FIGURE 1.

Generation and phenotypic analysis of a ΔpilD mutant strain of Synechocystis. (A) Strategy for deletion of pilD (slr1120) by replacement with the chloramphenicol acetyl transferase (cat) cassette. The position of screening primers used in panel (B) is shown. (B) Agarose gel showing PCR products amplified with the pilD_screen_F/pilD_screen_R primer pair with wild type (WT, lane 1) or ΔpilD (lane 2) genomic DNA as template. A larger 1.35 kb PCR product is observed for the ΔpilD mutant compared to the 1.23 kb WT band. Lane M = HyperLadder^TM 1 kb molecular weight marker (Bioline). (C) Growth of the WT, ΔpilD and ΔpilD* (suppressor mutant capable of photoautotrophic growth) in the absence or presence of 5 mM glucose. The originally isolated ΔpilD mutant cannot grow under photoautotrophic conditions; a ΔpsbB mutant that is also unable to grow photoautotrophically is included as a control. (D) Level of (pre)PilA1 in WT, ΔpilD and ΔpilD* in photomixotrophically grown whole-cell extracts determined by immunodetection with anti-PilA1 antibodies (upper panel). The accumulation of prePilA1 in the original mutant is decreased in the suppressor strain. The predicted molecular weights of pre- and processed PilA1 are indicated. The lower panel shows a duplicate Coomassie-stained SDS-PAGE gel to demonstrate approximately equal protein loading of each sample. (E) End-point RT-PCR analysis of pilA1 expression in WT and ΔpilD* showing the transcript is present in both strains. As expected, pilD transcripts were absent from the mutant; the rnpB housekeeping gene is included as a control. Reactions were performed in the presence (+) or absence (–) of reverse transcriptase.