Use of 23Na nuclear magnetic resonance spectroscopy to determine the true intracellular concentration of free sodium in a halophilic eubacterium

Abstract

We present new data obtained by 23Na nuclear magnetic resonance spectroscopy, which can distinguish free intracellular sodium from cell-bound sodium, showing that the intracellular concentration of Na+ the halophilic eubacterium Vibrio costicola is only 5 to 20% of that in the extracellular medium. Previous methods could not distinguish free intracellular Nn.' from that bound to cell structures, and it was believed that in halophilic eubacteria the total monovalent cation concentration inside matched that of the NaCl outside. Information obtained by the newer technology raises fundamental questions about the ways in which these organisms and others which live in hypersaline environments function and cope with osmotic stress.Halophilic bacteria belong to two major kingdoms. The archaebacterial extreme halophiles generally require 2 to 3 M NaCl and can grow in saturated brines (16,25). The composition of their cell envelopes differs from that of eubacterial cells, and they have high proportions of negatively charged proteins in their membranes, ribosomes, and other cell components. They are known to accumulate intracellular potassium ions to balance the extracellular sodium concentrations (16,25).The second group, the eubacterial halophiles, are far more widely distributed, both environmentally and among families and genera (19,22,32), and they are of great ecological and economic importance in agriculture, food industry, and other more specialized processes. The eubacterial halophiles also show more wide-ranging salt requirements and tolerances, i.e., they are more adaptable. The mechanisms and components which enable them to grow over such a wide range (from 3-to 10-fold, according to species) of salt concentrations are still largely unknown. One of the most important questions still unresolved involves the true intracellular ion concentrations relative to those in the medium and how the organisms deal with the osmotic and ionic gradients across their cell membranes. This problem has been highlighted by studies which showed that in vitro protein-synthesizing systems from such organisms (35) and most of their intracellular enzymes were inhibited by NaCl concentrations at which the cells had been grown (12,17,35). On the other hand, earlier studies of intracellular salt concentrations in such organisms suggested that the total concentration of intracellular cations was more or less equal to the cation concentration in the extracellular milieu (3,5,15,20,23,27,28,33

In Vivo 23 Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium Fibrobacter succinogenes S85

“…The intracellular free (visible) Na ϩ concentrations, measured by 23 Na NMR in Escherichia coli (3,12), in the halotolerant Brevibacterium sp. (12), or in the moderately halophilic Vibrio costicola (9), were similar to those measured in F. succinogenes for the same extracellular [Na ϩ ]. As a result, the calculated ⌬pNa values were comparable for these bacterial systems, although the ⌬pNa value was slightly higher in the case of E. coli (Ϫ76 mV for 75 mM extracellular [Na ϩ ]) (3).…”

23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli

“…Until the 23Na NMR method was applied to the Na' analyses in cells, analyses were restricted to the cellassociated ions, and the bound or attached sodium content was not distinguished (Ogino et al, 1983;Gilboa et al, 1991). According to our analyses, the concentrations of free and bound sodium in the cytosol were 0.13-0.14 and 0.23-0-26 pmol (mg protein)-', respectively.…”

“…I/. costicola NCMB 701 (from Dr H. Tokuda, University of Tokyo) was grown aerobically in proteose peptone plus 0.3 % (w/v) tryptone (PPT) medium (Gilboa et al, 1991) and harvested at the exponential growth phase for comparison. Bacterial pellets, collected by centrifugation, were diluted to 5-10 (mg cell protein) ml-' in the same growth medium, and were used for the analyses of free Na' concentration in the cells by NMR measurement.…”

Metabolic Enzymes from Psychrophilic Bacteria: Challenge of Adaptation to Low Temperatures in Ornithine Carbamoyltransferase from Moritella abyssi

“…The salt requirement of M. abyssi, which is a marine bacterium, can be met with half-strength seawater (57), but the intracellular salt concentration is not known. Assuming intracellular concentrations of free Na ϩ and K ϩ of between 0.15 and 0.3 M in moderate halophiles (23,49), it appears that in vivo, the main fraction of OCTase Mab is in the dodecameric form.…”