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The investigated haloarchaeal species, Halobacterium salinarum, Haloferax mediterranei, and H. volcanii, have all been shown to be polyploid. They contain several replicons that have independent copy number regulation, and most have a higher copy number during exponential growth phase than in stationary phase. The possible evolutionary advantages of polyploidy for haloarchaea, most of which have experimental support for at least one species, are discussed. These advantages include a low mutation rate and high resistance toward X-ray irradiation and desiccation, which depend on homologous recombination. For H. volcanii, it has been shown that gene conversion operates in the absence of selection, which leads to the equalization of genome copies. On the other hand, selective forces might lead to heterozygous cells, which have been verified in the laboratory. Additional advantages of polyploidy are survival over geological times in halite deposits as well as at extreme conditions on earth and at simulated Mars conditions. Recently, it was found that H. volcanii uses genomic DNA as genetic material and as a storage polymer for phosphate. In the absence of phosphate, H. volcanii dramatically decreases its genome copy number, thereby enabling cell multiplication, but diminishing the genetic advantages of polyploidy. Stable storage of phosphate is proposed as an alternative driving force for the emergence of DNA in early evolution. Several additional potential advantages of polyploidy are discussed that have not been addressed experimentally for haloarchaea. An outlook summarizes selected current trends and possible future developments.
Acinetobacter baumannii is a nosocomial pathogen which can persist in the hospital environment not only due to the acquirement of multiple antibiotic resistances, but also because of its exceptional resistance against disinfectants and desiccation. A suitable desiccation assay was established in which A. baumannii ATCC 19606T survived for ca. 1 month. The growth medium slightly influenced survival after subsequent desiccation. A significant effect could be attributed to the growth phase in which bacteria were dried: In exponential phase, cells were much more desiccation sensitive. The main focus of the present study was the elucidation of the role of compatible solutes, which are known to protect many bacteria under low water activity conditions, in desiccation survival of A. baumannii. Exogenous trehalose was shown to efficiently protect A. baumannii on dry surfaces, in contrast to other compatible solutes tested such as mannitol or glycine betaine. To analyze the importance of intracellularly accumulated solutes, a double mutant lacking biosynthesis pathways for mannitol and trehalose was generated. This mutant accumulated glutamate as sole solute in the presence of high NaCl concentrations and showed severe growth defects under osmotic stress conditions. However, no effect on desiccation tolerance could be seen, neither when cells were dried in water nor in the presence of NaCl.
Mannitol is the major compatible solute, next to glutamate, synthesized by the opportunistic human pathogen Acinetobacter baumannii under low water activities. The key enzyme for mannitol biosynthesis, MtlD, was identified. MtlD is highly similar to the bifunctional mannitol‐1‐phosphate dehydrogenase/phosphatase from Acinetobacter baylyi. After deletion of the mtlD gene from A. baumannii ATCC 19606T cells no longer accumulated mannitol and growth was completely impaired at high salt. Addition of glycine betaine restored growth, demonstrating that mannitol is an important compatible solute in the human pathogen. MtlD was heterologously produced and purified. Enzyme activity was strictly salt dependent. Highest stimulation was reached at 600 mmol/L NaCl. Addition of different sodium as well as potassium salts restored activity, with highest stimulations up to 41 U/mg protein by sodium glutamate. In contrast, an increase in osmolarity by addition of sugars did not restore activity. Regulation of mannitol synthesis was also assayed at the transcriptional level. Reporter gene assays revealed that expression of mtlD is strongly dependent on high osmolarity, not discriminating between different salts or sugars. The presence of glycine betaine or its precursor choline repressed promoter activation. These data indicate a dual regulation of mannitol production in A. baumannii, at the transcriptional and the enzymatic level, depending on high osmolarity.