Construction of the nfi-mutant and its use in shotgun sequencing. (A) Schematic of the nfi gene knockout strategy. The PCR product (donor DNA) containing 55 bp upstream homologous extensions of the nfi gene (H1) and 55 bp downstream homologous extensions of the nfi gene (H2) was prepared using the pKD3 plasmid as template. The nfi gene in the chromosome of E. coli DH5α is replaced with chloramphenicol resistant gene (cat) by Red recombination of H1 and H2. cat is then eliminated by subjecting the FLP recognition target (FRT) sites to FLP recombination; a single FRT site is retained. Nfi-F and Nfi-R are primers indicating the change in the nfi gene locus. The length of the region between Nfi-F and Nfi-R primers is 821 bp (nfi remain), 1,169 bp (nfi is replaced by cat), or 237 bp (cat is eliminated). (B) PCR verification using Nfi-F and Nfi-R primers. Lane 1. Wild-type E. coli DH5α (nfi remain). Lane 2. nfi is replaced with cat. Lane 3. cat is eliminated. (C) Distribution of eight newly obtained contigs in the PaP1 genome. These eight contigs were obtained by shotgun sequencing of the PaP1 genome using E. coli DH5α Δnfi as the host to construct shotgun library clones. The blue rectangular boxes represent contigs. The exact location of each contig is indicated by blue boxes.