Multicellular organisms developed the concept of specialized cells that perform specific functions. Examples are neurons and fibroblast to name just two out of more than 200. These cellular differences are established based on the same sequence information stored in the cell nucleus of all cells of an organism. The sequence information needs consequently different interpretations by the different cell types. During cellular development this interpretation of the genetic code has to be tightly regulated in space and time. Interpretation of the sequence information involves the controlled activation and silencing of specific genes so that certain proteins are made in one cell type but not in others. This involves an additional regulatory information layer beyond the pure base sequence. One aspect of this regulatory information layer relies on functional groups that are attached to the C(5) position of the canonical base dC. Currently four regulatory, non-canonical bases with a methyl (CH )-, a hydroxymethyl (CH OH)-, a formyl (CHO)- and a carboxyl (COOH)- group are known. While 5-methyl-cytidine is long recognised to be a regulatory base in the genome, the other three bases and the enzymes responsible for generating them, were just recently discovered.

The foliar pathogen Pseudomonas syringae pv. syringae exhibits an exceptional ability to survive on asymptomatic plants as an epiphyte. Intermittent wetting events on plants lead to osmotic and matric stresses which must be tolerated for survival as an epiphyte. In this study, we have applied bioinformatic, genetic, and biochemical approaches to address water stress tolerance in P. syringae pv. syringae strain B728a, for which a complete genome sequence is available. P. syringae pv. syringae B728a is able to produce the compatible solutes betaine, ectoine, N-acetylglutaminylglutamine amide (NAGGN), and trehalose. Analysis of osmolyte profiles of P. syringae pv. syringae B728a under a variety of in vitro and in planta conditions reveals that the osmolytes differentially contribute to water stress tolerance in this species and that they interact at the level of transcription to yield a hierarchy of expression. While the interruption of a putative gene cluster coding for NAGGN biosynthesis provided the first experimental evidence of the NAGGN biosynthetic pathway, application of this knockout strain and also a gfp reporter gene fusion strain demonstrated the small contribution of NAGGN to cell survival and desiccation tolerance of P. syringae pv. syringae B728a under in planta conditions. Additionally, detailed investigation of ectC, an orphan of the ectoine cluster (lacking the ectA and ectB homologs), revealed its functionality and that ectoine production could be detected in NaCl-amended cultures of P. syringae pv. syringae B728a to which sterilized leaves of Syringa vulgaris had been added.Any bacterium growing in an environment with variable external wetness conditions will need a method of adjusting its own intracellular osmolarity. In order to counteract the deleterious effects of high osmolarity and a lack of water itself on cell physiology and the resulting loss of cytosolic water (dehydration), many bacteria rapidly synthesize or take up compatible solutes. These osmotically active small molecules are compatible with cellular function even at high concentrations and serve to maintain cell turgor by balancing the osmotic pressure across the cellular membrane without compromising protein folding or other processes which could be inhibited by high levels of cytosolic salts (26,38,42,65,66,74).Because they are necessarily inert, there are only a limited variety of compounds that can serve as compatible solutes, including certain polyols, amino acids, amino acid derivatives, and peptides. Typical prokaryotic osmolytes include glycine betaine, carnitine, proline, L-␣-glutamate, mannitol, trehalose (␣-D-glucopyranosyl-␣-D-glucopyranoside), N-acetylglutaminylglutamine amide (NAGGN), glucosylglycerol, ectoine, and hydroxyectoine (65). These osmolytes can be produced de novo but are often accumulated by uptake from the environment to reduce the energetic cost to the cell. It is unclear what proportion of its compatible solutes an epiphyte must produce de novo, although it has been hypothesized that choline is widel...

We report on the presence of a functional hydroxyectoine biosynthesis gene cluster, ectABCD-ask, in Pseudomonas stutzeri DSM5190T and evaluate the suitability of P. stutzeri DSM5190 T for hydroxyectoine production. Furthermore, we present information on heterologous de novo production of the compatible solute hydroxyectoine in Escherichia coli. In this host, the P. stutzeri gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose when hydroxyectoine genes were expressed, as well as a remarkable inhibitory effect of externally applied betaine on hydroxyectoine synthesis. The specific heterologous production rate in E. coli under the conditions employed exceeded that of the natural producer Pseudomonas stutzeri and, for the first time, enabled effective hydroxyectoine production at low salinity (2%), with the added advantage of simple product processing due to the absence of other cosolutes.As reviewed extensively elsewhere, halotolerant and halophilic microorganisms can adapt to extreme salinity by accumulation or biosynthesis of a range of osmolytes (8,14,21,32,36). Extreme halophiles like the archaeon Halobacterium salinarum usually recruit inorganic ions, while others, in particular, highly adaptive organisms, make use of a more flexible strategy and employ compatible solutes (also called extremolytes) to obtain osmotic equilibrium. Unlike inorganic ions and even at molar concentrations, these organic low-molecularweight osmolytes do not interfere negatively with normal cell metabolism, even at molar concentrations. In addition to their function as osmoprotectants for whole living cells, compatible solutes also protect biological macromolecules against physical stress in vitro. Therefore, they have found their way into a range of biochemical applications (25, 33) and even skin care products (5).The compatible solute ectoine (12) is often detected as the main solute in chemoheterotrophic eubacterial halophiles of broad salt tolerance.

Compatible solutes are small organic osmolytes including but not limited to sugars, polyols, amino acids, and their derivatives. They are compatible with cell metabolism even at molar concentrations. A variety of organisms synthesize or take up compatible solutes for adaptation to extreme environments. In addition to their protective action on whole cells, compatible solutes display significant effects on biomolecules in vitro. These include stabilization of native protein and nucleic acid structures. They are used as additives in polymerase chain reactions to increase product yield and specificity, but also in other nucleic acid and protein applications.Interactions of compatible solutes with nucleic acids and protein-nucleic acid complexes are much less understood than the corresponding interactions of compatible solutes with proteins. Although we may begin to understand solute/nucleic acid interactions there are only few answers to the many questions we have. I summarize here the current state of knowledge and discuss possible molecular mechanisms and thermodynamics.