Fluorescent methods to study DNA, RNA, proteins and cytoplasmic membrane polarization in the pentachlorophenol-mineralizing bacterium Sphingomonas sp. UG30 during nutrient starvation in water.
The effect of sodium pentachlorophenolate (NaPCP) exposure on the nutrient-starved pentachlorophenol (PCP)-mineralizing bacterium Sphingomonas sp. UG30 was assessed using fluorescent methods to measure DNA, RNA, total cellular protein, and cytoplasmic membrane proteins. UG30 cells were inoculated into sterilized Speed River (Guelph, ON, Canada) water samples in the presence of 50, 100, and 250 ppm NaPCP. No marked changes were observed in the total cellular DNA, RNA or protein levels over 90 d, indicating the macromolecular composition of UG30 was not affected by both nutrient limitation and NaPCP. Total cell counts as determined by DAPI staining also did not change over 90 d. Over the same period, viable counts decreased with increasing concentrations of NaPCP. At 250 ppm NaPCP, viable cell counts decreased over 6 orders of magnitude after 1 hr exposure. Cell numbers partially recovered once NaPCP was degraded. The UG30 cytoplasmic membrane polarization ratio also decreased after NaPCP was depleted. The decreased polarization value at the end of the study period suggested the UG30 membrane was more fluid and that this increase in fluidity was due to nutrient starvation effects rather than exposure to NaPCP. These results indicated that UG30 is a robust organism that is able to degrade NaPCP even under adverse conditions and fluorescent methods are useful for determining macromolecular concentrations and cytoplasmic membrane polarization values.
Denich
T J
TJ
Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1.
Beaudette
L A
LA
Lee
H
H
Trevors
J T
JT
D001426:Bacterial Proteins / Q000032:analysis*; D001483:Base Sequence; D015169:Colony Count, Microbial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D005618:Fresh Water; D010416:Pentachlorophenol / Q000378:metabolism*; D012329:RNA, Bacterial / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D020578:Sphingomonas / Q000378:metabolism*; D014871:Water Microbiology
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D010416:Pentachlorophenol