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Transcriptional response of the sulfur chemolithoautotroph Thiomicrospira crunogena to dissolved inorganic carbon limitation.

ABSTRACT: The hydrothermal vent gammaproteobacterium Thiomicrospira crunogena inhabits an unstable environment and must endure dramatic changes in habitat chemistry. This sulfur chemolithoautotroph responds to changes in dissolved inorganic carbon (DIC) (DIC = CO(2) + HCO(3)(-) + CO(3)(-2)) availability with a carbon-concentrating mechanism (CCM) in which whole-cell affinity for DIC, as well as the intracellular DIC concentration, increases substantially under DIC limitation. To determine whether this CCM is regulated at the level of transcription, we resuspended cells that were cultivated under high-DIC conditions in chemostats in growth medium with low concentrations of DIC and tracked CCM development in the presence and absence of the RNA polymerase inhibitor rifampin. Induction of the CCM, as measured by silicone oil centrifugation, was hindered in the presence of rifampin. Similar results were observed for carboxysome gene transcription and assembly, as assayed by quantitative reverse transcription-PCR (qRT-PCR) and transmission electron microscopy, respectively. Genome-wide transcription patterns for cells grown under DIC limitation and those grown under ammonia limitation were assayed via microarrays and compared. In addition to carboxysome genes, two novel genes (Tcr_1019 and Tcr_1315) present in other organisms, including chemolithoautotrophs, but whose function(s) has not been elucidated in any organism were found to be upregulated under low-DIC conditions. Likewise, under ammonia limitation, in addition to the expected enhancement of ammonia transporter and P(II) gene transcription, the transcription of two novel genes (Tcr_0466 and Tcr_2018) was measurably enhanced. Upregulation of all four genes (Tcr_1019, 4-fold; Tcr_131, ?7-fold; Tcr_0466, >200-fold; Tcr_2018, 7-fold), which suggests that novel components are part of the response to nutrient limitation by this organism, was verified via qRT-PCR.

SUBMITTER: Dobrinski KP

PROVIDER: S-EPMC3318485 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Transcriptional response of the sulfur chemolithoautotroph Thiomicrospira crunogena to dissolved inorganic carbon limitation.

Dobrinski Kimberly P KP Enkemann Steven A SA Yoder Sean J SJ Haller Edward E Scott Kathleen M KM

Journal of bacteriology 20120210 8

The hydrothermal vent gammaproteobacterium Thiomicrospira crunogena inhabits an unstable environment and must endure dramatic changes in habitat chemistry. This sulfur chemolithoautotroph responds to changes in dissolved inorganic carbon (DIC) (DIC = CO(2) + HCO(3)(-) + CO(3)(-2)) availability with a carbon-concentrating mechanism (CCM) in which whole-cell affinity for DIC, as well as the intracellular DIC concentration, increases substantially under DIC limitation. To determine whether this CCM ...[more]

PMID: 22328671

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Project description:The hydrothermal vent gammaproteobacterium Thiomicrospira crunogena inhabits an unstable environment and must endure dramatic sweeps in habitat chemistry. This sulfur chemolithoautotroph responds to changes in dissolved inorganic carbon (DIC; = CO2 + HCO3- + CO3-2) availability with a carbon concentrating mechanism (CCM), in which whole-cell affinity for DIC, as well as the intracellular DIC concentration, increase substantially under DIC-limitation. To determine whether this CCM is regulated at the level of transcription, cells cultivated under high-DIC conditions in chemostats were resuspended in growth medium with low concentrations of DIC, and CCM development was tracked in the presence and absence of RNA polymerase inhibitor rifampicin. The induction of the CCM, as measured by silicone oil centrifugation, was hindered in the presence of rifampicin. Similar results were observed for carboxysome gene transcription and assembly, as assayed by qRT-PCR and transmission electron microscopy, respectively. Genome-wide transcription patterns, assayed via microarrays, were compared for cells grown under DIC-limitation versus ammonia limitation. In addition to carboxysome genes, two novel genes (Tcr_1019 and Tcr_1315), present in other organisms including chemolithoautotrophs, but whose function(s) have not been elucidated in any organism, were found to be upregulated under low-DIC conditions. Likewise, under ammonia limitation, in addition to the expected enhancement of ammonia transporter and PII-gene transcription, the transcription of two novel genes was measurably enhanced (Tcr_0466 and Tcr_2018). Upregulation of all four genes was verified via qRT-PCR (Tcr_1019: 4-fold; Tcr_1315: ~7-fold; Tcr_0466: >200-fold; Tcr_2018: 7-fold), suggesting novel components are part of the response to nutrient limitation by this organism. In this study Thiomicrospira crunogena cells were grown under inorganic carbon limitation and ammonia limitation (high inorganic carbon concentrations) to examine genome wide transcription responses

Project description:Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) with CO2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotroph Thiomicrospira crunogena has a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes of T. crunogena cultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, were at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded by Tcr_0854; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853-encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla.IMPORTANCE DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among the Bacteria and Archaea In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotroph T. crunogena were identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla in Bacteria and also in one phylum of Archaea, the Euryarchaeota Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genus Thiomicrospira.

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Project description:Candidatus Pelagibacter ubique is the most abundant marine microorganism, but is unable to utilize inorganic sulfur compounds that are plentiful in the ocean. To investigate how these cells adapt to organic sulfur limitation, batch cultures were grown in defined media containing either limiting or non-limiting amounts of dimethylsulfoniopropionate (DMSP) as the sole sulfur source. Protein and mRNA expression were measured during exponential growth, immediately prior to stationary phase, and in late stationary phase. Two distinct responses were observed: one as DMSP approached exhaustion, and another after the DMSP supply was depleted. The first response was characterized by increased transcription and translation of all Ca. P. ubique genes downstream of previously confirmed S-adenosyl methionine (SAM) riboswitches: bhmT, mmuM, and metY. These genes were up to 33 times more abundant during low DMSP conditions and shunt all available sulfur to methionine. The osmotically inducible organic hydroperoxidase OsmC was the most up-regulated protein as DMSP (an osmolyte) became scarce. The second response, during sulfur-depleted stationary phase, saw increased transcription of the heme c shuttle ccmC and two small genes of unknown function (SAR11_1163 and SAR11_1164) which were 6-10 times higher in sulfur-starved cultures. No known membrane transporters were up-regulated in response to sulfur limitation, suggesting that this bacterium's strategy for coping with sulfur stress focuses on intracellularly redistributing, rather than importing, organic sulfur compounds. This supports the conclusion that the few organosulfur molecules that Ca. P. ubique is able to metabolize are rarely limiting in the marine environment. Batch cultures of P. ubique were grown in a defined arificial seawater media. Five cultures were amended with a limiting concentration of DMSP as the sole sulfur source and another four control cultures were amended with a non-limiting DMSP concentration. Cultures were harvested for microarray analyses at multiple timepoints for the purpose of observing differences in gene expression related to sulfur limitation. Proteomic analyses were conducted in parallel and are available at https://www.ebi.ac.uk/pride/archive/projects/PXD003672 .

Dataset Information

Transcriptional response of the sulfur chemolithoautotroph Thiomicrospira crunogena to dissolved inorganic carbon limitation.

Publications

Transcriptional response of the sulfur chemolithoautotroph Thiomicrospira crunogena to dissolved inorganic carbon limitation.

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