Academic literature on the topic 'Thiomicrospira crunogena'

ABSTRACT A new autotrophic Thiomicrospira strain, MA-3, was isolated from the surface of a polymetal sulfide deposit collected at a Mid-Atlantic Ridge hydrothermal vent site. The DNA homology among three vent isolates, Thiomicrospira crunogena,Thiomicrospira sp. strain L-12, andThiomicrospira sp. strain MA-3, was 99.3% or higher, grouping them as the same species, T. crunogena (type strain, ATCC 35932). The fact that T. crunogena andThiomicrospira sp. strain L-12 were isolated from Pacific vent sites demonstrates a cosmopolitan distribution of this species.

The occurrence of the different genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key enzyme of the Calvin–Benson–Bassham cycle of autotrophic CO2 fixation, was investigated in the members of the genus Thiomicrospira and the relative genus Thioalkalimicrobium, all obligately chemolithoautotrophic sulfur-oxidizing Gammaproteobacteria. The cbbL gene encoding the ‘green-like’ form I RubisCO large subunit was found in all analysed species, while the cbbM gene encoding form II RubisCO was present only in Thiomicrospira species. Furthermore, species belonging to the Thiomicrospira crunogena 16S rRNA-based phylogenetic cluster also possessed two genes of green-like form I RubisCO, cbbL-1 and cbbL-2. Both 16S-rRNA- and cbbL-based phylogenies of the Thiomicrospira–Thioalkalimicrobium–Hydrogenovibrio group were congruent, thus supporting its monophyletic origin. On the other hand, it also supports the necessity for taxonomy reorganization of this group into a new family with four genera.

Enrichments at 2 M NaCl and pH 7.5–8, with thiosulfate or sulfide as electron donor, inoculated with sediments from hypersaline chloride–sulfate lakes of the Kulunda Steppe (Altai, Russia) resulted in the domination of two different groups of moderately halophilic, chemolithoautotrophic, sulfur-oxidizing bacteria. Under fully aerobic conditions with thiosulfate, bacteria belonging to the genus Halothiobacillus dominated while, under microaerophilic conditions, a highly motile, short vibrio-shaped phenotype outcompeted the halothiobacilli. Three genetically and phenotypically highly similar vibrio-shaped isolates were obtained in pure culture and one of them, strain HL 5T, was identified as a member of the Thiomicrospira crunogena cluster by 16S rRNA gene sequencing. The new isolates were able to grow with thiosulfate as electron donor within a broad salinity range from 0.5 to 3.5 M NaCl with an optimum at 1.5 M and within a pH range from 6.5 to 8.5 with an optimum at pH 7.5–7.8. Comparative analysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) gene sequences demonstrated that strain HL 5T possessed two genes, cbbL-1 and cbbL-2, of the form I RuBisCO and a cbbM gene of the form II RuBisCO, similar to the other members of the Thiomicrospira crunogena cluster. On the basis of phenotypic and genetic comparison, the new halophilic isolates are proposed to be placed into a novel species, Thiomicrospira halophila sp. nov. (type strain HL 5T=DSM 15072T=UNIQEM U 221T).

ABSTRACT The hydrothermal vent chemolithoautotroph Thiomicrospira crunogena grows rapidly in the presence of low concentrations of dissolved inorganic carbon (DIC) (= CO2 + HCO3 − + CO3 −2). Its genome encodes α-carbonic anhydrase (α-CA), β-CA, carboxysomal β-like CA (CsoSCA), and a protein distantly related to γ-CA. The purposes of this work were to characterize the gene products, determine whether they were differentially expressed, and identify those that are necessary for DIC uptake and fixation. When expressed in Escherichia coli, CA activity was detectable for α-CA, β-CA, and CsoSCA but not for the γ-CA-like protein. α-CA and CsoSCA but not β-CA were inhibited by sulfonamide inhibitors. CsoSCA was also inhibited by dithiothreitol. When grown under DIC limitation in chemostats, T. crunogena transcribed csoSCA more frequently than when ammonia limited, while genes encoding α-CA and β-CA were not differentially transcribed under these conditions. Cell extracts from T. crunogena grown under both DIC- and ammonia-limited conditions had CA activity that was strongly inhibited by sulfonamides, though extracts from nitrogen-limited cells had some CA activity that was resistant, perhaps due to a higher level of β-CA activity. Based on predictions from the SignalP software program, subcellular location when expressed in E. coli, and carbonic anhydrase assays conducted on intact T. crunogena cells, α-CA is located in the periplasm. However, inhibition of α-CA by acetazolamide had only a minor impact on rates of DIC uptake or fixation. Conversely, inhibition of CsoSCA with ethoxyzolamide inhibited carbon fixation but not DIC uptake, consistent with this enzyme functioning to facilitate DIC interconversion and fixation within carboxysomes.

A novel thermotolerant bacterium, designated strain I78T, was isolated from a self-temperature-recording in situ colonization system deployed in a hydrothermal diffusing flow (maximal temperature 78 °C) at the TOTO caldera in the Mariana Arc, Western Pacific. Cells were highly motile curved rods with a single polar flagellum. Growth was observed at 15–55 °C (optimum 35–40 °C; 60 min doubling time) and pH 5·0–8·0 (optimum pH 6·0). The isolate was a microaerobic chemolithomixotroph capable of using thiosulfate, elemental sulfur or sulfide as the sole energy source, and molecular oxygen as the sole electron acceptor. The isolate was able to grow chemolithoautotrophically with carbon dioxide. Various organic substrates such as complex proteinaceous compounds, carbohydrates, organic acids, amino acids and sugars could also support growth as the carbon source instead of carbon dioxide with sulfur oxidation. The G+C content of the genomic DNA was 43·8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genus Thiomicrospira and was most closely related to Thiomicrospira crunogena strain TH-55T and Thiomicrospira sp. strain L-12, while DNA–DNA hybridization demonstrated that the novel isolate could be genetically differentiated from previously described strains of Thiomicrospira. On the basis of its physiological and molecular properties the isolate is representative of a novel Thiomicrospira species, for which the name Thiomicrospira thermophila sp. nov. is proposed (type strain, I78T=JCM 12397T=DSM 16397T).

Gammaproteobacterium Thiomicrospira crunogena thrives at deep-sea vents despite extreme oscillations in the environmental supply of dissolved inorganic carbon (DIC; =CO2 + HCO3- + CO3-2). Survival in this habitat is likely aided by the presence of a carbon concentrating mechanism (CCM). Though CCMs are well-documented in cyanobacteria, based on this study T. crunogena is the first chemolithoautotroph to have a physiologically characterized CCM. T. crunogena is capable of rapid growth in the presence of 20 micrometers DIC, has the ability to use both extracellular HCO3- and CO2, and generates intracellular DIC concentrations 100-fold greater than extracellular, all of which are consistent with a CCM analogous to those present in cyanobacteria. Interestingly, however, the T.crunogena genome lacks apparent orthologs of many of the components of the cyanobacteria CCM (e.g., HCO3- transporters). However, despite this lack, several candidate genes were identified during genome annotation as likely to play a role in DIC uptake and fixation (three carbonic anhydrase genes: alpha-CA, beta-CA, and csoSCA, as well as genes encoding three RubisCO enzymes: cbbLS, CScbbLS, and cbbM, which encode a cytoplasmic form I RubisCO, a carboxysomal form I RubisCO, and a form II RubisCO, respectively). In order to clarify their possible roles in DIC uptake and fixation, alpha-CA, beta-CA and csoSCA transcription by low-DIC and high-DIC T. crunogena were assayed by qRT PCR, heterologous expression in E. coli, and potentiometric assays of low-DIC and high-DIC T. crunogena. Transcription of alpha-CA and beta-CA were not sensitive to the DIC concentration available during growth. When overexpressed in E.coli, carbonic anhydrase activity was detectable, and it was possible to measure the effects of the classical carbonic anhydrase inhibitors ethoxyzolamide and acetazolamide, as well as dithiothreitol (DTT; recently determined to be a carboxysomal CA inhibitor). The alpha-CA was sensitive to both of the classical inhibitors, but not DTT. Beta-CA was insensitive to all inhibitors tested, and the carboxysomal carbonic anhydrase was sensitive to both ethoxyzolamide and DTT. The observation that the CA activity measureable potentiometrically with intact T. crunogena cells is sensitive to classical inhibitors, but not DTT, strongly suggests the alpha-CA is extracellular. The presence of carbonic anhydrase activity in crude extracts of high-DIC cells that was resistant to classical inhibitors suggests that beta-CA may be more active in high-DIC cells. Incubating cells with ethoxyzolamide (which permeates cells rapidly) resulted in inhibition of carbon fixation, but not DIC uptake, while incubation with acetazolamide (which does not permeate cells rapidly) had no apparent effect on either carbon fixation or DIC uptake. The observations that inhibition of alpha-CA has no effect on DIC uptake and fixation, and that the beta-CA is not transcribed more frequently under low-DIC conditions, make it unlikely that either play a role in DIC uptake and fixation in low-DIC cells. Further studies are underway to determine the roles of alpha-CA and beta-CA in T. crunogena. To assay the entire genome for genes transcribed more frequently under low-DIC conditions, and therefore likely to play a role in the T. crunogena CCM, oligonucleotide arrays were fabricated using the T. crunogena genome sequence. RNA was isolated from cultures grown in the presence of both high (50 mM) and low (0.05 mM) concentrations of DIC, directly labeled with cy5 fluorophore, and hybridized to microarrays. Genes encoding the three RubisCO enzymes present in this organism demonstrated differential patterns of transcription consistent with what had been observed previously in Hydrogenovibrio marinus. Genes encoding two conserved hypothetical proteins were also found to be transcribed more frequently under low-DIC conditions, and this transcription pattern was verified by qRT-PCR. Knockout mutants are currently being generated to determine whether either gene is necessary for growth under low-DIC conditions. Identifying CCM genes and function in autotrophs beyond cyanobacteria will serve as a window into the physiology required to flourish in microbiallydominated ecosystems where noncyanobacterial primary producers dominate.

Thiomicrospira crunogena, a deep-sea hydrothermal vent chemolithoautotroph, uses the Calvin-Bensen-Bassham cycle to fix carbon. To meet its biosynthetic needs for oxaloacetate, oxoglutarate, and succinyl-coA, one would expect that this obligately autotrophic Gammaproteobacterium would use a ‘wishbone’ version of the citric acid cycle (CAC) to synthesize the intermediates necessary for biosynthesis, instead of the fully oxidative version to minimize carbon loss as carbon dioxide. However, upon examination of its complete genome sequence, it became apparent that this organism did not fulfill this expectation. Instead of a wishbone pathway, T. crunogena appears to run a fully oxidative CAC. The cycle is ‘locked’ in the oxidative direction by replacement of the reversible enzyme malate dehydrogenase with malate: quinone oxidoreductase, which is capable only of operation in the oxidative direction. Furthermore, oxoglutarate decarboxylation is catalyzed by oxoglutarate: acceptor oxidoreductase. The presence of both oxidoreductases was confirmed via assays on T. crunogena cell extracts. To determine whether this peculiar CAC was novel, complete genome sequences of ~340 Proteobacteria were examined via BLAST and COG searches in the Integrated Microbial Genome database. Genes catalyzing steps in the CAC were collected from each organism and vetted for paralogs that had adopted an alternative, ‘non-CAC’ function through genome context and cluster analysis. Alignments were made with the remaining sequences and were verified by comparing them to curated alignments at Pfam database and examination of active site residues. Phylogenetic trees were constructed from these alignments, and instances of horizontal gene transfer were determined by comparison to a 16S tree. These analyses verified that the CAC in T. crunogena is indeed unique, as it does not resemble any of the canonical cycles of the six classes of proteobacteria. Furthermore, three steps of the nine in its CAC appear to be catalyzed by enzymes encoded by genes that are likely to have been acquired via horizontal gene transfer. The gene encoding citrate synthase, and perhaps aconitase, are most closely affiliated with those present in the Cyanobacteria, while those encoding oxoglutarate: acceptor oxidoreductase cluster among the Firmicutes, and malate: quinone oxidoreductase clusters with the Epsilonproteobacteria.

Abstract The gammaproteobacterium Thiomicrospira crunogena XCL–2 is a hydrothermal vent chemolithoautotroph that has a carbon concentrating mechanism (CCM), which is functionally similar to that of cyanobacteria. At hydrothermal vents, dissolved inorganic carbon (DIC) concentrations and pH values fluctuate over time, with CO2 concentrations ranging from 20 μM to greater than 1 mM, therefore having a CCM would provide an advantage when CO2 availability is very low as CCMs generate intracellular DIC concentrations much higher than extracellular, thereby providing sufficient substrate for carbon fixation. The CCM in T. crunogena includes α–carboxysomes (intracellular inclusions containing form IA RubisCO and carbonic anhydrase), and also presumably requires at least one active HCO3 µ transporter to generate the elevated intracellular concentrations of DIC. To determine whether RubisCO itself might be adapted to low CO2 concentrations, the KCO2 for purified carboxysomal RubisCO was measured (250 μM SD ±; 40) and was much greater than that of whole cells (1.03 μM). This finding suggests that the primary adaptation by T. crunogena to low–DIC conditions has been to enhance DIC uptake, presumably by energy–dependent membrane transport systems that are either ATP–dependent and/or dependent on membrane potential (δ ψ). To determine the mechanism for active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis andδ ψ. After separate incubations with the ATP synthase inhibitor DCCD and the protonophore CCCP, intracellular ATP was diminished, as was the concentration of intracellular DIC and fixed carbon, despite the absence of an inhibitory effect on δ ψ in the DCCD–incubated cells. In some organisms, DCCD inhibits the NDH–1 and bc1 complexes so it was necessary to verify that ATP synthase was the primary target of DCCD in T. crunogena. Both electron transport complex activities were assayed in the presence and absence of DCCD and there was no significant difference between inhibited (309.0 μmol/s for NDH–1 and 3.4 μmol/s for bc1) and uninhibited treatments (271.7 μmol/s for NDH–1 and 3.6 μmol/s for bc1). These data support the hypothesis that an ATP–dependent transporter is primarily responsible for HCO3 µ transport in T. crunogena. The ATP–dependent transporter solute–binding protein gene (cmpA) from Synechococcus elongatus PCC 7942, was used to perform a BLAST query. Tcr_1153 was the closest match in the T. crunogena genome. However, the gene neighborhood and the result of a maximum likelihood tree suggest that Tcr_1153 is a nitrate transporter protein. Work is underway to find the genes responsible for this ATP–dependent transporter.