Fig. 1.
Functional analysis of the replication system of plasmid pIGRK. a Nucleotide sequence of the REP elements: single-strand initiation site (ssi), conserved region (CR), iteron-like sequences (IT), inverted repeats (IR), short direct repeats (DR) and proximal part of the repR gene. Predicted PrepR hexamers − 10 and − 35 (highlighted by single underlining) and the transcription start site (+ 1, in red) are indicated. The yellow arrow marks the IS1 integration site. The putative ribosome binding site is in bold and the RepR START codon (atg) is in bold and underlined. The double underlined sequence indicates the DNA region indispensable in cis for replication. The nucleotide coordinate numbering is compatible with GenBank accession AY543071.1. b Genetic organization of pIGRK. Elements indispensable in cis and in trans for replication are indicated. Operator and enhancer elements of the PrepR promoter are indicated. For more detail see [Additional file 1, Figure S1]. c Sequencing result for the repR 5′-RACE product. The chromatogram represents the repR template strand. The oligo d(G) primer sequence is indicated and the predicted repR transcription start site is marked by + 1. d Analysis of PrepR activity and regulation. Lines represent DNA fragments of pIGRK and their mutated versions. Yellow arrow marks the IS1 integration site. (T) – Tpro/Tlyz transcriptional terminator of P1, del. 4 bp – frameshift mutation introduced within the HindIII site, gtc(V69 V) – single nucleotide mutation (in red). pRS551 – “empty” test vector. e β-Galactosidase activity in strains carrying the constructs described in panel (d), reflecting the strength of the promoter