3.1.10. Ectothiorhodospiraceae

According to phylogeny of 16S rRNA and photosynthetic reaction center genes, the *Ectothiorhodospira* and *Halorhodospira* species form two clearly separated branches that might even require a separation at the family level [46]. This clear separation is also reflected in different options for osmotic adaptation and in different lineages of normal as well as B-type GMT sequences (Table 3, Figure 1, see Section 3.2.2).

The *Ectothiorhodospira* species lack ectoine biosynthesis, but can synthesize betaine from glycine (GMT-DMT-MAT), with the exception of *Ectothiorhodospira mobilis* and *Ectothiorhodospira marismortui* also from choline (*betABIproX*). They have a gene cluster including genes of the OpuA and BetT transport systems (*betT-opuAA,AB,AC*) and additional *betT* gene copies (except *Ectothiorhodospira vacuolata* and *Ectothiorhodospira shaposhnikovii*), but lack the otherwise common ProU transporter (Table 3). An exception is the *Ectothiorhodospira magna*, which obviously lacks the biosynthetic capabilities and entirely depends on the uptake of betaine and ectoine (*betT-opuAA,AB,AC*) (Table 3). *Ectothiorhodosinus mongolicus* and *Thiorhodospira sibirica* are exceptions among the *Ectothiorhodospiraceae*. While the first lacks genes for biosynthesis of betaine and ectoine and only has ProU (type W1) and BetT transport systems, the latter has no annotated osmotic stress genes at all. This limits the possible adaptation to elevated salt concentrations and suggests that alternative mechanisms/solutes are used to cope with the salt in the environment. Sucrose, N-carbamoyl-L-glutamine amide, or N-acetyl-glutaminylglutamine amide are possible candidates that were found to accumulate in other purple sulfur bacteria, though in marine and halophilic species, they are found only in addition to betaine [12].

The *Halorhodospira* species are the most halophilic and halotolerant phototrophic bacteria and can thrive even in saturated salt solutions [1,2]. All *Halorhodospira* species have complete gene clusters for ectoine (*ectABC*) and betaine biosynthesis from glycine, but lack genes for transformation of choline to glycine (Table 3). They are the only *Gammaproteobacteria* to include adenosylmethionine synthetase (MAT), adenosylhomocysteinase (SAHase) and 5,10-methylene tetrahydrofolate reductase (MTHFR) into a gene cluster, together with the two methyltransferase genes (GMT-DMT-MAT-SAHase-MTHFR). They are well equipped with the transport systems for osmolytes and have multiple copies of BetT, the secondary transporter OpuD (except *Halorhodospira neutriphila*) and the ProU (type W1) transport system (except *Halorhodospira halochloris*). These transport systems assure that osmolytes leaking out of the cells at very high, several molar cytoplasmic concentrations can be regained by the cells, and are not wasted to the environment. Therefore, it is assumed that the available transport systems are able to take up betaine and ectoine.

#### *3.2. Phylogeny of Glycine-Methyltransferase GMT*

The methyltransferases that transform glycine to betaine, glycine and sarcosine methyltransferase (GMT), and dimethylglycine methyltransferase (DMT) are present in a wide range of phototrophic bacteria, *Alpha*-, and *Gammaproteobacteria*, *Cyanobacteria* and green sulfur bacteria, as revealed by genome analysis using the SEED facility of the RAST platform [39]; shown in Tables 1–3. A phylogenetic tree of GMT methyltransferases, which in addition to phototrophic bacteria includes data from the genomes of chemotrophic bacteria as well as from BLAST searches, is shown in Figure 1. Though many of the deep branching points are poorly resolved and not supported by bootstrap values, it is obvious that the phylogeny of GMT is well depicted in a number of major phylogenetic branches. The phylogenetic grouping correlates well with differences in the gene clusters involved in betaine biosynthesis (Figure 1). The following major groups and distinct phylogenetic lineages are recognized.
