How Do Haloarchaea Synthesize Aromatic Amino Acids?
A, Illustration of the relative positions of the primers (P6, P7, P8) used in the PCR analysis. B, 1% agarose gel of chromosomal DNA from WT and ΔOE1477R using primers P6+P8 (lanes 2 and 4, respectively) and primers P7+P8 (lanes 3 and 5, respectively). MW markers are shown in lane 1. C and D, Southern blot analysis of WT and ΔOE1477R. Lane 1: SacI digested DNA from WT, lane 2: DIG VII size marker (in bp), lane 3: SacI digested DNA from ΔOE1477R, lane 4: HindIII digested pMG700 (containing flanking regions of OE1477R), and lane 5: HindIII digested pMG750 (containing the gene OE1477R). C, Hybridization with a DIG labeled probe of the US flanking region of OE1477R. D, Hybridization with a DIG labeled probe of part of the gene. Controls included DNA from plasmids pMG700 and pMG750, respectively, to demonstrate the substrate specificity. E, Phenotype of OE1477R compared to WT, growing on different plates. Cells were grown in liquid synthetic medium supplemented with 1.1 mM AroAA and 1.1 mM shikimate until OD600nm = 1.56, and 1.31 (deletion strain and WT, respectively). Cells were washed with basal salt solution before diluting 1/1000 and spotting 3 µl from each dilution on plates supplemented with 1.1 mM AroAA and 1.1 mM shikimate. The viability of the cells and the ability of the deletion strain to grow on these plates are demonstrated in the left and middle panels. While the growth of WT cells was clearly reduced but visible without AroAA (lower right panel), ΔOE1477R required the addition of AroAA.