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

Halophiles are found in all three domains of life. Within the Bacteria we know halophiles within the phyla Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, and Bacteroidetes. Within the Archaea the most salt-requiring microorganisms are found in the class Halobacteria. Halobacterium and most of its relatives require over 100–150 g/l salt for growth and structural stability. Also within the order Methanococci we encounter halophilic species. Halophiles and non-halophilic relatives are often found together in the phylogenetic tree, and many genera, families and orders have representatives with greatly different salt requirement and tolerance. A few phylogenetically coherent groups consist of halophiles only: the order Halobacteriales, family Halobacteriaceae (Euryarchaeota) and the anaerobic fermentative bacteria of the order Halanaerobiales (Firmicutes). The family Halomonadaceae (Gammaproteobacteria) almost exclusively contains halophiles. Halophilic microorganisms use two strategies to balance their cytoplasm osmotically with their medium. The first involves accumulation of molar concentrations of KCl. This strategy requires adaptation of the intracellular enzymatic machinery, as proteins should maintain their proper conformation and activity at near-saturating salt concentrations. The proteome of such organisms is highly acidic, and most proteins denature when suspended in low salt. Such microorganisms generally cannot survive in low salt media. The second strategy is to exclude salt from the cytoplasm and to synthesize and/or accumulate organic 'compatible' solutes that do not interfere with enzymatic activity. Few adaptations of the cells' proteome are needed, and organisms using the 'organic-solutes-in strategy' often adapt to a surprisingly broad salt concentration range. Most halophilic Bacteria, but also the halophilic methanogenic Archaea use such organic solutes. A variety of such solutes are known, including glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. The 'high-salt-in strategy' is not limited to the Halobacteriaceae. The Halanaerobiales (Firmicutes) also accumulate salt rather than organic solutes. A third, phylogenetically unrelated organism accumulates KCl: the red extremely halophilic Salinibacter (Bacteroidetes), recently isolated from saltern crystallizer brines. Analysis of its genome showed many points of resemblance with the Halobacteriaceae, probably resulting from extensive horizontal gene transfer. The case of Salinibacter shows that more unusual types of halophiles may be waiting to be discovered.

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Microbial life at high salt concentrations: phylogenetic and metabolic diversity

The osmoadaptation of most micro-organisms involves the accumulation of K+ ions and one or more of a restricted range of low molecular mass organic solutes, collectively termed ‘compatible solutes’. These solutes are accumulated to high intracellular concentrations, in order to balance the osmotic pressure of the growth medium and maintain cell turgor pressure, which provides the driving force for cell extension growth. In this review, I discuss the alternative roles which compatible solutes may also play as intracellular reserves of carbon, energy and nitrogen, and as more general stress metabolites involved in protection of cells against other environmental stresses including heat, desiccation and freezing. Thus, the evolutionary selection for the accumulation of a specific compatible solute may not depend solely upon its function during osmoadaptation, but also upon the secondary benefits its accumulation provides, such as increased tolerance of other environmental stresses prevalent in the organism’s niche or even anti-herbivory or dispersal functions in the case of dimethylsulfoniopropionate (DMSP). In the second part of the review, I discuss the ecological consequences of the release of compatible solutes to the environment, where they can provide sources of compatible solutes, carbon, nitrogen and energy for other members of the micro-flora. Finally, at the global scale the metabolism of specific compatible solutes (betaines and DMSP) in brackish water, marine and hypersaline environments may influence global climate, due to the production of the trace gases, methane and dimethylsulfide (DMS) and in the case of DMS, also couple the marine and terrestrial sulfur cycles.

Ecological significance of compatible solute accumulation by micro‐organisms: from single cells to global climate

Publisher Summary This chapter focuses on the osmoadaptation in bacteria. Bacterial ability to osmoadapt seems to be the norm rather than the exception. Under the right environmental circumstances (external osmolytes or precursors thereof), many bacteria, so far believed to be salt-sensitive, show considerable osmotolerance. Mechanisms for survival and growth in a high-osmolarity environment aim at maintaining osmotic equilibrium across the membrane. Compatible solutes used and produced by halo/osmophilic bacteria are useful tools to study and understand water solvent interactions, and to resolve principles of enzyme stabilization. They are bound to offer a range of technological applications as universal stress protectants. Among anaerobic halophilic eubacteria, attempts to find organic osmolytes have been unsuccessful; instead,high cytoplasmic amounts of potassium and sodium chloride were detected and proteins displayed an excess of acidic amino acids. As all enzymes investigated also showed maximal activity at high salt concentrations, there seems to be sufficient evidence that this eubacterial physiological group is adapted to an ionic cytoplasm. The salt-in-cytoplasm type of osmoadaptation is therefore apparently not confined to archaebacterial representatives, although NaCl rather than KCl seems to be the dominant cytoplasmic solute in these eubacteria. The best-studied model systems of osmotic adaptation are E. coli and S. typhimurium, which are discussed.

Osmoadaptation in Bacteria

Salt stress is primarily osmotic stress, and halophilic/halotolerant microorganisms have evolved two basic mechanisms of osmoadaplation: the KCI-type and the compatible-solute type, the latter representing a very flexible mode of adaptation making use of distinct stabilizing properties of compatible solutes. A comprehensive survey, using HPLC and NMR methods, has revealed the full diversity of euhacterial compatible solutes found in nature. With the exception of proline (a proteinogenic amino acid) they are characterized as amino acid derivatives of the following types: betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine. From our present knowledge of hiosynthetic pathways it appears that, apart from glycine betaine, all nitrogen-containing compatible solutes originate from two major pathways (the aspartate branch and the glutamate branch). Uptake of compatible solutes from the growth medium (environment) seems to have preference over de novo synthesis. Therefore in the natural ecosystem the solutes of primary producers (mainly glycine betaine), which are readily excreted upon dilution stress, certainly play an important role as a ‘preferred’ solute source for heterolrophic organisms, and as a ‘vital’ source for organisms unable to synthesize their own compatible solutes.

Microbial behaviour in salt‐stressed ecosystems

The aim of this investigation was to perform an extensive screening using HPLC and 13C-NMR spectroscopy to disclose the spectrum of osmolytes produced by aerobic heterotrophic and anoxygenic phototrophic eubacteria. The most predominant solutes detected within a wide range of marine and halophilic micro-organisms were two recently discovered tetrahydropyrimidines ectoine and hydroxyectoine, which were synthesized in response to osmotic stress.

The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria

Summary: A new family, the Halomonadaceae, has recently been proposed for members of the genera Deleya and Halomonas. The three strains investigated, Deleya halophila, Halomonas elongata and Flavobacterium halmephilum (reclassified as H. halmophila), are aerobic heterotrophic micro-organisms exhibiting an extreme salt tolerance. The major organic osmoregulatory solutes of these organisms were examined using 13C-nuclear magnetic resonance spectroscopy. The relative proportions of the solutes varied with respect to salt concentration, temperature and carbon source. The recently described amino acid ectoine was found to be a dominant solute. For the first time it could be shown for halophilic eubacteria that the intracellular concentration of solutes is sufficient to balance the osmotic pressure of the medium. Thus, there is no need to postulate a hypo-osmotic cytoplasm.

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The spectrum of compatible solutes in heterotrophic halophilic eubacteria of the family Halomonadaceae

On a first view there is apparently little correlation between taxonomy and halophily The distribution of different compatible solutes in the eubacterial phyla Proteobacteria and Firmacutes follows a pattern, however, that at least follows certain regularities: (1) All eubacteria that gain energy from photosynthesis or respiration and are capable of haloadaptation are able to accumulate and/or synthesize compatible solutes (2) When a eubacterium can grow in a non-complex medium, it usually can synthesize ectoine Growth on yeast extract does not necessarily exclude ectoine synthesis (3) Extreme halophily in eubacteria is always accompanied by glycine betaine synthesis The complete synthesis of glycine betaine from CO2 or simple carbon compounds has only been proven for cyanobacteria, Ectothiorhodospiraceae and Actinopolyspora halophila (4) A not yet identified compound “Y” occurs preferably in Firmacutes; Bacillus species accumulate and synthesize proline and glutamate as compatible solutes

Halophily, Taxonomy, Phylogeny and Nomenclature

In the course of a screening programme for novel natural substances acting as compatible solutes, an unusual amino acid derivative was detected in several halophilic Gram-positive eubacteria. The compound was isolated and subsequently identified as N
δ-acetylornithine using spectroscopic (NMR, MS) and chromatographic methods. So far, N
δ-acetylornithine has only been described as an excretion product of some bacteria under certain growth conditions. This report describes, for the first time, its intracellular accumulation and use as a compatible solute in halophilic/halotolerant eubacteria.

Axel Wohlfarth

Papers

The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria

The spectrum of compatible solutes in heterotrophic halophilic eubacteria of the family Halomonadaceae

Halophily, Taxonomy, Phylogeny and Nomenclature

Identification of N -acetylornithine as a novel osmolyte in some Gram-positive halophilic eubacteria