Effects of low temperature, cold shock and various carbon sources on the physiology of a psychrotrophic Acinetobacter sp.

Abstract

Growth of psychotrophic Acinetobacter sp. HH1-1 at low temperatures was investigated by monitoring cell membrane properties, enzyme activity, and protein synthesis during growth at 25*C and 5*C, and following a 25*C to 5*C decrease in growth temperature (cold shock). The effect of different carbon sources on the ability of HH1-1 to frow at low temperatures and respond to cold shock was also investigated. Cells were grown in bath cultures with acetate, Tween 80, or olive oil as the sole source of carbon. Membrane permeability was monitored after cold shock by measuring K^+ concentrations in culture supernatant. At various time points during growth at 25*C, 5*C, and after cold shock, cell membrane fluidity was examined by measuring the fluorescence polarization of the membrane-probe parinaric acid. The fatty acid composition of cells was determined using gas chromatography. Activities of the enzymes isocitrate lyase, esterase, and lipase were also monitored using spectrophotometric assays, and protein synthesis was assessed using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE).

Cell-membrane changes were observed after cold shock for all carbon sources. Cells became leaky and membranes less fluid. Acetate-grown cells responded more quickly to cold shock than did cells grown with either Tween 80 or olive oil by restoring membrane fluidity and by taking K^+ back into cells. Concentrations of the fatty acid palmitoleic acid (cis-^p-hexadecenoic, 16:1) increased 2 h after cold shock in acetate-grown cells and 24 h after cold shock in Tween 80- and olive oil-grown cells. In addition, an increased content of C16:1 fatty acid was observed in olive oil-grown cells during growth at 5*C relative to cells grown at 25*C. these data indicated that this fatty acid may be important for maintaining membrane fluidity at low temperatures.

Growth at low temperatures and cold shock had varying effects on isocitrate lyase, esterase, and lipase activities. During growth at 25*C, isocitrate lyase activity was measured in cell sonicate, but at 5*C and after cold shock, activity was measured primarily in cell culture supernatant. This response supported the conclusion that a loss in membrane permeability occurred at low temperatures. HH1-1 produced two cell-associated esterases and an extracellular esterase and lipase. Activities of the extracellular esterase and lipase were reduced at 5*C and after cold shock. These results indicated that the reduction in extracellular esterase and lipase activities may be related to inefficient transport of the enzymes across the cell membranes. In contrast, an increased synthesis of a 54-kDa cell-associated esterase observed 50 h after cold shock suggested a requirement for this enzyme at low temperatures.

HH1-1 responded to cold shock by synthesizing both cold shock proteins (csps) and cold acclimation proteins (caps). The synthesis of 3 csps (csps 89a, 36a, and 18) was increased 2 h after cold shock by all cells. An additional csp (csp12), with an estimated molecular mass of 12-kDa, was observed in olive oil-grown cells only. Csp12 was also induced when cells were grown at 30*C, this strain's maximal growth temperature, suggesting that csp12 may be a general stress protein rather than one required solely for cold-temperature growth. In addition to csps, fifteen cold acclimation proteins (caps) were observed at 72 h (acetate-grown cells) and at 140 h (Tween 80- and olive oil-grown cells) post cold shock. Two caps were common for all substrates whereas the other 13 proteins were unique to only one or two of the substrates tested.

The data collected in this study demonstrated that cells utilizing olive oil as the sole carbon source are affected by rapid decreases in temperature and growth at low temperatures to a greater extent than either acetate- or Tween 80-grown cells. Substrate effects on the physiology of Acinetobacter sp. HH1-1 would need to be considered if the low-temperature growth of this bacterium is to be used in industry, biotechnology, or for bioremediation schemes.