Standards in Genomic Sciences (2011) 4:23-35 DOI:10.4056/sigs.1483693

Complete genome sequence of “Thioalkalivibrio sulfidophilus” HL-EbGr7

Gerard Muyzer1*, Dimitry Yu Sorokin1,2, Konstantinos Mavromatis3, Alla Lapidus3, Alicia Clum3, Natalia Ivanova3, Amrita Pati3, Patrick d'Haeseleer4, Tanja Woyke3, Nikos C. Kyrpides3

1Department of Biotechnology, Delft University of Technology, Delft, The Netherlands 2Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia 3Joint Genome Institute, Walnut Creek, California, USA 4Joint Bioenergy Institute, California, USA *Corresponding author: Gerard Muyzer Keywords: haloalkaliphilic, sulfide, thiosulfate, -oxidizing (SOB)

“Thioalkalivibrio sulfidophilus” HL-EbGr7 is an obligately chemolithoautotrophic, haloalkali- philic sulfur-oxidizing bacterium (SOB) belonging to the . The strain was found to predominate a full-scale bioreactor, removing sulfide from biogas. Here we re- port the complete genome sequence of strain HL-EbGr7 and its annotation. The genome was sequenced within the Joint Genome Institute Community Sequencing Program, because of its relevance to the sustainable removal of sulfide from bio- and industrial waste gases.

Introduction “Thioalkalivibrio sulfidophilus” HL-EbGr7 is an obli- is stripped from the gas phase gately chemolithoautotrophic SOB using CO2 as a into an alkaline solution, which is subsequently carbon source and reduced inorganic sulfur com- transferred to a bioreactor where Thioalkalivibrio pounds as an energy source. It belongs to the genus oxidizes HS- almost exclusively to elemental sulfur Thioalkalivibrio. This genus is characterized by at a low red-ox potential [21]. Removal of toxic obligate haloalkaliphily and forms a monophyletic sulfide is needed, not only for a clean and healthy group within the family . environment, but also to protect gas turbines from The genus currently includes nine validly described corrosion. In contrast to chemical desulfurization [1] and many yet uncharacterized isolates processes, such as the ‘Claus-process’, biological [2,3]. The members are slow growing and well- removal is cheaper, cleaner and more sustainable, adapted to hypersaline (up to salt saturation) and as the produced hydrophilic bio-sulfur is a better alkaline (up to pH 10.5) conditions. They can oxid- fertilizer and fungicide than the chemically pro- ize sulfide, thiosulfate, elemental sulfur, sulfite and duced crystalline hydrophobic sulfur. polythionates (see Table 1 for summary). Moreo- To get insight into the molecular mechanism by ver, some species can reduce nitrate, nitrite or nitr- which Thioalkalivibrio strains adapt to haloalkaline ous oxide [17,18] or utilize thiocyanate (SCN-) as an conditions (i.e., pH 10 and up to 4 M of Na+) identifi- energy and source [19,20]. Genetic diver- cation of the genes that are involved in these adapta- sity analysis of 85 Thioalkalivibrio strains isolated tions is needed. The most important issues are sul- from different soda lakes located in Mongolia, fide specialization, carbon assimilation at high pH Kenya, California, Egypt and south Siberia, indi- and bioenergetic adaptation to high salt/high pH. In cated a high genetic diversity and an endemic cha- addition, information on the genome might help in racter, i.e., the majority of the genotypes (85.9%) optimizing the sulfur removal process. Here we were found to be unique to one region [15]. present a summary classification and a set of fea- Apart from their role in the sulfur cycle of soda tures for “T. sulfidophilus” HL-EbGr7, together with lakes, Thioalkalivibrio species also play a key role in the description of the genomic sequencing and anno- the sustainable removal of sulfide from wastewater tation. and gas streams. In this so-called ‘Thiopaq-process’, The Genomic Standards Consortium "Thioalkalivibrio sulfidophilus" HL-EbGr7 Classification and features has rod-shaped, elongated cells with a polar flagel- “T. sulfidophilus” HL-EbGr7 was isolated from a lum (Figure 1b and c). The strain is obligately al- full-scale Thiopaq bioreactor in the Netherlands kaliphilic with a pH optimum of 9.5. It can tolerate a salinity of 1.5 M (optimum at 0.4 M) of total so- used to remove H2S from biogas [21]. The reactor biomass had a very peculiar property, which made dium, sulfide concentrations up to 5 mM and a it different from the usual SOB biomass, i.e., an temperature up to 40°C. It utilizes ammonium and almost complete sulfide specialization and no thi- , but not nitrate or nitrite, as a N-source. On osulfate-oxidizing activity. This was probably the the basis of 16S rRNA gene sequencing the strain result of a very low red-ox potential at which the belongs to the Gammaproteobacteria with Thioal- reactor was operated. Therefore, the dominant kalivibrio denitrificans as the closest, described SOB could originally be enriched only with sulfide species (Figure 2). Despite this relation, strain HL- as substrate in cylinders with agarose-stabilized EbGr7 cannot grow anaerobically with NOx. Both medium containing opposing gradients of oxygen phylogeny and specific physiology indicate that and sulfide [Figure 1a, 22]. Subsequently, the this strain represents a novel species within the strain was purified using serial dilutions in gra- genus Thioalkalivibrio for which a tentative spe- dient cultures and finally from a colony on solid cies epithet “sulfidophilus” is proposed. medium with sulfide at micro-oxic conditions. It

Figure 1. a, Enrichment of “Thioalkalivibrio sulfidophilus” HL-EbGr7 in a gradient culture, whereby sulfide is diffusing from an agarose plug in

the bottom of the cylinder and O2 from the top. The bacterial cells ac- cumulate in a dense band at the most favorable sulfide and oxygen concentration. b, phase-contrast microphotograph; c, electron micro- scopy microphotograph of a total cell preparation contrasted with phosphotungstic acid.

24 Standards in Genomic Sciences Muyzer et al. Table 1. Features of “Thioalkalivibrio sulfidophilus” strain HL-EbGR7 according to the MIGS recommendations [4]. MIGS ID Property Term Evidence code Current classification Domain Bacteria TAS [5] Phylum TAS [6] Class Gammaproteobacteria TAS [7,8] Order Chromatiales TAS [7,9] Family Ectothiorhodospiraceae TAS [10] Genus Thioalkalivibrio TAS [11-13] Species “Thioalkalivibrio sulfidophilus” HL-EbGR7 NAS Gram stain negative TAS [2] Cell shape rod-shaped TAS [2] Motility motile TAS [2] Sporulation non-sporulating TAS [2] Temperature range Mesophile TAS [2] Optimum temperature 34 TAS [2] MIGS-6.3 Salinity range 0.2-1.5M Na+ (opt.0.4 M) TAS [2] MIGS-22 Oxygen requirement microaerophilic TAS [2] - Carbon source HCO3 TAS [2] Energy source Sulfide/polysulfide, thiosulfate, sulfur TAS [2] MIGS-6 Habitat Alkaline bioreactors; soda lakes TAS [2] MIGS-15 Biotic relationship free-living TAS [2] MIGS-14 Pathogenicity none NAS Biosafety level 1 TAS [14] Isolation Thiopaq bioreactor TAS [15] MIGS-4 Geographic location Eerbeek, The Netherlands TAS [15] MIGS-5 Sample collection time 2005 NAS MIGS-4.1 Latitude 52.11 TAS [16] MIGS-4.2 Longitude 6.07 TAS [16] MIGS-4.3 Depth Not applicable MIGS-4.4 Altitude Sea level NAS Evidence codes - IDA: Inferred from Direct Assay (first time in publication); TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

Genome sequencing information Genome project history Strain HL-EbGr7 was selected for sequencing in NC_011901. The genome project is listed in the the 2007 Joint Genome Institute Community Se- Genome OnLine Database (GOLD) [25] as project quencing Program, because of its relevance to Gc00934. Sequencing was carried out at the Joint bioremediation. A summary of the project infor- Genome Institute (JGI). Finishing was done by JGI- mation is presented in Table 2. The complete ge- Los Alamos National Laboratory (LANL) and ini- nome sequence was finished in December 2008. tial automatic annotation by JGI-Oak Ridge Na- The GenBank accession number for the project is tional Laboratory (ORNL). http://standardsingenomics.org 25 "Thioalkalivibrio sulfidophilus" HL-EbGr7

Figure 2. Phylogenetic tree based on 16S rRNA sequences showing the phylogenetic position of “Thioalkalivibrio sulfidophilus” HL-EbGr7. The sequence was aligned to sequences stored in the SILVA database using the SINA We- baligner [23]. Subsequently, the aligned sequences were imported into ARB [24], and a neighbor joining tree was constructed. Sequences of members from the Alphaproteobacteria were used as an outgroup, but were pruned from the tree. The scale bar indicates 1% sequence difference.

Growth conditions and DNA isolation After a long-term gradual adaptation on mixed at the JGI website [26]. Pyrosequencing reads substrate medium, the isolate was able to grow were assembled using the Newbler assembler solely with thiosulfate at micro-oxic conditions. version 1.1.02.15 (Roche). Large Newbler contigs The medium contained 40 mM thiosulfate as an were broken into 7,722 overlapping fragments of energy source and a standard sodium carbonate- 1,000 bp and entered into assembly as pseudo- bicarbonate buffer [2] (Sorokin et al., 2006) at pH reads. The sequences were assigned quality scores 10 and 0.6 M Na+. The cells were harvested by based on Newbler consensus q-scores with mod- centrifugation and stored at -80°C for DNA extrac- ifications to account for overlap redundancy and tion. Genomic DNA was obtained using phenol- adjust inflated q-scores. A hybrid 454/Sanger as- chloroform-isoamylalcohol (PCI) extraction. Brief- sembly was made using the Parallel Genome As- ly, the cell pellet was suspended in a Tris-EDTA sebler (PGA). Possible mis-assemblies were cor- buffer at pH 8, and lysed with a mixture of SDS rected and gaps between contigs were closed by and Proteinase K. The genomic DNA was extracted editing in Consed, or by custom primer walks of using PCI and precipitated with ethanol. The pellet sub-clones or PCR products. A total of 518 Sanger was dried under vacuum and subsequently dis- finishing reads were produced to close gaps, to solved in water. The quality and quantity of the resolve repetitive regions, and to raise the quality extracted DNA was evaluated using the DNA Mass of the finished sequence. The error rate of the Standard Kit provided by the JGI. completed genome sequence is less than 1 in Genome sequencing and assembly 100,000. Together, the combination of the Sanger The genome of “T. sulfidophilus” HL-EbGr7 was and 454 sequencing platforms provided a 31.49- sequenced using a combination of Sanger and 454 times coverage of the genome. The final assembly sequencing platforms. All general aspects of li- contains 32,486 Sanger reads and 390,057 pyro- brary construction and sequencing can be found sequencing reads.

26 Standards in Genomic Sciences Muyzer et al. Genome annotation Genome properties Genes were identified using Prodigal [27] as part The genome of strain HL-EbGr7 consists of a sin- of the Oak Ridge National Laboratory genome gle circular chromosome (Figure 3) with a size of annotation pipeline followed by a round of manual 3.46 Mbp. The G+C percentage determined from curation using the JGI GenePRIMP pipeline [28]. the genome sequence is 65.06%, which is a little The predicted CDSs were translated and used to higher than the G+C content determined by ther- search the National Center for Biotechnology In- mal denaturation (63.5 + 0.5 mol%). There are formation (NCBI) nonredundant database, Uni- 3,366 genes of which 3,319 are protein-coding Prot, TIGRFam, Pfam, PRIAM, KEGG, COG, and In- genes and the remaining 47 are RNA genes. 36 terPro, databases. Additional gene prediction pseudogenes were identified, constituting 1.07% analysis and functional annotation were per- of the total number of genes. The properties and formed within the Integrated Microbial Genomes statistics of the genome are summarized in Table Expert Review (IMG-ER) platform [29]. 3, and genes belonging to COG functional catego- ries are listed in Table 4.

Table 2. Genome sequencing project information MIGS ID Characteristic Details MIGS-28 Libraries used 6kb Sanger and 454 standard libraries MIGS-29 Sequencing platform ABI-3730, 454 GS FLX Titanium MIGS-31.2 Sequencing coverage 8.19 × Sanger, 23.3 × pyrosequence MIGS-31 Finishing quality Finished Sequencing quality Less than one error per 50kb MIGS-30 Assembler Newbler, PGA MIGS-32 Gene calling method Prodigal, GenePRIMP GenBank ID NC_011901 GenBank date of release December 29, 2008 GOLD ID Gc00934 NCBI project ID 29177 IMG Taxon ID 643348585 Personal culture collection, Winogradsky MIGS-13 Source material identifier Institute of Microbiology, Moscow Project relevance Bioremediation

Table 3. Genome statistics Attribute Value % of Total Genome size (bp) 3,464,554 100.00% DNA coding region (bp) 3,030,998 87.49% DNA G+C content (bp) 2,254,142 65.06% Number of replicons 1 Extrachromosomal elements 0 Total genes 3366 100.00% RNA genes 47 1.40% rRNA operons 3 0.09% Protein-coding genes 3319 98.06% Pseudogenes 36 1.07% Genes in paralog clusters 294 8.73% Genes assigned to COGs 2512 74.36% Genes assigned Pfam domains 2653 78.82% Genes with signal peptides 719 21.36% CRISPR repeats 2

http://standardsingenomics.org 27 "Thioalkalivibrio sulfidophilus" HL-EbGr7

Figure 3. Graphical circular map of the chromosome of “Thioalkalivibrio sulfidophilus” HL-EbGr7. From outside to the center: Genes on the forward strand (Colored by COG categories), Genes on the reverse strand (colored by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

28 Standards in Genomic Sciences Muyzer et al. Table 4. Number of genes associated with the general COG functional categories. Code value %age COG category E 180 6.39 Amino acid transport and metabolism G 94 3.34 Carbohydrate transport and metabolism D 49 1.74 Cell cycle control, cell, division, chromosome partitioning N 109 3.87 Cell motility M 199 7.06 Cell wall/membrane/envelope biogenesis B 2 0.07 Chromatin structure and dynamics H 139 4.93 Coenzyme transport and metabolism Z 0 0 Cytoskeleton V 37 1.31 Defense mechanism C 184 6.53 Energy production and conversion W 0 0 Extracellular structures S 270 9.58 Function unknown R 310 11.00 General function prediction only P 152 5.39 Inorganic ion transport and metabolism U 117 4.15 Intracellular trafficking, secretion, and vesicular transport I 70 2.48 Lipid transport and metabolism Y 0 0 Nuclear structure F 58 2.06 Nucleotide transport and metabolism O 142 5.04 Posttranslational modification, protein turnover, chaperones A 2 0.07 RNA processing and modification L 145 5.15 Replication, recombination and repair Q 47 1.67 Secondary metabolites biosynthesis, transport and catabolism T 216 7.67 Signal transduction mechanisms K 134 4.76 Transcription J 162 5.75 Translation, ribosomal structure and biogenesis - 854 25.37 Not in COGs

Insights from the genome sequence Autotrophic growth One of the major problems of autotrophic growth at 1,5-bisphosphate carboxylase/oxygenase (RuBis- high pH is the assimilation of inorganic carbon (Ci); CO) and carbonic anhydrase, the key enzymes in carbon dioxide concentrations are very low and CO2 fixation, are located in close proximity for an most inorganic carbon is present as HCO3- or even efficient carbon fixation [32]. The genome of strain as CO32- at pH values of 10 and higher. The latter is HL-EbGr7 contains the genes for the large (rbcL) not available to the cell, which is the main reason and small subunit (rbcS) of RuBisCO form 1Ac, and for growth limitation of haloalkaliphilic SOB at pH for the synthesis of a-carboxysomes, including above 10.5, since their energy-generating respira- csoSCA (formerly know as csoS3) encoding a car- tory system is still active up to pH 11-11.5 [2]. In- boxysome shell carbonic anhydrase. The latter is side the cells, where Ci assimilation occurs, the pH necessary to convert the transported HCO3- into is around 8.5, which means that HCO3- must be tak- CO2 – the actual substrate of RuBisCO. In contrast to en up as a substrate at an exterior pH of 10. This Thiomicrospira crunogena, the genomes of Thioal- demands active transport by means of a Na+/HCO3- kalivibrio are lacking genes for RuBisCO form 1Aq symporter, such as StbA, which has been found in and form II, which has been confirmed by Tourova the alkaliphilic cyanobacterium Synechocystis sp. et al. [33]. Expression studies at different CO2 con- strain PCC6803 [30]. However, genes encoding centrations in the chemolithoautotroph Hydroge- StbA have not been detected in strain HLEbGr7. novibrio marinus indicated the preferential expres- Another means of growth at limited CO2 concentra- sion of RuBisCO form 1Ac at low CO2 concentra- tions is the use of a carbon-concentrating mechan- tions and RuBisCO form 1Aq and/or form II at ism (CCM), which has been described for other higher CO2 concentrations [34]. This result indi- autotrophic microorganisms [31]. Part of the CCM cates that our strain is indeed adapted to low CO2 is the presence of carboxysomes, in which ribulose- concentrations. http://standardsingenomics.org 29 "Thioalkalivibrio sulfidophilus" HL-EbGr7

Figure 4. Hypothetical pathways for oxidation of sulfur compounds in “Thioalkalivibrio sulfidophilus” HL-EbGr7.

Sulfur metabolism Thioalkalivibrio species can oxidize reduced sulfur sisting of soxXAYZB, but lacking soxCD, leads to the compounds, such as sulfide and thiosulfate, to formation of elemental sulfur as an intermediate elemental sulfur and subsequently to sulfate. [35], and also gives the organism the ability to However, little is known about the enzymes that oxidize the sulfane moiety of thiosulfate, which are involved in the sulfur metabolism of these has been confirmed by culture studies. The soxXA organisms. Figure 4 shows the pathway of sulfur genes were present in 4 copies; the sox YZ genes metabolism in strain HL-EbGr7 inferred from the in 2 copies. Subsequently, elemental sulfur is oxi- genes found in the genome. Sulfide is oxidized by dized to sulfite by a reversed dissimilatory sulfite flavocytochrome c/sulfide dehydrogenase; both reductase pathway, consisting of dsrA- genes, encoding the small subunit A (fccA), and the BEFHCMKLJOPNR. In addition, we found genes large subunit B (fccB) were present in 3 copies. (hdrABC) encoding a heterodisulfide reductase Although sulfide:quinone oxidoreductase (SQR) complex, which was highly similar to that found in activity had been found in Thioalkalivibrio species, Acidithiobacillus ferrooxidans and was hypothe- the sqr gene could not be detected. A similar result sized to work in reverse [36]. Sulfite can be oxi- has also been found for Allochromatium vinosum dized to sulfate, either directly by sulfite dehydro- [35]. The presence of a truncated Sox cluster con- genase (sorA) or indirectly via adenosine-5′- 30 Standards in Genomic Sciences Muyzer et al. phosphosulfate (APS [37]) by APS reductase en- depending flagellar motor PomA/B. Apart from coded by aprBA and ATP sulfurylase encoded by the genes encoding the proton-translocating sat. Obviously, the two alternative pathways may NADH dehydrogenase (nuoABCDEFGHIJKLMN), we operate depending on the red-ox potential: (i) a also found genes (rnfABCDGE) that are homolog- sulfide-dependent microoxic pathway at very low ous to the nqr genes encoding the sodium- red-ox conditions, such as present in the Thiopaq translocating NADH:quinone oxidoreductase (Na+- reactor, involving the ‘reversed sulfidogenic’ NQR [40], ). Na+-NQR was first discovered in the pathway, and (ii) an aerobic sulfide/thiosulfate marine bacterium Vibrio alginolyticus [41]. It is oxidation pathway at high red-ox potential, such coupled to the respiratory chain, and oxidizes as in batch cultures with thiosulfate, involving the NADH with ubiquinone as electron acceptor. The truncated Sox cluster and SorA. The presence of free energy released is used to generate a sodium different copies of genes involved in the sulfur motive force at the FAD-quinone coupling site. metabolism might indicate the adaptation of this The presence of both a proton- and sodium- organism to a highly specialized sulfide oxidation translocating NADH:quinone oxidoreductase in lifestyle. one organism was described previously by Takada et al [42]. They showed that both pumps were Energy metabolism very active in a psychrophilic bacterium, Vibrio sp. Although we are gaining some insight into the strain ABE-1, growing at low temperatures. It is, of bioenergetics of alkaliphilic heterotrophs, such as course, not clear what the role either pump is in Bacillus species [38], it is a complete mystery how our strain, but it is tempting to speculate that they haloalkaliphilic chemolithoautotrophic bacteria are a special adaptation to generate enough ener- obtain enough energy for growth. To generate gy for growth under these extreme conditions. NADH for CO2 fixation, chemolithoautotrophic Future transcriptomic and proteomic studies are bacteria, using inorganic compounds (e.g. H2S or necessary to validate this speculation. NhaP is a + + NH3) as electron donors, have to transport elec- Na /H -antiporter (a secondary sodium pump), trons against the thermodynamic gradient (‘re- which plays a role in the regulation of the internal verse electron transport’), which is an energy- pH of the cell; it pumps sodium out of the cell and requiring process. In addition, those that are living leaves protons and ensuing energy generated by at high salt concentrations, have to invest extra the respiratory chain. Furthermore, we found all 7 energy in the production of organic compatible genes (mnhA-G) for the multisubunit Na+/H+- solutes. And thirdly, bacteria that live at high pH antiporter Mrp, which may play a similar role as have to invest additional energy to maintain their NhaP. Apart from genes encoding proton-driven pH homeostasis. flagellar motors (motA/B), we also found genes So, to obtain enough energy for growth, the ha- encoding sodium-driven flagellar motors (po- loalkaliphilic chemolithoautotrophic SOB must mA/B). Phylogenetic analysis of the motA/B and have a specially adaptated bioenergetics. The pomA/B grouped them with sequences of other most obvious solution would be the presence of bacteria, such as Halorhodospira halophila and primary sodium pumps, such as a sodium-driven Alkalilimnicola ehrlichii (results are not shown). ATP synthase, but genes for this could not be de- We have also found genes for the production of tected; instead we found all the genes for a pro- cardiolipin (cardiolipin synthase) and of squalene ton-driven F0F1-type ATP synthase (i.e., subunit (squalene synthase), confirming the high concen- A, B, and C of the F0 subcomplex, and subunit al- trations of these compounds in the cell membranes pha, beta, gamma, delta, and epsilon of the F1 sub- of another Thioalkalivibrio strain, strain ALJ15 [43]. complex). The presence of a proton-driven ATP These compounds contribute indirectly to an effi- synthase instead of a sodium-driven ATP synthase cient energy metabolism, as the negatively charged has been found in all genomes of so far studied cardiolipids might trap protons at the cell mem- aerobic alkaliphilic bacteria studied thus far [39]. brane preventing them from diffusing into the en- However, we could detect several genes encoding vironment [44], and squalene lowers the proton different sodium-dependent pumps, such as the permeability of the lipid bilayer [45]. From the primary sodium pump Rnf and secondary pumps, genes that we found, we made the following con- such as the Na+/H+ antiporters NhaP and Mrp, a ceptual model (Figure 5). sodium:sulfate symporter (SulP), and the sodium- http://standardsingenomics.org 31 "Thioalkalivibrio sulfidophilus" HL-EbGr7

Figure 5. Conceptual model of the different proton and sodium primary pumps, secondary transporters and flagellar motors in “Thioalkalivibrio sulfidophilus” HL-EbGr7.

Compatible solutes Thioalkalivibrio species are characterized by their synthesis from glycine in a three-step methylation tolerance to high salt concentrations, which can be process, i.e., glycine -> sarcosine -> dimethylgly- up to 4.3M total sodium [2,17]. To withstand these cine -> betaine. The sequences of the 2 enzymes hypersaline conditions, these species synthesize have high similarities to sequences found in the glycine-betaine as the main compatible solute. In close relatives Halorhodospira halophila and Ni- one of the high-salt Thioalkalivibrio strains, Banciu trococcus mobilis. Apart from glycine-betaine et al. [43] showed a positive correlation between Thioalkalivibrio species also produce sucrose as a salinity and the intracellular glycine-betaine con- minor compatible solute (up to 2.5% of cell dry centration, and found that glycine-betaine consti- weight at 2M of sodium) [43]. The genome of tuted 9% of cell dry weight at 4M of sodium in the strain HL-EbGr7 contains genes coding for the culture medium. In most cases, betaine is synthe- enzymes sucrose synthase and sucrose phosphate sized from choline by a two-step oxidation path- synthase, which both play a role in the synthesis way [46]. However, an alternative route is the of sucrose. In contrast to other members of the synthesis of betaine by a series of methylation Ectothiorhodospiraceae, i.e., Alkalilimnicola ehrli- reactions [47]. The genome of strain HL-EbGr7 chii, and Halorhodospira halodurans, no genes contains genes coding for glycine sarcosine N- were found for ectoine synthesis in the genome of methyltransferase and sarcosine dimethylglycine HL-EbGr7. methyltransferase, that are catalyzing betaine Acknowledgments This work was performed under the auspices of the US AC52-07NA27344, and Los Alamos National Laborato- Department of Energy Office of Science, Biological and ry under contract No. DE-AC02-06NA25396, UT- Environmental Research Program, and by the Universi- Battelle and Oak Ridge National Laboratory under con- ty of California, Lawrence Berkeley National Laborato- tract DE-AC05-00OR22725. DS was supported finan- ry under contract No. DE-AC02-05CH11231, Lawrence cially by RFBR grant 10-04-00152. Livermore National Laboratory under Contract No. DE-

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