Glycine betaine (N,N,N-trimethylglycine) has been shown to be a very efficient osmolyte found in a wide range of bacterial and plant species, where it is accumulated at high cytoplasmic concentrations in response to osmotic stress.
Was shown that S. meliloti (in contrast to Escherichia coli, Bacillus subtilis, and other bacteria), can use glycine betaine not only as osmoprotectants but as carbon, nitrogen, and energy sources as well.
Glycine betaine either can be taken up directly from the environment by specific transport systems or synthesized from choline by a two-step pathway with betaine aldehyde as intermediate. This pathway appears to be conserved in bacteria and plants, but shows divergence in the enzymes involved. Gram-positive bacteria such as Arthrobacter pascens and A. globiformis and the fungus Cylindrocarpon didymun use a soluble choline oxidase to catalyze both steps .
Higher plants and Gram-negative bacteria both are using a conserved betaine aldehyde dehydrogenase to catalyze the betaine aldehyde to glycine betaine reaction. The choline-to-betaine aldehyde reaction, however, is catalyzed by a choline monooxygenase in plants and by a choline dehydrogenase in bacteria such as E. coli, Pseudomonas aeruginosa, and S. meliloti.
In most organisms, betaine is synthesized as a result of the two-step oxidation of choline via betaine aldehyde, a toxic intermediate.The glycine betaine biosynthesis pathway has been characterized at the molecular level in E. coli and Bacillus subtilis.
In several higher plants from taxonomically unrelated families, the relevant enzymes are choline monooxygenase (CMO), a ferredoxin-dependent soluble Rieske-type protein, and betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8), a soluble NAD+-dependent enzyme. These enzymes are found mostly in the chloroplast stroma and their activities, as well as levels of betaine, increase in response to salt stress. BADH has also been found in several plants that barely accumulate any betaine. In mammalian cells and in microorganisms such as Escherichia coli, betaine is synthesized by choline dehydrogenase (EC 1.1.99.1), a membrane-bound oxygen-dependent enzyme, in combination with betaine aldehyde dehydrogenase (EC 1.2.1.8).
Some bacteria, such as Synrhizobium meliloti and Pseudomonas aeruginosa, can degrade choline-O-sulfate to choline, which is then converted to glycine betaine and catabolized further into ammonia and pyruvate. The key enzyme for this process is choline sulfatase, encoded by the betC gene.
In contrast to these two pathways that each involve two enzymes, the biosynthesis of betaine is catalysed by a single flavoenzyme, choline oxidase (EC 1.1.3.17), in certain microorganisms, such as the soil bacterium Arthrobacter globiformis. As far as is known, to date this enzyme has only been found in microorganisms.

=========Variant codes:==========

1.0 - choline monooxygenase/betaine aldehyde dehydrogenase pathway (betaine is synthesized as a result of the two-step pathway with betaine aldehyde as intermediate);
1.1 – variant 1 + choline-O-sulfate degradation pathway;
2.0 - choline oxidase pathway;
3.0 – only Glycine betaine transport systems are present, no synthesis;
_3 - any of synthesis + transport system
( like in Escherichia coli – variant 1.13 -Choline-sulfatase + choline monooxygenase/betaine aldehyde dehydrogenase pathway + transport system);
-1 - indicates a genome without a functioning subsystem;
x - indicates that the genome possesses a functioning system, but a gene or genes that performs a role has not yet been identified.

Since many microorganisms are not capable of de novo glycine betaine synthesis and instead they use a two-step oxidation of choline via the intermediate glycine betaine aldehyde for its production, and thus depend on the efficient uptake of this precursor for the osmoregulatory glycine betaine production – I extended this SS to include the choline uptake systems.

===== Choline oxidase===========

Choline oxidase (E.C. 1.1.3.17) catalyzes the four-electron oxidation of choline to glycine betaine (N,N,N-trimethylglycine; betaine) via betaine aldehyde as intermediate. Molecular oxygen acts as primary electron acceptor in the reaction.
Choline oxidase has been purified from Cylindrocarpon didymum M-1, Alcaligenes sp., and Arthrobacter globiformis. Based on amino acid sequence comparisons, the enzyme can be grouped in the GMC oxidoreductase enzyme superfamily, which comprises enzymes like glucose oxidase, cholesterol oxidase, or cellobiose dehydrogenase, that utilize FAD as cofactor for catalysis and non-activated primary alcohols as substrate.

====== Glycine betaine uptake systems:============

The presence of uptake systems for glycine betaine has been reported for a variety of gram-negative and gram-positive bacteria and also in members of the Archaea.

------ ProV, ProW, and ProX :-------------

One of the most extensively studied uptake systems is the osmoregulatory locus known as proU, which is an operon that encodes a high-affinity ATP-binding cassette (ABC) transport system consisting of three proteins (ProV, ProW, and ProX), that is found both in E. coli and Salmonella enterica serovar Typhimurium:
- ProV is a peripheral membrane protein found on the cytoplasmic side which shares considerable sequence identity with ATP-binding proteins from other ABC systems.
- ProW is the integral membrane component of the transport system, and
- ProX represents the periplasmic glycine betaine-binding protein (GBBP).

---------- OpuA, OpuB and OpuC ABC uptake systems:--------------

Within the gram-positive bacteria, molecular aspects of glycine betaine uptake have been recently studied in details in B. subtilis. OpuA and OpuC are members of the superfamily of prokaryotic and eukaryotic ABC uptake systems.
The OpuA system is the predominant transporter for glycine betaine and consists of three components: an ATPase (OpuAA), an integral membrane protein (OpuAB), and a hydrophilic polypeptide (OpuAC) which functions as the GBBP. The OpuC glycine betaine uptake system is related to OpuA but contains an additional integral inner membrane component . Both OpuA and OpuC exhibit structural and functional similarities to the ProU system from E. coli. Except for OpuA which is highly specific for glycine betaine, the transport capacity of ProU and OpuC could be extended to other substrates. Upon osmotic stress, ProU is also involved in proline and proline betaine uptake, which both play a role in osmoadaptation in E. coli. OpuC is less specific since besides glycine betaine, choline, choline-O-sulfate, carnitine, crotonobetaine, and ectoine can enter the cell via this ABC transporter. In addition to these multicomponent binding protein-dependent systems, glycine betaine transporters composed of only one integral membrane protein have also been reported in many bacteria, such as ProP in the enteric bacteria and OpuD in B. subtilis.

OpuD is essential for glycine betaine uptake and osmoprotection in E. coli. OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity.

There are two evolutionarily closely related ABC transport systems (OpuB and OpuC) for high-affinity, osmotically stimulated uptake of choline in B. subtilis.

===========B. subtilis gbsAB operon and Choline uptake (Ref.5)============================

B. subtilis possesses several dedicated transport systems for osmoprotectants, such as the osmoregulated proline transporter OpuE (osmoprotectant uptake) and the glycine betaine uptake systems OpuA, OpuC and OpuD. Physiological studies have shown that the OpuC system also serves as sole uptake route for the osmoprotectants ectoine, carnitine, crotonobetaine and choline-O-sulphate.
The first step in glycine betaine synthesis in B. subtilis and E. coli is performed by two different types of enzymes. A soluble, metal-containing, type III alcohol dehydrogenase (GbsB) functions in B. subtilis to convert choline into glycine betaine aldehyde, whereas this reaction is catalysed in E. coli by a FAD-containing, membrane-bound choline dehydrogenase, which can also oxidize glycine betaine aldehyde to glycine betaine at the same rate.

In contrast to the genetic arrangement for the glycine betaine biosynthetic genes in E. coli, the B. subtilis gbsAB operon (type III alcohol dehydrogenase (GbsB) and a glycine betaine aldehyde dehydrogenase (GbsA)) is not flanked by genes that could potentially encode a system mediating osmotically stimulated choline uptake.Choline uptake in B. subtilis is mediated by two evolutionarily highly conserved multicomponent ABC transporters that probably evolved through the duplication of a primordial gene cluster. Despite their close relatedness, these transporters display striking differences in their substrate specificity and play different physiological roles in the osmostress response of B. subtilis.



=======REFERENCES:============

1. Mandon K, Osteras M, Boncompagni E, Trinchant JC, Spennato G, Poggi MC, Le Rudulier D. The Sinorhizobium meliloti glycine betaine biosynthetic genes (betlCBA) are induced by choline and highly expressed in bacteroids. Mol Plant Microbe Interact. 2003 Aug;16(8):709-19. PMID: 12906115.

2. Osteras M, Boncompagni E, Vincent N, Poggi MC, Le Rudulier D. Presence of a gene encoding choline sulfatase in Sinorhizobium meliloti bet operon: choline-O-sulfate is metabolized into glycine betaine. Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11394-9.
PMID: 9736747.

4. Boncompagni E, Dupont L, Mignot T, Osteras M, Lambert A, Poggi MC, Le Rudulier D. Characterization of a Snorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake.J Bacteriol. 2000 Jul;182(13):3717-25.PMID: 10850986.

5.Kappes R.M., Kempf B., Kneip S., Boch J., Gade J., Meier-Wagner J.,
Bremer E. Two evolutionarily closely related ABC transporters mediate the
uptake of choline for synthesis of the osmoprotectant glycine betaine
in Bacillus subtilis. Mol. Microbiol. 32:203-216(1999). PMID: 10216873

6. Wargo MJ, Szwergold BS, Hogan DA. Identification of two gene clusters and a transcriptional regulator required for Pseudomonas aeruginosa glycine betaine catabolism. J Bacteriol. 2008 Apr;190(8):2690-9. PMID: 17951379