MOESM2 of An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species

Additional file 2. Schematic representation and comparison of the original and modified VFAE procedures

Bacterial strains and plasmids used in this study.

a<p>phenol⁺, growth on phenol; phenol⁻, no growth on phenol; Ap^R, ampicillin resistance; Tc^R, tetracycline resistance; Km^R, kanamycin resistance.</p

Primers used in this study.

a<p>Restriction sites for BamHI (5′-GGATCC-3′), HindIII (5′-AAGCTT-3′), EcoRI (5′-GAATTC-3′), and SalI (5′-GTCGAC-3′) are shown in bold and italic.</p

Analysis of the promoter region and transcriptional start site for mphK.

<p>(A) Intergenic region between <i>mphR</i> and <i>mphK</i>. The first nucleotide in the transcript is shown in bold. Arrows and dashed arrows indicate incomplete inverted repeats (named IR1, IR2, and IR3) and the translational start codons for <i>mphR</i> and <i>mphK</i>, respectively. Double-underlines mean the putative σ⁵⁴-dependent −12/−24 promoter consensus sequence and the σ⁷⁰-dependent −10/−35 promoter consensus sequences. An arrowhead with +1 indicates the transcriptional start site for <i>mphK</i>. A grey-shaded box indicates the putative ribosome-binding site for the <i>mphK</i> transcription. (B) Mapping of the transcriptional start site for <i>mphK</i> by primer extension. A sequence ladder was generated with the same primer (lanes T, A, C, and G).</p

General features of the <i>D. gobiensis</i> genome.

<p>General features of the <i>D. gobiensis</i> genome.</p

Unrooted Deinococcales neighbor-joining phylogenetic tree deduced from the nucleotide acid sequences of the orthologous proteins that occur in all 14 sequenced strains from the phylum Deinococcus-Thermus.

<p><i>D. gobiensis</i> and <i>D. radiodurans</i> are most closely related. Numbers indicate bootstrap values below 100.</p

<i>D. gobiensis</i> I-0 genome structure.

<p>The seven replicons were opened at sequence position 1 and concatenated. Circle 1, red, chromosome (3.1 Mb); violet, plasmid 1 (P1, 433 kb); indigo, P2 (425 kb); blue, P3 (232 kb); light blue, P4 (72 kb); dark green, P5 (55 kb); light green, P6 (53 kb). Circles 2 and 3, predicted protein coding sequences (CDSs) clockwise and anticlockwise, respectively. Coloring is according to COG. Circle 4, Fold change in the immediate global transcriptional response to UV irradiation for each gene: green, upgulated; red, down-regulated; yellow, not changed significantly. Circle 5, red, rRNA; purple, tRNA; green, ncRNAs (noncoding). Circle 6, blue, genes with homologues in other <i>Deinococcus</i> genomes; red, genes found only in <i>D. gobiensis</i> I-0; other colors, genes with closest homologues in other phyla. Circle 7, deviation from the average 69.15% total genomic GC content: red, higher; blue, lower. Circle 8, previously reported genes that are involved in DNA repair and stress-responses. Circle 9, location of the 23 genomic islands. Circle 10, Mb scale.</p