acidocaldarius (strain SK-1 [[DELTA]pyrE [DELTA]suaI]) electrocompetent cells for transformation with a shuttle vector and via homologous recombination were prepared from a late log to stationary phase culture ([OD.sub.600 [??] 0.7) and an early to midlog phase culture ([OD.sub.600] = 0.1-0.4) incubated in XTU medium, respectively.
UV-irradiated cultures were immediately inoculated into 6 mL of XTU liquid medium to yield an initial [OD.sub.600] of 0.005.
To compare the growth properties of strain SK-5 in the presence or absence of agmatine, overnight cultures (late log to stationary phase) were inoculated into 6 mL of XTU liquid medium supplemented with 100-1000 [micro]g/mL agmatine to yield an initial [OD.sub.600] of 0.005.
A total of 2.3 x [10.sup.8] DP-1 Int cells were spread on XTU plates containing 5-FOA for pop-out recombination.
A total of 3.4 x [10.sup.8] SK-5 Int cells were spread on XTU plates containing 5-FOA and 1 mg/mL agmatine for pop-out recombination.
The growth of the argD-deficient strain SK-5 ([DELTA]pyrE [DELTA]suaI [DELTA]argD) was studied using XTU liquid culture in the presence or absence of agmatine (Figure 7).
Host strain SK-5 ([DELTA]pyrE [DELTA]suaI [DELTA]argD) cells harvested at early to midlog phase ([OD.sub.600] = 0.308) were transformed with 8 ng of plasmid DNA (1 [micro]L) by electroporation (15 kV/cm, 9 ms) and spread on XTU plates (regeneration in recovery buffer was not conducted).
Wt (SK-1) and [DELTA]phr (DP-1) cells were irradiated with UV light (1200 J/[m.sup.2]) and cultivated in XTU liquid medium under light or dark conditions for viability testing.
Wt (SK-1) and [DELTA]argD (SK-5) were cultivated in XTU liquid medium with or without agmatine at 75[degrees]C with shaking.