusions with IPTG (1 mM). Note that the abscissa is a logarithmic scale. (C) Reproducibility of derepression. Four independent transformants from each of the above transformations were streaked on minimal medium plates supplemented with 100 nM biotin. Colonies from these plates were then used to inoculate overnight cultures on the same medium which in turn were diluted 1:100 into fresh medium containing 100 nM biotin and IPTG. These cultures were allowed to grow to late log phase prior to β-galactosidase assays. (D) Biotin attachment to the fusion proteins. Biotinylation of the acceptor proteins was assayed by incorporation of [³H]-biotin into trichloroacetic acid insoluble material (Abdel-Hamid and Cronan, 2007; Chapman-Smith et al., 1999; Cronan, 1988) 4 h after IPTG induction of expression of the maltose binding protein fusions. In the strain carrying the vector plasmid the attachment of biotin is to the essential chromosomally encoded AccB protein.