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Qin Sheng

Advisor: Malcolm Winkler, Ph.D.

In Escherichia coli, D-1-deoxyxylulose-5′-phosphate (DXP) is the biosynthetic precursor to isopentenyl diphosphate, thiamin, and pyridoxine. DXP synthase is encoded by the dxs gene located at 9 min on the chromosomal map of E. coli. We demonstrated the essentiality of dxs to E. coli growth by dxs+/dxs:: WCmr merodiploid segregation experiments. We also constructed and characterized a conditional dxs mutant in which the chromosomal dxs was disrupted by a WCmr cassette, and a prototrophic dxs+ was cloned under the control of a tightly regulated PBAD promoter on a plasmid. In this way, when the PBAD promoter is induced by arabinose, the mutant behaves like a prototrophic strain, whereas when PBAD is repressed by glucose, the mutant behaves like a null mutant. This construct required either D-1-deoxyxylulose or a combination of thiamin, pyridoxine, and dimethyl allyl alcohol for growth in enriched minimal medium containing glucose. This is the first genetic evidence showing DXP is the precursor to three essential biosynthetic pathways. recAdxs, pcnB1dxs double mutants or recApcnB1dxs triple mutant were constructed to reduce the effect of incomplete repression of PBAD promoter and high plasmid copy number, as well as to prevent the homologous recombination between prototrophic dxs+ on plasmid and disrupted dxs on chromosome. The appearance of a few mutant colonies under repression conditions suggests the existence of a putative bypass pathway for DXP biosynthesis. DXP biosynthetic pathway is widespread in bacteria including opportunistic pathogens or innocuous species related to well-known pathogens. Therefore DXP synthase potentially represents a novel target for antibacterial drugs.

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Research Info

Construction and Physiological Study of D-1-Deoxyxylulose-5′-Phosphate Synthase Mutant in Escherichia coli K-12