Hindawi Publishing Corporation ISRN Microbiology Volume 2014, Article ID 597418, 11 pages http://dx.doi.org/10.1155/2014/597418 Research Article Function and Evolution of the Sox Multienzyme Complex in the Marine Gammaproteobacterium Congregibacter litoralis Stefan Spring Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany Correspondence should be addressed to Stefan Spring; [email protected] Received 29 November 2013; Accepted 23 January 2014; Published 31 March 2014 Academic Editors: D. A. Saffarini and J. Tang Copyright © 2014 Stefan Spring. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Core sets of sox genes were detected in several genome sequenced members of the environmental important OM60/NOR5 clade of marine gammaproteobacteria. However, emendation of media with thiosulfate did not result in stimulation of growth in two of these −1 strains and cultures of Congregibacter litoralis DSM 17192T did not oxidize thiosulfate to sulfate in concentrations of one mmol L or above. On the other hand, a significant production of sulfate was detected upon growth with the organic sulfur compounds, cysteine and glutathione. It was found that degradation of glutathione resulted in the formation of submillimolar amounts of thiosulfate in the closely related sox-negative strain Chromatocurvus halotolerans DSM 23344T. It is proposed that the Sox multienzyme complex in Congregibacter litoralis and related members of the OM60/NOR5 clade is adapted to the oxidation of submillimolar amounts of thiosulfate and nonfunctional at higher concentrations of reduced inorganic sulfur compounds. Pelagic bacteria thriving in the oxic zones of marine environments may rarely encounter amounts of thiosulfate, which would allow its utilization as electron donor for lithoautotrophic or mixotrophic growth. Consequently, in evolution the Sox multienzyme complex in some of these bacteria may have been optimized for the effective utilization of trace amounts of thiosulfate generated from the degradation of organic sulfur compounds. 1. Introduction of thiosulfate to sulfate encoded by sox genes in bacteria is discussed. In a previous study the distribution of sox Aerobic marine gammaproteobacteria affiliated to the genes among members of the OM60/NOR5 clade was OM60/NOR5 clade are widespread in saline environments revealed by analyses of sequenced genomes and detection and of ecological importance in several euphotic coastal of the soxB gene (representing the key enzyme sulfate environments [1]. It is thought that aerobic anoxygenic thiohydrolase) with specific PCR primers [6]. It turned photoheterotrophy provides some members of this clade out that sox genes are present mainly in members of with a selective advantage against competing obligate the OM60/NOR5 clade that encode also genes enabling chemoheterotrophic bacteria [2]. Besides light energy, the aerobic anoxygenic photoheterotrophy, like Congregibacter oxidation of reduced inorganic sulfur compounds to sulfate 17192T is utilized by a large number of heterotrophic proteobacteria litoralis (C. litoralis)DSM , Congregibacter sp. 22749T as energy yielding process for mixotrophic growth. Several strain NOR5-3, Luminiphilus syltensis DSM ,or pathways for the oxidation of reduced sulfur compounds the isolate HTCC2080. However, there is no stringent to sulfate are known in bacteria, but most knowledge exists correlation of genes encoding Sox proteins and subunits of about a thiosulfate oxidizing multienzyme complex, which the photosynthetic apparatus, because the isolate IMCC3088 is encoded by a set of sulfur oxidizing (sox) genes [3]. It encodes sox genes, but no photosynthetic apparatus [7], was found that the genes soxA,B,C,D,X,Y,and Z are whereas the bacteriochlorophyll a-containing strains present in most, if not all, bacteria that are able to oxidize Pseudohaliea rubra DSM 19751T and Chromatocurvus thiosulfate to sulfate without forming a free intermediate halotolerans DSM 23344T do not encode a soxB gene [4, 5]. Hence, a common mechanism for the direct oxidation representing a Sox multienzyme complex. Recently, it was 2 ISRN Microbiology shown that in C. litoralis emendation of cultivation media determined in cultures of C. litoralis and Chromatocurvus with thiosulfate did not stimulate growth [8]. This was halotolerans growninLSorSFmediumbythebarium unexpected, because in marine members of the Roseobacter chloride turbidity method of Madsen and Aamand [11]. clade that encode a complete set of sox genes mixotrophic Uninoculated controls were incubated in parallel and used growth with thiosulfate as additional energy source could to determine a possible chemical production of sulfate. be demonstrated [9]. Hence, aerobic marine bacteria may Values below 50 M sulfate could not be exactly determined benefit from sox genes in several ways that are independent with this method due to a nonlinearity of the slope of the of the well-known lithotrophic oxidation of thiosulfate to barium sulfate assay in this concentration range. Thiosulfate sulfate. To get a clue about a yet unknown function of sox concentrations were estimated in culture supernatants by the genes in aerobic marine gammaproteobacteria a study was cyanolysis method as described by Kelly et al. [12]andsulfite initiatedinwhichthesulfurmetabolisminC. litoralis was was quantified by the method proposed by Denger et al. [13]. analyzed in detail and compared with closely related species lacking a Sox complex. 2.3. Semiquantitative Detection of soxB Transcripts Using PCR. RNA was extracted from early stationary phase 2. Materials and Methods cultures of C. litoralis DSM 17192T grown under var- ious conditions using the RNeasy Midi kit of Qiagen 2.1. Used Strains and Cultivation Conditions. The following (Hilden, Germany) as described previously [2]. Reverse type strains were used in this study and taken from the transcriptase-PCR (RT-PCR) of mRNA was performed with collection of the Leibniz Institute DSMZ (Braunschweig, the OneStep RT-PCR kit of Qiagen following the instruc- Germany): C. litoralis DSM 17192T, Luminiphilus syltensis tions given by the manufacturer and using 0.5 gofRNA. 22749T 23344T For the semiquantitative detection of transcripts of the C. DSM , Chromatocurvus halotolerans DSM , 19751T litoralis soxB gene the forward primer KT71 soxB-F (5 - and Pseudohaliea rubra DSM . For routine cultivation allstrainsweregrowninSYPHCcomplexmedium[6] TCCAGGCGATAGTTGAATCC-3 ) and the reverse primer ∘ under air atmosphere at 28 C. The preparation of defined KT71 soxB-R (5 -AGCTTCGACCAGCTCATTGT-3 )were marine media and the generation of distinct gas atmospheres used.TheresultingPCRproducthadasizeof290bp. for growing strains in batch cultures under semiaerobic Amplification of transcripts, visualization of PCR products, incubation conditions have been described elsewhere [6, 10]. and normalization of mRNA levels were done as reported The SYPHC complex medium and defined marine medium previously [2]. −1 contained 35.0 g L sea salts (Sigma S9883) resulting in a sulfate concentration of around 25 mM. The growth and 2.4. Phylogenetic Analyses of Sox Proteins. Amino acid sulfate production of C. litoralis and Chromatocurvus halo- sequences of the proteins SoxB, SoxC, and SoxA were tolerans with various sulfur compounds were determined in obtained from the UniProt database (release 2013 08) and a carbonate-buffered saline medium devoid of sulfate. The alignedusingtheClustalWalgorithmimplementedinthe basalsaltsolutionwasnamedSFandhadthefollowing ARB package [14]. Phylogenetic trees based on aligned composition (per liter demineralized water): 21.0 g NaCl, 2.5 g protein sequences were reconstructed using the ARB dis- MgCl2 × 6H2O,1.0gKCl,0.2gCaCl2 × 2H2O, 0.1 g NH4Cl, tance matrix (neighbor-joining) program with the PAM 0.05 g KH2PO4,2.5gNaHCO3, and 1 mL vitamins solution correction and maximum likelihood (RAxML) program with (see DSMZ medium 503). The vitamins, KH2PO4,NaHCO3, the PROTGAMMA option and BLOSUM62 as amino acid and any additional substrates were added to the basal substitution model. No filter or weighting masks were used medium after autoclaving from stock solutions sterilized by to constrain the used positions of the alignment. filtration. The pH of the completed medium was adjusted to pH 7.5. In most experiments a sulfate-poor medium des- 3. Results and Discussion ignatedLSwasusedthatwasobtainedbytransferringan inoculum size of 1 vol% from defined marine medium to SF 3.1. Effect of Thiosulfate on the Growth Response of Members medium resulting in an initial sulfate concentration of around of the OM60/NOR5 Clade. The growth response of the type 250 M, which was sufficient to prevent growth inhibition by strains of C. litoralis and Luminiphilus syltensis both encoding sulfate limitation. Cultures in essentially sulfate-free media a core set of sox genes (soxA,B,C,D,X,Y,andZ)andthesox- were obtained by two successive transfers in SF medium negative strains Chromatocurvus halotolerans DSM 23344T containing L-glutamate as carbon source. All used chemicals and Pseudohaliea rubra DSM 19751T was tested in defined were obtained from Sigma-Aldrich (Taufkirchen, Germany) and complex marine media with and without thiosulfate and complex nutrients from DIFCO BBL (Becton Dickinson; under various incubation conditions.