Dataset: Pipeline for phylogenetic analysis of the GlcDEF, GOX/LOX, and tsar genes

Strains

            Six clones each of the open ocean Synechococcus strain WH8102 and the coastal Synechococcus strain CC9311 were obtained by dilution to extinction in SN media [1]. The parent cultures of each organism were obtained from the National Center for Marine Algae (Boothbay Harbor, Maine) and were axenic upon receipt. Six clones of Alteromonas sp. strain EZ55 and Prochlorococcus MIT9312 were also previously obtained and cryopreserved at -80 °C [2]. The EZ55 clones used in our Synechococcus co-cultures were the same 6 clones used in our previous transcriptomic study of MIT9312 [2] in order to maximize the comparability of results between that study and the present study. Co-cultures were initiated by mixing each of the six clones of CC9311 and WH8102 with one of the EZ55 clones.

Culture conditions

            Synechococcus cultures were grown under similar conditions to those described in our previous experiment with Prochlorococcus [2]. Briefly, all cultures were prepared in acid-washed conical-bottom glass centrifuge tubes containing 13 mL of artificial seawater (ASW) amended with nutrient stocks [1] and with acid and/or base to control pCO₂. ASW (per L: 28.41 g NaCl, 0.79 g KCl, 1.58 g CaCl2*2H2O, 7.21 g MgSO4*7H2O, 5.18 g MgCl2*6H2O) was sterilized in acid-washed glass bottles, amended with 2.325 mM (final concentration) of filter-sterilized sodium bicarbonate, then bubbled with sterile air overnight. Synechococcus cultures were grown in SEv (per L: 32 μM NaNO₃, 2 μM NaH₂PO₄, 20 μL SN trace metal stock, and 20 μL F/2 vitamin stock). The primary differences between this medium and the PEv medium used in our earlier Prochlorococcus study are the nitrogen source (NO₃^- vs. NH₄⁺, with molar concentration of N and N:P ratios identical to PEv) and the addition of F/2 vitamins [1]. Carbonate chemistry of each media batch was determined prior to pCO₂ manipulations by measuring alkalinity and pH by titration and colorimetry, respectively [2, 3] and then using the oa function in seacarb package in R to determine how much hydrochloric acid and bicarbonate (for 800 ppm pCO₂) or sodium hydroxide (for 400 ppm pCO₂) was needed to achieve desired experimental conditions [4]. Acid and base amendments were introduced immediately prior to inoculation. Cultures were grown in a Percival growth chamber at 21º C under 150 μmol photons m^-2 s^-1 on a 14:10 light:dark cycle. Synechococcus cultures were grown on a rotating tissue culture wheel at approximately 60 rpm.

For "EZ55 growth experiments with photorespiration metabolites"  and "RNA library preparation and sequencing" details see the related dataset "Synechococcus growth and genetic sequence accessions from pCO2 experiments" https://www.bco-dmo.org/dataset/882390

Detection of glycolate utilization genes

            Several genes involved in the bacterial glycolate utilization pathway (glycolate/lactate oxidase, the 3 subunits of glycolate dehydrogenase, and tartronate semialdehyde reductase) were not annotated in the reference genomes for our organisms so we specifically sought to detect them using a reciprocal BLAST analysis. We retrieved any sequences from each of the four reference genomes with high similarity (E-value < 0.001) to the relevant genes from Escherichia coli and/or Synechococcus elongatus using blastp [7] and then back-matched each retrieved sequence to the E. coli or S. elongatus reference genome. If the reciprocal match was the same gene used in the original BLAST search, we considered the match significant.