**4. Conclusions**

The comprehensive analysis of more than 130 genomes of phototrophic bacteria gives insight into their ability to synthesize the compatible solutes betaine and ectoine, and to take up these compounds. The potential to accumulate these compatible solutes and the kind of solute accumulated, clearly define the range of salt concentrations and the habitats where these bacteria can develop. The data sugges<sup>t</sup> that betaine is the primary compatible solute at high salt concentrations and the most ancient one in evolutionary terms. All halophilic phototrophic bacteria rely on betaine synthesis, and only few of them have additional options of ectoine biosynthesis, or betaine synthesis from choline. Ectoine synthesis as a sole compatible solute is only found in some marine bacteria, in particular *Rhodobacteraceae* and the acidiphilic *Acidiphilum* species. This is in accord with its potential to achieve osmotic protection in moderately halotolerant marine bacteria. As information on the presence of choline in marine and hypersaline habitats is missing, it is unclear whether the transformation of choline to betaine is of relevance for osmotic adaptation in

these environments. Only few marine species of the *Rhizobiales,* exclusively rely on this pathway, but in addition have several transport systems (betT, ProU) to take up betaine and ectoine from the environment. Therefore, these bacteria are more limited than others to adapt to marine salt concentrations, and their occurrence is restricted to habitats where appropriate solutes are available.

The possibility to take up and accumulate compatible solutes or their precursors from the environment and also from the culture media, was been largely neglected in previous studies on salt relations of marine and halophilic species. In order to consider osmotic adaptation in an ecological context and to characterize salt relations of individual species, the potential of uptake needs to be considered. In complex microbial communities such a microbial mats, the study of synthesis, release and uptake at the community level would be rewarding and would shed light on possible complex syntrophic interactions based on the exchange of compatible solutes between members of the community. In view of the variety and the presence of multiple transport systems in several species, characterization of their catalytic properties and regulation is necessary to identify their functional roles. Quite rewarding from both an evolutionary point of view and from a functional context, should be the analysis of chimeric transport systems as they occur in *Prosthecochloris vibrioformis* (*proVopuAB,AC*) and in the *Roseiflexus* species (*opuCBproXV*).

Despite the fact that synthesis and accumulation of betaine is common to all known halophilic phototrophic bacteria, there is a considerable variation in gene arrangemen<sup>t</sup> and formation of gene clusters. In addition, fused GMT-DMT genes occur in some groups of the studied bacteria and a second type called the B-type of GMT sequences is found in several species. The phylogeny of the biosynthesis pathway (of GMT), suggests that the roots are manifested early in bacterial evolution and are most likely before diversification of bacteria as we know today. The recognition of two major phylogenetic branches of *Cyanobacteria* and their relations to others suggests that they represent one of the most ancient betaineproducing bacterial phyla and betaine biosynthesis and might have originated in one of their early ancestor. In another very ancient phylum of phototrophic bacteria, the *Chlorobi* (green sulfur bacteria), betaine biosynthesis was found only in *Chloroherpeton thalassium*. This is one of the most ancient representatives of green sulfur bacteria that is known and the deeply rooted branch of its GMT sequence points out that it might represent one of the most ancient betaine producers as well. Other green sulfur bacteria such as the *Chlorobium* and *Chlorobaculum* species and their relatives might have lost the capability of betaine synthesis during adaptation to freshwater habitats. Most remarkable is the occurrence of B-type GMT sequences, which are phylogenetically distant to all other GMT sequences, and might represent a much older system of betaine biosynthesis. Such a GMT gene is included in a gene cluster for betaine biosynthesis only in the *Rhodovibrio* species. Therefore, studies of betaine biosynthesis of the *Rhodovibrio* species should be quite rewarding.

In addition to these few examples, data are presented form a comprehensive basis for more detailed studies on osmotic adaptation of phototrophic bacteria.

**Supplementary Materials:** The Supplementary Material for this article is available online at https: //www.mdpi.com/2076-2607/9/1/46/s1.

**Author Contributions:** Cultivation of bacterial cultures, DNA extraction and purification were performed by T.R.; genomic sequencing and quality assurance by S.K. and A.K.; sequence assembly by S.C.N.; sequence annotation, retrieval of sequences from annotated genomes and databases, phylogenetic calculations and design of the study, as well as writing of the manuscript was done by J.F.I. All authors contributed to and approved the work for publication. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable. **Data Availability Statement:** Data on gene and genome sequences and accession numbers are contained within supplementary material of the article. All sequence data are available in a publicly accessible repository of gene bank.

**Conflicts of Interest:** The authors declare no conflict of interest.
