3.1.3. Chloroflexi

Genes for the synthesis of ectoine and betaine were not found in the *Chloroflexus* and *Roseiflexus* species (Table 1). Additionally, transport systems for osmotica are absent from the *Chloroflexus* species, which characterizes them as strict freshwater bacteria. An ABC transport system (annotated as *opuCBproXV* and an ABC transport protein) found in the *Roseiflexus castenholzii* and *Roseiflexus* species RS-1 (Table 1) might as well represent an ancient evolutionary form of osmolyte transport and betaine uptake from the environment. Thereby, the *Roseiflexus* species might gain limited possibility for osmotic adaptation. As these bacteria are among the oldest mat-forming phototrophic bacteria and *Roseiflexus* might be able to take up betaine or other osmolytes, they or relatives thereof are expected to be found in marine microbial mats. It would be interesting to see, whether marine relatives have possibilities of compatible solute biosynthesis. The species known and characterized so far are expected to have lost such possibilities.

## 3.1.4. Marine Rhizobiales

Freshwater *Rhizobiales*(species of *Rhodoblastus*, *Rhodopseudomonas*, *Blastochloris*, *Rhodomicrobium*, and *Rhodoplanes*) lack both, biosynthesis genes for ectoine and betaine (from choline and glycine) as well as transport systems for these osmolytes (Table 2). In a group of marine *Rhizobiales* (*Fulvimarina pelagi, Hoeflea phototrophica, Rhodobium orientis, Afifella* species), the choline-dependent biosynthesis of betaine (BetABI) and a BetT transporter are present (Table 2). In addition, one or more copies/versions of the ProU system are found. Therefore, osmotic adaptation of these bacteria to the marine environment can be achieved by uptake of betaine or choline. In the absence of these compounds in the environment, other compatible solutes might be accumulated. For *Afifella marina* (formerly *Rhodopseudomonas marina*), the accumulation of trehalose was demonstrated [12].

#### 3.1.5. Marine and Halotolerant Rhodobacteraceae

The *Rhodobacteraceae* include freshwater, marine and halophilic species. With the exception of the freshwater *Rhodobacter* species, all of them have a complete gene cluster for ectoine biosynthesis (*ectRectABCask-ect*). In addition, the extremely halotolerant *Roseivivax halodurans*, *Roseivivax roseus*, *Rhodosalinus sediminis*, and *Roseovarius nitratireducens* (not *Roseivivax halotolerans* and *Roseovarius halotolerans*) have genes of betaine biosynthesis from

glycine, with gene clusters of varying composition (Table 2). In these bacteria, the GMT and DMT methyltransferase genes are fused, as in some of the halophilic *Rhodospirillaceae* (see below). Most of the *Rhodobacteraceae* have several transport systems for osmolytes. All have the BetT transport system, some strains have multiple copies. With the exception of *Rhodobaculum claviforme* and *Rhodovulum imhoffii*, they have several ProU systems, which according to the ProW sequences are phylogenetically distinct (Table 2 and see below under Section 3.5).
