Steady-state cell culture from each replicate chemostat (45 ml) was rapidly fixed with 5 ml of phenol–ethanol solution (5% vol/vol). Cells were collected by centrifugation at 4,500 × g and the supernatant was removed. Loose cell pellets were frozen at −70 degree celcius.
Growth protocol
Ruegeria pomeroyi strain DSS-3 was grown in 320 ml custom-made chemostats with a culture volume of 200 ml. Steady-state biomass was limited by ammonium (NH4Cl, 0.26 mM) or potassium phosphate (KH2PO4, 9.2 μM). Cell cultures limited by ammonium were supplied with an excess of D-ribose-5-phosphate (C5H9Na2O8P*2H2O, 0.5 mM), methyphosphonic acid (CH5PO3, 0.5 mM), or potassium phosphate (KH2PO4, 0.5 mM); those limited by potassium phosphate were supplied with an excess of ammonium (NH4Cl, 2.8 mM). Other non-limiting nutrients were provided in identical amounts for all cultures. The culture medium was buffered with 10 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane and added with vitamins and trace metals (see Table A1 in the Appendix in Chan et al., 2012, Frontiers in Microbiology, 3:159. doi: 10.3389/fmicb.2012.00159 for components of cell culture medium). The feed medium was added to each chemostat culture at a constant rate (8.4 ml per hour), and spent medium and cells were removed at the same rate. During the incubation, cell cultures were mixed by constant stirring. Growth temperature was maintained at 30 degree celsius using a circulating water bath. Salinity was constant at 25. Oxygen was bubbled into the culture at a flow rate of 2 ml per minute through an aquarium air pump, controlled by a flow meter. The pH ranged from 6.6 to 7.0 during all growth regimes. Cell cultures were considered to be at steady-state when the change in OD600 was less than or equal to 10% between two successive measurements. All cultures reached steady-state after three volume exchanges. Cells were harvested after five volume exchanges. At steady-state, R. pomeroyi DSS3-3 has a doubling time of 16.5 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA from thawed steady-state cell culture previously fixed with phenol-ethanol solution was purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s procedures. DNA was depleted by TURBO DNase treatment (Applied Biosystems/Ambion, Austin, TX, USA). Ribosomal RNA was depleted with MicrobeExpress (Applied Biosystems/Ambion), according to manufacturer's recommendations. Purified mRNA was amplified with MessageAmp II-Bacteria Kit (Applied Biosystems/Ambion) with procedures as described in the manufacturer's manual.
Label
AlexaFluor dye 647 (red emission)
Label protocol
Amplified mRNA was labeled with AlexaFluor 647 dye (Invitrogen, Carlsbad, CA, USA; red emmision) according to manufacturer's recommended procedures. Amplified and labeled mRNA was purified with the MEGAclear kit (Applied Biosystems/Ambion, Austin, TX, USA) and concentrated by ethanol precipitation.
Hybridization protocol
Labeled and purified mRNA samples were hybridized to custom microarray slides manufactured by Combimatrix (Mukilteo, WA, USA; GEO platform ID GPL4067) and imaged according to the procedures described in the manufacturer's protocol. Hybridized slides were stripped according to Combimatrix's protocol and re-used up to three times.
Scan protocol
Axon GenePix 4000B microarray scanner (Molecular Devices Corporation, Sunnyvale, CA, USA) at 5 µm resolution (450-550 PMT).
Description
Biological replicate 1 of 3 from the potassium phosphate limited growth regime
Data processing
The procedures were modified from Bürgmann et al., 2007, Environmental Microbiology, 9: 2742-2755. Each gene was represented by up to two probes and each probe was spotted up to three times on the array (Bürgmann et al., 2007; see platform ID GPL4067 for the array design). Each hybridized spot was manually examined in GenePix Pro 6.0 Software (Molecular Devices, Sunnyvale, CA, USA) for quality. The median fluorescence value (F635 Median; see raw data files in the Supplement) for each hybridized spot was subtracted from the median fluorescence value of the five closest empty spots (B635 Median; see raw data files in the Supplement), resulted in the values specified in the PRE_VALUE columns in the Data Tables. Spots were flagged as bad with Acuity 4.0 (Molecular Devices) when they were unevenly hybridized (determined manually by checking the hybridization circularity), when the detected fluorescence minus the background fluorescence was less than two standard deviations of the background fluorescence (F635 Median-B635 Median <2 SD(B635); see raw data files in the Supplement), or when the signal to noise ratio (SNR635; see raw data files in the Supplement) was <3. Flagged spots are indicated by 'null' in the Data Tables. For the remaining spots with good hybridization signals, those targeting 5S rRNA, 16S rRNA, 23S rRNA, mismatch probes, and manufacturer's control probes (see platform ID GPL4067 for the array design) were removed. Then, fluorescence values from all spots (up to three) targeting a same probe were averaged and combined and used for normalization. The six arrays described in this accession were analyzed together with seven arrays previously deposited: GEO Series GSE27032 (platform GPL4067), samples GSM665914-GSM665917 (NH4Cl-limited, 0.26 mM; KH2PO4 excess, 0.50 mM; three biological replicates and one technical replicate) and samples GSM665918-GSM665920 (NH4Cl excess, 2.8 mM; KH2PO4-limited, 9.20 uM; three biological replicates); these seven samples were obtained from steady-state R. pomeroyi DSS-3 cells cultured using identical protocols and medium, but with different ammonium and phosphorus compounds and concentrations. Quality controls of the microarray hybridizations for these seven samples were performed with procedures identical to the six arrays described here. After quality controls, fluorescence values of probes from the two ammonium-limited, KH2PO4 excess technical replicates (samples GSM665914 and GSM665915) were averaged and combined. In total, the analysis resulted in 12 samples from four treatment regimes. Probes were removed across all 12 arrays if flagged spots were present in all triplicate samples within a treatment regime. This filtering step resulted in 5,650 probes for all 12 arrays and their fluorescence values (originated from everaging and combining the PRE_VALUEs from spots representing the same probes) were used for quantile normalization (Bolstad et al., 2003, Bioinformatics 19:185-193), resulted in the normalized fluorescence values (VALUE) in Data Tables. Some consecutively listed spots (each spot is represented by a unique ID_REF) in Data Tables are indicated with 'null' becuase these are spots that either did not pass quality controls or spots that passed quality controls but their normalized fluorescence values are listed with their corresponding set of probes in the rows immediately above. ID_REF- Each ID_REF represents a spot on the array (as specified in column 1 in the platform GPL4067). ID_REFs targeting 5S rRNA, 16S rRNA, 23S rRNA, mismatch probes, and manufacturer's control probes were removed. Each probe was spotted up to three times on the array; thus, a probe_ID can have up to three different ID_REFs. probe_ID- Each probe_ID represents a probe (as specified in column 2 in the platform GPL4067). Each probe was spotted up to three times on the array. PRE_VALUE- F635 Median minus B635 Median fluorescence values (not normalized). 'Null' indicates spots that did not pass quality controls. In the ammonium-limited, potassium phosphate excess biological replicate 1 of 3 sample, values for both technical replicate microarray hybridizations (PRE_VALUE_1, originated from IPErep1_1.gpr, and PRE_VALUE_2, originated from IPErep1_2.gpr) are shown. VALUE- Quantile-normalized fluorescence values. Fluorescence values of probes from 12 arrays originated from four treatment regimes were used for normalization (see procedures described in Data processing). Prior to normalization, good quality fluorescence values from all spots (up to three) targeting the same probes were averaged and combined. Fields for some consecutively listed ID_REFs (each ID_REF represents a spot on the array) are indicated with 'null' becuase these are spots that either did not pass quality controls or spots that passed quality controls but their normalized values are listed with their corresponding set of probes in the rows immediately above.
Global gene expression of Ruegeria pomeroyi DSS-3 during ammonium-limited, ribose phosphate-, methylphosphonate-, or potassium phosphate-excess growth regimes or during potassium phosphate-limited, ammonium-excess regime
