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. 2023 Jul 5;13:10876. doi: 10.1038/s41598-023-37341-4

Figure 6.

Figure 6

Influence of ISs in the long-term and short-term adaptation of 2A sublineage strain CCM 4253 to an energy substrate switch. (A) Ordered CCM 4253 pseudochromosome and genome resequencing Circos maps showing replicative transposition events (1) occurring during the growth of iron- (green) and sulfur-adapted (purple) cultures. Concentric lanes represent the reads coverage of (2) original sequencing, (3) iron-grown cultures resequencing, technical replicates, and (4) sulfur-grown resequencing cultures, technical replicates. ISs (grey, Supplementary Table 5A), except ISAfe1, highlighted in orange. Supporting alignments in bam format and coverage values are provided in Supplementary Table 5C and Figshare (https://doi.org/10.6084/m9.figshare.20523591). (B) Standard organization of the pstI and pstII operons and mutant ∆pstII operon in the A. ferrooxidans CCM 4253 chromosome. The position of the ISAfe1 element inserted into the open reading frame of the pstC2 gene is indicated. Putative promoter regions (− 10 and − 35), PhoB-binding sites, ribosome binding site (RBS), and direct repeats (DR) are indicated. Lowercase letters indicate the sequence of the pstC2 gene, while uppercase letters indicate the sequence of the IS element. The scales under each operon indicate the sequence size (bp). (C) PCR evaluation of the transpositional mutation in the long-term iron- and sulfur-adapted cultures, as shown in Supplementary Fig. 2 (Experiment 1). Lane M represents a 1-kb DNA ladder; lane Fe2+ represents the PCR product of the pstC2 locus from the iron-adapted culture; lane S0 represents the PCR product of the pstC2 locus from the sulfur-adapted culture. The gel image has been cropped for display. The original gel image is shown in Supplementary Fig. 2D. (D) PCR evaluation of reversal of the transpositional mutation during subsequent culture transfers, as shown in Supplementary Fig. 2 (Experiment 3). Lane M represents a 1-kb DNA ladder; lanes 1–6 represent the PCR product of the pstC2 locus from the first to sixth individual iron passages in Fig. 6E. The gel image has been cropped for display. The original gel image is shown in Supplementary Fig. 2E. (E) The long-term sulfur-adapted A. ferrooxidans CCM 4253 mutant strain ΔpstC2 passaged on ferrous iron repeatedly, as shown in Supplementary Fig. 2 (Experiment 3). The first (purple circle), second (dark blue triangle), third (light blue square), fourth (green diamond), fifth (light green inverted triangle), and sixth (yellow circle) iron passage.

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