The moderately halophilic heterotrophic aerobic bacteria form a diverse group of microorganisms. The property of halophilism is widespread within the bacterial domain. Bacterial halophiles are abundant in environments such as salt lakes, saline soils, and salted food products. Most species keep their intracellular ionic concentrations at low levels while synthesizing or accumulating organic solutes to provide osmotic equilibrium of the cytoplasm with the surrounding medium. Complex mechanisms of adjustment of the intracellular environments and the properties of the cytoplasmic membrane enable rapid adaptation to changes in the salt concentration of the environment. Approaches to the study of genetic processes have recently been developed for several moderate halophiles, opening the way toward an understanding of haloadaptation at the molecular level. The new information obtained is also expected to contribute to the development of novel biotechnological uses for these organisms.

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Biology of moderately halophilic aerobic bacteria

Two general strategies exist for the growth and survival of prokaryotes in environments of elevated osmolarity. The ‘salt in cytoplasm’ approach, which requires extensive structural modifications, is restricted mainly to members of the Halobacteriaceae. All other species have convergently evolved to cope with environments of elevated osmolarity by the accumulation of a restricted range of low molecular mass molecules, termed compatible solutes owing to their compatibility with cellular processes at high internal concentrations. Herein we review the molecular mechanisms governing the accumulation of these compounds, both in Gram-positive and Gram-negative bacteria, focusing specifically on the regulation of their transport/synthesis systems and the ability of these systems to sense and respond to changes in the osmolarity of the extracellular environment. Finally, we examine the current knowledge on the role of these osmostress responsive systems in contributing to the virulence potential of a number of pathogenic bacteria.

Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence

Microorganisms that adapt to moderate and high salt environments use a variety of solutes, organic and inorganic, to counter external osmotic pressure. The organic solutes can be zwitterionic, noncharged, or anionic (along with an inorganic cation such as K+). The range of solutes, their diverse biosynthetic pathways, and physical properties of the solutes that effect molecular stability are reviewed.

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Organic compatible solutes of halotolerant and halophilic microorganisms

Two major types of environment provide habitats for the most xerophilic organisms known: foods preserved by some form of dehydration or enhanced sugar levels, and hypersaline sites where water availability is limited by a high concentration of salts (usually NaCl). These environments are essentially microbial habitats, with high-sugar foods being dominated by xerophilic (sometimes called osmophilic) filamentous fungi and yeasts, some of which are capable of growth at a water activity (a(w)) of 0.61, the lowest a(w) value for growth recorded to date. By contrast, high-salt environments are almost exclusively populated by prokaryotes, notably the haloarchaea, capable of growing in saturated NaCl (a(w) 0.75). Different strategies are employed for combating the osmotic stress imposed by high levels of solutes in the environment. Eukaryotes and most prokaryotes synthesize or accumulate organic so-called 'compatible solutes' (osmolytes) that have counterbalancing osmotic potential. A restricted range of bacteria and the haloarchaea counterbalance osmotic stress imposed by NaCl by accumulating equivalent amounts of KCl. Haloarchaea become entrapped and survive for long periods inside halite (NaCl) crystals. They are also found in ancient subterranean halite (NaCl) deposits, leading to speculation about survival over geological time periods.

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Life at low water activity.

The accumulation of compatible solutes is a prerequisite for the adaptation of microorganisms to osmotic stress imposed by salt or organic solutes Two types of strategies exist to cope with high external solute concentrations; one strategy is found in the extremely halophilic Archaea of the family Halobacteriaceae and the Bacteria of the order Haloanaerobiales involving the accumulation of inorganic ions The other strategy of osmoadaptation involves the accumulation of specific organic solutes and is found in the vast majority of microorganisms The organic osmolytes range from sugars, polyols, amino acids and their respective derivatives, ectoines and betaines The diversity of these organic solutes has increased in the past few years as more organisms, especially thermophilic and hyperthermophilic Bacteria and Archaea, have been examined The term compatible solute can also be applied to solutes that protect macromolecules and cells against stresses such as high temperature, desiccation and freezing The mechanisms by whihh compatible solutes protect enzymes, cell components and cells are still a long way from being thoroughly elucidated, but there is a growing interest in the utilization of these solutes to protect macromolecules and cells from heating, freezing and desiccation

An overview of the role and diversity of compatible solutes in Bacteria and Archaea

A novel biotechnological process called "bacterial milking" has been established for the production of compatible solutes using the Gram-negative bacterium Halomonas elongata. Following a high-cell-density fermentation which provided biomass up to 48 g cell dry weight per liter, we applied alternating osmotic shocks in combination with crossflow filtration techniques to harvest the compatible solutes ectoine and hydroxyectoine. H. elongata, like other halophilic or halotolerant microorganisms, produces compatible solutes in response to the salinity of the medium. When transferred to a low salinity medium (osmotic downshock), H. elongata cells rapidly released their solutes to achieve osmotic equilibrium. Subsequent reincubation in a medium of higher salt concentration resulted in resynthesis of these compatible solutes and-after a defined regeneration time-the procedure could be repeated. By repeatedly performing this "bacterial milking" process (at least nine times) we were able to produce large amounts of ectoines with a biomass productivity of 155 mg of ectoine per cycle per gram cell dry weight. Further purification of the products was achieved by a simple two-step procedure based on cation exchange chromatography and crystallization. The principles described in this article may also be useful for the production of other low-molecular-weight compounds.

Bacterial milking: A novel bioprocess for production of compatible solutes.

Production of hydroxyectoine: high cell-density cultivation and osmotic downshock of Marinococcus strain M52

Marine, halotolerant microorganisms have great potential for the production of organic osmolytes (compatible solutes), which typically attract water and stabilize biological structures in vivo and in vitro [Galinski, 1995]. In this study we describe the exploitation of the marine, extremely halotolerant Halomonas elongata for the semi-continuous production of the tetrahydropyrimidines ectoine and S,S-β-hydroxyectoine, both novel compatible solutes with applications as stress protectants for enzymes, DNA and whole cells [Lippert & Galinski, 1992; Lapidot et al., 1993; Louis et al., 1994] (Fig. 1).

Production and Application of Natural Stabilizing Compounds from Halotolerant Bacteria

L'invention concerne un procede permettant d'obtenir in vivo des constituants de cellules, selon lequel les cellules concernees passent brusquement d'un premier etat dans lequel regnent des conditions quasi-stationnaires entre la cellule et le milieu ambiant, a un second etat, ce qui oblige les cellules a reagir en degageant leurs constituants dans l'environnement afin de permettre a la cellule de s'adapter aux conditions regnant dans le second etat.

Thomas Sauer

Papers

Bacterial milking: A novel bioprocess for production of compatible solutes.

Production of hydroxyectoine: high cell-density cultivation and osmotic downshock of Marinococcus strain M52

Production and Application of Natural Stabilizing Compounds from Halotolerant Bacteria

Procede permettant d'obtenir in vivo des constituants de cellules