Growth and mutant construction.

All cyanobacterial strains used in this study are listed in Table 2. Synechocystis sp. PCC 6803 strains were grown in 100-ml flasks containing BG-11 medium (15), whereas Cyanothece sp. ATCC 51142 (16) was grown in ASP2 medium (17, 18), both with shaking at ∼125 rpm with illumination of ∼30 to 40 μmol of photons m⁻²s⁻¹ of light from cool white fluorescent bulbs. For experimental procedures, cultures were inoculated in identical media and, when stated, modified by subculturing in nitrate-free medium (BG-11 NF) or in medium with 0.01× nitrate. Growth was assayed using absorbance at 730 nm (Perkin-Elmer UV-Vis Lambda 40 spectrophotometer).

Bacterial strains and plasmids used in this study

To generate mutations, genomic DNA was extracted from 100 ml of culture (∼1 × 10⁸ cells/ml). A Braun homogenizer and 0.5 ml of glass beads (0.1 to 0.2 mm) were used to lyse cells with four 30-s bursts at high speed, followed by centrifugation. The supernatant was then transferred to new tubes and sequentially washed by vortexing with 500 μl of phenol and chloroform. DNA was precipitated by adding 2 vol of ice-cold absolute ethanol. For the branching-enzyme constructs, pUC19 was used as a cloning backbone for sequential insertion of ∼500-nucleotide (nt) fragments located immediately upstream of the native start codon and downstream of the coding sequence of the branching-enzyme gene sll0158. First, the upstream fragment was amplified using primers BEup/F and BEup/R and inserted in the PstI and XmaI sites. The downstream region, amplified using primers BEdown/F and BEdown/R, was inserted into the XmaI and EcoRI sites. The coding sequence of sll0158 was PCR amplified with forward primers BE97/f and BE112/f and reverse primer BE/R1. These primers amplified the gene region corresponding to the 97th or 112th amino acid, introducing a start codon in either amino acid position 97 (yielding mutant gene sll0158Δ97) or 112 (yielding mutant gene sll0158Δ112), respectively. Mutant sll0158 fragments were inserted between the upstream and downstream fragments using XmaI and BglII sites. A kanamycin resistance cassette from pRL448, amplified using primers BEKanF and BRKanR, was used for positive selection and inserted between the truncated branching-enzyme gene and downstream region.

A procedure similar to that described above was used to replace the native debranching enzyme (DBE) encoded by the gene slr0237 with the DBE from Cyanothece sp. PCC 8801. Upstream and downstream fragments of the native DBE were amplified using primers U0237F/R and D0237F/R and inserted via the BamHI and SalI sites and SalI and PstI sites in pUC19, respectively. The coding sequence for the DBE from Cyanothece sp. PCC 8801 was amplified using the primer pair 8801-1045F/8801-1045R and inserted in between the upstream and downstream fragments via a SalI site. Next, a spectinomycin cassette (∼2 kb), isolated from the plasmid pRL453, was inserted between the DBE gene, PCC8801_1045, and the downstream fragment of slr0237 at the EcoRI site. Synechocystis sp. PCC 6803 was transformed using standard transformation protocols (19). Transformed colonies were selected on antibiotic plates and transferred over 2 to 4 months for segregation. Full segregation of the mutants was confirmed by PCR and at regular intervals thereafter. Sequences for primers used can be found in Table 3.

Primer sequences used in study