| There have been numerous studies on the need for the sodium cation for the growth of haloarchaea, but minimal studies on the effects of the anion on haloarchaeal growth and metabolism. Two haloarchaeal isolates, Halorubrum californiense and Halorubrum saccharovorum - strain 218, have been studied to determine whether other anions can substitute for the chloride anion. These isolates grow optimally at 3.5 M NaCl. When the isolates were subjected to total NaCl substitution with the sodium salts of nitrate, acetate, carbonate, and sulfate there was no growth. In addition neither isolate could grow at 1.5M NaCl. However, both grew when the ionic strength of the medium was adjusted to 3.5 M by the addition of 2 M sodium nitrate or sodium acetate. Growth was also observed when 2M potassium nitrate and potassium acetate were substituted which indicated that it was not the sodium concentration alone that supported the growth, but that the anion was equally important. This suggests that the anion can be substituted to maintain osmotic balance, but chloride is still required at a minimum level to support critical cellular processes. To understand better the effects of these anion replacements, protein expression patterns were studied. One dimensional SDS-PAGE gel electrophoresis and Matrix Assisted Laser Desorption Ionization Mass Spectroscopy were used to evaluate changes in protein expression. The treated samples were grown in a complex medium containing 1.5 M NaCl and supplemented with 2 M sodium acetate or sodium nitrate. Controls were also grown at the optimum 3.5 M NaCl for comparison. The results of these growth experiments showed that there were several up regulated proteins for the treated isolates compared to the proteins produced under optimal growth conditions. All anion substituted groups had lower mass ribosomal proteins in the 7-8.5 kDa range indicating a constitutive group for cell function. At higher molecular weights, above 10 kDa, there was significant variability among the isolates for the salt treatments. For the acetate substitution, Hrr. californiense showed four up regulated and three down regulated proteins in the 10 kDa to 20 kDa range while in the nitrate treatment only five up regulated proteins and four down regulated proteins were noted Additionally, anion substitution suppressed the expression of several translocase-type proteins. These data suggest that the observed changes in protein expression are associated with stress adaptation as the cell adjusts to loss of the chloride ion while still maintaining osmotic balance with other anions. |
