Open Access

Complete genome sequence of Thioalkalivibrio sp. K90mix

  • Gerard Muyzer,
  • , Dimitry Y. Sorokin,
  • , Konstantinos Mavromatis
  • , Alla Lapidus
  • , Brian Foster
  • , Hui Sun
  • , Natalia Ivanova
  • , Amrita Pati
  • , Patrik D'haeseleer
  • , Tanja Woyke
  • and Nikos C. Kyrpides
Corresponding author

DOI: 10.4056/sigs.2315092

Received: 23 December 2011

Published: 31 December 2011

Abstract

Thioalkalivibrio sp. K90mix is an obligately chemolithoautotrophic, natronophilic sulfur-oxidizing bacterium (SOxB) belonging to the family Ectothiorhodospiraceae within the Gammaproteobacteria. The strain was isolated from a mixture of sediment samples obtained from different soda lakes located in the Kulunda Steppe (Altai, Russia) based on its extreme potassium carbonate tolerance as an enrichment method. Here we report the complete genome sequence of strain K90mix 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 wastewater and gas streams.

Keywords:

natronophilicsulfidethiosulfatesulfur-oxidizing bacteriasoda lakes

Introduction

Thioalkalivibrio sp. K90mix is an obligately chemolithoautotrophic SOxB using CO2 as a carbon source and reduced inorganic sulfur compounds as an energy source. It belongs to the genus Thioalkalivibrio. This genus represents a dominant SOxB type in soda lakes – extremely alkaline and saline habitats - and is the first example of an obligate chemolithoautotroph capable of growing in saturated sodium carbonate brines. It forms a monophyletic group within the family Ectothiorhodospiraceae of the Gammaproteobacteria. The genus currently includes nine validly published species [1] and around 70, yet uncharacterized strains that are extremely salt-tolerant and genetically different from the characterized isolates recovered from hypersaline soda lakes [2-4]. The members are slow growing obligate autotrophs, well adapted to hypersaline (up to salt saturation) and alkaline (up to pH 10.5) conditions. Members of the genus Thioalkalivibrio have versatile metabolic capabilities, including oxidation of reduced sulfur compounds [1,2], denitrification [5,6] and thiocyanate utilization [7,8].

Apart from playing an important role in the sulfur cycle of soda lakes, Thioalkalivibrio species also are being used for the sustainable removal of sulfide from wastewater and gas streams [9,10]. In this process hydrogen sulfide is absorbed to a high salt alkaline solution, which is subsequently transferred to a bioreactor in which Thioalkalivibrio spp. oxidize HS- to elemental sulfur. The produced biosulfur can then be used as a fertilizer or fungicide [9].

To get a comprehensive understanding of the molecular mechanism by which Thioalkalivibrio sp. K90mix oxidize sulfur compounds and adapts to extreme alkaline (up to pH 10.5) and hypersaline conditions (up to 4 M of Na+ or 3.6 M of K+) it is necessary to identify the genes that are involved in these adaptations. The most important issues in this are the mechanism of sulfide oxidation, carbon assimilation at high pH, and bioenergetic adaptation to high salt and high pH. Here we present a summary classification and a set of features for Thioalkalivibrio sp. K90mix together with the description of the genomic sequencing and annotation.

Classification and features

Because of limited solubility of sodium carbonates in the biodesulfurization process, we made a series of enrichment cultures with an increasing ratio of potassium to sodium carbonate (potassium carbonates have a 2-5 times higher solubility than sodium carbonates). Thioalkalivibrio sp. K90mix was isolated from a culture that was inoculated with a mixture of sediment samples from different hypersaline soda lakes and was grown at the maximal possible substitution of sodium for potassium, 3.6 M K+/0.4 M Na+ (90% substitution).

Thioalkalivibrio sp. K90mix has rod-shaped cells with a polar flagellum (Figure 1), that elongate at high concentrations of K+ (Figure 1b). The strain is obligately alkaliphilic with a pH optimum of 10 (Table 1). It can tolerate a salinity of 4.0 M total Na+, but has an optimum of 0.3 M, sulfide concentrations up to 1 mM and a temperature up to 40°C. It has a preference for carbonate and sulfate as counter-anions over chloride and, therefore must be called “natronophilic”, instead of “haloalkaliphilic”. It utilizes ammonia, nitrate and nitrite as a nitrogen source. On the basis of 16S rRNA gene sequencing the strain belongs to the genus Thioalkalivibrio within the Gammaproteobacteria with T. thiocyanoxidans and T. nitratis as the closest described species (Figure 2). Most of the yet undescribed Thioalkalivibrio isolates from hypersaline lakes of Siberia and Mongolia also belong to this core genetic cluster of the genus Thioalkalivibrio.

Figure 1

Phase contrast micrographs of the cell morphology of Thioalkalivibrio sp. K90mix grown at pH 10 and 4 M Na+ (a), or 3.6 M K+/0.4 M Na+ (b).

Table 1

Classification and general features of Thioalkalivibrio sp. K90mix according to the MIGS recommendations [11].

MIGS ID

Property

Term

Evidence code

Current classification

Domain Bacteria

TAS [12]

Phylum Proteobacteria

TAS [13]

Class Gammaproteobacteria

TAS [14,15]

Order Chromatiales

TAS [16]

Family Ectothiorhodospiraceae

TAS [17]

Genus Thioalkalivibrio

TAS [18-21]

Species Thioalkalivibrio sp. K90mix

NAS

Gram stain

negative

TAS [2]

Cell shape

rod-shaped

TAS [2]

Motility

motile

TAS [2]

Sporulation

non-sporulating

TAS [2]

Temperature range

Mesophile; maximum at 41oC

TAS [2]

Optimum temperature

34oC

TAS [2]

MIGS-6.3

Salinity range

0.2-4.0 M Na+ (opt. 0.3 M)

TAS [2]

MIGS-22

Oxygen requirement

aerobic

TAS [2]

Carbon source

HCO3-

TAS [2]

Energy source

Sulfide/polysulfide, thiosulfate, sulfur, sulfite

TAS [2]

MIGS-6

Habitat

soda lakes

TAS [2]

MIGS-15

Biotic relationship

free-living

TAS [2]

MIGS-14

Pathogenicity

none

NAS

Biosafety level

1

TAS [22]

Isolation

Soda lake sediments

TAS [4]

MIGS-4

Geographic location

Kulunda Steppe, Altai, Russia

TAS [4]

MIGS-5

Sample collection time

2005

NAS

MIGS-4.1

Latitude

51.41

TAS [23]

MIGS-4.2

Longitude

79.48

TAS [23]

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.

Figure 2

Phylogenetic tree based on 16S rRNA sequences showing the phylogenetic position of Thioalkalivibrio sp. K90mix. The sequence was aligned to sequences stored in the SILVA database using the SINA Webaligner [24]. Subsequently, the aligned sequences were imported into ARB [25], and a neighbor joining tree was constructed. Sequences of members from the Alphaproteobacteria were used as outgroup, but were pruned from the tree. The scale bar indicates 1% sequence difference.

Genome sequencing information

Genome project history

Strain K90mix was selected for sequencing in the 2007 Joint Genome Institute Community Sequencing Program, because of its relevance to bioremediation. A summary of the project information is presented in Table 2. The complete genome sequence was finished in February 2010. The GenBank accession numbers are NC_013889 and NC_013930 for the chromosome and plasmid, respectively. The genome project is listed in the Genome OnLine Database (GOLD) [26] as project Gc01217. Sequencing was carried out at the Joint Genome Institute (JGI) Finishing was done by JGI-Los Alamos National Laboratory (LANL) and initial automatic annotation by JGI-Oak Ridge National Laboratory (ORNL).

Table 2

Genome sequencing project information

MIGS ID

Characteristic

Details

MIGS-28

Libraries used

6kb and 40kb Sanger and 454 standard libraries

MIGS-29

Sequencing platform

ABI-3730, 454 GS FLX Titanium

MIGS-31.2

Sequencing coverage

10.0× Sanger, 32.1× pyrosequence

MIGS-31

Finishing quality

Finished

Sequencing quality

Less than one error per 100 kb

MIGS-30

Assembler

Newbler, PGA

MIGS-32

Gene calling method

Prodigal, GenePRIMP

GenBank ID

NC_013889

GenBank date of release

February 21, 2010

GOLD ID

Gc01217

NCBI project ID

30759

IMG Taxon ID

646564584

MIGS-13

Source material identifier

Personal culture collection, Winogradsky Institute of Microbiology, Moscow

Project relevance

Bioremediation

Growth conditions and DNA isolation

Thioalkalivibrio sp. K90mix was grown with 40 mM thiosulfate as an energy source in standard sodium carbonate-bicarbonate medium at pH 10 and 2 M Na+ [2] at 35oC with shaking at 200 rpm. The cells were harvested by centrifugation and stored at minus 80°C for DNA extraction. Genomic DNA was obtained using phenol-chloroform-isoamylalcohol (PCI) extraction. The genomic DNA was extracted using PCI and precipitated with ethanol. The pellet was dried under vacuum and subsequently dissolved in water. The quality and quantity of the extracted DNA was evaluated using the DNA Mass Standard Kit provided by the JGI.

Genome sequencing and assembly

The genome of Thioalkalivibrio sp. K90mix was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [27]. Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 3,292 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the PGA assembler. Possible mis-assemblies were corrected and gaps between contigs were closed by editing in Consed, by custom primer walks from sub-clones or PCR products. A total of 181 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. Illumina reads were used to improve the final consensus quality using an in-house developed tool (the 'Polisher' [28]). The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Sanger and 454 sequencing platforms provided 42.1× coverage of the genome. The final assembly contains 28,443 Sanger reads (10.0×) and 419,015 pyrosequencing reads (32.1×).

Genome annotation

Genes were identified using Prodigal [29] as part of the Oak Ridge National Laboratory genome annotation pipeline followed by a round of manual curation using the JGI GenePRIMP pipeline [30]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro, databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [31].

Genome properties

The genome of strain K90mix consists of a circular chromosome with a size of 2.74 Mbp (Figure 3) and a linear plasmid of 240 Kbp. The G+C percentage determined from the genome sequence is 65.54%, which is similar to the value determined by thermal denaturation (65.8±0.5 mol%). There are 2942 genes of which 2888 are protein-coding genes and the remaining 54 are RNA genes. 33 pseudogenes were identified, constituting 1.12% of the total number of genes. The genome is smaller than that of “Thioalkalivibrio sulfidophilus” HL-EbGr7 [32], 2.98 Mbp versus 3.46 Mbp, but has a similar percentage of protein-coding genes (98.16% versus 98.06%). The properties and statistics of the genome are summarized in Table 3, and genes belonging to COG functional categories are listed in Table 4.

Figure 3

Graphical circular map of the chromosome of Thioalkalivibrio sp. strain K90mix. 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.

Table 3

Genome statistics

Attribute

Value

% of Total

Genome size (bp)

2,985,056

100.00%

DNA coding region (bp)

2,673,521

89.56%

DNA G+C content (bp)

1,956,383

65.54%

Number of replicons

1

Extrachromosomal elements (plasmid)

1

Total genes

2,942

100.00%

RNA genes

54

1.84%

rRNA operons

3

0.10%

Protein-coding genes

2,888

98.16%

Pseudogenes

33

1.12%

Genes in paralog clusters

267

9.08%

Genes assigned to COGs

2,139

72.71%

Genes assigned Pfam domains

2,292

77.91%

Genes with signal peptides

934

31.75%

CRIPR repeats

2

Table 4

Number of genes associated with the general COG functional categories.

Code

Value

%age

  Description

J

   156

  6.57

  Translation, ribosomal structure and biogenesis

A

1

.04

  RNA processing and modification

K

86

3.62

  Transcription

L

164

6.91

  Replication, recombination and repair

B

2

0.08

  Chromatin structure and dynamics

D

41

1.73

  Cell cycle control, mitosis and meiosis

Y

0

0.0

  Nuclear structure

V

35

1.47

  Defense mechanisms

T

166

6.99

  Signal transduction mechanisms

M

176

7.41

  Cell wall/membrane biogenesis

N

94

3.96

  Cell motility

Z

0

0.0

  Cytoskeleton

W

0

0.0

  Extracellular structures

U

97

4.09

  Intracellular trafficking and secretion

O

110

4.63

  Posttranslational modification, protein turnover, chaperones

C

146

6.15

  Energy production and conversion

G

79

3.33

  Carbohydrate transport and metabolism

E

155

6.53

  Amino acid transport and metabolism

F

66

2.78

  Nucleotide transport and metabolism

H

146

6.15

  Coenzyme transport and metabolism

I

65

2.74

  Lipid transport and metabolism

P

119

5.01

  Inorganic ion transport and metabolism

Q

42

1.77

  Secondary metabolites biosynthesis, transport and catabolism

R

240

10.11

  General function prediction only

S

188

7.92

  Function unknown

-

803

27.29

  Not in COGs

Insights from the genome sequence

Autotrophic growth

As mentioned before, [32] autotrophic growth at extremely high pH is a problem, because inorganic carbon is mainly present as carbonate (with bicarbonate as a minor fraction) at pH values above 10. This would demand active transport of bicarbonate into the cell. We found a gene related to stbA encoding a Na+/HCO3- symporter in the marine cyanobacterium Synechocystis sp. strain PCC 6803 [33]. Figure 4 shows a phylogenetic tree of different sequences related to StbA and the hydrophobicity profiles of StbA of Synechocystis sp. PCC 6803 and Thioalkalivibrio sp. K90mix. In addition, we have found genes for the large (TK90_0858) and small subunit (TK90_0859) of RuBisCO form 1Ac, and for the synthesis of α-carboxysomes (TK90_0860 – TK90_0866), including csoSCA encoding a carboxysome shell alpha-type carbonic anhydrase, which was also found in genomic analysis of “Thioalkalivibrio sulfidophilus HL-EbGr7 [32].

Figure 4

Phylogeny and hydropathy of StbA from different microorganisms. The blue boxes indicate sequences belonging to the cyanobacteria. The dots on the branches are bootstrap values between 50% and 75% (open dots) or between 75% and 100% (closed dots) The scale bar indicated 10% sequence difference. The sequence of Thioalkalivibrio sp. K90mix is indicated in bold. Hydropathy profiles were made for the StbA protein sequences of Synechocystis sp. PCC6803 and Thioalkalivibrio sp. K90mix to indicate transmembrane spanning domains.

Sulfur metabolism

Thioalkalivibrio sp. K90mix can oxidize sulfide/polysulfide, thiosulfate, sulfite (in vitro) and elemental sulfur to sulfate. Elemental sulfur is formed as an intermediate during sulfide and thiosulfate oxidation at oxygen limitation and near-neutral pH. Figure 5 shows a schematic overview of the different genes that are involved in the oxidation of sulfur compounds. The genome of Thioalkalivibrio sp. K90mix contains genes for flavocytochrome c/sulfide dehydrogenase (TK90_0236), which oxidizes sulfide to elemental sulfur. It contains an incomplete set of sox genes including soxYZ (TK90_0123 and TK90_0124), soxAX (TK90_0432 and TK90_0433) and two copies of soxB (TK90_0627 and TK90_1150), but is lacking soxCD, which would allow oxidizing the sulfane atom of thiosulfate to the state of elemental sulfur, but no further. However, it does not contain the reverse dissimilatory sulfite reduction pathway to oxidize sulfur to sulfite, which has been found in the genome of “Thioalkalivibrio sulfidophilus HLEbGr7 [32]. Absence of dsr genes has also been found for the green sulfur bacterium Chloroherpeton thalassium that can oxidize sulfide to elemental sulfur, but subsequently can only oxidize the produced sulfur very slowly [34], probably due to the absence of dsr. Frigaard and Dahl [35] suggested that the presence of a RuBisCo-like protein (RLP) might be involved in sulfur oxidation [36]. Genes encoding for the RuBisCo-like protein were not found, nor were genes encoding sulfur dioxygenase or sulfur oxygenase-reductase, which can oxidize or disproportionate sulfur in several acidophilic bacteria and archaea [37]. However, we found a gene cluster encoding two sulfur transferases (rhd, TK90_0630; sirA, TK90_0631) and a heterodisulfide reductase complex (TK90_0632 - TK90_0637) consisting of hdrA, hdrB, and hdrC (Figure 6). dsrE was missing in this cascade, but was present at 3 other places in the genome (TK_0511, TK_0639, TK90_1244).

Figure 5

Schematic overview of the different genes that are involved in the oxidation of sulfur compounds, although the role of the Hdr complex has not been proven yet. The genes encoding the reverse dissimilatory sulfite reductase (dsr), which are present in the genome of Thioalkalivibrio sulfidophilus, are absent in the genome of Thioalkalivibrio sp. K90mix.

Figure 6

Comparison of the hdr cluster of A. ferroooxidans ATCC 23270 (AF), Thioalkalivibrio sp. K90mix (K90) and Thioalkalivibrio sulfidophilus HL-EbGR7 (HL). The heterodisulfide reductase complex consists of the genes encoding HdrC1, HdrB1, HdrA, orf2, HdrC2 and HdrB2. The accessory proteins are Rhd, and TusA. DsrE was not found in this order, but was present at other places in the genome. The percentage of amino-acid similarity is indicated.

The Hdr complex plays a function in the energy metabolism of methanogens [38] and sulfate-reducing prokaryotes [39]. In methanogens, the enzyme complex catalyzes the reversible reduction of the disulfide (CoM-S-S-CoB) of the two methanogenic thiol-coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH); in sulfate reducing microorganisms the substrate (X-S-S-X) is not known. Recently, the genes encoding the Hdr complex have also been detected in the genomes of the acidophilic sulfur oxidizing bacteria Acidithiobacillus ferrooxidans [40] and Acidithiobacillus caldus [41]. Quantrini and co-workers [40] hypothesized that Hdr, like the dissimilatory sulfite reductase (dsr), is working in reverse, whereby sulfur (i.e., sulfane atom) is oxidized to sulfite, and electrons are donated to the quinone pool. Although the role of the Hdr complex in the sulfur metabolism still has to be confirmed by expression analysis, Hdr genes have not yet been detected in the genomes of the neutrophilic chemolithotrophic sulfur-oxidizing bacteria Thiomicrospira crunogena and Thiobacillus denitrificans or of phototrophic sulfur-oxidizing bacteria Allochromatium vinosum and Halorhodospira halophila. The absence of a reverse dsr pathway in Thioalkalivibrio sp. K90mix might be the reason why it can only tolerate sulfide concentrations up to 1 mM. Sulfite can be oxidized further to sulfate, either directly by sulfite dehydrogenase (sorA; TK90_0686) or indirectly via adenosine-5’-phosphosulfate (APS) by APS reductase encoded by aprBA (TK90_0064 and TK90_0065) and ATP sulfurylase encoded by sat (TK90_0062) [42]. All these genes are present in the investigated genome.

Energy metabolism and pH homeostasis

At this time, it is not clear how Thioalkalivibrio sp. K90mix can withstand the harsh conditions of high pH and salinity. The difference between the pH of the environment (pH 10) and the pH in the cell (pH 8) causes a reversed ΔpH and consequently lowers the proton motive force (PMF). Therefore, Thioalkalivibrio requires a special molecular mechanisms to obtain enough energy for growth. It certainly needs this energy, because the production of osmolytes, to withstand the high concentrations of salts, costs 55 molecules of ATP for one molecule of glycine betaine, and 110 molecules of ATP for 1 molecule of sucrose [43]. In addition, the chemolithoautotrophic life style of CO2 fixation is energetically very expensive. The redox potential of the substrate couple S°/HS- (-260 mV) is more positive that the potential of NAD+/NADH (-340 mV) and therefore the direct reduction of NAD+ in order to supply reducing equivalents for CO2 fixation is not possible. Reverse electron transport is necessary in order to produce enough NADH, necessary for CO2 fixation, which costs extra energy. In addition, because of the large pH gradient over the cell membrane Thioalkalivibrio needs special mechanisms to keep the intracellular pH around neutral (pH homeostasis), which again is an energy requiring process.

The genome has revealed genes encoding similar proteins as those found for “Thioalkalivibrio sulfidophilus HL-EbGr7 [32]. We found genes for a proton-driven F0F1-type ATP synthase (i.e., subunit A TK90_2593, B TK90_2591, and C TK90_2592of the F0 subcomplex, and subunit alpha (TK90_2589), beta (TK90_2587), gamma (TK90_2588), delta (TK90_2590), and epsilon (TK90_2586) of the F1 subcomplex), genes encoding the proton-translocating NADH dehydrogenase (nuoABCDEFGHIJKLMN) (TK90_0708 to TK90_0721), as well as the genes for a putative primary sodium pump Rnf [44] (rnfABCDGE) (TK90_1790 to TK90_1795). In addition, we found several genes encoding different secondary sodium-dependent pumps, such as the Na+/H+ antiporters NhaP (TK90_1831) and Mrp (mnhA-G) (TK90_0748 to TK90_0752), which according to Padan et al. [45] both play an essential role in alkaline pH homeostasis. In addition, we found genes encoding transporters belonging to the SulP family (TK90_0019, TK90_0897, TK90_0985). Transporters of this group could be involved in the low affinity, but high flux of bicarbonate uptake [46]. In addition, genes encoding the sodium-depending flagellar motor PomA/B (TK90_1180 and TK90_1181) are also present in the genome (see below for more details). As Thioalkalivibrio sp. K90mix can stand high concentrations of potassium, we also searched for K+-transporters and found genes encoding the following transporters: TrkA-C (TK90_0502), TrkA-N (TK90_2266) and TrkH (TK90_2267) that are part of the potassium uptake system [47].

Chemotaxis and motility

We found different genes encoding methyl-accepting chemotaxis sensory transducers (TK90_0580, TK90_0949, TK90_1402, TK90_2562, TK90_2397) that are involved in chemotaxis. One of these genes, Aer (TK90_0580), encodes a redox sensor involved in aerotaxis. In E. coli, Aer regulates the motility behavior in gradients of oxygen, redox potential and certain nutrients by interacting with the CheA-CheW complex. We found genes encoding several different proteins of this complex, CheA (TK90_1178), CheW (TK90_1183 and TK90_1184), CheY (TK90_1176), CheZ (TK90_1177), CheB (TK90_1179), CheV (TK90_0924) and CheR (TK90_0925). Chemotaxis consists of a complex cascade of different reactions: the redox sensor Aer senses a difference in redox potential induced by a change in the environmental oxygen concentration, which leads to the autophosphorylation of the histidine protein kinase CheA. CheA phosphorylates CheY, which will switch on the flagellar motor (see [48] for a detailed overview). CheW acts as an adaptor protein, while CheB, CheR, CheZ, and CheV are involved in feedback regulation.

Thioalkalivibrio sp. K90mix has all the genes that are indispensable for the production of flagellar proteins (FlgA, TK90_0923; FlgB, TK90_0926; FlgC, TK90_0927; FlgD, TK90_0928; FlgE, TK90_0929; FlgF, TK90_0930; FlgG, TK90_0931; FlgH, TK90_0932; FlgI, TK90_0933; FlgK, TK90_0935; FlgL, TK90_0936; FlgM, TK90_0922; FlgN, TK90_0921; FliC, TK90_1400, 1448, 1450; FliD, TK90_1447; FliE, TK90_1157; FliF, TK90_1158; FliG, TK90_1159; FliH, TK90_1160; FliI, TK90_1161; FliJ, TK90_1162; FliK, TK90_1163; FliM, TK90_1165; FliN, TK90_1166; FliO, TK90_1167; FliP, TK90_1168; FliQ, TK90_1169; FliR, TK90_1170; FliS, TK90_1446; FlhA, TK90_1172; FlhB, TK90_1171; see Table 1 in [49]). Phylogenetic analysis of sequences encoding different flagellar motors showed, with significant bootstrap values, genes encoding both proton-driven motors (TK90_0578, TK90_0577) related to E. coli MotA and MotB, as well as sodium-driven motors (TK90_1180, TK1181) related to Vibrio cholera PomA and PomB and Bacillus subtilis MotP and MotS (Figure 7). The results also show the presence of proton- and sodium-driven flagellar motors in the Ectothiorhodospiraceae, “Thioalkalivibrio sulfidophilus, Halorhodospira halophila, and Alkalilimnicola ehrlichei, as well as in Achromatium vinosum, Acidithiobacillus caldus, Vibrio alginolyticus and Vibrio parahaemolyticus. Vibrio cholerae, Bacillus subtilis, B. halodurans, and Nitrococcus mobilis only have sodium-driven flagellar motors. Grouping of the proton-driven MotA and sodium-driven PomA has also been found by Krulwich et al. [50].

Figure 7

Phylogenetic tree based on protein sequences of different flagellar motors. The sequences of the proton-driven flagellar motor MotAB from E. coli (ECDH10B_2031, ECDH10B_2030) and of the sodium-driven flagellar motor PomAB from V. cholerae (VC0892, VC0893) were used as reference proteins. Other proteins were selected after BLAST analysis. Subsequently, the selected protein sequences were aligned used Clustal W, and a neighbor joining tree was drawn using MEGA 5. Ac, Acidithiobacillus caldus ATCC 51756; Ae, Alkalilimnicola ehrlichei MLHE-1; Av, Allochromatium vinosum DSM 180; Bh, Bacillus halodurans C125; Bs, Bacillus subtilis strain 168; Ec, Escherichia coli DH10B; Hh, Halorhodospira halophila SL1; Nm, Nitrococcus mobilis NB-231; Ts, Thioalkalivibrio sulfidophilus HLEbGR7; Tv-K90, Thioalkalivibrio sp. K90mix; Va, Vibrio alginolyticus 12G01; Vc, Vibrio cholera O1; Vp, Vibrio parahaemolyticus RIMD2210633. The sequences of Thioalkalivibrio sp. K90mix are shown in bold type. The bar indicates 20% sequence difference. Numbers on the branches indicate percentage bootstrap values from 1000 iterations; only those values are shown that distinguish the different flagellar motors. The CheW sequence of Thioalkalivibrio sp. K90mix (TK90_1183) was used as an outgroup, but was pruned from the tree.

Transposases and environmental stress

Comparative analysis of the genomes of “Thioalkalivibrio sulfidophilus” HL-EbGr7 [32] and Thioalkalivibrio sp. K90mix showed a greater abundance of genes encoding different transposases (i.e., COG2801, COG3328, COG3547) in the latter. Transposases are enzymes that can move specific sequences of DNA, known as transposons or transposable elements, within the genome. Krulwich [51] found that the genome of the alkaliphilic bacterium Bacillus halodurans C125 contained 112 transposase genes as compared to 10 in the genome of its closest non-alkaliphilic relative B. subtilis. She suggested that this might be one of the mechanisms of alkaliphilic adaptation at the genome level. Although strains HL-EbGr7 and K90mix are both obligately alkaliphilic, they differ in salt tolerance. HL-EbGr7 can tolerate only low (up to 1.5 M) salt concentrations, while K90mix can tolerate high (up to 4 M) salinities.

Capy et al. [52] mentioned that environmental stress might stimulate transposition and consequently increase the genetic variability, which can be beneficial for the adaptation to novel environmental conditions. Foti et al. [4] used rep-PCR [53] to study the genetic diversity within the genus Thioalkalivibrio and found a relatively high diversity of 56 genotypes among 85 strains that were isolated from different soda lakes in Africa and Asia. In addition, preliminary enrichment experiments with potassium carbonate instead of sodium carbonate and higher concentrations of chloride selected populations of high salt-tolerant Thioalkalivibrio strains with different rep-PCR patterns (unpublished results), which might be an indication that transposition might occur more frequently in strains with a wide range of salt tolerance.

Oxidative stress

Reactive oxygen species (ROS), such as superoxides (O2-) and hydrogen peroxidase (H2O2), are naturally produced at hypersaline conditions and are deleterious to cellular macromolecules. To protect themselves from this oxidative stress, Thioalkalivibrio sp. K90mix and “Thioalkalivibrio sulfidophilus HL-EbGr7 have several defense mechanisms. Superoxides are converted to oxygen and hydrogen peroxide by the enzyme superoxide dismutase (TK90_0947, Tgr7_2463), while hydrogen peroxide is converted to oxygen by hydroperoxidase (TK90_0947, Tgr7_1107) or to H2O by the cytochrome C peroxidase (TK90_0812, Tgr7_2739). In addition, Thioalkalivibrio sp. K90mix produces high concentrations of a specific membrane-bound yellow pigment named ‘natronochrome’ [54]. The pigment has a high degree of unsaturation and might also play a role in the protection against reactive oxygen species (ROS). The gene(s) responsible for the synthesis of this anti-oxidant remains to be identified.

Osmotic stress

Thioalkalivibrio sp. K90mix is an extremely salt-tolerant bacterium. It can grow in saturated sodium and potassium carbonate and sodium sulfate brines containing up to 4 M Na+/K+ but, in contrast to halo-alkaliphiles, it is inhibited by high concentrations of chloride. So, a more proper term for such an extremophile would be an “extreme natronophile”. To withstand these extreme salinities, it synthesizes glycine betaine as the main compatible solute; the genome contains the genes for glycine sarcosine N-methyltransferase (TK90_0179) and sarcosine dimethylglycine methyltransferase (TK90_0180). In addition, the genome contains the gene for sucrose phosphate synthase (TK90_2312) to produce sucrose as a compatible solute. Genes for ectoine were not found.

Declarations

Acknowledgments

This work was performed under the auspices of the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, UT-Battelle and Oak Ridge National Laboratory under contract DE-AC05-00OR22725. Dimitry Sorokin was supported financially by RFBR grant 10-04-00152.


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

  1. Sorokin DY and Kuenen JG. Haloalkaliphilic sulfur-oxidizing bacteria from soda lakes. FEMS Microbiol Rev. 2005; 29:685-702 View ArticlePubMed
  2. Sorokin DY, Banciu H, Robertson LA, Kuenen JG. Haloalkaliphilic sulfur-oxidizing bacteria. In: The Prokaryotes. Volume 2: Ecophysiology and Biochemistry 2006; pp. 969-984. Dworkin, M., Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E. (Ed's). Springer, New York.
  3. Sorokin DY, van den Bosch PLF, Abbas B, Janssen AJH and Muyzer G. Microbiological analysis of the population of extremely haloalkaliphilic sulfur-oxidizing bacteria dominating in lab-scale sulfide-removing bioreactors. Appl Microbiol Biotechnol. 2008; 80:965-975 View ArticlePubMed
  4. Foti M, Ma S, Sorokin DY, Rademaker JLW, Kuenen GJ and Muyzer G. Genetic diversity and biogeography of haloalkaliphilic sulfur-oxidizing bacteria beloning to the genus Thioalkalivibrio. FEMS Microbiol Ecol. 2006; 56:95-101 View ArticlePubMed
  5. Sorokin DY, Kuenen JG and Jetten M. Denitrification at extremely alkaline conditions in obligately autotrophic alkaliphilic sulfur-oxidizing bacterium “Thioalkalivibrio denitrificans”. Arch Microbiol. 2001; 175:94-101 View ArticlePubMed
  6. Sorokin DY, Antipov AN and Kuenen JG. Complete denitrification in a coculture of haloalkaliphilic sulfur-oxidizing bacteria from a soda lake. Arch Microbiol. 2003; 180:127-133 View ArticlePubMed
  7. Sorokin DY, Tourova TP, Lysenko AM and Kuenen JG. Microbial thiocyanate utilization under high alkaline conditions. Appl Environ Microbiol. 2001; 67:528-538 View ArticlePubMed
  8. Sorokin DY, Tourova TP, Antipov AN, Muyzer G and Kuenen JG. Anaerobic growth of the haloalkaliphilic denitrifying sulfur-oxidizing bacterium Thialkalivibrio thiocyanodenitrificans sp. nov. with thiocyanate. Microbiology. 2004; 150:2435-2442 View ArticlePubMed
  9. Janssen AJ, Lens PN, Stams AJ, Plugge CM, Sorokin DY, Muyzer G, Dijkman H, Van Zessen E, Luimes P and Buisman CJ. Application of bacteria involved in the biological sulfur cycle for paper mill effluent purification. Sci Total Environ. 2009; 407:1333-1343 View ArticlePubMed
  10. van den Bosch PLF, Sorokin DY, Buisman CJN and Janssen AJH. The effect of pH on thiosulfate formation in a new biotechnological process for the removal of hydrogen sulfide from gas streams. Environ Sci Technol. 2008; 42:2637-2642 View ArticlePubMed
  11. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  12. Woese CR, Kandler O and Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA. 1990; 87:4576-4579 View ArticlePubMed
  13. Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 1.
  14. . Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 106. Int J Syst Evol Microbiol. 2005; 55:2235-2238 View Article
  15. Garrity GM, Bell JA, Lilburn T. Class III. Gammaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 1.
  16. Imhoff J. Order I. Chromatiales ord. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 1-3.
  17. Imhoff JF. Reassignment of the genus Ectothiorhodospira Pelsh 1936 to a new family, Ectothiorhodospiraceae fam. nov., and emended description of the Chromatiaceae Bavendamm 1924. Int J Syst Bacteriol. 1984; 34:338-339 View Article
  18. Sorokin DY, Lysenko AM, Mityushina LL, Tourova TP, Jones BE, Rainey FA, Robertson LA and Kuenen GJ. Thioalkalimicrobium aerophilum gen. nov., sp. nov. and Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus gen. nov., sp. nov., Thioalkalivibrio nitratis sp.nov., novel and Thioalkalivibrio denitrificancs sp. nov., novel obligately alkaliphilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria from soda lakes. Int J Syst Evol Microbiol. 2001; 51:565-580PubMed
  19. Banciu H, Sorokin DY, Galinski EA, Muyzer G, Kleerebezem R and Kuenen JG. Thialkalivibrio halophilus sp. nov., a novel obligately chemolithoautotrophic, facultatively alkaliphilic, and extremely salt-tolerant, sulfur-oxidizing bacterium from a hypersaline alkaline lake. Extremophiles. 2004; 8:325-334 View ArticlePubMed
  20. . Notification that new names and new combinations have appeared in volume 51, part 2, of the IJSEM. Int J Syst Evol Microbiol. 2001; 51:795-796 View ArticlePubMed
  21. De Vos P, Trüper HG and Tindall BJ. Judicial Commission of the International Committee on Systematics of Prokaryotes Xth International (IUMS) Congress of Bacteriology and Applied Microbiology. Minutes of the meetings, 28, 29 and 31 July and 1 August 2002, Paris, France. Int J Syst Evol Microbiol. 2005; 55:525-532 View Article
  22. Classification of Bacteria and Archaea in risk groups. TRBA 466.Web Site
  23. Pruesse E, Quast C, Knittel K, Fuchs B, Ludwig W, Peplies J and Glöckner FO. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 2007; 35:7188-7196 View ArticlePubMed
  24. Ludwig W, Strunk O, Westram R, Richter L and Meier H. Yadhukumar, Buchner A, Lai T, Steppi S, Jobb G. ARB: a software environment for sequence data. Nucleic Acids Res. 2004; 32:1363-1371 View ArticlePubMed
  25. Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Hugenholtz P, Markowitz VM and Kyrpides NC. The Genomes OnLine Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2010; 38:D346-D354 View ArticlePubMed
  26. . Web Site
  27. Lapidus A, LaButti K, Foster B, Lowry S, Trong S, Goltsman E. POLISHER: An effective tool for using ultra short reads in microbial genome assembly and finishing. AGBT, Marco Island, FL, 2008.
  28. Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  29. Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  30. Markowitz VM, Szeto E, Palaniappan K, Grechkin Y, Chu K, Chen IM, Dubchak I, Anderson I, Lykidis A and Mavromatis K. The integrated microbial genomes (IMG) system in 2007: data content and analysis tools extensions. Nucleic Acids Res. 2008; 36:D528-D533 View ArticlePubMed
  31. Muyzer G, Sorokin DY, Mavromatis K, Lapidus A, Clum A, Ivanova N, Pati A, d'Haeseleer P, Woyke T and Kyrpides NC. Complete genome sequence of “Thioalkalivibrio sulfidophilus” HL-EbGR7. Stand Genomic Sci. 2011; 4:23-35 View ArticlePubMed
  32. Shibata M, Katoh H, Sonoda M, Ohkawat H, Shimoyama M, Fukuzawa H, Kaplan A and Ogawa T. Genes essential to sodium-dependent bicarbonate transport in cyanobacteria. J Biol Chem. 2002; 277:18658-18664 View ArticlePubMed
  33. Gibson J, Pfennig N and Waterbury JB. Chloroherpeton thalassium gen. nov. et spec. nov., a non-filamentous, flexing and gliding green sulfur bacterium. Arch Microbiol. 1984; 138:96-101 View ArticlePubMed
  34. Frigaard N-U, Dahl C. Sulfur metabolism in phototrophic sulfur bacteria. Advances in Microbial Physiology, 2009; 54: 1-3-200.
  35. Hanson TE and Tabita FR. A ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress. Proc Natl Acad Sci USA. 2001; 98:4397-4402 View ArticlePubMed
  36. Rohwerder T and Sand W. The sulfane sulfur of persulfides is the actual substrate of the sulfur-oxidizing enzymes from Acidithiobacillus and Acidiphillum spp. Microbiology. 2003; 149:1699-1710 View ArticlePubMed
  37. Hedderich R, Hamann N and Bennati M. Heterodisulfide reductase from methanogenic archaea: a new catalytic role of iron-sulfur cluster. Biol Chem. 2005; 386:961-970 View ArticlePubMed
  38. Mander GJ, Pierik AJ, Huber H and Hedderich R. Two distinct heterodisulfide reductase-like enzymes in the sulfate-reducing archaeon Archaeoglobus profundus. Eur J Biochem. 2004; 271:1106-1116 View ArticlePubMed
  39. Quatrini R, Appia-Ayme C, Denis Y, Jedlicki E, Holmes DS and Bonnefoy V. Extending the model for iron and sulfur oxidation in the extreme acidophile Acidithiobacillus ferrooxidans. BMC Genomics. 2009; 10:394 View ArticlePubMed
  40. Mangold S, Valdés J, Holmes DS and Dopson M. Sulfur metabolism in the extreme acidophile Acidithiobacillus caldus. Front Microbiol. 2011; 2:1-13PubMed
  41. Kappler U. Bacterial sulfite-oxidizing enzymes. Biochim Biophys Acta 2011; 1807: 1-10.
  42. Oren A. Thermodynamic limits to microbial life at high salt concentrations. [PubMed]. Environ Microbiol. 2011; 11:1908-1923PubMed
  43. Biegel E and Müller V. Bacterial Na+-translocating ferridoxins: NAD+ oxidoreductase. Proc Natl Acad Sci USA. 2010; 107:18138-18142 View ArticlePubMed
  44. Padan E, Bibi E, Ito M and Krulwich TA. Alkaline pH homeostasis in bacteria: New insights. Biochim Biophys Acta. 2005; 1717:67-88 View ArticlePubMed
  45. Price GD, Woodger FJ, Badger MR, Howitt SM and Tucker L. Identification of a SulP-type bicarbonate transporter in marine cyanobacteria. Proc Natl Acad Sci USA. 2004; 101:18228-18233 View ArticlePubMed
  46. Corratgé-Faillie C, Jabnoune M, Zimmerman S, Véry AA, Fizames C and Sentenac H. Potassium and sodium transport in non-animals cells: the Trk/Ktr/HKT transporter system. Cell Mol Life Sci. 2010; 67:2511-2532 View ArticlePubMed
  47. Porter SL, Wadhams GH and Armitage JP. Signal processing in complex chemotaxis pathways. Nat Rev Microbiol. 2011; 9:153-165 View ArticlePubMed
  48. Pallen MJ and Matzke NJ. From the origin of species to the origin of bacterial flagella. Nat Rev Microbiol. 2006; 4:784-790 View ArticlePubMed
  49. Krulwich TA. Bioenergetic adaptations that support alkaliphily. 2007. In: Physiology and biochemistry of extremophiles. Eds. Gerday C, Glansdorff N. ASM Press, Washington, D.C. pp. 311-329.
  50. Krulwich TA. Alkaliphily. 2003. In: Extremophiles (Life under extreme conditions) [Eds. Gerday C, Glansdorff N], in Encyclopedia of Life Support Systems (EOLSS). UNESCO-EOLSS.
  51. Capy P, Gasperi G, Biémont C and Bazin C. Stress and transposable element: co-evolution or useful parasites? Heredity. 2000; 85:101-106 View ArticlePubMed
  52. Versalovic J, de Bruijn FJ and Lupski JR. Genomic fingerprinting of bacteria using repetitive sequence based PCR 9rep-PCR). Meth Cell Mol Biol. 1994; 5:25-40
  53. Takaichi S, Maoka T, Akimoto N, Sorokin DY, Banciu H and Kuenen JG. Two novel yellow pigments natronochrome and chloronatronochrome from the natrono(alkali)philic sulfur-oxidizing bacterium Thialkalivibrio versutus ALJ 15. Tetrahedron Lett. 2004; 45:8303-8305 View Article