Transduction of Genes From a Lipopolysaccharide Secondary Mutant

Graves, James, and Jacob Wingate

Lipopolysaccharide (LPS) mutations in gram-negative bacteria are associated with increased sensitivity to antimicrobial agents and decreased expression of proteins. Moreover, secondary mutants can frequently be distinguished from poor growing LPS mutants by a change in colony appearance and increase in size. Transduction, a method by which genes can be transferred from one strain to another by a bacterial virus (phage), was used to study LPS mutants of Escherichia coli. Donor strains harbored a copy of the tetracycline resistance transposon Tn10 linked to the region of the chromosome that contained the genes for LPS synthesis (rfa) to serve as a selectable marker. Because phage reproduced poorly on LPS mutants when examined by the cross streak method, a lysogen of thermoinducible generalized transducing phage P1cml,clr100 was used from which to isolate LPS mutants. The phage genome contained a gene for chloramphenicol resistance as a marker. Strains that contained phage P1cml,clr100 or produced altered LPS grew poorly at 42oC. Attempts to produce a heat induced lysate of the phage in broth culture from a selected LPS mutant were unsuccessful. An antibiotic disc test of the LPS mutant revealed an increased sensitivity to chloramphenicol which suggested that the P1cml,clr100 may have been cured. A selected secondary LPS mutant was discovered to be sensitive to infection by the transducing phage P1kc but demonstrated increased sensitivity to erythromycin in comparison to a strain with wild type LPS. When tested, a P1kc lysate prepared on the secondary LPS mutant by agar overlay propagation produced about 2.8 X 102 tetracycline resistant transductants/ml of the cell-phage mixture. Transductants tested exhibited wild type sensitivity to LPS specific phage U3. Transduction of genes from the region of the chromosome associated with LPS synthesis could help determine if secondary mutations are linked and increase understanding of cell membranes and permeability.