The complex transcriptional response of Acaryochloris marina to different oxygen levels

Ancient oxygenic photosynthetic prokaryotes produced oxygen as a waste product, but existed for a long time under an oxygen-free (anoxic) atmosphere, before an oxic atmosphere emerged. The change in oxygen levels in the atmosphere influenced the chemistry and structure of many enzymes that contained prosthetic groups that were inactivated by oxygen. In the genome of Acaryochloris marina, multiple gene copies exist for proteins that are normally encoded by a single gene copy in other cyanobacteria. Using high throughput RNA sequencing to profile transcriptome responses from cells grown under microoxic and hyperoxic conditions, we detected 8446 transcripts out of the 8462 annotated genes in the Cyanobase database. Two thirds of the 50 most abundant transcripts are key proteins in photosynthesis. Microoxic conditions negatively affected the levels of expression of genes encoding photosynthetic complexes, with the exception of some subunits. In addition to the known regulation of the multiple copies of psbA, we detected a similar transcriptional pattern for psbJ and psbU, which might play a key role in the altered components of photosystem II. Furthermore, regulation of genes encoding proteins important for reactive oxygen species-scavenging is discussed at genome level, including, for the first time, specific small RNAs having possible regulatory roles under varying oxygen levels. Overall design: Acaryochloris marina MBIC11017 was routinely kept in a culture room at 27°C under 15–30 µmol photons•m?2•s?1 of cool white light. Sterilized K+ES (artificial seawater), buffered with 25 mM TES at pH 8.0 was used as the culture medium for all three treatment groups. To make sure photosynthesis was not limited by CO2, NaHCO3 was dissolved in a small volume of autoclaved media, and injected into the enclosed culture flasks every two days (yielding an initial concentration of 0.375 mM). The initial cell density of all culture groups was adjusted to an optical density at 750 nm of 0.2. The cultures were shaken on an orbital flat-bed shaker at ~90 rpm. Cultures under normal O2 levels were inoculated in 1-l Erlenmeyer glass flask containing 500 ml of medium, capped with a cotton stopper that permitted gas exchange. Thus, the O2 concentration of the gas phase inside the bottle was similar to atmospheric levels ~21% (v/v). Microoxic conditions were achieved by using a 500-ml two-necked round-bottom flask sealed tightly by a rubber stopper. Cultures were vacuumed, and refilled with pure nitrogen gas (99.95% purity) to ensure normal atmospheric pressure. We repeated this process several times, yielding a final O2 concentration of <0.2% inside the sealed culture flask. To maintain a microoxic condition, a positive pressure was created by bubbling nitrogen through the culture. A similar set-up was used for hyperoxic conditions, with the exception that pure O2 gas was used to refill the flask after vacuuming. Because the cells generate O2 under illumination, ongoing input of O2 gas to maintain the high-oxygen concentration was not required. However, to avoid pressure build-up, the flask was re-vacuumed and refilled with O2 gas every 48 h, as described above. The O2 concentration inside the flask remained within the range of 65–75% (v/v) during the experiment. Acaryochloris cultures were harvested after five days from all three different treatments. The experiment was triplicated under ambient conditions, and duplicated under microoxis and hyperoxis conditions. The harvested cell pellets were mixed with TRIzol (TRIzol® Reagent, Life Technologies, Australia), and frozen immediately using liquid nitrogen. They were stored at ?80°C for at least 60 min. The frozen samples were thawed in a water bath of 37°C, and spun down at 16,000×g for 5 min to eliminate cellular debris. This supernatant was mixed at a volume ratio of 4:1, with chloroform and spun down at 16,000×g for 10 min at 4°C. The upper layer, containing RNA and DNA, was carefully transferred to a new tube, without disturbing the white middle layer of solid components. The RNA was precipitated by addition of an equal volume of isopropanol and incubated at ?20°C for at least 45 min. RNA pellets were washed with 70% (v/v) ethanol and the DNA removed using the Baseline-ZERO™ DNase kit (Epicentre, WI, USA), following the manufacturer''s instructions. Prior to RNA quality assessment, the absence of DNA was confirmed by polymerase chain reaction (data not shown). The quality of RNA was assessed on a 2100 Bioanalyzer (Agilent Technologies, CA, USA), using a RNA 6000 Nano RNA Kit (Agilent Technologies) to obtain an RNA integrity number of >8.0. Transcripts corresponding to rRNAs (5S, 16S and 23S) were reduced from the samples with a Ribo-Zero Kit (Epicentre), following the manufacturer''s instructions. Further processing, including quality assessment, was undertaken prior to RNAseq analysis by Beijing Genomics Institution, China.