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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:Synechocystis 6803 cells was grown photoautotrophically at 32 °C buffered in BG-11 and bubbled with 3% CO2. A relatively mild Ci stress was applied by switching the aeration from 3% CO2 to air alone. After incubation under designated conditions, a 100-ml aliquot of culture was immediately combined with an equal volume of ice-cold mixture of phenol and ethanol (1:10, w/v) in an ice bath. The resultant cells were collected by centrifugation at 1000 x g for 10 min at 4 °C. Total RNA was isolated with RNeasy Midi Kit (Qiagen, Valencia, CA) and further treated with the DNA-free kit (Ambion, Austin, TX). Fluorescently labeled cDNA was produced via a two-step indirect procedure involving cDNA synthesis from 16 µg of total RNA in a reverse transcriptase reaction incorporating aminoallyl-modified deoxynucleotide, followed by the second step involving chemical coupling of fluorescent dye to the introduced amino moieties of the synthesized cDNA. Labeled cDNA were adjusted to 14.75 µl, and the remainder of the hybridization components containing 2.5 µl of 10 µg µl-1 salmon sperm DNA, 8.75 µl of 20x SSC, 0.25 µl of 10% SDS, and 8.75 µl of formamide were added. The mixture was then heated for 2 min at 99 °C and maintained at 42 °C until hybridization. Hybridizations were preformed in a static incubator at 42 °C for 12-16 h then washed by placing in a 250-ml solution of 2x SSC and 0.1% SDS at 42 °C for 5 min with gentle agitation provided by rotation of a magnetic stir bar. The slide was transferred quickly to a 250-ml solution of 0.1x SSC and 0.1% SDS, incubated for 10 min at room temperature with gentle agitation, and washed five additional times in 0.1x SSC for 1 min at room temperature. Hybridization signals from the microarray were quantified using GenePix Pro 4.1 (Axon Instruments, Union City, CA). The quality control procedures were conducted in the image analysis software, and then data were saved to Acuity 3.1 (Axon Instruments). Keywords: time-course

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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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