3.4.4. Modification of LPS

The disruption of polymyxin interactions with negatively charged phosphate groups of lipid A of LPS is a commonly observed mechanism of COL-R [34]. Several genes involved in the modification of LPS were observed for both lifestyles through selection, including *mgrB*, *phoQ*, *phoP*, and *pmrB*. Two-component transduction systems PmrAB and PhoPQ regulate the modifications of LPS in response to colistin or a decline or rise in Mg2+ and Fe3+ levels, respectively [35]. Activation of the PhoPQ signaling system leads to the synthesis of small regulatory transmembrane protein MgrB, which functions as a negative feedback regulator of PhoPQ systems [36]. SNPs in *phoQ* were acquired early in planktonic KP, by days 2, 3, and 27 for populations 1–3. A SNP in *pmrB* was acquired by day 2 and persisted until day 27 in population 3, while another SNP in *pmrB* was generated by day 36 in population 1. Mutations in *phoP* were generated by days 27 and 15 for populations 1 and 3. A single SNP in *mgrB* was observed on day 27 for population 1 at 71% frequency (Table 3). *ArnC*, which encodes undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase, was also

modified in planktonic population 3 by day 36 (81%). ArnC is one of several enzymes involved in adding an amino sugar L-Ara4N to lipid A, which disrupts the interaction of cationic peptides with LPS, leading to rapid colistin resistance [37].

**Figure 4.** Timing of colistin-resistant mutations by functional role for independent KP lifestyles. Mutations are listed in relation to the first appeared time during evolution and color-coded by functional role to compare COL-R resistance patterns in planktonic and biofilm lifestyles. The timing in which shared mutations are generated and their corresponding functions can be compared between planktonic and biofilm-evolved KP. The colors in the bars are used to identify the individual or combinations of mutations generated at a similar timeframe throughout colistin selection. There is no connection between the color scheme used here and in other figures in this manuscript.

For biofilm populations, SNPs in *phoQ* and *pmrB* were generated by day 6 in populations 1 and 3, while a single SNP in *phoP* was generated on day 36 in population 3. Multiple SNPs in *phoQ* were generated in biofilm population 2 at unique positions, with each additional mutation aligning with a substantial rise in MIC (Table 4). A single deletion in *mgrB*–*kdgR* was generated on day 30 and was subsequently fixed. Overall, it appears that mutations involved in LPS modifications are observed early on (by days 2–6) in selection and reappear at later timepoints (days 27–36) for both planktonic and biofilm populations. Additionally, mutations involved in LPS biosynthesis are generated around or shortly following modifications in LPS (Figure 4).
