ISSN 0026-2617, Microbiology, 2017, Vol. 86, No. 5, pp. 666–669. © Pleiades Publishing, Ltd., 2017. Original Russian Text © N.A. Chernyh, I.V. Kublanov, M.I. Prokof’eva, N.V. Pimenov, E.N. Frolov, A.V. Mardanov, A.A. Khvashchevskaya, N.V. Guseva, A.V. Lebedinskii, E.A. Bonch-Osmolovskaya, 2017, published in Mikrobiologiya, 2017, Vol. 86, No. 5, pp. 651–654.
SHORT COMMUNICATIONS
Production of Organic Matter and Diversity of the Ribulose Bisphosphate Carboxylase Genes in Sediments of the Solnechny Spring, Uzon Caldera, Kamchatka N. A. Chernyha, *, I. V. Kublanova, M. I. Prokof’evaa, N. V. Pimenova, E. N. Frolova, A. V. Mardanovb, A. A. Khvashchevskayac, N. V. Gusevac, A. V. Lebedinskiia, and E. A. Bonch-Osmolovskayaa a
Winogradsky Institute of Microbiology, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia b Institute of Bioengineering, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia c Tomsk Polytechnical University, Tomsk, Russia *e-mail:
[email protected] Received December 12, 2016
DOI: 10.1134/S0026261717050071
The main producers of organic matter in the hot springs of Yellowstone National Park and Kamchatka are considered to be bacteria of the phylum Aquificae and archaea of the family Thermoproteaceae (Reysenbach et al., 2000, Chernyh et al., 2015), which assimilate inorganic carbon via the reductive tricarboxylic acid cycle and the dicarboxylate/4-hydroxybutyrate cycle, respectively. The main energy substrate for them is molecular hydrogen, which is oxidized either aerobically (Aquificae) or anaerobically in the course of sulfur reduction (Thermoproteaceae). Members of Aquificae are also capable of anaerobic respiration or aerobic utilization of reduced sulfur compounds and, less frequently, of reduced iron and arsenic compounds. The microorganisms that have the Calvin– Benson cycle are usually not considered as producers of organic matter in hydrothermal vents due to the thermolability of some of its intermediates, the critical temperature thought to be 70–75°C (Berg, 2011). In the present work, we have considered the community of the Solnechny Spring (Uzon Caldera, Kamchatka) with a temperature of 61–64°C as a habitat where the primary production of organic matter may involve the Calvin–Benson cycle. The Solnechny Spring (61–64°C, pH 6.1, Eh –34, 54°29994199 N, 159°59953099 E) is located at a distance from massive hydrothermal manifestations. The spring is a funnel about 1 m in diameter, with active gas release, and is located on the bottom of a shallow lake. Abundant red flakes are formed in the spring. The DNA sampled in 2011 from the Solnechny Spring was an object of metagenomic analysis (Menzel et al., 2015; the spring name Solnechny was translated in that paper: Sun Spring). The samples for hydrochemical analysis and radioisotopic studies were taken in August 2015. The contents of NH 4+, NO 3−, SO 24 −, Cl–,
Br–, Ca2+, Mg2+, Na+, K+, Li+ were determined by the method of ion chromatography using an ICS-5000 Dual Channel Reagent-Free Ion Chromatography Hybrid Bundle with conductometric detection (Dionex – Thermo Scientific, United States) and a common autosampler. Anions were identified by using an IonPac AS19 analytical column (2 × 250 mm) and an IonPac AG19 protective column (2 × 50 mm). The device was calibrated by using the basic combined calibration solutions (Dionex, United States). The spring water contained (mg/L): O2, 1.0; CO2, 105; CO2– 3 , 0;
HCO 3−, 464; SO 24 − , 57; Сl–, 140; NO–3 < 0.1; NH+4, 2.1; Ca2+, 64; Mg2+, 19; Na+, 186; K+, 22; F–, 0.17; Br–, 0.26; Li+, 0.39; total mineralization, 954. The concentration of HCO 3− was 7.5 mM; the content of HS– was below the sensitivity threshold of the method. The gas released from the spring contained (%): CO2, 64.7; O2, 7.4; N2, 27.9; CH4, 0.01. The rate of inorganic carbon assimilation was measured by the radioisotopic technique with 14C-labeled NaHCО3 (packaging of 40 MBq, specific activity of 806 GBq/mol) according to the method described previously (Pimenov, Bonch-Osmolovskaya, 2006). All experiments were carried out in three replicates. The rate of Cinorg assimilation into biomass is presented in Fig. 1. Aerobic conditions stimulated the process by nearly an order of magnitude. The maximum rate, attained under aerobic conditions after 10-h incubation, was 0.46 μmol/dm3/h. Under anaerobic conditions, the highest rate of Cinorg assimilation (0.12 μmol/dm3/h) was observed in the first hours of incubation and then decreased, indicating the activity of aerobic autotrophs utilizing the remains of dissolved oxygen in the aerobic sample.
666
mmol/dm3/h
PRODUCTION OF ORGANIC MATTER AND DIVERSITY
0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0
667
1
2
3
7
13
25 Hours
Fig. 1. Effects of aeration and incubation time on the rate of inorganic carbon assimilation by sediment samples from the Solnechny Spring (Uzon Caldera, Kamchatka). (1) Incubation under aerobic conditions; (2) incubation under anaerobic conditions.
Thus, the rate of inorganic carbon assimilation in the Solnechny Spring is within the range of values determined previously for different springs of the Uzon Caldera: 0.02 to 3.9 μmol/dm3/h (Pimenov, 2011; Chernyh et al., 2015). Prokaryotes are characterized by a great diversity of mechanisms for autotrophic assimilation of inorganic carbon: there are six currently known pathways. All of them occur in thermophilic prokaryotes too; however, the most widespread pathways in the latter are the reductive tricarboxylic acid cycle, typical of bacteria from the phylum Aquificae, and the Wood–Ljungdahl pathway, typical of most of autotrophic Firmicutes. Hyperthermophilic archaea of the family Thermoproteaceae assimilate inorganic carbon via the dicarboxylate/4-hydroxybutyrate cycle (Berg, 2011). Our total analysis of metagenomic reads showed that members of Aquificae and Thermoproteaceae comprise no more than two per cent of the total community (Menzel et al., 2015). In this context, and with regard to the rather high rate of inorganic carbon assimilation into biomass revealed by radioisotopic technique, we analyzed the occurrence frequency of the genes of the key enzyme of the Calvin–Benson cycle, RuBisCO, in the metagenome. To assess the share of microorganisms fixing CO2 via the Calvin–Benson cycle in the community, raw data of metagenomic sequencing (reads, ncbi.nlm.nih.gov/sra/ SRR5574655) were analyzed. To reveal reads containing RuBisCO gene fragments, an actual (release 2016_05) sample of domains of the large subunit of this protein in the Pfam database (PF00016, _RuBisCO_large, rp15 database) was used as a query for the search of the desired reads by tBLASTn algorithm implemented in Geneious 8.1.8 (parameters: word = 2, e-val = 0.01, max hits = 1000, other = default). After the filtration of numerous repeated hits of query components to the same reads, 139 reads with significant hits were revealed. Further MICROBIOLOGY
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verification of these reads (using the BLASTx algorithm and with thorough analysis of the results) showed that 109 of them indeed contained RuBisCO gene fragments. The total length of reads of the RuBisCO gene fragments was calculated to be 28374 nt (the total length of all reads was 241225273 nt). If we assume the absence of any selectivity of the sequencing process, the share of RuBisCO reads in the total volume of reads must be directly proportional to the share of genomes with the RuBisCO genes in the entire environmental metagenome and directly proportional to the share covered by the RuBisCO gene in individual genomes of its owners. Assuming, for the sake of simplicity, that the sizes of genomes of the RuBisCO gene owners are equal, the sizes of the RuBisCO genes are equal, and the RuBisCO gene, if present, is always present in a single copy, we obtain: RubiscoReads/TotalReads = x(RubiscoSize/GenomeSize), where x is the sought quantity, i.e., the share of genomes with the RuBisCO gene in the entire environmental metagenome. Hence, x = RubiscoReads/TotalReads/ (RubiscoSize/GenomeSsize). If we take the genome sizes of RuBisCO gene owners to be equal to 3.78 Mb (the average size of complete prokaryotic genomes in the IMG database, December 15, 2016, 1732 genomes) and the RuBisCO gene size to be equal to 1413 nt (the average size of the RuBisCO gene calculated from SwisProt proteins), we obtain x = 28374/241225273/1413 × 3.78 × 10 6 = 0.31. Thus, 31% of all genomes in the community of the Solnechny Spring carried the RuBisCO gene in their genomes. Four RuBisCO forms are known, but only for forms I and II has the involvement in the Calvin–Ben-
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Table 1. Analysis of RuBisCO gene reads in the metagenome of the Solnechny Spring (Uzon Caldera, Kamchatka) Gene reads Total
Number of reads
Total length of reads
Portion of owners in the community, %
Phylogenetic affiliations of genes
666042
241225273
100
109
28374
31
Form I
4
780
Form II
0
0
0
–
Form III
66
16 467
18
Ammonifex Desulfurococcus Methanosaeta “Candidatus Bathyarchaeota”
1
351
38
10776
Any RuBisCO form
Form II/III Form IV
son cycle been shown. In order to determine the share in the community of microorganisms containing particular RuBisCO forms, the Pfam PF00016, _RuBisCO_large, rp15 sequences, as well as sequences of biochemically characterized RuBisCO, were aligned with MAFFT v. 7, and a Maximum Likelihood phylogenetic tree was constructed (not shown). The revealed 109 reads containing RuBisCO gene fragments were in silico translated and aligned by MAFFT v. 7 with the alignment containing all reference sequences. Using the ARB-parsimony algorithm, the short sequences of RuBisCO gene fragments from the reads were positioned in the Maximum Likelihood tree constructed for the virtually complete reference sequences. Proceeding from the positions in the resulting tree, the RuBisCOs corresponding to the translated reads were referred to particular forms, and the phylogenetic positions of the organisms owning them were determined. The portion of the owners of particular RuBisCO forms in the community under study was calculated in a way analogous to the above calculation of the portion of microorganisms with any RuBisCO form. The results are shown in Table 1. A lot of studies have been devoted to the diversity of RuBisCO forms in prokaryotes, inasmuch as this protein plays a highly important role in the life of our planet. The most widespread form is Form I, which is present in all plants and in many prokaryotes, mainly in mesophiles but also in thermophilic cyanobacteria and some thermophilic representatives of Firmicutes. Our database analysis has not revealed any thermophiles among the microorganisms possessing Form II RuBisCO. Form III occurs in many archaea (including thermophilic and hyperthermophilic) and in uncultivated members of the bacterial phyla Parcubacteria (OD1), WS6, and Microgenomates (OP11), known from the results of environmental metagenome sequencing. This enzyme, however, does not seem to be involved in the Calvin–Benson cycle in these organisms but functions in the metabolism of nucleo-
0.9
0.4 12
Thermus
Methanosalsum zhilinae Bacteria and Archaea
tides (Tabita et al., 2007; Wrighton et al., 2016). The gene encoding Form III RuBisCO was also found in the genome of the thermophilic bacterium Ammonifex degensii from the Firmicutes phylum (Berg et al., 2011); however, due to the presence in the genome of the genes of another pathway of autotrophic CO2 assimilation (the Wood–Ljungdahl pathway), it is still unknown whether this Form III RuBisCO functions in the Calvin–Benson cycle. Form II/III occurs in some methanogens (Methanosalsum, Methanococcoides), as well as in uncultivated members of the bacterial phyla Peregrinibacteria, Microgenomates, and WS6, known from the results of sequencing of environmental metagenomes (Wrighton et al., 2016). So far, this form has not been shown to participate in the Calvin–Benson cycle; in some cases, its involvement in nucleotide metabolism has been established (Wrighton et al., 2016; Kono et al., 2017). Form IV is widespread in prokaryotes, but it is not involved in carboxylation reactions (Tabita et al., 2007). Among the RuBisCO genes found in the Solnechny Spring, there were no Form II genes. The revealed Form I genes unexpectedly turned out to belong to bacteria of the genus Thermus (Table 1). RuBisCO Form III in the Solnechny Spring was represented by the genes of archaea of the genera Desulfurococcus, Methanosaeta, and members of Candidatus Bathyarchaeota; the first of these microorganisms is an organotroph, the second one is an aceticlastic methanogen, and the third taxon is represented only by uncultivated organisms, the genomes of which were assembled based on the results of metagenomic sequencing. However, among the RuBisCO Form III sequences there was also the gene of this enzyme belonging to a bacterium of the genus Ammonifex. At present, this genus is represented by only two species, one of them, Ammonifex thiophilus, being an isolate from the Treshchinny Spring of the Uzon Caldera (Miroshnichenko et al., 2008). The analysis of its complete genome sequenced in collaboration with the MICROBIOLOGY
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Immanuel Kant Baltic Federal University has shown that it contains the RuBisCO Form III gene (Chernyh et al., unpublished data). In contrast to archaea, the genome of A. thiophilus contains genes encoding all enzymes of the Calvin–Benson cycle, which may allow this microorganism to participate in the assimilation of inorganic carbon via this pathway. Thus, the portion of microorganisms with the RuBisCO gene in the Solnechny Spring community is about 31%. The population carrying RuBisCO Form I (one of the two forms with proven involvement in the Calvin–Benson cycle) is insignificant, only about 1% of the community. However, given that the share of the aerobic autotrophs from Aquificae and Thermoproteaceae in this community is as low as 2%, and the Wood-Ljungdahl pathway is oxygen-sensitive, the substantial fixation of CO2 revealed by the radioisotopic technique may be evidence of the involvement of other RuBisCO forms in autotrophic CO2 fixation. ACKNOWLEDGMENTS This work was supported by the Russian Science Foundation (project no. 14-24-00165). REFERENCES Berg, I.A., Ecological aspects of the distribution of different autotrophic CO2 fixation pathways, Appl. Environ. Microbiol, 2011, vol. 77, pp. 1925–1936. Chernyh, N.A., Mardanov, A.V., Gumerov, V.M., Miroshnichenko, M.L., Lebedinsky, A.V., Merkel, A.Y., Crowe, D., Pimenov, N.V., Rusanov, I.I., Ravin, N.V., Moran, M.A., and Bonch-Osmolovskaya, E.A., Microbial life in Bourlyashchy, the hottest thermal pool of Uzon Caldera, Kamchatka, Extremophiles, 2015, vol. 19, pp. 1157– 1171. Huang, Y., Niu, B., Gao, Y., Fu, L., and Li, W., CD-HIT Suite: a web server for clustering and comparing biological sequences, Bioinformatics, 2010, vol. 26, pp. 680–682. Kono, T., Mehrotra, S., Endo, C., Kizu, N., Matusda, M., Kimura, H., Mizohata, E., Inoue, T., Hasunuma, T.,
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Translated by E. Makeeva
