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, ϳ7-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.

Functions, Compositions, and Evolution of the Two Types of Carboxysomes: Polyhedral Microcompartments That Facilitate CO 2 Fixation in Cyanobacteria and Some Proteobacteria

“…It has been speculated that extracellular or cytoplasmic CA enzymes may play a role in CO 2 fixation by facilitating the interconversion of carbon species. However, in T. crunogena, noncarboxysomal ␣-CA and ␤-CA enzymes (note that the ␣-, ␤-, and ␥-classes of CA enzymes mentioned in this review are a separate classification system from that for carboxysomes and cyanobacteria) were recently shown not to have a direct role in CO 2 fixation (72). Similarly, the EcaB ␤-CA enzyme from Synechocystis strain PCC 6803 is probably not involved in CO 2 fixation, though its periplasmic targeting suggests that it may play some role in external exchange of Ci species (73).…”

Proteomic and Mutant Analysis of the CO 2 Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena

“…In most cases, these proteins corresponded to genes with elevated transcript abundances under low-DIC conditions (Table 5) (20). Proteins corresponding to three abundant transcripts (Tcr_0843, 0845, 0853) could not be detected in the proteome (Table 5); either their expression was posttranscriptionally regulated, or they were present but not quantified due to analytical limitations.…”

“…Cells were cultivated under either DIC or NH 3 limitation, as previous work had demonstrated that DIC limitation induced the expression of a CCM in this organism while NH 3 limitation did not (15,20,22). To cultivate cells under DIC limitation, TASW was prepared with 2 mM NaHCO 3 and the growth chamber was pulsed with O 2 to maintain oxygen tensions at 5 to 20% atmospheric saturation using the BioFlo dO 2 controller.…”

“…In T. crunogena, the genes encoding the carboxysomal components of the CCM (shell proteins, carboxysomal RubisCO, and carbonic anhydrase) are carried in its genome (10), and these genes are upregulated under low-DIC conditions (20). In contrast, the DIC uptake mechanism is not apparent from the genome sequence.…”

“…In contrast, the DIC uptake mechanism is not apparent from the genome sequence. Orthologs to cyanobacterial genes encoding HCO 3 Ϫ transporters are absent (10,21), and upregulation of genes likely to encode membrane transporters was not apparent when assayed by oligonucleotide microarrays (20). The T. crunogena genome encodes a periplasmic carbonic anhydrase, which does not appear to play a role in DIC uptake under low-DIC conditions.…”