Transcriptional analysis and mutation of a dnaA-like gene in Synechocystis sp. strain PCC 6803

Abstract

Transcription of the dnaA gene of the cyanobacterium Synechocystis sp. strain PCC 6803 is light dependent and yields a monocistronic mRNA, as determined by Northern analysis. Surprisingly, mutants with inactivated dnaA were viable. In batch cultures under standard conditions, the mutants grew like the wild type and did not show an aberrant phenotype. We conclude that, unlike the situation in other bacteria, dnaA of Synechocystis sp. cannot have an essential function, such as initiation of DNA replication.

FIG. 1

Northern analysis of dnaA, orf134, and psbDC from Synechocystis sp. under light and dark conditions. Samples for RNA isolation were collected at the end of a 12-h dark period and at 2-h intervals during a following 12-h light period. (A) RNA is a negative image of total RNA stained with SYBRgreen; sizes of rRNA fragments are indicated (14). dnaA, orf134, and psbC are images of Northern blots with total RNA of the samples that were probed by ³²P-labeled antisense transcripts of dnaA, orf134, and psbC. Arrowheads indicate the locations of full-length transcripts. Images were generated by ImageQuant software. (B) Comparison of the relative mRNA levels from dot blots in which total RNA of a 6-h light sample was hybridized by dnaA, orf134, and psbC antisense transcripts.

FIG. 2

Physical map and Southern analysis of a dnaA knockout mutant of Synechocystis sp. (A) Restriction map of the dnaA region and the Km^r cassette (aph gene) insertion site. dnaA is a dark gray box, aph is a light gray box, and orf134 (upstream of dnaA) is a blank box. The fragment which is replaced in the mutant is shown as a blank box with dotted borders. Fragments used as probes in Southern analysis are represented by bars with the same shading. (B) Southern analysis of the dnaA region. Southern blots with cleaved chromosomal DNAs from the wild type and the mutant were hybridized with the following probes: full-length dnaA fragment, internal dnaA fragment and Km^r cassette. Lanes: 1 to 4, wild-type DNA cleaved with HindIII (lane 1), HindIII/EcoRV (lane 2), HindIII/XbaI (lane 3), or XbaI (lane 4); 5 to 8, mutant DNA cleaved with HindIII (lane 5), HindIII/EcoRV (lane 6), HindIII/XbaI (lane 7), or XbaI (lane 8). M, digoxigenin-labeled DNA molecular weight marker III (Boehringer). Fragment (frag.) sizes in kilobases are indicated on the left.