ABSTRACT
More than 50 symbiotic mutants of Rhizobium japonicum were isolated by purported plasmid-curing techniques. Wild-type R. japonicum strains were grown in liquid culture at 28 or 36 degrees C in different concentrations of acridine orange, ethidium bromide, or sodium dodecyl sulfate for selection of mutants. The symbiotic traits of 133 isolates from nine treatment groups were determined. Forty-two isolates were Nod- Nif+, seven were Nod+ Nif-, and two were Nod- Nif-. The nifDH genes were deleted in three mutants and consequently showed no hybridization to a nifDH probe. None of these mutants showed any detectable loss of plasmid DNA.
Subject(s)
Mutation , Nitrogen Fixation , Rhizobium/genetics , Sodium Dodecyl Sulfate , Acridine Orange/toxicity , Ethidium/toxicity , Fatty Alcohols/toxicity , Plant Physiological Phenomena , Rhizobium/drug effects , Rhizobium/metabolismABSTRACT
A flow chamber has been developed which collects about 60% of the total cell fluorescence for analysis compared to about 2.5% for conventional flow systems. The chamber, an ellipsoid of revolution, is gold-plated for increased reflectivity. Fluorochrome-stained cells enter the flow cell directly above the primary focus of the ellipsoid at the rate of 1000 cell/sec. A focused argon-ion laser beam enters the flow cell parallel to the semiminor axis and intersects the cell stream at the primary focus. Fluorescent light emanating from this point is reflected toward the secondary focus, where it exits the chamber for analysis. The high efficiency flow cytometer has been used to obtain nucleotide fluorescence distributions from samples of Micrococcus glutamicus bacteria stained with propidium iodide and of spermatozoa stained by the acriflavine-Feulgen procedure.
