Project description:To study mixotrophy, it is desirable to have an organism capable of growth in the presence and absence of both organic and inorganic carbon sources, as well as organic and inorganic energy sources. Metallosphaera sedula is an extremely thermoacidophilic archaeon which has been shown to grow in the presence of inorganic carbon and energy source supplements (autotrophy), organic carbon and energy source supplements (heterotrophy), and in the presence of organic carbon and inorganic energy source supplements. The recent elucidation of M. sedulaâ??s inorganic carbon fixation cycle and its genome sequence further facilitate its use in mixotrophic studies. In this study, we grow M. sedula heterotrophically in the presence of organic carbon and energy sources (0.1% tryptone), autotrophically in the presence of inorganic carbon and energy sources (H2 + CO2), and â??mixotrophicallyâ?? in the presence of both organic and inorganic carbon and energy sources (0.1% tryptone + H2 + CO2 ) to characterize the nature of mixotrophy exhibited. Two 3 slide loops joined at equivalent conditions (8 slides total) for Mse cells includes 3 conditions tested in duplicate (biological repeats): heterotrophy (H1 and H2), autotrophy (A1 and A2), and mixotrophy (M1 and M2). Half of an RNA sample for one condition was labeled with Cy3 while the other half was labeled with Cy5. The two differently labeled samples were run on different slides. Each probe is spotted on each slide 5 times (5 replicates; spot intensities for all replicates on slide provided in associated raw data file).
Project description:To study mixotrophy, it is desirable to have an organism capable of growth in the presence and absence of both organic and inorganic carbon sources, as well as organic and inorganic energy sources. Metallosphaera sedula is an extremely thermoacidophilic archaeon which has been shown to grow in the presence of inorganic carbon and energy source supplements (autotrophy), organic carbon and energy source supplements (heterotrophy), and in the presence of organic carbon and inorganic energy source supplements. The recent elucidation of M. sedula’s inorganic carbon fixation cycle and its genome sequence further facilitate its use in mixotrophic studies. In this study, we grow M. sedula heterotrophically in the presence of organic carbon and energy sources (0.1% tryptone), autotrophically in the presence of inorganic carbon and energy sources (H2 + CO2), and “mixotrophically” in the presence of both organic and inorganic carbon and energy sources (0.1% tryptone + H2 + CO2 ) to characterize the nature of mixotrophy exhibited.
Project description:Metallosphaera sedula is an extremely thermoacidophilic archaeon that grows heterotrophically on peptides, and chemolithoautotrophically on hydrogen, sulfur, or reduced metals as energy sources. During autotrophic growth, carbon dioxide is incorporated into cellular carbon via the 3-hydroxypropionate /4-hydroxybutyrate cycle (3HP/4HB). To date, all of the steps in the pathway have been connected to enzymes encoded in specific ORFs, except for the one responsible for ligation of coenzyme A (CoA) to 4-hydroxybutyrate (4HB). While several candidates for this step have been identified through bioinformatic analysis of the M. sedula genome, none have been shown to catalyze this biotransformation. Transcriptomic analysis of cells grown under strict H2-CO2 autotrophy was used elucidate additional candidate genes involved in carbon fixation and identify the genes which encode for 4HB-CoA synthetase. Three slide loop for Mse cells includes 3 conditions tested in duplicate (biological repeats from tandem fermentors): autotrophic carbon limited (ACL), autotrophic carbon rich (ACR), and heterotrophic (HTR). Half of an RNA sample for one condition was labeled with Cy3 while the other half was labeled with Cy5. The two differently labeled samples were run on different slides. Each probe is spotted on each slide 5 times (5 replicates; spot intensities for all replicates on slide provided in associated raw data file).
Project description:Metallosphaera sedula is an extremely thermoacidophilic archaeon that grows heterotrophically on peptides, and chemolithoautotrophically on hydrogen, sulfur, or reduced metals as energy sources. During autotrophic growth, carbon dioxide is incorporated into cellular carbon via the 3-hydroxypropionate /4-hydroxybutyrate cycle (3HP/4HB). To date, all of the steps in the pathway have been connected to enzymes encoded in specific ORFs, except for the one responsible for ligation of coenzyme A (CoA) to 4-hydroxybutyrate (4HB). While several candidates for this step have been identified through bioinformatic analysis of the M. sedula genome, none have been shown to catalyze this biotransformation. Transcriptomic analysis of cells grown under strict H2-CO2 autotrophy was used elucidate additional candidate genes involved in carbon fixation and identify the genes which encode for 4HB-CoA synthetase.
Project description:Extremely thermoacidophilic members of the Archaea such as the lithoautotroph, Metallosphaera sedula, are among the most acid resistant forms of life and are of great relevance in bioleaching. Here, adaptive laboratory evolution was used to enhance the acid resistance of this organism while genomics and transcriptomics were used in an effort to understand the molecular basis for this trait. Unlike the parental strain, the evolved derivative, M. sedula SARC-M1, grew well at pH of 0.90. Enargite (Cu3AsS4) bioleaching conducted at pH 1.20 demonstrated SARC-M1 leached 23.78% more copper relative to the parental strain. Genome re-sequencing identified two mutations in SARC-M1 including a nonsynonymous mutation in Msed_0408 (an amino acid permease) and a deletion in pseudogene Msed_1517. Transcriptomic studies by RNA-seq of wild type and evolved strains at various low pH values demonstrated there was enhanced expression of genes in M. sedula SARC-M1 encoding membrane complexes and enzymes that extrude protons or that catalyze proton-consuming reactions. In addition, M. sedula SARC-M1 exhibited reduced expression of genes encoding enzymes that catalyze proton-generating reactions. These unique genomic and transcriptomic features of M. sedula SARC-M1 support a model for increased acid resistance arising from enhanced control over cytoplasmic pH.
Project description:Thermomacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, a M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supra-normal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to up-regulation of 55 genes. Genome re-sequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism or transport. These mutations included 7 non-synonymous substitutions, 4 insertions and 1 deletion. One of the insertion mutations mapped to pseudogene, Msed_1517, and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that include the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula was naturally lacking this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low affinity high velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated spontaneous arsenate resistant mutants derived from CuR1 all underwent mutation in pitA and non-selectively became copper resistant. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching.