# A coevolution experiment between Flavobacterium johnsoniae and Burkholderia thailandensis reveals parallel mutations that reduce antibiotic susceptibility

John Chodkowski, Ashley Shade

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John Chodkowski, Ashley Shade. A coevolution experiment between Flavobacterium johnsoniae and Burkholderia thailandensis reveals parallel mutations that reduce antibiotic susceptibility. Microbiology, 2023, 169 (2), pp.001267. 10.1099/mic.0.001267. hal-04277801

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Submitted on 14 Nov 2023

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1 A coevolution experiment between *Flavobacterium johnsoniae* and

2 *Burkholderia thailandensis* reveals parallel mutations that reduce antibiotic

3 susceptibility

4

5 John L. Chodkowski¹ and Ashley Shade¹,²,³*

6 ¹Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA

7 ²Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA

8 ³Program in Ecology, Evolution and Behavior, Michigan State University, East Lansing, MI 48824, USA

9 * Corresponding author and material requests. Email: *shadeash@msu.edu*; ORCID: 0000-0002-7189-3067

10

11 Acknowledgements

12 We thank Dr. Mark McBride for sending us the plasmid pYT354 in DH5αMCR used for recombinant
13 construction and strain HB101 used for triparental conjugation. We also thank Dr. Yongtao Zhu for his consultation
14 in creating *F. johnsoniae* recombinants. Lastly, we thank Dr. Colin Manoil and members of the Manoil laboratory
15 for providing transposon mutants.

16

17 Abstract

18 One interference mechanism of bacterial competition is the production of antibiotics. Bacteria exposed to
19 antibiotics can resist antibiotic inhibition through intrinsic or acquired mechanisms. Here, we performed a
20 coevolution experiment to understand long-term consequences of antibiotic production and antibiotic susceptibility
21 for two environmental bacterial strains. We grew five independent lines of the antibiotic-producing environmental
22 strain, *Burkholderia thailandensis* E264, and the antibiotic-inhibited environmental strain, *Flavobacterium
23 johnsoniae* UW101, together and separately on agar plates for 7.5 months (1.5 month incubations), transferring each
line five times to new agar plates. We observed that the *F. johnsoniae* ancestor could tolerate the *B. thailandensis*-produced antibiotic through efflux mechanisms, but that the co-evolved lines had reduced susceptibility. We then sequenced genomes from the coevolved and monoculture *F. johnsoniae* lines, and uncovered mutational ramifications to the long-term antibiotic exposure. The coevolved genomes from *F. johnsoniae* revealed four potential mutational signatures of reduced antibiotic susceptibility that were not observed in the evolved monoculture lines. Two mutations were found in *tolC*: one corresponding to a 33 bp deletion and the other corresponding to a nonsynonymous mutation. A third mutation was observed as a 1 bp insertion coding for a RagB/SusD nutrient uptake protein. The last mutation was a G83R nonsynonymous mutation in acetyl-coA carboxylase carboxyltransferase subunit alpha (AccA). Deleting the 33 bp from *tolC* in the *F. johnsoniae* ancestor reduced antibiotic susceptibility, but not to the degree observed in coevolved lines. Furthermore, the *accA* mutation matched a previously described mutation conferring resistance to *B. thailandensis*-produced thailandamide. Analysis of *B. thailandensis* transposon mutants for thailandamide production revealed that thailandamide was bioactive against *F. johnsoniae*, but also suggested that additional *B. thailandensis*-produced antibiotics were involved in the inhibition of *F. johnsoniae*. This study reveals how multi-generational interspecies interactions, mediated through chemical exchange, can result in novel interaction-specific mutations, some of which may contribute to reductions in antibiotic susceptibility.

Key words: Burkholderia thailandensis E264, Flavobacterium johnsoniae UW101, coevolution, TolC, efflux, thailandamide, interference competition

42

## 43 Introduction

44 Cultivation-independent sequencing of environmental microbial DNA has revealed the prevalence of 45 antibiotic resistance genes in pristine environments (Allen at al. 2010), indicating that antibiotics and their 46 corresponding resistance mechanisms have long-evolved in natural environments that predate their use in medicine 47 (D'Costa et al. 2006). For example, glycopeptide antibiotics and resistance mechanisms have been present in 48 bacterial genomes for at least 150 million years (Waglechner et al. 2019). Thus, it is expected that understanding the
evolution of antibiotic resistance in “natural” settings will provide insights into emerging mechanisms of antibiotic
resistance that may be useful for addressing resistance in clinical settings (Walsh 2013).

Microbial antibiotic production in the environment is typically viewed through the lens of competition (van der Meij et al. 2017). Bacteria produce antibiotics that interfere directly with competitors by inflicting cell damage (Hibbing et al. 2010). The DNA blueprints for antibiotics are organized in biosynthetic gene clusters (BSGC), in which locally proximal genes collectively encode the pathway for molecule production (Medema et al. 2015). The activation of BSGCs is typically tied to stress regulation (Baral et al. 2018), suggesting antibiotic production can be deployed as a survival strategy when conditions are not optimal for growth (Granato et al. 2019).

Bacteria can survive antibiotic exposure through the upregulation of intrinsic resistance mechanisms and can achieve antibiotic resistance through acquired mechanisms (e.g. Mutation or horizontal gene transfer; Arzanlou et al. 2017). Multidrug efflux pumps are particularly interesting because they can provide both intrinsic and acquired resistance mechanisms (Poole 2004). For example, low-levels of antibiotic exposure can upregulate intrinsic mechanisms of resistance, such as efflux pumps (Du et al. 2018; Frimodt-Møller and Løbner-Olesen 2019). The survival of bacteria to low levels of antibiotics can facilitate adaptive resistance (Ebbensgaard et al. 2020). This has been demonstrated by clinically relevant antibiotic resistance achieved via efflux pumps (Blair et al. 2014).

Therefore, studying multigenerational interactions between antibiotic-producing and antibiotic-inhibited environmental isolates may provide insight into evolutionary dynamics driving antibiotic resistance.

We were interested in the competitive ability of two environmental strains, *B. thailandensis* and *F. johnsoniae*. A previous study examined the competitive ability of *F. johnsoniae* against *B. thailandensis* through contact-dependent mechanisms involving the type VI secretion system (Russel et al. 2014). Given the nature of *B. thailandensis* as a prolific secondary metabolite producer, including various antibiotics (Mao et al. 2017), and the various TolC efflux systems observed in *F. johnsoniae*, we wanted to expand the exploration of interspecies competition between these strains by focusing on contact-independent mechanisms. Preliminary results suggested that *F. johnsoniae* was susceptible to a *B. thailandensis*-produced antibiotic(s), but intrinsic mechanisms, such as efflux systems, allowed *F. johnsoniae* to tolerate the presence of the antibiotic(s) and permit growth on agar. We then tested whether these intrinsic mechanisms of antibiotic(s) tolerance would pave the way for mutational acquisition in *F. johnsoniae* that would make it less susceptible to antibiotic(s).
We performed an agar-based experimental coevolution with *B. thailandensis* and *F. johnsoniae*. These strains were co-plated together and, in parallel, also plated in monocultures on M9 minimal medium agar plates containing 0.2% glucose (M9-glucose; % v/v) for over the span of 7.5 months. *B. thailandensis* and *F. johnsoniae* were co-plated such that *B. thailandensis* antibiotic inhibition of *F. johnsoniae* could occur without intergrowth of the colonies. By comparing outcomes of the coevolved lines to the evolved monoculture lines, we asked: What are the genetic and phenotypic repercussions of coevolution and how consistent are they across independent, replicate lines? What are the genetic signatures of *F. johnsoniae* of reduced antibiotic susceptibility? What is the antibiotic produced by *B. thailandensis* that inhibits *F. johnsoniae*? We found that coevolved *F. johnsoniae* lines reduced susceptibility to the *B. thailandensis*-produced antibiotic while evolved monoculture lines remained susceptible. A 33 bp deletion in *tolC* and a nonsynonymous mutation in *accA* suggested two different paths to the evolution of reduced antibiotic susceptibility in *F. johnsoniae*. The ancestor *F. johnsoniae* strain became less susceptible after we deleted the *tolC* 33 bp region, but not to an equivalent level observed in the coevolved lines. This result suggests that multiple mutations may contribute to the antibiotic susceptibility. Though genomics from *B. thailandensis* did not further inform as to the bioactive compound(s), a mutation in *F. johnsoniae* provided evidence that thailandamide, a previously described antibiotic that inhibits fatty acid synthesis (Wu and Seyedsayamdost 2018), was among the bioactive compounds that inhibited *F. johnsoniae*. Our data also suggest that multiple antibiotics produced by *B. thailandensis* contributed to the observed inhibition of *F. johnsoniae*.

93

## 94 Results

### 95 *F. johnsoniae* is inhibited by a bioactive compound(s) produced by *B. thailandensis*

We first observed that *B. thailandensis* inhibited *F. johnsoniae* when co-plated on M9-agar plates (Fig. 1). *B. thailandensis* exhibited radial growth on all edges along the circumference of the colony, while the *F. johnsoniae* colony proximal to *B. thailandensis* was inhibited. However, the distal end of the *F. johnsoniae* colony grew away from *B. thailandensis*, suggesting that *F. johnsoniae* may have intrinsic mechanisms to reduce antibiotic susceptibility. We note that, despite efforts to do so, we were unable to purify and directly link the antibiotic(s) that inhibited *F. johnsoniae*. We still use the terminology antibiotic(s) to refer to any number of compounds that may be
102 inhibiting *F. johnsoniae*, whether that was an antibiotic derived from a BSGC or a by-product of *B. thailandensis*
103 metabolism that was bioactive against *F. johnsoniae*.

104

105 Efflux allows *F. johnsoniae* to grow in the presence of a *B. thailandensis*-produced antibiotic

106 Given the growth pattern of *F. johnsoniae* when co-plated with *B. thailandensis*, we hypothesized that
107 efflux contributed to *F. johnsoniae* as an intrinsic mechanism to reduce antibiotic susceptibility. We collected the
108 organic fraction from spent supernatant of *B. thailandensis* grown in monoculture, which contained the antibiotic(s).
109 We then treated *F. johnsoniae* cultures with the supernatant alone and in combination with daidzein, an efflux pump
110 inhibitor (Aparna et al. 2014). While the supernatant inhibited *F. johnsoniae*, daidzein did not, and the supernatant
111 with daidzein significantly inhibited *F. johnsoniae* more than the supernatant alone (Fig. 2, Supplementary Fig. 1).
112 This suggested that *F. johnsoniae* had intrinsic mechanisms to reduce antibiotic susceptibility via efflux. extrusion
113 and, these intrinsic mechanisms of antibiotic tolerance could allow for mutational acquisition to further decrease
114 antibiotic susceptibility. We tested this hypothesis by performing a coevolution experiment.

115

116

117 Coevolutionary outcomes of *B. thailandensis*-*F. johnsoniae* interactions

118 To better understand the underlying factors contributing to *F. johnsoniae* antibiotic susceptibility, we next
119 asked how the susceptibility to antibiotic(s) in *F. johnsoniae* would change when coevolved with *B. thailandensis*.
120 We performed an agar-based coevolution experiment with 10 µL volume of two liquid overnight cultures spotted 14
121 mm apart to allow for extracellular chemical interactions, allowed to grow into colonies, and passaged onto another
122 plate before intergrowth of the colonies could occur (Fig 3). The colonies spotted were either one of each of *B*.
123 *thailandensis* and *F. johnsoniae* ("coevolved"), two of the same *B. thailandensis* colonies, or two of the same *F*.
124 *johnsoniae* colonies ("monoculture"). All monoculture controls were grown in parallel to the co-evolved lines (Fig.
125 3). We observed that the radial growth increased between the 5th plate passage and the 1st plate passage within both
126 the monoculture evolved and coevolved lines (Wilcoxon Rank-Sum; p-values < 0.01), suggesting increased growth
127 capabilities on the M9-glucose medium over time. The radial growth of *F. johnsoniae* generally increased with each
successive plate passage (Fig. 4, Supplementary Fig. 2). In addition, *B. thailandensis* substantially inhibited *F. johnsoniae* on the first plate (Supplementary Fig. 2), as the radial growth of the coevolved lines was significantly less than the radial growth of the monoculture lines (q-value = 0.039). In successive plate passages, the radial growth in coevolved lines were insignificantly different to the monoculture evolved lines (q-values >=0.15) with the exception of plate passage 4 (q-value= 0.039). This suggested that co-evolved lines of *F. johnsoniae* were becoming less susceptible to *B. thailandensis*-produced antibiotics. Indeed, when a freezer stock from a coevolved *F. johnsoniae* line and a freezer stock from the corresponding monoculture *F. johnsoniae* line were plated with the *B. thailandensis* ancestor, *F. johnsoniae* from the coevolved line displayed more radial growth and thus less antibiotic susceptibility as compared to the evolved monoculture control (Supplementary Fig. 3). Interestingly, colonies from the coevolved *F. johnsoniae* lines were also able to resist colony invasion by *B. thailandensis*, while colonies from the evolved monoculture lines could not (Supplementary Fig. 4). Overall, these results suggest that coevolved *F. johnsoniae* evolved decreased susceptibility to antibiotics as an outcome of long-term exposure to and engagement with *B. thailandensis*.

We performed whole genome sequencing to discover mutations that may have contributed to reduced antibiotic susceptibility in the *F. johnsoniae* coevolved lines. We sequenced a clonal isolate from the *F. johnsoniae* ancestor and clonal isolates from each of the 5 independent coevolved and monoculture evolved lines from the final (fifth) plate passage. We detected mutations in all coevolved and monoculture evolves isolate genomes compared to the ancestor (Table 1, Supplemental File 1). *F. johnsoniae* lines coevolved with *B. thailandensis* acquired mutations that were distinct from acquired mutations in *F. johnsoniae* lines grown in monocultures (Table 2, Supplementary Fig. 5).

<table><caption>**Table 1.** Summary of mutation types observed in *F. johnsoniae* from the (co)evolution experiment. Mutations are from five representative clonal isolates after the final (5th) plate passage and the ancestor.</caption><tr><td><i>F. johnsoniae</i> isolate</td><td>Total</td><td>Insertion</td><td>Deletion</td><td>Nonsynonymous</td><td>Synonymous</td><td>Nonsense</td></tr></table>
<table><tr><td>Coevolved rep 1</td><td>6</td><td>2</td><td>1</td><td>3</td><td>0</td><td>0</td></tr><tr><td>Coevolved rep 2</td><td>5</td><td>0</td><td>2</td><td>3</td><td>0</td><td>0</td></tr><tr><td>Coevolved rep 3</td><td>6</td><td>0</td><td>0</td><td>5</td><td>1</td><td>0</td></tr><tr><td>Coevolved rep 4</td><td>5</td><td>1</td><td>1</td><td>3</td><td>0</td><td>0</td></tr><tr><td>Coevolved rep 5</td><td>5</td><td>1</td><td>2</td><td>2</td><td>0</td><td>0</td></tr><tr><td>Evolved monoculture rep 1</td><td>6</td><td>1</td><td>3</td><td>2</td><td>0</td><td>0</td></tr><tr><td>Evolved monoculture rep 2</td><td>7</td><td>1</td><td>3</td><td>2</td><td>0</td><td>1</td></tr><tr><td>Evolved monoculture rep 3</td><td>6</td><td>0</td><td>4</td><td>2</td><td>0</td><td>0</td></tr><tr><td>Evolved monoculture rep 4</td><td>6</td><td>2</td><td>1</td><td>3</td><td>0</td><td>0</td></tr><tr><td>Evolved monoculture rep 5</td><td>3</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td></tr><tr><td>Ancestor<sup>a</sup></td><td>2</td><td>0</td><td>1</td><td>1</td><td>0</td><td>0</td></tr></table>

F. johnsoniae evolved isolates harbored mutations in an efflux outer membrane protein, TolC, and could be evidence of an acquired mutation that would reduce antibiotic susceptibility (Table 2). A *tolC* mutation was observed in 4 out of the 5 coevolved lines. While *F. johnsoniae* contains 16 *tolC* genes (Supplementary Table 1), the same gene was mutated in 4 independent replicates. Though, the TolC harboring mutations in coevolved isolates (FJOH_RS06580) only had 24.2% or less protein sequence identity with all other TolC proteins in *F. johnsoniae* (Supplementary Table 2). Furthermore, there was evidence of parallel evolution at the nucleotide level, as 3/5 of the coevolved lines had the same *tolC* 33 bp deletion at the same *tolC* locus (Supplemental File 1). This deletion was from nucleotides 261-293 in the FJOH_RS06580 coding sequence, resulting in an *in-frame* deletion (Fig. 5). This deletion affected one of the extracellular loops of TolC. The WT sequence revealed a 11 bp direct repeat, occurring before the deletion and representing the last 11 bp of the 33 bp deletion. This suggests that the deletion may have

<footnote><sup>a</sup>Ancestor mutations that were also observed in evolved line(s) were not included in the tally of evolved line mutations.</footnote>
occurred during a replication deletion event. The other mutation in FJOH_RS06580 (coevolved line 3) was a single
nucleotide polymorphism (G247A in the coding sequence) that resulted in a nonsynonymous mutation (G83R).
Protein modeling suggested that this mutation resulted in a decreased diameter of the efflux channel (Supplementary
Fig. 6). Coevolved line 4 did not harbor a mutation in FJOH_RS06580 but instead had a unique bp insertion in a
*ragB/susD* nutrient uptake outer membrane protein (FJOH_RS24865). Guanine was inserted between nucleotide
positions 792 and 793 in the coding sequence for FJOH_RS24865. This frameshift mutation resulted in a premature
termination codon 6 bp downstream of the insertion, likely rendering the protein nonfunctional. Mutations were also
uniquely found in regulators of coevolved isolates including, a small insertion in a TetR/AcrR family transcriptional
regulator in coevolved isolate 1, a nonsynonymous mutation in an OmpR family response regulator in coevolved
isolate 2, and a nonsynonymous mutation in a LytTR family response regulator within the same loci of coevolved
isolates 2 and 5. These regulators may have indirect effects on antibiotic susceptibility (e.g. up/downregulation of
efflux pumps).

**Table 2.** Distinctions and overlaps of loci with mutations unique to the *F. johnsoniae* lines that were coevolved with *B. thailandensis*. These mutations were detected (+) in at least one coevolved line and not present (-) in any of the evolved *F. johnsoniae* monocultures
<table><thead><tr><th>Locus</th><th>Annotation</th><th>Rep 1</th><th>Rep 2</th><th>Rep 3</th><th>Rep 4</th><th>Rep 5</th></tr></thead><tbody><tr><td>FJOH_RS00255</td><td>acetyl-CoA carboxylase<br>carboxyltransferase subunit alpha</td><td>-</td><td>-</td><td>C479A</td><td>-</td><td>-</td></tr><tr><td>FJOH_RS02320</td><td>aminomethyl-transferring glycine<br>dehydrogenase</td><td>-</td><td>-</td><td>-</td><td>-</td><td>Δ1 bp<br>(504)</td></tr><tr><td>FJOH_RS04780</td><td>hypothetical protein</td><td>G329C</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>FJOH_RS06580</td><td>TolC family protein</td><td>Δ33 bp<br>(261-293)</td><td>Δ33 bp<br>(261-293)</td><td>G247A</td><td>-</td><td>Δ33 bp<br>(261-293)</td></tr><tr><td>FJOH_RS07830</td><td>NAD(P)/FAD-dependent<br>oxidoreductase</td><td>-</td><td>C148A</td><td>-</td><td>-</td><td>-</td></tr><tr><td>FJOH_RS09515</td><td>LyTR family DNA-binding<br>domain-containing protein</td><td>-</td><td>G46A</td><td>-</td><td>-</td><td>C44T</td></tr><tr><td>FJOH_RS09520</td><td>Histidine kinase</td><td>825insG</td><td>-</td><td>G496A</td><td>Δ1 bp<br>(621)</td><td>-</td></tr><tr><td>FJOH_RS11170</td><td>Gfo/Idh/MocA family<br>oxidoreductase</td><td>-</td><td>-</td><td>G273C</td><td>-</td><td>-</td></tr><tr><td>FJOH_RS12240</td><td>AIR synthase-related protein</td><td>-</td><td>-</td><td>C791T</td><td>-</td><td>-</td></tr><tr><td>FJOH_RS14175</td><td>response regulator transcription<br>factor</td><td>-</td><td>C269T</td><td>-</td><td>-</td><td>-</td></tr><tr><td>FJOH_RS20510</td><td>TetR/AcrR family transcriptional<br>regulator</td><td>201insT</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>FJOH_RS21875</td><td>glycoside hydrolase</td><td>T1489C</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>FJOH_RS24865</td><td>RagB/SusD family nutrient uptake<br>outer membrane protein</td><td>-</td><td>-</td><td>-</td><td>793insG</td><td>-</td></tr><tr><td>FJOH_RS25290</td><td>phosphoribosylanthranilate<br>isomerase</td><td>-</td><td>-</td><td>-</td><td>T50C</td><td>-</td></tr></tbody></table>

Mutations were also observed in (co)evolved *B. thailandensis* lines (Supplemental File 1). Most mutations were unique to individually sequenced isolates. Exact mutational parallelism was present, (e.g. flagellar assembly protein FilH, TetR/AcrR transcriptional regulator, and efflux periplasmic adaptor subunit) but was observed across both monoculture evolved lines and coevolved lines, indicating that these mutations were not acquired due to the presence of *F. johnsoniae*.

One mutation of interest was observed in *B. thailandensis* coevolved line 4: a 7 bp insertion in BTH_II1238. This gene codes for *btaP*, a metallo-β-lactamase involved in the production of the bactobolin
antibiotic (Duerkop et al. 2009). The insertion resulted in a premature stop codon, rendering the protein non-functional. A previous study showed abrogated bactobolin production in a *btaP* knockout (Carr et al. 2011), meaning that the insertion observed in our study likely abrogated bactobolin production as well. To ascertain that *F. johnsoniae* inhibition persists in the presence of *B. thailandensis* with abrogated bactobolin production, we co-plated *F. johnsoniae* with a *B. thailandensis btaK::T23* transposon mutant. We had possession of this transposon mutant, and, similar to the *btaP* mutant, the *btaK* (BTH_II1233) mutant has been confirmed to abrogate bactobolin production (Duerkop et al. 2009). As expected, we found that the *B. thailandensis btaK::T23* transposon mutant inhibited *F. johnsoniae* despite the inability to produce bactobolin (Supplementary Fig. 7), suggesting either that bactobolin was ineffective against *F. johnsoniae* or that multiple antibiotics were involved in *F. johnsoniae* inhibition. Overall, there was no mutational genomic evidence to help to inform the *B. thailandensis*-produced antibiotic(s) that inhibits *F. johnsoniae*.

201

## The FJOH_RS06580 *tolC* 33 bp deletion reduces antibiotic susceptibility

We asked whether the mutations in *tolC* or the mutation in *ragB/susD* observed in coevolved *F. johnsoniae* lines would reduce antibiotic susceptibility to the *B. thailandensis*-produced antibiotic(s). These mutations were amplified from the coevolved *F. johnsoniae* lines and recombined into the ancestor so that the ancestor would only harbor one of these mutations and not any of the additional mutations observed in the coevolved lines. Strains and plasmids used to make recombinant *F. johnsoniae* are outlined in Table 3. Ancestor *B. thailandensis* inhibited the *F. johnsoniae* ancestor and recombinant strains *reG247A_tolC* and *re792_793insG_ragB* (Fig. 6). In fact, *reG247A_tolC* appeared more inhibited compared to the *F. johnsoniae* ancestor, suggesting that a decreased diameter to the TolC efflux channel (inferred from protein structure predicted by SWISS-MODEL) is detrimental to fitness. In contrast, recombinant strain *reΔ33_tolC* was the least inhibited when plated with ancestor *B. thailandensis*, suggesting that the 33 bp deletion reduces susceptibility to an antibiotic(s) (Fig. 6B). Thus, it appears that *F. johnsoniae* uses intrinsic mechanisms to reduce antibiotic susceptibility (e.g. efflux pumps, Fig. 2) but also acquired a mutation in the outer membrane protein of an efflux system that further reduced susceptibility to a *B. thailandensis*-produced antibiotic(s). The acquired mutation evolved independently in 3/5 of the coevolved isolates at the same *tolC* locus. However, the 33 bp deletion in *tolC* alone did not reduce antibiotic susceptibility to the same degree observed in the coevolved lines, as the coevolved lines harboring the *tolC* 33 bp deletion with
additional mutations less susceptible to *B. thailandensis*-produced antibiotic(s) compared to the recombinant strain reΔ33_tolC (Supplementary Fig. 8). This suggests that coevolved lines harboring the *tolC* 33 bp mutation may contain synergistic mutations contributing to further reductions in antibiotic susceptibility.

**Table 3.** Strains and plasmids used in this study.

²²³ *ªAbbreviations: Ap*<sup>r</sup>-ampicillin resistance, 100 micrograms per ml; Km*<sup>r</sup>* kanamycin resistance 30 micrograms per

<table><thead><tr><th>Strain or plasmid</th><th>Description<sup>a</sup></th><th>Source or reference</th></tr></thead><tbody><tr><td><em>Escherichia coli</em> strains</td><td></td><td></td></tr><tr><td>DH5αMCR</td><td>Strain used for general cloning</td><td>Life Technologies (Grand Island, NY)</td></tr><tr><td>HB101</td><td>Strain used with pRK2013 for triparental conjugation</td><td>Bolivar and Backman 1979; Figurski and Helinski 1979</td></tr><tr><td>DH5αMCR_pYT354</td><td>pYT354 in DH5amcr; sacB-containing suicide vector</td><td>Zhu et al. 2017</td></tr><tr><td><em>B. thailandensis</em> strain</td><td></td><td></td></tr><tr><td>E264 (ATCC 700388)</td><td>Wild type</td><td>Brett et al. 1998</td></tr><tr><td><em>F. johnsoniae</em> strains</td><td></td><td></td></tr><tr><td>UW101 (ATCC 17061)</td><td>Wild type</td><td>McBride et al. 2009</td></tr><tr><td>CoE_Δ33_tolC</td><td>Coevolved strain containing a 33 bp (261-293) deletion in tolC (FJOH_RS06580)</td><td>This study</td></tr><tr><td>CoE_G247A_tolC</td><td>Coevolved strain containing a nonsynonymous mutation (G247A) in tolC (FJOH_RS06580)</td><td>This study</td></tr><tr><td>CoE_792_793insG_ragB</td><td>Coevolved strain containing an insertion (G) between bps 792 and 793 in ragB/susD (FJOH_RS24865)</td><td>This study</td></tr><tr><td>reΔ33_tolC</td><td>tolC (FJOH_RS06580) 33 bp deletion placed in ancestor</td><td>This study</td></tr><tr><td>reG247A_tolC</td><td>tolC (FJOH_RS06580) G247A mutation placed in ancestor</td><td>This study</td></tr><tr><td>re792_793insG_ragB</td><td>rtragB/susD (FJOH_RS24865) bp (G) insertion placed between bps 792 and 793 in ancestor</td><td>This study</td></tr><tr><td>Plasmids</td><td></td><td></td></tr><tr><td>pYT354</td><td>sacB-containing suicide vector; Ap<sup>r</sup> (Em<sup>r</sup>). pYT354 is modified from pYT313 with different multiple cloning site</td><td>Zhu et al. 2017</td></tr><tr><td>pJC101</td><td>Construct used to replace ancestral native tolC (FJOH_RS06580) with tolC 33 bp deletion</td><td>This study</td></tr><tr><td>pJC102</td><td>Construct used to replace ancestral native tolC (FJOH_RS06580) with tolC G247A</td><td>This study</td></tr><tr><td>pJC103</td><td>Construct used to replace ancestral native ragB/susD (FJOH_RS24865) with ragB/susD 792_793insG</td><td>This study</td></tr><tr><td>pRK2013</td><td>Helper plasmid for triparental conjugation; Km<sup>r</sup></td><td>Figurski and Helinski 1979</td></tr></tbody></table>

224 ml; (Em*<sup>r</sup>*-erythromycin resistance, 100 micrograms per ml for *F. johnsoniae*). Antibiotics resistance phenotypes

225 listed in parentheses (Em) are those expressed in *F. johnsoniae* but not in *E. coli*. Antibiotics resistance phenotypes
listed not in parentheses (Ap, Km) are those expressed in *E. coli* but not in *F. johnsoniae*.

We then quantified the extent of *F. johnsoniae* antibiotic susceptibility. We grew *B. thailandensis* in spent *F. johnsoniae* medium and extracted the organic fraction from the spent *B. thailandensis* medium. The organic layer contained unknown compound(s) that were bioactive against *F. johnsoniae*. A Kirby-Bauer disk diffusion assay was performed to quantify the zone of inhibition (ZOI) of our *F. johnsoniae* strains (Table 4). The ZOIs in coevolved strains were significantly less than the ZOI's in monoculture evolved strains (Wilcoxon Rank-Sum; p-value <0.001). However, the ZOIs in coevolved replicates were not significantly different from the ZOI in the ancestor (Wilcoxon Rank-Sum; q-values >= 0.6). In addition, the ZOI in the recombinant strain, reΔ33_tolC, did not significantly differ from the ZOI in the ancestor (Wilcoxon Rank-Sum; q-value = 1.0). Despite this observation, all coevolved strains and the reΔ33_tolC recombinant strain grew better than the ancestor when plated together on the same plate with *B. thailandensis* (Supplementary Fig. 9). Coevolved strains, but not the recombinant strain, grew better than the monoculture evolved strains, consistent with the Kirby-Bauer results. Taken together, these data suggest that coevolved strains are less susceptible to antibiotic(s) produced by *B. thailandensis*.

<table><caption>**Table 4.** Results of antimicrobial activity observed in spent *B. thailandensis* medium against *F. johnsoniae* strains represented by zone of inhibition (ZOI).</caption><thead><tr><th>Strain</th><th colspan="3">ZOI (mm)</th></tr></thead><tbody><tr><td>Ancestor</td><td>2.62</td><td>2.63</td><td>3.10</td></tr><tr><td>Evolved monoculture rep 1</td><td>5.50</td><td>5.39</td><td>6.20</td></tr><tr><td>Evolved monoculture rep 2</td><td>4.90</td><td>5.55</td><td>4.61</td></tr><tr><td>Evolved monoculture rep 3</td><td>5.90</td><td>5.53</td><td>3.25</td></tr><tr><td>Evolved monoculture rep 4</td><td>3.96</td><td>4.72</td><td>4.75</td></tr><tr><td>Evolved monoculture rep 5</td><td>5.41</td><td>5.17</td><td>5.14</td></tr><tr><td>Coevolved rep 1</td><td>1.31</td><td>2.14</td><td>1.33</td></tr><tr><td>Coevolved rep 2</td><td>2.00</td><td>2.00</td><td>2.17</td></tr><tr><td>Coevolved rep 3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Coevolved rep 4</td><td>2.06</td><td>2.04</td><td>2.07</td></tr><tr><td>Coevolved rep 5</td><td>1.97</td><td>3.08</td><td>2.31</td></tr><tr><td>reΔ33_tolC</td><td>2.48</td><td>3.17</td><td>3.11</td></tr></tbody></table>

**Thailandamide is one of the bioactive molecules that inhibits *F. johnsoniae***

The nonsynonymous mutation in *F. johnsoniae* coevolved line 3 *tolC* did not reduce susceptibility to the *B. thailandensis*-produced antibiotic(s). But, *F. johnsoniae* coevolved line 3 displayed reduced antibiotic(s)
susceptibility despite harboring this mutation (Fig. 7A). This *F. johnsoniae* coevolved line 3 also contained a unique nonsynonymous mutation in acetyl-CoA carboxylase carboxyltransferase subunit alpha (*accA*, Table 2). This was a C479A nonsynonymous mutation in the coding sequence of *accA* that resulted in a P160Q alteration in AccA. This mutation was similar to a P164Q mutation in AccA from *Salmonella enterica* serovar Typhimurium strain LT2 that conferred resistance to thailandamide from *B. thailandensis* (Wozniak et al. 2018). The P164Q mutation from *S. enterica* aligned with the P160Q mutation observed in our coevolved line (Fig. 7). Thus, we hypothesized that thailandamide was one of the antibiotics responsible for inhibition of *F. johnsoniae*.

We plated the *F. johnsoniae* ancestor with *B. thailandensis thaF* (BTH_II1675), *thaA* (BTH_II1681), and *atsR* (BTH_I0633) transposon mutants. *thaF* encodes a polyketide synthetase trans-AT domain directly involved in the biosynthesis of thailandamide, *thaA* encodes a LuxR-type regulator in the thailandamide biosynthetic gene cluster, and *astR* encodes a global regulator. ThaA positively regulates the thailandamide biosynthetic gene cluster while AtsR negatively regulates the thailandamide biosynthetic gene cluster. We found that *thaF::T23* and *thaA::T23* mutants decreased inhibition of *F. johnsoniae* (Fig. 7B, Fig. 7C) while the *atsR::T23* mutant increased inhibition of *F. johnsoniae* (Fig. 7D). This suggests that thailandamide is bioactive against *F. johnsoniae*. However, *F. johnsoniae* was still slightly inhibited when plated with *thaF::T23* and *thaA::T23* mutants, which also suggests that in addition to thailandamide, there may be another *B. thailandensis*-produced antibiotic that is inhibiting *F. johnsoniae*. However, we were unable to determine this molecule.

264

## Discussion

We performed an experimental coevolution study between a strain capable of antibiotic production (*B. thailandensis*) and an antibiotic-susceptible strain (*F. johnsoniae*). Our findings show how long-term interspecies interactions facilitated through chemical exchange via diffusion in agar can result in the acquisition of mutations that reduce antibiotic susceptibility. Analysis of mutations of *F. johnsoniae* coevolved lines as compared to lines evolved in monoculture revealed that reduced antibiotic susceptibility was achieved via a mutation in an efflux outer membrane protein. Specifically, a 33 bp deletion in *tolC*, which eliminated 11 amino acids that were part of an extracellular loop in TolC, reduced antibiotic susceptibility in coevolved *F. johnsoniae* lines. Parallel evolution was
observed for this mutation, as 3/5 coevolved isolates harbored the same 33 bp deletion within the same *tolC* locus.
Decreased antibiotic susceptibility was also achieved through a nonsynonymous mutation in *accA*. While the *accA*
mutation was not directly assessed for antibiotic susceptibility in our study, the same mutation was spontaneously
derived in a *Salmonella* strain that conferred resistance to *B. thailandensis*-derived thailandamide (Wozniak et al.
2018). This led us to hypothesize that thailandamide was the antibiotic inhibiting *F. johnsoniae*. *B. thailandensis*
transposon mutants with abrogated thailandamide production confirmed that thailandamide was bioactive against *F.*
*johnsoniae*, but slight inhibition was observed, again suggesting that multiple antibiotics are inhibiting *F.*
*johnsoniae*.

Performing the experimental coevolution on agar plates created a heterogenous environment that facilitated
the evolution of decreased antibiotic susceptibility. *F. johnsoniae* growth at the distal end of the colony was
permitted because of an antibiotic concentration gradient that was established via diffusion. Low-dose antibiotics
likely upregulated intrinsic mechanisms of resistance (e.g. efflux pumps) that conferred low-levels of resistance
(Sandoval-Motta and Aldana 2016). The ability to survive exposure to antibiotics via intrinsic mechanisms
(Frimodt-Møller et al. 2018; Meouche & Dunlop 2018) can provide the opportunity for mutational acquisition of
resistance (Frimodt-Møller and Løbner-Olesen 2019; Ebbensgaard et al. 2020). This was demonstrated
experimentally in a seminal study that found that a heterogenous environment increased the rate of adaptation to
antibiotics with as few at 100 bacteria in the initial inoculum (Zhang et al. 2011). This approach has been expanded
to show how the initial adaptation to low levels of antibiotics facilitate adaptations to high levels of resistance
(Baym et al. 2016). Thus, evolutionary adaptations to antibiotic resistance can be fostered in heterogenous
environments that would otherwise not be achieved in a uniform environment (Hermsen and Hwa 2010; Hermsen et
al. 2012).

Reduced susceptibility to the *B. thailandensis* antibiotic(s) was observed in *F. johnsoniae* coevolved lines
that had a 33 bp deletion in *tolC*. The occurrence of the 11 bp directed repeats may support a replication
misalignment event that led to the 33 bp deletion (Kong and Masker 1994; Bzymek and Lovett 2001). TolC forms
the outer membrane channel part of resistance nodulation division (RND) efflux transporters (Nishino et al. 2003;
Anes et al. 2015). *tolC* (*FJOH_RS06580*) is located in an operon that includes the remaining components necessary
for a functional efflux pump. This includes a TetR/AcrR family transcriptional regulator (*FJOH_RS06575*), an
300 efflux transporter periplasmic adaptor subunit (FJOH_RS06585), and a multidrug efflux pump permease
301 subunit (FJOH_RS0690). We note that we were unable to confirm the RND-type transport system of this operon.
302 Elimination of 11 amino acids from an extracellular loop in TolC may result in TolC more frequently adopting an
303 open conformation, which could increase the rate of antibiotic extrusion. “Leaky” TolC mutants have been
304 characterized, but these mutations occurred at the periplasmic end of TolC (Bavro et al. 2008; Pei et al. 2011). In
305 fact, mutational studies of the TolC extracellular loops appear uncommon but may present a novel mechanism for
306 antibiotic resistance (Krishnamoorthy et al. 2013). One study carried out a mutational analysis in OprM, a TolC
307 homolog. Two separate insertion mutants were created in external loops of the OprM β-barrel. Modest reductions in
308 channel conductance were observed for these mutants, but this was correlated with either unchanged or reduced
309 MICs to an array of antimicrobials. Regardless, the authors demonstrated that external loops contribute to the
310 control of passage of certain substrates (Wong et al. 2001).

311 The *F. johnsoniae* recombinant strain harboring the 33 bp deletion in *tolC* had reduced antibiotic
312 susceptibility, but not to the same degree as the *F. johnsoniae* coevolved lines to the *B. thailandensis*-produced
313 antibiotic(s). Additional mutations in the coevolved lines are likely to provide further reductions in antibiotic
314 susceptibility. For example, coevolved line 1 also harbored a 1 bp insertion (T) at position 4734734 in
315 FJOH_RS20510, annotated as a TetR/AcrR family transcriptional regulator. This results in a nonsense mutation that
316 would render the protein nonfunctional. TetR regulators are typically negative regulators, so a nonfunctional TetR
317 regulator would lead to increased expression of efflux pump systems (Cuthbertson and Nodwell 2013). We note that
318 this occurred in FJOH_RS20510 and not FJOH_RS06575, but some TetR regulators have multiple targets and TetR
319 from FJOH_RS20510 could also be negatively regulating the FJOH_RS06580-FJOH_RS06585-FJOH_RS06590
320 efflux system (Colclough et al. 2019). The remaining coevolved lines with the 33 bp deletion in *tolC* (line 2 and line
321 5) also had mutations in transcriptional regulators (OmpR and LytTR). Mutations in OmpR may have a potential
322 link to antibiotic resistance by altering expression of the major porin, OmpF, or other genes controlled under the
323 EnvZ/OmpR two-component regulatory system (Card et al. 2021). The potential contributions of mutations in
324 LytTR to reduced antibiotic susceptibility is unknown.

325 While it was clear that differences in antibiotic susceptibility emerged between the monoculture and
326 coevolved lines, there were both congruencies and discrepancies between Kirby-Bauer assay results (Table 4) and
qualitative plate assays (Supplementary Fig. 9). To start, the decreased antibiotic susceptibility in coevolved strains was consistent between both assays. Quantitatively, the coevolved strains had a modest decrease in ZOIs compared to the ancestor and a significant decrease compared to monoculture evolved strains. Qualitatively, the coevolved strains were the least susceptible to the *B. thailandensis*-produced antibiotic(s). The monoculture evolved strains had a modest increase in ZOIs compared to the ancestor despite qualitatively being less susceptible to the *B. thailandensis* antibiotic(s). Lastly, the ZOI of the recombinant strain, reΔ33_tolC was not different from the ancestor but was qualitatively less susceptible to the *B. thailandensis* antibiotic(s). These differences in quantitative vs qualitative outcomes could have arisen for a few reasons. The Kirby-Bauer assays were performed from antibiotic(s) concentrated from spent liquid culture. The antibiotic profile of *B. thailandensis* in liquid culture could be different from the profile on agar medium. Similarly, the spent supernatant from the Kirby-Bauer assay was obtained from strains that did not directly interact with one another. There could be a dynamic, chemical interactions between *F. johnsoniae* and *B. thailandensis* when co-plated that leads to a unique antibiotic profile not achieved by growing *B. thailandensis* in filtered spent *F. johnsoniae* culture. Lastly, the Kirby-Bauer assays were performed on TSA50 medium and not M9-glucose agar (see Materials and Methods section: Extraction of *B. thailandensis* bioactive compound(s) and Kirby-Bauer assays). Differences in nutrient composition between TSA50 and M9-glucose could have affected the overall expression of and/or functionality of mechanisms to reduce antibiotic susceptibility *F. johnsoniae*.

TolC also has a multifaceted role beyond antibiotic export. TolC has been found to be the outer membrane protein part of type I secretion systems (Lee et al. 2012), involved in the export of siderophores (Bleuel et al. 2005), and an importer of toxins such as bacteriocins (Housden et al. 2021). Furthermore, mutations that make TolC non-functional have pleiotropic consequences. For example, a gene expression analysis of a non-functional TolC mutant showed upregulation of nutrient transporters, stress response genes, and central metabolism, while genes involved in nitrogen metabolism and transport were downregulated (Santos et al. 2010). We cannot completely rule out these alternative explanations, such as nutrient acquisition, as a reason why *F. johnsoniae* could grow better in the presence of *B. thailandensis*. The Kirby-Bauer results show that there was a significant difference between monoculture evolved and coevolved lines ZOIs. This result provides credence to mutational acquisition in coevolved lines related to decreased antibiotic susceptibility. Additional functional assays would need to be performed, specifically with the *tolC* 33 bp mutant, to better understand its role in antibiotic susceptibility. In addition, further
effots to isolate the bioactive compound(s) and test their effects on strains in isolation would improve MIC assays
and establish mutations that lead to resistance.

A nonsynonymous mutation in *accA* in coevolved line 3 also reduced susceptibility to the *B. thailandensis*-
produced antibiotic(s). In addition, this mutation guided our efforts to uncover that thailandamide was one of the
antibiotics produced in *B. thailandensis* that inhibited *F. johnsoniae*. Thailandamide resistance was characterized in
spontaneous mutants from *S. enterica* (Wozniak et al. 2018). Wozniak and colleagues found 6 unique mutations for
3 different amino acid positions in AccA that conferred thailandamide resistance. One of these mutations was also
spontaneously generated in our study (P160Q in *F. johnsoniae*, P164Q in *S. enterica*). Thailandamide is speculated
to inhibit fatty acid synthesis by competitively inhibiting the binding of carboxy-biotin to acetyl-CoA carboxylase
(Wu and Seyedsayamdost 2018). Protein modeling shows that the P164Q mutation in *S. enterica* is located in a loop
region of AccA that forms the channel to the active site where carboxy-biotin binds. The authors speculate that this
mutation may alter the shape of the channel, thereby limiting the accessibility of thailandamide to the active site
(Wozniak et al. 2018).

*B. thailandensis* transposon mutants with abrogated thailandamide production effectively reduced
inhibition of *F. johnsoniae*. But, we still observed inhibition with the thailandamide transposon mutants. It is likely
that *F. johnsoniae* was subjected to multiple antibiotics but other mutations conferring reduced antibiotic
susceptibility in the coevolved lines did not provide insight into other *B. thailandensis*-produced antibiotics affecting
*F. johnsoniae*. Furthermore, genome sequencing of *B. thailandensis* coevolved isolates did not provide insights into
the *B. thailandensis*-produced antibiotic(s) affecting *F. johnsoniae*. The only possible mutation of interest was a 7 bp
insertion that abrogated bactobolin production. But, a *B. thailandensis* transposon mutant with abrogated bactobolin
production still inhibited *F. johnsoniae*, suggesting that there are other bioactive compounds inhibiting *F. johnsoniae*.

*F. johnsoniae* coevolved line 4 did not harbor a mutation in *tolC* or *accA*. However, the *ragB/susD*
mutation was unique to coevolved line 4 and we hypothesized that this may provide an alternative mechanism for
reduced antibiotic susceptibility. Some antibiotics enter bacterial cells via nutrient transporters (Yoneyama and
Nakae 1993; Castañeda-García et al. 2009). Since the *ragB/susD*, rendered the protein nonfunctional, reduced
antibiotic susceptibility could have been conferred to coevolved line 4 by the reduction of antibiotic uptake. We did
not find this to be the case, as the *F. johnsoniae* recombinant strains with the *ragB/susD* nonsynonymous mutation had equivalent susceptibility to the *B. thailandensis*-produced antibiotic(s) as the *F. johnsoniae* ancestor. The other mutations in coevolved line 4 may reduce antibiotic susceptibility or, it is possible that mutations did not fixate in the population, and we chose an isolate for sequencing that did not acquire a mutation conferring reduced antibiotic susceptibility. Bacterial population sequencing could shed light on this discrepancy.

The diversity of antibiotics (Rutledge and Challis 2015) and corresponding resistance mechanisms (Mungan et al. 2020) demonstrate the breadth of genetic novelties that have arisen from millions of years of bacterial competition. Our results provide a case study of bacterial interspecies chemical engagements that can lead to evolution in a heterogeneous environment, with insights into understanding the complex and potentially interacting mutational paths to decreased antibiotic susceptibility.

## 392 Materials and Methods

### 393 Extraction of *B. thailandensis* supernatant containing antibiotic activity

A freezer stock of *B. thailandensis* was plated on 50% trypticase soy agar (TSA50) and grown for ~16 h at 27 °C. A loopful of lawn growth was transferred to 7 mL of M9 minimal salts-0.2% glucose (M9-glucose; % v/v) medium to achieve an initial OD590 of ~0.2 as measured on an Evolution 60S UV-Visible Spectrophotometer (Thermo Scientific). The culture was incubated with shaking at 200 rpm for ~16 h at 27 °C. The next day, 1 mL of culture was transferred to 50 mL fresh M9-glucose medium. The culture was incubated with shaking at 200 rpm for 24 h at 27 °C. The culture was transferred to a falcon tube and centrifuged at 5000 rpm for 20 min at 4 °C. The supernatant was removed, filtered with a 0.22 µM PES filter, and transferred to a separatory funnel. Ten mL of dichloromethane (DCM) was added to the separatory funnel. The separatory funnel was agitated three times and the DCM layer was removed. The addition, agitation, and removal of DCM was repeated twice more. The collected DCM layer was dried under nitrogen gas. The dried DCM extracts were reconstituted in 1 mL of a 50:50 methanol:water (% v/v) mixture. This mixture was used for the efflux pump inhibitor experiment.

### 406 Efflux pump inhibitor experiment
A freezer stock of *F. johnsoniae* was plated on TSA50 and grown for ~16 h at 27 °C. Five individual colonies were then inoculated in 5 mL of 50% trypticase soy broth (TSB50) and incubated with shaking at 200 rpm for ~16 h at 27 °C. The following day, 50 µL of culture was diluted into 4.95 mL of fresh TSB50 for each independent replicate. Then, 650 µL aliquots were dispensed in each of seven tubes for each independent replicate. Seven conditions were prepared across the seven tubes as follows: 1) *F. johnsoniae* culture control 2) *F. johnsoniae* with DMSO control 3) *F. johnsoniae* with 50:50 methanol:water (% v/v) control 4) *F. johnsoniae* with DMSO + 50:50 methanol:water (% v/v) control 5) *F. johnsoniae* with daidzein + 50:50 methanol:water (% v/v) 6) *F. johnsoniae* with DMSO + *B. thailandensis* supernatant and 7) *F. johnsoniae* with daidzein + *B. thailandensis* supernatant. A volume of 1.04 µL was added to tubes containing Daidzein (10 mg/ml) or DMSO and a volume of 16.25 µL was added to tubes containing *B. thailandensis* supernatant or 50:50 methanol:water (% v/v). After additions of solvent or components, 200 µL aliquots from each tube was placed in a 96 well plate (Supplementary Fig. 1). The plate contained 5 independent replicates, 2 technical replicates/independent replicate for conditions 1-3 and 3 tech reps/independent replicate for conditions 4-7 (90 samples). For the remaining 6 wells, 200 µL TSB50 was added to each well as a negative control. An initial OD590 reading was measured on a Tecan Infinite® F500 Multimode Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland). The plate was then incubated with shaking at 200 rpm for 24 h at 27 °C. A final OD590 reading was taken after 24 h of incubation. A Wilcoxon Rank-Sum test was used to compare all the controls to the *F. johnsoniae* culture control. All comparisons were insignificantly different (q-values >= 0.82). For this reason, we only used the *F. johnsoniae* culture control (with no added solvents) to serve as a control for a follow-up Wilcoxon Rank-Sum test with FDR correction that compared final OD590 across test conditions. Analyses were performed in R using the stats package (R Core Team 2021). Measurements were uploaded to R to generate boxplots using ggplot2 (Wickham 2016).

428

429 **Experimental evolution**

B. thailandensis E264 and F. johnsoniae UW101 were plated from freezer stocks onto TSA50. Plates were incubated for ~16 h at 27 °C. A single, isolated colony of B. thailandensis and of F. johnsoniae were inoculated as separate cultures in 7 mL of TSB50 to serve as the ancestral cultures. Cultures were incubated with shaking at 200 rpm for ~16 h at 27 °C. The following day, the cultures were pelleted by centrifugation at 5000 rpm for 10 min at room temperature, the supernatant was removed, and the cultures were resuspended in 1X phosphate buffered saline

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435 (PBS). This process was repeated once more. The cultures were resuspended in PBS at a final volume of 5 mL.
436 Ancestral freezer stocks were prepared by adding 750 mL of each overnight culture to 750 mL of 70% glycerol (%
437 v/v). All freezer stocks were stored at -80 °C. From the remaining cultures containing ancestral *B. thailandensis* and
438 ancestral *F. johnsonaie*, the OD590 was measured on an Evolution 60S UV-Visible Spectrophotometer (Thermo
439 Scientific) and each culture was diluted to an OD590 of 0.1 in PBS to prepare the evolution experiments. Ten µL of
440 a culture (OD590 0.1) was spotted onto M9 minimal salts agar plates containing 0.2% glucose (M9-glucose; % v/v).
441 Both strains were plated in isolation and co-plated (Fig. 3). When co-plated together, *B. thailandensis* and *F.*
442 *johnsoniae* were spotted 14 mm apart. This allowed sufficient time for the strains to chemically interact without
443 intergrowth of colonies. Five independent replicates were prepared, resulting in 15 plates total. The plates were
444 wrapped with parafilm and incubated at 27 °C for 1.5 months.

445 After incubation, we performed a plate passage. Sterilized toothpicks were used for the collection of lawn
446 growth. These cells were resuspended in 1 mL of PBS. For *B. thailandensis*, we preferentially collected radial
447 colony growth that was growing toward *F. johnsoniae*. A section of radial colony growth was collected for *B.*
448 *thailandensis* colonies in monocultures. The entirety of the *F. johnsoniae* was removed from each plate. First plate
449 passage freezer stocks were prepared by adding 500 mL of each resuspended culture to 500 mL of 70% glycerol (%
450 v/v). From the remaining cultures, the OD590 was measured on an Evolution 60S UV-Visible Spectrophotometer
451 (Thermo Scientific) and each culture was diluted to an OD590 of 0.1 in PBS. The plating scheme was repeated as
452 previously described while preserving the previous replicate partnerships of the last coevolved experiment (e.g. *B.*
453 *thailandensis* coevolved replicate 1 was re-plated with *F. johnsoniae* coevolved replicate 1). The plates were
454 wrapped with parafilm and incubated at 27 °C for 1.5 months. This process was repeated for a total of 5 plate
455 passages, resulting in a total experimentation time of 7.5 months.

456

457 Measurements of radial colony growth of (co)evolved plates

458 Prior to a setting up another plate passage (after a 1.5 mos incubation period), plates were imaged using an
459 Epson Perfection V370 Photo Scanner. A ruler was placed in the scanner to scale pixels to mm for radial growth
460 measurements. Images were uploaded to ImageJ2 for analysis (Rueden et al. 2017; Schneider et al. 2012). Radial
growth was determined by measuring the distance from the center of the colony to the furthest point of radial growth. Measurements were uploaded to R to generate boxplots using ggplot2 (Wickham 2016).

**Whole genome sequencing**

All *F. johnsoniae* replicates (monoculture and coevolution experiments) from the 5th plate passage and the *F. johnsoniae* ancestor were plated from freezer stocks onto TSA50 (11 freezer stocks total). Plates were incubated for ~16 h at 27 °C. The following night, isolated colonies were inoculated into TSB50 medium and incubated with shaking at 200 rpm for ~16 h at 27 °C. The following morning, DNA was extracted from all 11 cultures using the E.Z.N.A.® Bacterial DNA Kit (Omega Bio-tek, Norcross, GA) according to the manufacturer's instructions. DNA integrity was assessed from 260/280 and 260/230 ratios using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). DNA samples were sent to the Microbial Genome Sequencing Center (MiGS, Pittsburgh, PA) for whole genome sequencing. Illumina DNA library preparations were performed at the MiGS facility according to standard operating protocols. Sequencing (2x151bp) was performed on a NextSeq 2000 platform. A minimum of 200 Mbp of sequencing data was obtained with >Q30 reads. Low-quality bases were removed with Trimmomatic (Bolger et al. 2014) using a sliding-window approach; reads were trimmed if the average Phred quality score was <20 in a 4-bp window. Reads were also removed if read length was < 70 bp. Average Phred quality scores of trimmed reads were assessed using FastQC (Andrews 2010). Mutations were identified using *breseq* version0.37.0 with default parameters (Deatherage and Barrick 2014) *B. thailandensis* E264 (accession numbers NC_007651 & NC_007650) and *F. johnsoniae* UW101 (accession number NC_009441) were used as reference sequences. The summary files from post-*breseq* analysis were used to determine total reads, percentage of reads mapped to reference sequences, and average genome coverage. Sequencing statistics from whole genome sequencing are provided in Supplemental File 2.

484

TolC model prediction

The *F. johnsoniae* TolC protein sequence of interest (FJOH_RS06580) was downloaded from NCBI. The protein FASTA file was manually edited to remove 11 amino acids associated with the 33 bp deletion (amino acids
87-97). The protein FASTA file was also manually edited to create the nonsynonymous mutation (G83R). Then all three files were placed into SWISS-MODEL to model the protein structure of TolC (Waterhouse et al. 2018). The template used for rendering all models was SWISS-MODEL Template Library (SMTL) ID: 6wx1.1 (Budiardjo et al. 2022). The model from TolC wild-type was downloaded and uploaded into Swiss-PdbViewer to highlight amino acids associated with the 33 bp deletion (Guex and Peitsch 1997). The SWISS-MODEL homology target-template modeling reports are available at [https://github.com/ShadeLab/Paper_Chodkowski_Coevolution_2022](https://github.com/ShadeLab/Paper_Chodkowski_Coevolution_2022).

494

## Construction of mutants in *F. johnsoniae* ancestral strain

Single mutations of interest observed in the coevolved lines were engineered into the genome of the ancestral strain. These mutants were constructed following the previously described method (Zhu et al. 2017), with the exception a nested PCR step. All primers used in this study are listed in Supplementary Table 3.

499

## Nested PCR

DNA extracted from coevolved lines for whole genome sequencing was used as templates for PCR. Coevolved line 1 (CoE_Δ33_tolC) was used to amplify the 33 bp deletion in tolC (*F. johnsoniae* locus FJOH_RS06580; 261-293 in coding sequence), coevolved line 3 (CoE_G247A_tolC) was used to amplify the nonsynonymous mutation in tolC (*F. johnsoniae* locus FJOH_RS06580; G247A in coding sequence), and coevolved line 4 (CoE_792_793insG_ragB) was used to amplify the base insertion sequence in *ragB/susD* (*F. johnsoniae* locus FJOH_RS24865; 792_793insG in coding sequence). Nested PCR was performed to obtain enough DNA for downstream methods. For the first round of nested PCR, a 3.3-kbp fragment containing tolCΔ33 or tolC(G247A) was amplified using primers 1001 and 1002. A 3.5-kbp fragment containing *ragB792_793insG* was amplified using primers 1010 and 1011. The PCR reactions contained reagents and volumes outlined in Supplementary Table 4. PCR conditions were as follows: 98 °C for 30 s, 98 °C for 10 s, 56 °C for 15 s, and 72°C for 70 s, repeated 29 times from step 2, followed by 72°C for 10 min and hold at 4 °C. PCR products were run on a 0.8% agarose (% w/v) gel at 100V for 60 min. PCR bands at the correct fragment sizes were excised from the gel and extracted using Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, Madison WI). PCR products were quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA).
For the second round of PCR, a 3.2-kbp fragment containing *tolCΔ33* or *tolC* (G247A) was amplified using primers 1003 (engineered XbaI site) and 1004 (engineered BamHI site). A 3.1-kbp fragment containing *ragB792_793insG* was amplified using primers 1012 (engineered XbaI site) and 1013 (engineered BamHI site). The PCR reactions contained reagents and volumes outlined in Supplementary Table 5. PCR conditions were as follows: 98 °C for 30 s, 98 °C for 10 s, 56 °C for 15 s, and 72°C for 70 s, repeated 29 times from step 2, followed by 72°C for 10 min and hold at 4 °C. PCR products were run on a 0.8% agarose (% w/v) gel at 100V for 60 min. PCR bands at the correct fragment sizes were excised from the gel and extracted using Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, Madison WI). PCR products were quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA).

524

## Plasmid isolation and purification

A *E. coli* DH5α*mcr_pYT354* freezer stock was plated on lysogeny broth (LB) agar with ampicillin (100 µg/mL) and incubated for ~16 h at 37 °C. A single colony was inoculated into 5 mL LB with ampicillin (100 µg/mL) and incubated with shaking at 200 rpm for ~16 h at 37 °C. Plasmid *pYT354* was extracted *E. coli* DH5α*mcr_pYT354*and purified the following morning using the E.Z.N.A.® Plasmid DNA Mini Kit I Q-spin (Omega Bio-tek, Norcross, GA) according to the manufacturer's instructions.

531

## Restriction enzyme digestion and ligation

Separately prepared restriction enzyme double digestions were performed on purified PCR products from nested PCR round 2 and *pYT354*. The reaction reagents and volumes are outlined in Supplementary Table 6. Reactions were incubated at 37 °C for 15 min. Reactions were then run on a 0.8% agarose (% w/v) gel at 100V for 60 min. Bands at the correct fragment sizes were excised from the gel and extracted using Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, Madison WI). PCR products were quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA).

The PCR fragment containing *tolCΔ33* was ligated into *pYT354* to form pJC101, the PCR fragment containing *tolC* (G247A) was ligated into *pYT354* to form pJC102, and the PCR fragment containing
541 ragB792_793insG was ligated into pYT354 to form pJC103. Ligation reagents are outlined in Supplementary Table
542 7. The reactions were incubated at room temperature for 10 min and then heat inactivated for 10 min at 65 °C.

543
544 **Preparation of heat shock competent cells**
545
A *E. coli* DH5αmcr freezer stock was plated on lysogeny broth (LB) agar and incubated ~16 h at 37 °C. A
546 single colony was inoculated into 5 mL LB and incubated with shaking at 200 rpm for ~16 h at 37 °C. One mL of
547 the overnight culture was diluted into 100 mL LB and incubated with shaking at 37 °C until the OD590 reached 0.3
548 (~2 h), as measured on an Evolution 60S UV-Visible Spectrophotometer (Thermo Scientific). The culture was
549 placed on ice for 15 min. Two 45 mL aliquots were placed into 2, 50 mL falcon tubes and centrifuged at 4000 rpm
550 for 10 min at 4 °C. Supernatant was decanted, and the pellets were resuspended in 45 mL ice-cold 0.1 M CaCl₂. The
551 resuspended cultures were placed on ice for 30 min. Cultures were then centrifuged at 4000 rpm for 10 min at 4 °C.
552 Supernatant was decanted, and the pellets were resuspended in 4.5 mL ice-cold 0.1 M CaCl₂ with 15% glycerol (%
553 v/v). The cultures were then distributed as 50 µL aliquots into microcentrifuge tubes. Competent cells were stored as
554 freezer stocks at -80 °C until ready for use.

555
556 **Heat shock transformation**
557
Heat shock competent cells (50 µL) were removed from the freezer and placed on ice for 20 min (4 tubes, 1
558 for each ligation product and 1 pYT354 plasmid control). Two µL of ligation products (and 1 µL of 10 ng/µL
559 pYT354 plasmid) were added to each tube and placed on ice for an additional 20 min. Cells were then heat shocked
560 for 45 s at 42 °C. Heat shocked cells were placed on ice for 2 min. One mL of super optimal broth (SOC) medium
561 was added to each tube and the tubes were incubated with shaking at 200 rpm at 37 °C for 1 h. Cells were pelleted
562 by centrifugation at 4000 g for 2 min at 4 °C. The supernatant was removed (950 µL) and the remaining culture was
563 plated on LB agar containing ampicillin (100 µg/mL). Plates were incubated ~16 h at 37 °C. Successful
564 transformants were inoculated into LB containing ampicillin (100 µg/mL) and incubated ~16 h at 37 °C. Freezer
565 stocks were made the following morning for triparental conjugation.

566
567 Triparental conjugation and recombinant confirmation

568 pJC101, pJC103, and pJC104 in recombinant *E. coli* DH5αMCR needed to be transferred to the *F. johnsoniae* ancestral strain. This was introduced into *F. johnsoniae* by triparental conjugation as previously described (Rhodes et al. 2011) using recombinant *E. coli* DH5αMCR, *F. johnsoniae* ancestor, and *E. coli* HB101 (carrying the helper plasmid pRK2013), except that the *sacB* and *ermF*-containing suicide vector was used to select for successful *F. johnsoniae* recombinants (Zhu et al. 2017). F. johnsoniae recombinants (reΔ33_tolC, reG247A_tolC, re792_793insG_ragB) were confirmed by Sanger sequencing at the Michigan State Genomics Core using primers 1005 and 1006 for tolCΔ33 or tolC(G247A) recombinants and primers 1014 and 1014 for the *ragB*792_793insG recombinant.

576

577 Acetyl-CoA carboxylase carboxyl transferase subunit alpha (AccA) protein alignment

578 AccA sequences were downloaded from *F. johnsoniae* UW101 (protein ID: WP_011921560.1) and from
579 *Salmonella enterica* serovar Typhimurium strain LT2 (protein ID: NP_459237.1) on NCBI and concatenated as a
text file. The text file was uploaded to T-Coffee (Version_11.00) for protein alignment using default parameters (Di
580 Tommaso et al. 2011). The FASTA alignment file from T-Coffee output was downloaded and used as input for
581 BoxShade (version 3.21) using default parameters. The protein alignment was then uploaded to Inkscape for final
582 edits.
583

584

585 Extraction of *B. thailandensis* bioactive compound(s) and Kirby-Bauer assays

586 A freezer stock of *F. johnsoniae* was plated on TSA50 and grown for ~16 h at 27 °C. A loopful of lawn
587 growth was transferred to 7 mL of M9 minimal salts-0.2% glucose (M9-glucose; % v/v) medium to achieve an
588 initial OD590 of ~0.2 as measured on an Evolution 60S UV-Visible Spectrophotometer (Thermo Scientific). The
589 culture was incubated with shaking at 200 rpm for ~16 h at 27 °C. The next day, 5 mL of culture was transferred to
590 50 mL fresh M9-glucose medium in a 250 mL Erlenmeyer flask. The culture was incubated with shaking at 200 rpm
591 for 48 h at 27 °C. On the second day of the *F. johnsoniae* 48 h incubation, a freezer stock of *B. thailandensis* was
592 plated on TSA50 and grown for ~16 h at 27 °C. A loopful of *B. thailandensis* lawn growth was transferred to 7 mL
593 of M9-glucose medium to achieve an initial OD590 of ~0.2 as measured on an Evolution 60S UV-Visible
Spectrophotometer (Thermo Scientific). The *B. thailandensis* culture was incubated with shaking at 200 rpm for ~16 h at 27 °C.

At the completion of the *F. johnsoniae* 48 h incubation, the culture was transferred to a falcon tube and centrifuged at 5000 rpm for 20 min at 4 °C. The supernatant was removed, filtered with a 0.22 µM PES filter, and ~50 mL was transferred to a 250 mL Erlenmeyer flask. Five mL of the overnight *B. thailandensis* culture was transferred to the 50 mL filtered spent *F. johnsoniae* M9-glucose medium. In addition, 250 µL 40% glucose (% w/v) was added to the culture to re-achieve ~0.2% glucose (% v/v) in the M9-medium. The culture was incubated with shaking at 200 rpm for 48 h at 27 °C. At the completion of the *B. thailandensis* 48 h incubation in the filtered spent *F. johnsoniae* medium, the culture was transferred to a falcon tube and centrifuged at 5000 rpm for 20 min at 4 °C. The supernatant was removed, filtered with a 0.22 µM PES filter, and transferred to a separatory funnel. Ten mL of dichloromethane (DCM) was added to the separatory funnel. The separatory funnel was agitated three times and the DCM layer was removed. The addition, agitation, and removal of DCM was repeated twice more. The collected DCM layer was dried under nitrogen gas. The dried DCM extracts were reconstituted in 1 mL of a 50:50 methanol:water (% v/v) mixture. This mixture was used for the Kirby-Bauer assays.

Freezer stocks of the *F. johnsoniae* ancestor, co(evolved) strains from the fifth plate passage, and recombinant strain, reΔ33_tolC, were plated on TSA50 and grown for ~16 h at 27 °C. A loopful of lawn growth was transferred and resuspended in 500 µL of 1X PBS. The cultures were pelleted by centrifugation at 5000 rpm for 10 min at room temperature, the supernatant was removed, and the cultures were resuspended in 500 µL 1X PBS. The OD590 of the cultures were measured on an Evolution 60S UV-Visible Spectrophotometer (Thermo Scientific). From the OD590 values, we calculated the volume of PBS to remove to achieve an OD590 of 10. The cultures were pelleted by centrifugation at 5000 rpm for 10 min at room temperature, the calculated volumes of supernatant were removed, and the cultures were resuspended in the remaining volume of 1X PBS. One-hundred µL of each culture was placed on TSA50 using a cell spreader. TSA50 plates were used instead of M9-glucose agar plates because *F. johnsoniae* was incapable of growing overnight on this medium. Once dried, 3, 6 mm blank paper discs (BD BBL™) were placed on each plate. Each disc received 20 µL of one of the following solutions: 1) *B. thailandensis* filtered spent medium grown in *F. johnsoniae* filtered spent medium (described above), 2) 50:50 methanol:water (%
620 v/v) control, or 3) water control. Plates were incubated at ~16 h at 27 °C. Three independent replicates were prepared for each strain.

622 The following day, each plate was imaged using an Epson Perfection V370 Photo Scanner. A ruler was placed in the scanner to scale pixels to mm for zone of inhibition (ZOI) measurements. Images were uploaded to ImageJ2 for analysis (Rueden et al. 2017; Schneider et al. 2012). ZOIs were measured as the length between the outer perimeter of the disc to the point of observable growth. Measurements were uploaded to R to test for differences in ZOIs between strains using Wilcoxon Rank-Sum test. Plate images have been uploaded to GitHub [https://github.com/ShadeLab/Paper_Chodkowski_Coevolution_2022].

628

629 **Re-plating experiments**

630 Strains of interest ((co)evolved strains, ancestor strains, recombinants, and transposon mutants; see Figs. 6 & 7 and Supplementary Figs. 3,4, 8, and 9) were plated from freezer stocks onto TSA50. Plates were incubated for ~16 h at 27 °C. A loopful of lawn growth for each strain was inoculated as separate cultures in 7 mL TSB50.
631
632 Cultures were incubated with shaking at 200 rpm for ~16 h at 27 °C. The following day, the cultures were pelleted by centrifugation at 5000 rpm for 10 min at room temperature, the supernatant was removed, and the cultures were resuspended in 1X PBS. This process was repeated once more. The cultures were resuspended in PBS at a final volume of 5 mL. The OD590 was measured on an Evolution 60S UV-Visible Spectrophotometer (Thermo Scientific) and each culture was diluted to an OD590 of 0.1 in PBS. Ten µL of a culture (OD590 0.1) was spotted onto M9 minimal salts agar plates containing 0.2% glucose (M9-glucose; % v/v). Strains were either plated in isolation or co-plated together. When co-plated together, strains were spotted 14 mm apart. Plates were incubated at 27 °C for various amounts of time (see Figs. 6 & 7 and Supplementary Figs. 3,4,8, and 9 legends for details). *B. thailandensis* transposon mutants were acquired from the Manoil lab (Gallagher et al. 2013).

630
631
632
633
634
635
636
637
638
639
640
641
642

Strains of interest ((co)evolved strains, ancestor strains, recombinants, and transposon mutants; see Figs. 6 & 7 and Supplementary Figs. 3,4, 8, and 9) were plated from freezer stocks onto TSA50. Plates were incubated for ~16 h at 27 °C. A loopful of lawn growth for each strain was inoculated as separate cultures in 7 mL TSB50.

Cultures were incubated with shaking at 200 rpm for ~16 h at 27 °C. The following day, the cultures were pelleted by centrifugation at 5000 rpm for 10 min at room temperature, the supernatant was removed, and the cultures were resuspended in 1X PBS. This process was repeated once more. The cultures were resuspended in PBS at a final volume of 5 mL. The OD590 was measured on an Evolution 60S UV-Visible Spectrophotometer (Thermo Scientific) and each culture was diluted to an OD590 of 0.1 in PBS. Ten µL of a culture (OD590 0.1) was spotted onto M9 minimal salts agar plates containing 0.2% glucose (M9-glucose; % v/v). Strains were either plated in isolation or co-plated together. When co-plated together, strains were spotted 14 mm apart. Plates were incubated at 27 °C for various amounts of time (see Figs. 6 & 7 and Supplementary Figs. 3,4,8, and 9 legends for details). *B. thailandensis* transposon mutants were acquired from the Manoil lab (Gallagher et al. 2013).

642

643 **Statements and Declarations**

644

645 **Funding**
646 This work was supported by Michigan State University and NSF CAREER award DEB #1749544.

647

648 Conflicts of interest

649 The authors declare that there is no conflict of interest

650

651 Data and code availability

652 Supplemental files, Sanger sequencing files, and our breseq pipeline are available at

653 [https://github.com/ShadeLab/Paper_Chodkowski_Coevolution_2022]. *F. johnsoniae* and *B. thailandensis* whole

654 genome raw sequences files are deposited in the NCBI Sequence Read Archive (BioProject ID PRJNA812898).

655

656 Authors' contributions

657 JLC and AS conceived the project. JLC carried out the experiments and data analysis. JLC and AS wrote the

658 manuscript.

659

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842

843

844

845 Figure legends

846 **Fig. 1 A bioactive compound is produced by B. thailandensis** that inhibits *F. johnsoniae*. *B. thailandensis* (right) and *F. johnsoniae* (left) were co-plated on M9-glucose agar at a distance that allowed for chemical interactions. An unidentified antibiotic(s) inhibited *F. johnsoniae*.

849

850 **Fig. 2** *F. johnsoniae* efflux system contributes to the extrusion of a *B. thailandensis*-produced antibiotic(s). An end-
851 point growth measurement (n=5) was taken after *F. johnsoniae* 24 h incubation with either *B. thailandensis* culture
852 supernatant (Supernatant), with the efflux pump inhibitor (Daidzein), or with a combination of the supernatant and
853 daidzein. An untreated *F. johnsoniae* culture (Culture control), a *F. johnsoniae* culture with dimethyl sulfoxide
854 (DMSO), a *F. johnsoniae* culture with methanol (MeOH), a *F. johnsoniae* culture with both solvents (DMSO +
855 MeOH), and blank TSB50 medium served as controls. The bottom and top of each boxplot are the first and third
856 quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to
857 the largest value (top), and smallest value (bottom) was no further away than 1.5 times the interquartile range. Black
858 points on a boxplot represent outliers that were greater than 1.5 times the interquartile range. AWilcoxon Rank-Sum
859 test was performed comparing all treatments to the culture control. \* q-value <0.05, n.s.: not significant.
860

861 **Fig. 3** Schematic of (co)evolution experiment.

862

863 **Fig. 4** *F. johnsoniae* trends toward increased growth success with each plate passage in the presence of *B. thailandensis*. Images were taken at the end of each plate passage (1.5 mos) to quantify radial growth for both *F. johnsoniae* monoculture evolved (gray bars) lines and *F. johnsoniae* coevolved (white bars) lines (n=5 independent

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replicated per time point). Radial colony growth was measured from the center of the colony to the point of furthest growth on the agar plate. The bottom and top of each box are the first and third quartiles, respectively, and the line inside the box is the median. The whiskers extend from their respective hinges to the largest value (top), and smallest value (bottom) no further away than 1.5× the interquartile range. Black points on a boxplot represent outliers that were greater than 1.5 times the interquartile range.

871

872 **Fig. 5** A *tolC* 33 bp deletion is located on a TolC extracellular loop. The TolC protein (A) contains a α-helical trans-
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The yellow box represents the inset in (B), where the 11 amino acids corresponding to the 33 bp deletion are
highlighted in blue. These amino acids are part of one of the extracellular loops. The nucleotide sequence is shown
below the image, representing bps 247-294 (amino acids 83-98) in the FJOH_RS06580 coding sequence. The
corresponding amino acids are shown above each codon. Nucleotides highlighted in blue represent the 33 bp
deletion. Nucleotides highlighted in orange show how the deletion was in-frame. The 11 bp direct repeats are in bold
and underlined. The nucleotide highlighted in green is associated with the nonsynonymous mutation (G247A in the
coding sequence, G83R in TolC). TolC from *E. coli* was used as the template (SMTL ID: 6wxi.1) to construct the
target *F. johnsoniae* WT TolC model (SWISS-MODEL: GMQE= 0.6, Seq ID=19.06) shown in the figure. The *F.
johnsoniae* TolC model generated from removal of 33 bp still maintains equivalent modeling quality (SWISS-
MODEL: GMQE=0.61, Seq ID=19.06).

884

885 **Fig. 6** The *tolC* 33 bp deletion reduces antibiotic susceptibility. The ancestor (A) and recombinant strains of *F.*

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33 bp deletion (B) is the least inhibited by *B. thailandensis* compared to the *F. johnsoniae* ancestor and other *F. johnsoniae* recombinant strains. Plates were imaged after a week of incubation.

**Fig. 7** Thailandamide is bioactive against *F. johnsoniae*. While the nonsynonymous mutation in *tolC* did not reduce antibiotic susceptibility (present in *F. johnsoniae* coevolved line 3), *F. johnsoniae* coevolved line 3 still displayed a reduction in antibiotic susceptibility at the end of plate passage five (A). *B. thailandensis* transposon mutants in *thaF* (B) and *thaA* (C) have decreased inhibition toward *F. johnsoniae* while an *atsR* transposon mutant has increased inhibition of *F. johnsoniae* (D). Below the panels is an amino acid sequence alignment between the *F. johnsoniae* AccA and S. enterica AccA. The asterisks indicate positions within the proteins that were identical. The blue box highlights the alignment of P160 in *F. johnsoniae* and P164 in S. enterica.

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /><figcaption>Fig. 1 B. thailandensis-produced antibiotic(s) inhibits F. johnsoniae. B. thailandensis (right) and F. johnsoniae (left) were co-plated on M9-glucose agar at a distance that allowed for chemical interactions. An unidentified antibiotic(s) inhibited F. johnsoniae.</figcaption></figure>
<figure><img src="image_15.png" /></figure>

Fig. 2 F. johnsoniae efflux system contributes to the extrusion of a B. thailandensis-produced antibiotic(s). An end-point growth measurement was taken after F. johnsoniae incubation with B. thailandensis culture supernatant, with the efflux pump inhibitor (daidzein), or with a combination of the supernatant and daidzein. An untreated F. johnsoniae culture, a F. johnsoniae culture with dimethyl sulfoxide (DMSO), a F. johnsoniae culture with methanol (MeOH), a F. johnsoniae culture with combined solvents, and blank medium served as controls. An ANOVA was performed comparing all treatments to the culture control. A Tukey HSD post-hoc analysis was performed for pairwise comparisons. Data shown are representative of 5 independent experiments; \* p<0.05, \*\* p<0.001, \*\*\*p<0.0001, n.s.: not significant
<figure><img src="image_16.png" /><figcaption>Fig. 3 Schematic of (co)evolution experiment.</figcaption></figure>
<figure><img src="image_17.png" /></figure>

Fig. 4 F. johnsoniae trends toward increased growth success with each plate passage in the presence of B. thailandensis. Radial colony growth of coevolved F. johnsoniae was measured from the center of the colony to point of furthest growth on the agar plate. Measurements were taken at the end of each plate passage
<figure><img src="image_18.png" /></figure>

Fig. 5 A toIC 33 bp deletion is located on a ToIC extracellular loop. The ToIC protein (A) contains a α-helical trans-periplasmic tunnel, a β-barrel channel embedded in the outer membrane, and extracellular loops at the cell surface. The yellow box represents the inset in (B), where the 11 amino acids corresponding to the 33 bp deletion are highlighted in blue. These amino acids are part of one of the extracellular loops. The nucleotide sequence is shown below the image, representing bps 247-294 in the FJOH_RS06580 coding sequence. The corresponding amino acids are shown above each codon. Nucleotides highlighted in blue represent the 33 bp deletion. Nucleotides highlighted in orange show how the deletion was in-frame. The 11 bp direct repeats are in bold and underlined. The nucleotide highlighted in green is associated with the nonsynonymous mutation (G247A in the coding sequence, G83R in ToIC).
<figure><img src="image_19.png" /></figure>

Fig. 6 The toIC 33 bp deletion confers antibiotic resistance. The ancestor (A) and recombinant strains of F. johnsoniae ancestor (C-D) were co-plated with B. thailandensis. The recombinant strain with the toIC 33 bp deletion (B) is the least inhibited by B. thailandensis compared to the ancestor and other recombinant strains. Plates were imaged after a week of incubation.
<figure><img src="image_20.png" /><figcaption>Fig. 7 Thailandamide is bioactive against F. johnsoniae. While the nonsynonymous mutation in toIC did not confer antibiotic resistance (present in F. johnsoniae coevolved line 3), F. johnsoniae coevolved line 3 still displayed antibiotic resistance at the end of plate passage five (A). B. thailandensis transposon mutants in thaF (B) and thaA (C) have decreased inhibition toward F. johnsoniae while an atsR transposon mutant has increased inhibition of F. johnsoniae (D). Below the panels is an amino acid sequence alignment between the F. johnsoniae AccA and S. enterica AccA. The asterisks indicate positions within the proteins that were identical. The blue box highlights the alignment of P160 in F. johnsoniae and P164 in S. enterica.</figcaption></figure>
1 Supplementary material for

2

3 A coevolution experiment between *Flavobacterium johnsoniae* and *Burkholderia*

4 *thailandensis* reveals parallel mutations that reduce antibiotic susceptibility

5

6 John L. Chodkowski¹ and Ashley Shade¹,²,³*

7 ¹Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA

8 ²Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA

9 ³Program in Ecology, Evolution and Behavior, Michigan State University, East Lansing, MI 48824, USA

10 * Corresponding author and material requests. Email: *shadeash@msu.edu*; ORCID: 0000-0002-7189-3067

11

12 Data and code availability: [https://github.com/ShadeLab/Paper_Chodkowski_Coevolution_2022](https://github.com/ShadeLab/Paper_Chodkowski_Coevolution_2022)

13
14 Supplementary Tables

15
<table><caption>16 Supplementary Table 1 Summary of *tolC* loci in *F. johnsoniae*</caption><thead><tr><td>Locus</td><td>Protein ID</td><td>Top blastp hit</td><td>AA length</td></tr></thead><tbody><tr><td>FJOH RS06580</td><td>WP_012023347.1</td><td>TolC family protein</td><td>444</td></tr><tr><td>FJOH RS07030</td><td>WP_012023433.1</td><td>“</td><td>451</td></tr><tr><td>FJOH RS08665</td><td>WP_012023747.1</td><td>“</td><td>461</td></tr><tr><td>FJOH RS14165</td><td>WP_012024794.1</td><td>“</td><td>415</td></tr><tr><td>FJOH RS15250</td><td>WP_012025000.1</td><td>“</td><td>436</td></tr><tr><td>FJOH RS15955</td><td>WP_008463753.1</td><td>“</td><td>415</td></tr><tr><td>FJOH RS16725</td><td>WP_012025212.1</td><td>“</td><td>469</td></tr><tr><td>FJOH RS16800</td><td>WP_012025225.1</td><td>“</td><td>484</td></tr><tr><td>FJOH RS17335</td><td>WP_044047818.1</td><td>“</td><td>426</td></tr><tr><td>FJOH RS20485</td><td>WP_012025935.1</td><td>“</td><td>472</td></tr><tr><td>FJOH RS22150</td><td>WP_044048008.1</td><td>“</td><td>461</td></tr><tr><td>FJOH RS22200</td><td>WP_012026267.1</td><td>“</td><td>412</td></tr><tr><td>FJOH RS22240</td><td>WP_012026275.1</td><td>“</td><td>472</td></tr><tr><td>FJOH RS23175</td><td>WP_012026451.1</td><td>“</td><td>417</td></tr><tr><td>FJOH RS25120</td><td>WP_012026826.1</td><td>“</td><td>479</td></tr><tr><td>FJOH RS25325</td><td>WP_012026867.1</td><td>“</td><td>439</td></tr></tbody></table>

17

18
<table><caption>**Supplementary Table 2** Percent identity matrix for all TolC proteins annotated in *F. johnsoniae*. Mutations in coevolved isolates were found in RS06580. Multiple sequence alignments were made using Clustal Omega</caption><tr><td>RS06580</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RS15250</td><td>24.2</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RS20485</td><td>15.99</td><td>15.5</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RS15955</td><td>13.94</td><td>11.86</td><td>13.51</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RS16800</td><td>16.46</td><td>15.21</td><td>14.84</td><td>16.19</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RS25120</td><td>15.15</td><td>14.65</td><td>12.29</td><td>18.49</td><td>31.99</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RS16725</td><td>19.07</td><td>18.78</td><td>11.62</td><td>18.09</td><td>32.03</td><td>29.68</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RS22240</td><td>15.46</td><td>15.08</td><td>13.89</td><td>16.71</td><td>28.6</td><td>34.48</td><td>40.51</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RS17335</td><td>17.71</td><td>19.84</td><td>14.68</td><td>16.48</td><td>15.38</td><td>13.95</td><td>15.61</td><td>15.3</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RS22150</td><td>18.02</td><td>17.5</td><td>15.11</td><td>11.83</td><td>14.15</td><td>15.82</td><td>15.92</td><td>15.42</td><td>16.67</td><td>100</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RS23175</td><td>16.84</td><td>15.3</td><td>13.35</td><td>14.48</td><td>18.53</td><td>17.26</td><td>15.84</td><td>15.32</td><td>15.95</td><td>13.57</td><td>100</td><td></td><td></td><td></td><td></td></tr><tr><td>RS14165</td><td>17.36</td><td>14.82</td><td>13.66</td><td>16.49</td><td>17.05</td><td>18.09</td><td>14.51</td><td>14.78</td><td>13.06</td><td>18.25</td><td>23.08</td><td>100</td><td></td><td></td><td></td></tr><tr><td>RS22200</td><td>17.05</td><td>17.69</td><td>12.37</td><td>17.21</td><td>15.37</td><td>14.11</td><td>14.8</td><td>15.82</td><td>18.68</td><td>15.17</td><td>22.19</td><td>28.1</td><td>100</td><td></td><td></td></tr><tr><td>RS07030</td><td>20.15</td><td>18.72</td><td>14.77</td><td>14.66</td><td>14.63</td><td>14.11</td><td>14.5</td><td>14</td><td>17.95</td><td>16.1</td><td>15.38</td><td>12.94</td><td>14.21</td><td>100</td><td></td></tr><tr><td>RS25325</td><td>20.54</td><td>19.55</td><td>15.84</td><td>12.87</td><td>13.51</td><td>13.51</td><td>16.12</td><td>15.62</td><td>12.5</td><td>21.22</td><td>16.71</td><td>15.4</td><td>14.86</td><td>24.65</td><td>100</td></tr><tr><td>RS08665</td><td>22</td><td>21.84</td><td>17.36</td><td>14.33</td><td>14.47</td><td>16.2</td><td>19.01</td><td>16.41</td><td>18.59</td><td>18.76</td><td>15.03</td><td>18.16</td><td>18.59</td><td>20.15</td><td>20.35</td><td>100</td></tr></table>
<table><caption>22 Supplementary Table 3 Primers used in this study</caption><thead><tr><td>Primer</td><td>Sequence (5' &gt; 3')</td><td>Description</td></tr></thead><tbody><tr><td>1001</td><td>TTGCTTATTTGGGAG GAACAACA</td><td>Used to amplify tolC for nested PCR round 1</td></tr><tr><td>1002</td><td>CATCTGCTTTTGCGAG CGATGA</td><td>Used to amplify tolC for nested PCR round 1</td></tr><tr><td>1003</td><td>GCTAGTCTAGAGCA TCAGTTGAGTTTTCA CTGGA</td><td>Used for nested PCR round 2 to construct pJC101 and pJC102; XbaI site underlined</td></tr><tr><td>1004</td><td>GCTAGGGATCCAAG CTTGCAACCTGGCTT TC</td><td>Used for nested PCR round 2 to construct pJC101 and pJC102; BamHI site underlined</td></tr><tr><td>1005</td><td>AAATGACGGTCCCA TCTCAAA</td><td>Used to amplify tolC to confirm successful mutant construction</td></tr><tr><td>1006</td><td>CCCATGTAAAAACTTC AATGCGT</td><td>Used to amplify tolC to confirm successful mutant construction</td></tr><tr><td>1010</td><td>TGAGAACCAAAGGC TGGGAA</td><td>Used to amplify ragB/susD for nested PCR round 1</td></tr><tr><td>1011</td><td>GGTACATTGTTTTTCG GCGCA</td><td>Used to amplify ragB/susD for nested PCR round 1</td></tr><tr><td>1012</td><td>GCTAGTCTAGATGG GGATTAACCAGCGA CAG</td><td>Used for nested PCR round 2 to construct pJC103; XbaI site underlined</td></tr><tr><td>1013</td><td>GCTAGGGATCCTTCA CCTGCATCGGCAGTT C</td><td>Used for nested PCR round 2 to construct pJC103; BamHI site underlined</td></tr><tr><td>1014</td><td>ATGCTCCCGCAAAA CCAAGA</td><td>Used to amplify ragB/susD to confirm successful mutant construction</td></tr><tr><td>1015</td><td>ATCAGGACCAGTTG TTGCCG</td><td>Used to amplify ragB/susD to confirm successful mutant construction</td></tr></tbody></table>

23

24
<table><caption>25 **Supplementary Table 4** PCR conditions for nested PCR round 1</caption><thead><tr><td>Reagent</td><td>Volume (µL)</td></tr></thead><tbody><tr><td>Template (6.25 ng/µL)</td><td>10</td></tr><tr><td>Forward/Reverse primers (10 µM)</td><td>2.5</td></tr><tr><td>10 mM dNTPs (Sigma-Aldrich, St. Louis, MO)</td><td>1</td></tr><tr><td>Phusion DNA polymerase (New England BioLabs, Ipswich, MA)</td><td>0.5</td></tr><tr><td>Phusion 5X buffer (HF buffer for <i>tolC</i> and GC buffer for <i>ragB/susD</i>)</td><td>9.5</td></tr><tr><td>Nuclease-free water</td><td>24</td></tr></tbody></table>

26

27
<table><caption>28 **Supplementary Table 5** PCR conditions for nested PCR round 2</caption><thead><tr><td>Reagent</td><td>Volume (µL)</td></tr></thead><tbody><tr><td>Template (1 ng/µL; PCR product from R1)</td><td>0.5</td></tr><tr><td>Forward/Reverse primers (10 µM)</td><td>2.5</td></tr><tr><td>10 mM dNTPs</td><td>1</td></tr><tr><td>Phusion DNA polymerase</td><td>0.5</td></tr><tr><td>Phusion 5X buffer (HF buffer for <i>tolC</i> and GC buffer for <i>ragB/susD</i>)</td><td>9.5</td></tr><tr><td>Nuclease-free water</td><td>33.5</td></tr></tbody></table>

29

30
<table><caption>31 Supplementary Table 6 Reagents and reaction volumes for restriction enzyme digestion</caption><thead><tr><td>Reagent</td><td>Volume (µL)</td></tr></thead><tbody><tr><td>Nested PCR R2 products or pYT354 (1 µg/µL)</td><td>1</td></tr><tr><td>10X cutsmart buffer (New England BioLabs, Ipswich, MA)</td><td>5</td></tr><tr><td>BamHI-HF (New England BioLabs, Ipswich, MA)</td><td>1 (20 units)</td></tr><tr><td>XbaI (New England BioLabs, Ipswich, MA)</td><td>1 (20 units)</td></tr><tr><td>Nuclease-free water</td><td>42</td></tr></tbody></table>

32

33
<table><caption>34 **Supplementary Table 7** Reagents and reaction volumes/mass for ligation reactions</caption><thead><tr><td>Reagent</td><td>Volume/Mass</td></tr></thead><tbody><tr><td>Insert (~3.2 for <i>tolC</i>, ~3.1 kbp for <i>ragB/susD</i>)</td><td>Varied<sup>a</sup></td></tr><tr><td>Vector (~7.7 kbp)</td><td>50 ng</td></tr><tr><td>T4 DNA ligase (New England BioLabs, Ipswich, MA)</td><td>1 µL</td></tr><tr><td>10 X T4 DNA ligase buffer (New England BioLabs, Ipswich, MA)</td><td>2 µL</td></tr><tr><td>Nuclease-free water</td><td>Up to 20 µL</td></tr></tbody></table>

35 <sup>a</sup>To achieve a 1:3 vector:insert molar ratio, 61.49 ng was used from *tolC*-containing PCR products and 59.37 ng used from *ragB/susD*-containing PCR products.

37
38 Supplementary Figures

39
<figure><img src="image_21.png" /><figcaption>**Supplementary Fig. 1** Schematic of preparation for efflux pump inhibitor experiment.</figcaption></figure>

40

41

42
<figure><img src="image_22.png" /></figure>
45

46 Supplementary Fig. 2 Colony morphologies and growth success over the (co)evolution experiment. Plate images

47 were taken at 1.5 months after each plate passage. Shown are colony morphologies and growth success of *B.*

48 *thailandensis* monoculture (column 1), co-plated *B. thailandensis-F. johnsoniae* (column 2), and *F. johnsoniae*

49 monoculture (column 3) for a representative independent replicate (rep 3). Each row is plate passage

50
F. johnsoniae
coevolved

<figure><img src="image_23.png" /></figure>

B. thailandensis
ancestor

F. johnsoniae
coevolved

<figure><img src="image_24.png" /></figure>

F. johnsoniae
monoculture evolved

F. johnsoniae
monoculture evolved

<figure><img src="image_25.png" /></figure>

B. thailandensis
ancestor

<figure><img src="image_26.png" /></figure>

B. thailandensis
ancestor

<figure><img src="image_27.png" /></figure>

F. johnsoniae
ancestor

<figure><img src="image_28.png" /></figure>
**Supplementary Fig. 3** Coevolved *F. johnsoniae* has reduced susceptibility to a *B. thailandensis*-produced antibiotic(s). Coevolved *F. johnsoniae* can grow better in the presence of *B. thailandensis* (column 1, top row) compared to the monoculture evolved *F. johnsoniae* (column 1, middle row). Monocultures are shown as a growth control (column 2, top and middle rows). Shown are evolved lines from one of the independent replicates (rep 3) from the fifth plate passage. Ancestor *F. johnsoniae* and ancestor *B. thailandensis* are shown as additional monoculture controls (bottom row). Images were taken after incubation for 1.5 months

59
<figure><img src="image_29.png" /><figcaption>**Supplementary Fig. 4** Coevolved *F. johnsoniae* can resist colony invasion. On each plate, *F. johnsoniae* is on the left and *B. thailandensis* (right) is on the right. The *B. thailandensis* ancestor was co-plated with *F. johnsoniae* evolved monoculture (left plate) and *F. johnsoniae* coevolved (right plate) from the fifth plate passage. Plates were incubated for 2.5 months to allow the chance for physical interactions to occur</figcaption></figure>

60

61

62

63

64

65
<figure><img src="image_30.png" /><figcaption>**Supplementary Fig. 5** Coevolved lines acquire unique mutations as a result of interspecies interactions. Shown is a Venn diagram comparing distinctions and overlaps of gene loci where mutations were observed in the ancestor, monoculture evolved lines, and coculture evolved lines</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>B)</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>C)</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>D)</figcaption></figure>

<figure><img src="image_34.png" /></figure>

71

**Supplementary Fig. 6** A nonsynonymous mutation in TolC narrows the opening of the efflux channel. A model of TolC (A) with the G83R nonsynonymous mutation. TolC is rotated +90 about the x-axis in panels B-D such that TolC is viewed from top looking down the channel. The G83R residue (B) is located on one of the extracellular loops of TolC. The opening of the efflux channel in WT TolC (C) is predicted to narrow due to the G83R mutation (D). TolC from *E. coli* was used as the template (SMTL ID: 6wxi.1) to construct the target *F. johnsoniae* WT TolC (SWISS-MODEL: GMQE=0.6, Seq ID=19.06) and G83R TolC (SWISS-MODEL: GMQE= 0.59, Seq ID=19.06) models.
<figure><img src="image_35.png" /><figcaption>79
80
81
82
83
84</figcaption></figure>

**Supplementary Fig. 7** A *B. thailandensis* btaK::T23 mutant with abrogated bactobolin production still inhibits *F. johnsoniae*. *B. thailandensis* WT (top) and *B. thailandensis* btaK::T23 (bottom) was co-plated with *F. johnsoniae* WT. Strains were also plated outside the interspecies interaction zone as controls
<figure><img src="image_36.png" /></figure>

85

**Supplementary Fig. 8** *F. johnsoniae* recombinants display a reduction in antibiotic susceptibility, but not to the same degree observed in coevolved lines. The 33 bp deletion in FJOH_RS06580 was placed into the *F. johnsoniae* ancestor and co-plated with *B. thailandensis*. Two confirmed successful recombinants (reΔ33_tolC, replicates 1&2)
are less inhibited by *B. thailandensis* compared to the *F. johnsoniae* ancestor but are more inhibited compared to the coevolved line from which FJOH_RS06580 was amplified to create the recombinants. All strains were co-plated with the *B. thailandensis* ancestor. Plates were imaged after a month of incubation

92
<figure><img src="image_37.png" /><figcaption>**Supplementary Fig. 9** Growth success of *F. johnsoniae* strains when co-plated with *B. thailandensis*. *F. johnsoniae* strains (yellow) were plated in the vicinity of the *B. thailandensis* ancestor (beige, middle colony) to observe growth inhibition. In each panel, the *F. johnsoniae* ancestor (Anc; panels A-F), the *F. johnsoniae* coevolved replicates from the 5th plate passage (C1, panels A-F; C2-C5, panels A-E), the *F. johnsoniae* evolved monocultures from the 5th plate passage (M1-M5, panels A-E), and the *F. johnsoniae* recombinant strain, reΔ33_tolC (Δ33, panel F), were plated in a triangular formation around *B. thailandensis*. Duplicate colonies were spotted at the exterior of the plates as a growth control. Plates were imaged after a week of incubation</figcaption></figure>

93

94

95

96

97

98

99

100
Summary of results from breseq

<table><thead><tr><th>Organism</th><th>Evolved line and replicate</th><th>gene_name</th></tr></thead><tbody><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td>FJOH_RS04780</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td>FJOH_RS06580</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td>FJOH_RS09520</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td>FJOH_RS14155</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td>FJOH_RS20510</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td>FJOH_RS21875</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 2</td><td>FJOH_RS06580</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 2</td><td>FJOH_RS07830</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 2</td><td>FJOH_RS09515</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 2</td><td>FJOH_RS14175</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 2</td><td>FJOH_RS15020</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>FJOH_RS00255</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>FJOH_RS06580</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>FJOH_RS09520</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>FJOH_RS11170</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>FJOH_RS12240</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>FJOH_RS14155</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 4</td><td>FJOH_RS09520</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 4</td><td>FJOH_RS14155</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 4</td><td>FJOH_RS24860</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 4</td><td>FJOH_RS24865</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 4</td><td>FJOH_RS25290</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 5</td><td>gcvP</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 5</td><td>FJOH_RS06580</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 5</td><td>FJOH_RS07435</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 5</td><td>FJOH_RS09515</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 5</td><td>FJOH_RS14155</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td>[FJOH_RS00780]</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td>FJOH_RS07435</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td>FJOH_RS07490</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td>FJOH_RS12480</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td>FJOH_RS14155</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td>FJOH_RS26065</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td>FJOH_RS01300/ffh</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td>rpsL</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td>FJOH_RS07435</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td>FJOH_RS13650</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td>FJOH_RS14155</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td>FJOH_RS26355</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td>FJOH_RS24860</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td>FJOH_RS07435</td></tr></tbody></table>
<table><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td>FJOH_RS07495</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td>rpoC</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td>FJOH_RS14155</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td>FJOH_RS15020</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td>FJOH_RS24860</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td>FJOH_RS05090</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td>FJOH_RS07435</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td>FJOH_RS07495</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td>FJOH_RS14155</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td>FJOH_RS20925</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td>FJOH_RS24860</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 5</td><td>FJOH_RS02110</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 5</td><td>FJOH_RS14170</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 5</td><td>FJOH_RS15030/FJC</td></tr><tr><td>F. johnsoniae</td><td>tolc 33 bp deltion recombi</td><td>FJOH_RS06580</td></tr><tr><td>F. johnsoniae</td><td>tolc 33 bp deltion recombi</td><td>FJOH_RS19625/FJC</td></tr></table>
<table><thead><tr><th>gene_position</th><th>gene_product</th></tr></thead><tbody><tr><td></td><td>329 hypothetical protein</td></tr><tr><td>coding (261-293/1335 nt)</td><td>TolC family protein</td></tr><tr><td>coding (825/999 nt)</td><td>histidine kinase</td></tr><tr><td></td><td>2476 CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td>coding (201/594 nt)</td><td>TetR/AcrR family transcriptional regulator</td></tr><tr><td></td><td>1489 glycoside hydrolase</td></tr><tr><td>coding (261-293/1335 nt)</td><td>TolC family protein</td></tr><tr><td></td><td>148 NAD(P)/FAD-dependent oxidoreductase</td></tr><tr><td></td><td>46 LytTR family DNA-binding domain-containing protein</td></tr><tr><td></td><td>269 response regulator transcription factor</td></tr><tr><td>coding (747-758/3714 nt)</td><td>SusC/RagA family TonB-linked outer membrane protein</td></tr><tr><td></td><td>479 acetyl-CoA carboxylase carboxyltransferase subunit alpha</td></tr><tr><td></td><td>247 TolC family protein</td></tr><tr><td></td><td>496 histidine kinase</td></tr><tr><td></td><td>273 Gfo/Idh/MocA family oxidoreductase</td></tr><tr><td></td><td>791 AIR synthase-related protein</td></tr><tr><td></td><td>278 CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td>coding (621/999 nt)</td><td>histidine kinase</td></tr><tr><td></td><td>2476 CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td></td><td>328 TonB-dependent receptor</td></tr><tr><td>coding (793/1734 nt)</td><td>RagB/SusD family nutrient uptake outer membrane protein</td></tr><tr><td></td><td>50 phosphoribosylanthranilate isomerase</td></tr><tr><td>coding (504/2850 nt)</td><td>aminomethyl-transferring glycine dehydrogenase</td></tr><tr><td>coding (261-293/1335 nt)</td><td>TolC family protein</td></tr><tr><td>coding (353/2184 nt)</td><td>carboxy terminal-processing peptidase</td></tr><tr><td></td><td>44 LytTR family DNA-binding domain-containing protein</td></tr><tr><td></td><td>695 CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td></td><td>[FJOH_RS00780]</td></tr><tr><td>coding (786/2184 nt)</td><td>carboxy terminal-processing peptidase</td></tr><tr><td>coding (584-597/747 nt)</td><td>response regulator transcription factor</td></tr><tr><td>coding (719/774 nt)</td><td>carboxypeptidase-like regulatory domain-containing protein</td></tr><tr><td></td><td>1369 CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td></td><td>838 sigma-54 dependent transcriptional regulator</td></tr><tr><td>Intergenic (-39/+50)</td><td>bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase</td></tr><tr><td></td><td>147 30S ribosomal protein S12</td></tr><tr><td>coding (817/2184 nt)</td><td>carboxy terminal-processing peptidase</td></tr><tr><td>coding (1064/1260 nt)</td><td>site-specific integrase</td></tr><tr><td></td><td>2297 CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td></td><td>3992 T9SS type A sorting domain-containing protein</td></tr><tr><td>coding (2054-2056/3288 nt)</td><td>TonB-dependent receptor</td></tr><tr><td>coding (995/2184 nt)</td><td>carboxy terminal-processing peptidase</td></tr></tbody></table>
<table><tr><td>coding (103/1731 nt)</td><td>ATP-binding protein</td></tr><tr><td>2071</td><td>DNA-directed RNA polymerase subunit beta'</td></tr><tr><td>2476</td><td>CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td>coding (973-1245/3714 nt)</td><td>SusC/RagA family TonB-linked outer membrane protein</td></tr><tr><td>coding (603-605/3288 nt)</td><td>TonB-dependent receptor</td></tr><tr><td>124</td><td>DUF4837 family protein</td></tr><tr><td>coding (735-779/2184 nt)</td><td>carboxy terminal-processing peptidase</td></tr><tr><td>coding (1606/1731 nt)</td><td>ATP-binding protein</td></tr><tr><td>2476</td><td>CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td>coding (137/540 nt)</td><td>tail fiber protein</td></tr><tr><td>1673</td><td>TonB-dependent receptor</td></tr><tr><td>coding (3112-3204/3252 nt)</td><td>SusC/RagA family TonB-linked outer membrane protein</td></tr><tr><td>158</td><td>HAMP domain-containing histidine kinase</td></tr><tr><td>intergenic (-108/+243)</td><td>sigma-70 family RNA polymerase sigma factor/hypothetical protein</td></tr><tr><td>coding (261-293/1335 nt)</td><td>ToIC family protein</td></tr><tr><td>intergenic (-402/-451)</td><td>hypothetical protein/Crp/Fnr family transcriptional regulator</td></tr></table>
<table><thead><tr><th>mutation_ca</th><th>new_seq</th><th>position</th><th>position_end</th><th>position_star</th><th>size</th><th>SNP_type</th></tr></thead><tbody><tr><td>snp_nonsyn</td><td>C</td><td>1032329</td><td>1032329</td><td>1032329</td><td></td><td>nonsynonymous</td></tr><tr><td>small_indel</td><td></td><td>1442876</td><td>1442908</td><td>1442876</td><td>33</td><td></td></tr><tr><td>small_indel</td><td>T</td><td>2115383</td><td>2115383</td><td>2115383</td><td></td><td></td></tr><tr><td>snp_nonsyn</td><td>A</td><td>3254087</td><td>3254087</td><td>3254087</td><td></td><td>nonsynonymous</td></tr><tr><td>small_indel</td><td></td><td>4734734</td><td>4734734</td><td>4734734</td><td></td><td></td></tr><tr><td>snp_nonsyn</td><td>C</td><td>5103192</td><td>5103192</td><td>5103192</td><td></td><td>nonsynonymous</td></tr><tr><td>small_indel</td><td></td><td>1442876</td><td>1442908</td><td>1442876</td><td>33</td><td></td></tr><tr><td>snp_nonsyn</td><td>A</td><td>1735676</td><td>1735676</td><td>1735676</td><td></td><td>nonsynonymous</td></tr><tr><td>snp_nonsyn</td><td>T</td><td>2115191</td><td>2115191</td><td>2115191</td><td></td><td>nonsynonymous</td></tr><tr><td>snp_nonsyn</td><td>A</td><td>3260769</td><td>3260769</td><td>3260769</td><td></td><td>nonsynonymous</td></tr><tr><td>small_indel</td><td></td><td>3437168</td><td>3437179</td><td>3437168</td><td>12</td><td></td></tr><tr><td>snp_nonsyn</td><td>T</td><td>48770</td><td>48770</td><td>48770</td><td></td><td>nonsynonymous</td></tr><tr><td>snp_nonsyn</td><td>A</td><td>1442862</td><td>1442862</td><td>1442862</td><td></td><td>nonsynonymous</td></tr><tr><td>snp_nonsyn</td><td>T</td><td>2115712</td><td>2115712</td><td>2115712</td><td></td><td>nonsynonymous</td></tr><tr><td>snp_synonym</td><td>C</td><td>2593409</td><td>2593409</td><td>2593409</td><td></td><td>synonymous</td></tr><tr><td>snp_nonsyn</td><td>T</td><td>2825359</td><td>2825359</td><td>2825359</td><td></td><td>nonsynonymous</td></tr><tr><td>snp_nonsyn</td><td>G</td><td>3256285</td><td>3256285</td><td>3256285</td><td></td><td>nonsynonymous</td></tr><tr><td>small_indel</td><td></td><td>2115587</td><td>2115587</td><td>2115587</td><td>1</td><td></td></tr><tr><td>snp_nonsyn</td><td>A</td><td>3254087</td><td>3254087</td><td>3254087</td><td></td><td>nonsynonymous</td></tr><tr><td>snp_nonsyn</td><td>C</td><td>5823750</td><td>5823750</td><td>5823750</td><td></td><td>nonsynonymous</td></tr><tr><td>small_indel</td><td>G</td><td>5827528</td><td>5827528</td><td>5827528</td><td></td><td></td></tr><tr><td>snp_nonsyn</td><td>C</td><td>5916920</td><td>5916920</td><td>5916920</td><td></td><td>nonsynonymous</td></tr><tr><td>small_indel</td><td></td><td>461783</td><td>461783</td><td>461783</td><td>1</td><td></td></tr><tr><td>small_indel</td><td></td><td>1442876</td><td>1442908</td><td>1442876</td><td>33</td><td></td></tr><tr><td>small_indel</td><td>A</td><td>1639597</td><td>1639597</td><td>1639597</td><td></td><td></td></tr><tr><td>snp_nonsyn</td><td>A</td><td>2115193</td><td>2115193</td><td>2115193</td><td></td><td>nonsynonymous</td></tr><tr><td>snp_nonsyn</td><td>A</td><td>3255868</td><td>3255868</td><td>3255868</td><td></td><td>nonsynonymous</td></tr><tr><td>large_deletion</td><td></td><td>149160</td><td>149449</td><td>149160</td><td>290</td><td></td></tr><tr><td>small_indel</td><td></td><td>1640030</td><td>1640030</td><td>1640030</td><td>1</td><td></td></tr><tr><td>small_indel</td><td></td><td>1654068</td><td>1654081</td><td>1654068</td><td>14</td><td></td></tr><tr><td>small_indel</td><td>A</td><td>2874473</td><td>2874473</td><td>2874473</td><td></td><td></td></tr><tr><td>snp_nonsyn</td><td>A</td><td>3255194</td><td>3255194</td><td>3255194</td><td></td><td>nonsynonymous</td></tr><tr><td>snp_nonsyn</td><td>T</td><td>6085864</td><td>6085864</td><td>6085864</td><td></td><td>nonsynonymous</td></tr><tr><td>large_deletion</td><td></td><td>257856</td><td>257906</td><td>257856</td><td>51</td><td></td></tr><tr><td>snp_nonsyn</td><td>A</td><td>411121</td><td>411121</td><td>411121</td><td></td><td>nonsynonymous</td></tr><tr><td>small_indel</td><td>A</td><td>1640061</td><td>1640061</td><td>1640061</td><td></td><td></td></tr><tr><td>small_indel</td><td></td><td>3132257</td><td>3132257</td><td>3132257</td><td>1</td><td></td></tr><tr><td>snp_nonsyn</td><td>T</td><td>3254266</td><td>3254266</td><td>3254266</td><td></td><td>nonsynonymous</td></tr><tr><td>snp_nonsyn</td><td>A</td><td>4834921</td><td>4834921</td><td>4834921</td><td></td><td>nonsense</td></tr><tr><td>small_indel</td><td></td><td>5825476</td><td>5825478</td><td>5825476</td><td>3</td><td></td></tr><tr><td>small_indel</td><td></td><td>1640239</td><td>1640239</td><td>1640239</td><td>1</td><td></td></tr></tbody></table>
<table><tr><td>small_indel</td><td>1656294</td><td>1656294</td><td>1656294</td><td>1</td></tr><tr><td>snp_nonsyno A</td><td>2249614</td><td>2249614</td><td>2249614</td><td>nonsynonymous</td></tr><tr><td>snp_nonsyno A</td><td>3254087</td><td>3254087</td><td>3254087</td><td>nonsynonymous</td></tr><tr><td>large_deletion</td><td>3436681</td><td>3436953</td><td>3436681</td><td>273</td></tr><tr><td>small_indel</td><td>5824025</td><td>5824027</td><td>5824025</td><td>3</td></tr><tr><td>snp_nonsyno C</td><td>1091086</td><td>1091086</td><td>1091086</td><td>nonsynonymous</td></tr><tr><td>small_indel</td><td>1639979</td><td>1640023</td><td>1639979</td><td>45</td></tr><tr><td>small_indel T</td><td>1654791</td><td>1654791</td><td>1654791</td><td></td></tr><tr><td>snp_nonsyno A</td><td>3254087</td><td>3254087</td><td>3254087</td><td>nonsynonymous</td></tr><tr><td>small_indel GG</td><td>4828379</td><td>4828379</td><td>4828379</td><td></td></tr><tr><td>snp_nonsyno C</td><td>5825095</td><td>5825095</td><td>5825095</td><td>nonsynonymous</td></tr><tr><td>large_deletion</td><td>416734</td><td>416826</td><td>416734</td><td>93</td></tr><tr><td>snp_nonsens T</td><td>3260203</td><td>3260203</td><td>3260203</td><td>nonsense</td></tr><tr><td>small_indel</td><td>3440151</td><td>3440164</td><td>3440151</td><td>14</td></tr><tr><td>small_indel</td><td>1442876</td><td>1442908</td><td>1442876</td><td>33</td></tr><tr><td>small_indel CTAA</td><td>4493481</td><td>4493481</td><td>4493481</td><td></td></tr></table>
Type

<table><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>INS</td></tr><tr><td>SNP</td></tr><tr><td>INS</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>INS</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>DEL</td></tr><tr><td>INS</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>DEL</td></tr><tr><td>DEL</td></tr><tr><td>INS</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>SNP</td></tr><tr><td>INS</td></tr><tr><td>DEL</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>DEL</td></tr></table>
<table><tr><td>DEL</td></tr><tr><td>SNP</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>DEL</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>INS</td></tr><tr><td>SNP</td></tr><tr><td>INS</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>SNP</td></tr><tr><td>DEL</td></tr><tr><td>DEL</td></tr><tr><td>INS</td></tr></table>
Summary of results from breseq

<table><thead><tr><th>Organism</th><th>Evolved line and replicate</th><th>aa_new_seq</th><th>aa_position</th><th>aa_ref_seq</th><th>codon_new_seq</th></tr></thead><tbody><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td>A</td><td></td><td>110 G</td><td>GCA</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td>C</td><td></td><td>826 R</td><td>TGT</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 1</td><td>H</td><td></td><td>497 Y</td><td>CAT</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 2</td><td>T</td><td></td><td>50 P</td><td>ACA</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 2</td><td>T</td><td></td><td>16 A</td><td>ACG</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 2</td><td>F</td><td></td><td>90 S</td><td>TTC</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>Q</td><td></td><td>160 P</td><td>CAG</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>R</td><td></td><td>83 G</td><td>AGA</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>R</td><td></td><td>166 G</td><td>AGA</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>G</td><td></td><td>91 G</td><td>GGC</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>L</td><td></td><td>264 S</td><td>TTA</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 3</td><td>S</td><td></td><td>93 L</td><td>TCA</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 4</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 4</td><td>C</td><td></td><td>826 R</td><td>TGT</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 4</td><td>P</td><td></td><td>110 S</td><td>CCT</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 4</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 4</td><td>A</td><td></td><td>17 V</td><td>GCA</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 5</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 5</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 5</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 5</td><td>L</td><td></td><td>15 P</td><td>CTG</td></tr><tr><td>F. johnsoniae</td><td>Coevolved replicate 5</td><td>V</td><td></td><td>232 G</td><td>GTT</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td>S</td><td></td><td>457 A</td><td>TCT</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 1</td><td>T</td><td></td><td>280 A</td><td>ACA</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td>I</td><td></td><td>49 M</td><td>ATT</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td>Q</td><td></td><td>766 R</td><td>CAA</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td>*</td><td></td><td>1331 S</td><td>TAA</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td>S</td><td></td><td>691 G</td><td>AGT</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td>C</td><td></td><td>826 R</td><td>TGT</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 3</td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td>E</td><td>42</td><td>Q</td><td>GAA</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td>C</td><td>826</td><td>R</td><td>TGT</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 4</td><td>P</td><td>558</td><td>H</td><td>CCC</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 5</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 5</td><td>*</td><td>53</td><td>L</td><td>TAG</td></tr><tr><td>F. johnsoniae</td><td>Monoculture replicate 5</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>tolc 33 bp deltion recombinant strain</td><td></td><td></td><td></td><td></td></tr><tr><td>F. johnsoniae</td><td>tolc 33 bp deltion recombinant strain</td><td></td><td></td><td></td><td></td></tr></table>
codon_numb codon_positi codon_ref_se gene_name

<table><tr><td>110</td><td>2</td><td>GGA</td><td>FJOH_RS04780</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS06580</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS09520</td></tr><tr><td>826</td><td>1</td><td>CGT</td><td>FJOH_RS14155</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS20510</td></tr><tr><td>497</td><td>1</td><td>TAT</td><td>FJOH_RS21875</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS06580</td></tr><tr><td>50</td><td>1</td><td>CCA</td><td>FJOH_RS07830</td></tr><tr><td>16</td><td>1</td><td>GCG</td><td>FJOH_RS09515</td></tr><tr><td>90</td><td>2</td><td>TCC</td><td>FJOH_RS14175</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS15020</td></tr><tr><td>160</td><td>2</td><td>CCG</td><td>FJOH_RS00255</td></tr><tr><td>83</td><td>1</td><td>GGA</td><td>FJOH_RS06580</td></tr><tr><td>166</td><td>1</td><td>GGA</td><td>FJOH_RS09520</td></tr><tr><td>91</td><td>3</td><td>GGG</td><td>FJOH_RS11170</td></tr><tr><td>264</td><td>2</td><td>TCA</td><td>FJOH_RS12240</td></tr><tr><td>93</td><td>2</td><td>TTA</td><td>FJOH_RS14155</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS09520</td></tr><tr><td>826</td><td>1</td><td>CGT</td><td>FJOH_RS14155</td></tr><tr><td>110</td><td>1</td><td>TCT</td><td>FJOH_RS24860</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS24865</td></tr><tr><td>17</td><td>2</td><td>GTA</td><td>FJOH_RS25290</td></tr><tr><td></td><td></td><td></td><td>gcvP</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS06580</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS07435</td></tr><tr><td>15</td><td>2</td><td>CCG</td><td>FJOH_RS09515</td></tr><tr><td>232</td><td>2</td><td>GGT</td><td>FJOH_RS14155</td></tr><tr><td></td><td></td><td></td><td>[FJOH_RS00780]</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS07435</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS07490</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS12480</td></tr><tr><td>457</td><td>1</td><td>GCT</td><td>FJOH_RS14155</td></tr><tr><td>280</td><td>1</td><td>GCA</td><td>FJOH_RS26065</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS01300/ffh</td></tr><tr><td>49</td><td>3</td><td>ATG</td><td>rpsL</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS07435</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS13650</td></tr><tr><td>766</td><td>2</td><td>CGA</td><td>FJOH_RS14155</td></tr><tr><td>1331</td><td>2</td><td>TCA</td><td>FJOH_RS26355</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS24860</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS07435</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS07495</td></tr><tr><td>691</td><td>1</td><td>GGT</td><td>rpoC</td></tr><tr><td>826</td><td>1</td><td>CGT</td><td>FJOH_RS14155</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS15020</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS24860</td></tr></table>
<table><tr><td>42</td><td>1</td><td>CAA</td><td>FJOH_RS05090</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS07435</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS07495</td></tr><tr><td>826</td><td>1</td><td>CGT</td><td>FJOH_RS14155</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS20925</td></tr><tr><td>558</td><td>2</td><td>CAC</td><td>FJOH_RS24860</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS02110</td></tr><tr><td>53</td><td>2</td><td>TTG</td><td>FJOH_RS14170</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS15030/FJOH_RS15030</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS06580</td></tr><tr><td></td><td></td><td></td><td>FJOH_RS19625/FJOH_RS19625</td></tr></table>
gene_position

gene_product

<table><tr><td></td><td>329</td><td>hypothetical protein</td></tr><tr><td>coding (261-293/1335 nt)</td><td></td><td>TolC family protein</td></tr><tr><td>coding (825/999 nt)</td><td></td><td>histidine kinase</td></tr><tr><td></td><td>2476</td><td>CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td>coding (201/594 nt)</td><td></td><td>TetR/AcrR family transcriptional regulator</td></tr><tr><td></td><td>1489</td><td>glycoside hydrolase</td></tr><tr><td>coding (261-293/1335 nt)</td><td></td><td>TolC family protein</td></tr><tr><td></td><td>148</td><td>NAD(P)/FAD-dependent oxidoreductase</td></tr><tr><td></td><td>46</td><td>LytTR family DNA-binding domain-containing protein</td></tr><tr><td></td><td>269</td><td>response regulator transcription factor</td></tr><tr><td>coding (747-758/3714 nt)</td><td></td><td>SusC/RagA family TonB-linked outer membrane protein</td></tr><tr><td></td><td>479</td><td>acetyl-CoA carboxylase carboxyltransferase subunit alpha</td></tr><tr><td></td><td>247</td><td>TolC family protein</td></tr><tr><td></td><td>496</td><td>histidine kinase</td></tr><tr><td></td><td>273</td><td>Gfo/Idh/MocA family oxidoreductase</td></tr><tr><td></td><td>791</td><td>AIR synthase-related protein</td></tr><tr><td></td><td>278</td><td>CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td>coding (621/999 nt)</td><td></td><td>histidine kinase</td></tr><tr><td></td><td>2476</td><td>CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td></td><td>328</td><td>TonB-dependent receptor</td></tr><tr><td>coding (793/1734 nt)</td><td></td><td>RagB/SusD family nutrient uptake outer membrane protein</td></tr><tr><td></td><td>50</td><td>phosphoribosylanthranilate isomerase</td></tr><tr><td>coding (504/2850 nt)</td><td></td><td>aminomethyl-transferring glycine dehydrogenase</td></tr><tr><td>coding (261-293/1335 nt)</td><td></td><td>TolC family protein</td></tr><tr><td>coding (353/2184 nt)</td><td></td><td>carboxy terminal-processing peptidase</td></tr><tr><td></td><td>44</td><td>LytTR family DNA-binding domain-containing protein</td></tr><tr><td></td><td>695</td><td>CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td></td><td></td><td>[FJOH_RS00780]</td></tr><tr><td>coding (786/2184 nt)</td><td></td><td>carboxy terminal-processing peptidase</td></tr><tr><td>coding (584-597/747 nt)</td><td></td><td>response regulator transcription factor</td></tr><tr><td>coding (719/774 nt)</td><td></td><td>carboxypeptidase-like regulatory domain-containing protein</td></tr><tr><td></td><td>1369</td><td>CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td></td><td>838</td><td>sigma-54 dependent transcriptional regulator</td></tr><tr><td>intergenic (-39/+50)</td><td></td><td>bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase</td></tr><tr><td></td><td>147</td><td>30S ribosomal protein S12</td></tr><tr><td>coding (817/2184 nt)</td><td></td><td>carboxy terminal-processing peptidase</td></tr><tr><td>coding (1064/1260 nt)</td><td></td><td>site-specific integrase</td></tr><tr><td></td><td>2297</td><td>CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td></td><td>3992</td><td>T9SS type A sorting domain-containing protein</td></tr><tr><td>coding (2054-2056/3288 nt)</td><td></td><td>TonB-dependent receptor</td></tr><tr><td>coding (995/2184 nt)</td><td></td><td>carboxy terminal-processing peptidase</td></tr><tr><td>coding (103/1731 nt)</td><td></td><td>ATP-binding protein</td></tr><tr><td></td><td>2071</td><td>DNA-directed RNA polymerase subunit beta'</td></tr><tr><td></td><td>2476</td><td>CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td>coding (973-1245/3714 nt)</td><td></td><td>SusC/RagA family TonB-linked outer membrane protein</td></tr><tr><td>coding (603-605/3288 nt)</td><td></td><td>TonB-dependent receptor</td></tr></table>
<table><tr><td>124</td><td>DUF4837 family protein</td></tr><tr><td>coding (735-779/2184 nt)</td><td>carboxy terminal-processing peptidase</td></tr><tr><td>coding (1606/1731 nt)</td><td>ATP-binding protein</td></tr><tr><td>2476</td><td>CusA/CzcA family heavy metal efflux RND transporter</td></tr><tr><td>coding (137/540 nt)</td><td>tail fiber protein</td></tr><tr><td>1673</td><td>TonB-dependent receptor</td></tr><tr><td>coding (3112-3204/3252 nt)</td><td>SusC/RagA family TonB-linked outer membrane protein</td></tr><tr><td>158</td><td>HAMP domain-containing histidine kinase</td></tr><tr><td>intergenic (-108/+243)</td><td>sigma-70 family RNA polymerase sigma factor/hypothetical protein</td></tr><tr><td>coding (261-293/1335 nt)</td><td>TolC family protein</td></tr><tr><td>intergenic (-402/-451)</td><td>hypothetical protein/Crp/Fnr family transcriptional regulator</td></tr></table>
<table><tr><td>&gt;</td><td>FJOH_RS04780</td><td>FJOH_RS04780</td></tr><tr><td>&gt;</td><td>FJOH_RS06580</td><td>FJOH_RS065 FJOH_RS06580</td></tr><tr><td>&lt;</td><td>FJOH_RS09520</td><td>1 FJOH_RS09520</td></tr><tr><td>&lt;</td><td>FJOH_RS14155</td><td>FJOH_RS14155</td></tr><tr><td>&gt;</td><td>FJOH_RS20510</td><td>1 FJOH_RS205 FJOH_RS20510</td></tr><tr><td>&gt;</td><td>FJOH_RS21875</td><td>FJOH_RS21875</td></tr><tr><td>&gt;</td><td>FJOH_RS06580</td><td>FJOH_RS065 FJOH_RS06580</td></tr><tr><td>&gt;</td><td>FJOH_RS07830</td><td>FJOH_RS07830</td></tr><tr><td>&lt;</td><td>FJOH_RS09515</td><td>FJOH_RS09515</td></tr><tr><td>&lt;</td><td>FJOH_RS14175</td><td>FJOH_RS14175</td></tr><tr><td>&lt;</td><td>FJOH_RS15020</td><td>FJOH_RS150 FJOH_RS15020</td></tr><tr><td>&lt;</td><td>FJOH_RS00255</td><td>FJOH_RS00255</td></tr><tr><td>&gt;</td><td>FJOH_RS06580</td><td>FJOH_RS06580</td></tr><tr><td>&lt;</td><td>FJOH_RS09520</td><td>FJOH_RS09520</td></tr><tr><td>&gt;</td><td>FJOH_RS11170</td><td>FJOH_RS11170</td></tr><tr><td>&gt;</td><td>FJOH_RS12240</td><td>FJOH_RS12240</td></tr><tr><td>&lt;</td><td>FJOH_RS14155</td><td>FJOH_RS14155</td></tr><tr><td>&lt;</td><td>FJOH_RS09520</td><td>FJOH_RS095 FJOH_RS09520</td></tr><tr><td>&lt;</td><td>FJOH_RS14155</td><td>FJOH_RS14155</td></tr><tr><td>&gt;</td><td>FJOH_RS24860</td><td>FJOH_RS24860</td></tr><tr><td>&gt;</td><td>FJOH_RS24865</td><td>1 FJOH_RS248 FJOH_RS24865</td></tr><tr><td>&gt;</td><td>FJOH_RS25290</td><td>FJOH_RS25290</td></tr><tr><td>&gt;</td><td>gcvP</td><td>FJOH_RS023 FJOH_RS023</td></tr><tr><td>&gt;</td><td>FJOH_RS06580</td><td>FJOH_RS065 FJOH_RS06580</td></tr><tr><td>&gt;</td><td>FJOH_RS07435</td><td>1 FJOH_RS074 FJOH_RS07435</td></tr><tr><td>&lt;</td><td>FJOH_RS09515</td><td>FJOH_RS09515</td></tr><tr><td>&lt;</td><td>FJOH_RS14155</td><td>FJOH_RS14155</td></tr><tr><td>&gt;</td><td>FJOH_RS00780</td><td>[FJOH_RS007 FJOH_RS00780</td></tr><tr><td>&gt;</td><td>FJOH_RS07435</td><td>FJOH_RS074 FJOH_RS07435</td></tr><tr><td>&lt;</td><td>FJOH_RS07490</td><td>FJOH_RS074 FJOH_RS07490</td></tr><tr><td>&lt;</td><td>FJOH_RS12480</td><td>1 FJOH_RS12480</td></tr><tr><td>&lt;</td><td>FJOH_RS14155</td><td>FJOH_RS14155</td></tr><tr><td>&lt;</td><td>FJOH_RS26065</td><td>FJOH_RS26065</td></tr><tr><td>&lt;/&gt;</td><td>FJOH_RS01300</td><td>FJOH_RS01300/FJOH_RS01300</td></tr><tr><td>&lt;</td><td>rpsL</td><td>FJOH_RS02100</td></tr><tr><td>&gt;</td><td>FJOH_RS07435</td><td>1 FJOH_RS074 FJOH_RS07435</td></tr><tr><td>&gt;</td><td>FJOH_RS13650</td><td>FJOH_RS13650</td></tr><tr><td>&lt;</td><td>FJOH_RS14155</td><td>FJOH_RS14155</td></tr><tr><td>&gt;</td><td>FJOH_RS26355</td><td>FJOH_RS263 FJOH_RS26355</td></tr><tr><td>&gt;</td><td>FJOH_RS24860</td><td>FJOH_RS248 FJOH_RS24860</td></tr><tr><td>&gt;</td><td>FJOH_RS07435</td><td>FJOH_RS074 FJOH_RS07435</td></tr><tr><td>&lt;</td><td>FJOH_RS07495</td><td>FJOH_RS074 FJOH_RS07495</td></tr><tr><td>&gt;</td><td>rpoo</td><td>FJOH_RS10115</td></tr><tr><td>&lt;</td><td>FJOH_RS14155</td><td>FJOH_RS14155</td></tr><tr><td>&lt;</td><td>FJOH_RS15020</td><td>FJOH_RS150 FJOH_RS15020</td></tr><tr><td>&gt;</td><td>FJOH_RS24860</td><td>FJOH_RS248 FJOH_RS24860</td></tr></table>
<table><tr><td></td><td>FJOH_RS05090</td><td></td><td>FJOH_RS05090</td></tr><tr><td></td><td>FJOH_RS07435</td><td></td><td>FJOH_RS074 FJOH_RS074</td></tr><tr><td></td><td>FJOH_RS07495</td><td>1</td><td>FJOH_RS07495</td></tr><tr><td></td><td>FJOH_RS14155</td><td></td><td>FJOH_RS14155</td></tr><tr><td></td><td>FJOH_RS20925</td><td>1</td><td>FJOH_RS209 FJOH_RS209</td></tr><tr><td></td><td>FJOH_RS24860</td><td></td><td>FJOH_RS24860</td></tr><tr><td></td><td>FJOH_RS02110</td><td></td><td>FJOH_RS02110</td></tr><tr><td></td><td>FJOH_RS14170</td><td></td><td>FJOH_RS141 FJOH_RS141</td></tr><tr><td></td><td>FJOH_RS15030</td><td></td><td>FJOH_RS15030/FJOH_RS1</td></tr><tr><td></td><td>FJOH_RS06580</td><td></td><td>FJOH_RS065 FJOH_RS065</td></tr><tr><td></td><td></td><td>1</td><td>FJOH_RS19625/FJOH_RS1</td></tr></table>
<table><thead><tr><th>locuselo</th><th>locuselo</th><th>mutation_c</th><th>new_seq</th><th>position</th><th>position_end</th><th>position_star</th></tr></thead><tbody><tr><td>FJOH_RS04780</td><td></td><td>snp_nonsynoc</td><td>C</td><td>1032329</td><td>1032329</td><td>1032329</td></tr><tr><td>30</td><td></td><td>small_indel</td><td></td><td>1442876</td><td>1442908</td><td>1442876</td></tr><tr><td>FJOH_RS09520</td><td></td><td>small_indel</td><td>T</td><td>2115383</td><td>2115383</td><td>2115383</td></tr><tr><td>FJOH_RS14155</td><td></td><td>snp_nonsynoc</td><td>A</td><td>3254087</td><td>3254087</td><td>3254087</td></tr><tr><td>10</td><td></td><td>small_indel</td><td>T</td><td>4734734</td><td>4734734</td><td>4734734</td></tr><tr><td>FJOH_RS21875</td><td></td><td>snp_nonsynoc</td><td>C</td><td>5103192</td><td>5103192</td><td>5103192</td></tr><tr><td>30</td><td></td><td>small_indel</td><td></td><td>1442876</td><td>1442908</td><td>1442876</td></tr><tr><td>FJOH_RS07830</td><td></td><td>snp_nonsynoc</td><td>A</td><td>1735676</td><td>1735676</td><td>1735676</td></tr><tr><td>FJOH_RS09515</td><td></td><td>snp_nonsynoc</td><td>T</td><td>2115191</td><td>2115191</td><td>2115191</td></tr><tr><td>FJOH_RS14175</td><td></td><td>snp_nonsynoc</td><td>A</td><td>3260769</td><td>3260769</td><td>3260769</td></tr><tr><td>20</td><td></td><td>small_indel</td><td></td><td>3437168</td><td>3437179</td><td>3437168</td></tr><tr><td>FJOH_RS00255</td><td></td><td>snp_nonsynoc</td><td>T</td><td>48770</td><td>48770</td><td>48770</td></tr><tr><td>FJOH_RS06580</td><td></td><td>snp_nonsynoc</td><td>A</td><td>1442862</td><td>1442862</td><td>1442862</td></tr><tr><td>FJOH_RS09520</td><td></td><td>snp_nonsynoc</td><td>T</td><td>2115712</td><td>2115712</td><td>2115712</td></tr><tr><td>FJOH_RS11170</td><td></td><td>snp_synonymc</td><td>C</td><td>2593409</td><td>2593409</td><td>2593409</td></tr><tr><td>FJOH_RS12240</td><td></td><td>snp_nonsynoc</td><td>T</td><td>2825359</td><td>2825359</td><td>2825359</td></tr><tr><td>FJOH_RS14155</td><td></td><td>snp_nonsynoc</td><td>G</td><td>3256285</td><td>3256285</td><td>3256285</td></tr><tr><td>20</td><td></td><td>small_indel</td><td></td><td>2115587</td><td>2115587</td><td>2115587</td></tr><tr><td>FJOH_RS14155</td><td></td><td>snp_nonsynoc</td><td>A</td><td>3254087</td><td>3254087</td><td>3254087</td></tr><tr><td>FJOH_RS24860</td><td></td><td>snp_nonsynoc</td><td>C</td><td>5823750</td><td>5823750</td><td>5823750</td></tr><tr><td>35</td><td></td><td>small_indel</td><td>G</td><td>5827528</td><td>5827528</td><td>5827528</td></tr><tr><td>FJOH_RS25290</td><td></td><td>snp_nonsynoc</td><td>C</td><td>5916920</td><td>5916920</td><td>5916920</td></tr><tr><td>20</td><td></td><td>small_indel</td><td></td><td>461783</td><td>461783</td><td>461783</td></tr><tr><td>30</td><td></td><td>small_indel</td><td></td><td>1442876</td><td>1442908</td><td>1442876</td></tr><tr><td>35</td><td></td><td>small_indel</td><td>A</td><td>1639597</td><td>1639597</td><td>1639597</td></tr><tr><td>FJOH_RS09515</td><td></td><td>snp_nonsynoc</td><td>A</td><td>2115193</td><td>2115193</td><td>2115193</td></tr><tr><td>FJOH_RS14155</td><td></td><td>snp_nonsynoc</td><td>A</td><td>3255868</td><td>3255868</td><td>3255868</td></tr><tr><td>30</td><td></td><td>large_deletion</td><td></td><td>149160</td><td>149449</td><td>149160</td></tr><tr><td>35</td><td></td><td>small_indel</td><td></td><td>1640030</td><td>1640030</td><td>1640030</td></tr><tr><td>90</td><td></td><td>small_indel</td><td></td><td>1654068</td><td>1654081</td><td>1654068</td></tr><tr><td>FJOH_RS12480</td><td></td><td>small_indel</td><td>A</td><td>2874473</td><td>2874473</td><td>2874473</td></tr><tr><td>FJOH_RS14155</td><td></td><td>snp_nonsynoc</td><td>A</td><td>3255194</td><td>3255194</td><td>3255194</td></tr><tr><td>FJOH_RS26065</td><td></td><td>snp_nonsynoc</td><td>T</td><td>6085864</td><td>6085864</td><td>6085864</td></tr><tr><td>1305</td><td>FJOH_RS013</td><td>large_deletion</td><td></td><td>257856</td><td>257906</td><td>257856</td></tr><tr><td>FJOH_RS02100</td><td></td><td>snp_nonsynoc</td><td>A</td><td>411121</td><td>411121</td><td>411121</td></tr><tr><td>35</td><td></td><td>small_indel</td><td>A</td><td>1640061</td><td>1640061</td><td>1640061</td></tr><tr><td>FJOH_RS13650</td><td></td><td>small_indel</td><td></td><td>3132257</td><td>3132257</td><td>3132257</td></tr><tr><td>FJOH_RS14155</td><td></td><td>snp_nonsynoc</td><td>T</td><td>3254266</td><td>3254266</td><td>3254266</td></tr><tr><td>35</td><td></td><td>snp_nonsensc</td><td>A</td><td>4834921</td><td>4834921</td><td>4834921</td></tr><tr><td>50</td><td></td><td>small_indel</td><td></td><td>5825476</td><td>5825478</td><td>5825476</td></tr><tr><td>35</td><td></td><td>small_indel</td><td></td><td>1640239</td><td>1640239</td><td>1640239</td></tr><tr><td>95</td><td></td><td>small_indel</td><td></td><td>1656294</td><td>1656294</td><td>1656294</td></tr><tr><td>FJOH_RS10115</td><td></td><td>snp_nonsynoc</td><td>A</td><td>2249614</td><td>2249614</td><td>2249614</td></tr><tr><td>FJOH_RS14155</td><td></td><td>snp_nonsynoc</td><td>A</td><td>3254087</td><td>3254087</td><td>3254087</td></tr><tr><td>20</td><td></td><td>large_deletion</td><td></td><td>3436681</td><td>3436953</td><td>3436681</td></tr><tr><td>30</td><td></td><td>small_indel</td><td></td><td>5824025</td><td>5824027</td><td>5824025</td></tr></tbody></table>
<table><tr><td>FJOH_RS05090</td><td>snp_nonsyno</td><td>C</td><td>1091086</td><td>1091086</td><td>1091086</td></tr><tr><td>35</td><td>small_indel</td><td></td><td>1639979</td><td>1640023</td><td>1639979</td></tr><tr><td>FJOH_RS07495</td><td>small_indel</td><td>T</td><td>1654791</td><td>1654791</td><td>1654791</td></tr><tr><td>FJOH_RS14155</td><td>snp_nonsyno</td><td>A</td><td>3254087</td><td>3254087</td><td>3254087</td></tr><tr><td>25</td><td>small_indel</td><td>GG</td><td>4828379</td><td>4828379</td><td>4828379</td></tr><tr><td>FJOH_RS24860</td><td>snp_nonsyno</td><td>C</td><td>5825095</td><td>5825095</td><td>5825095</td></tr><tr><td>FJOH_RS02110</td><td>large_deletion</td><td></td><td>416734</td><td>416826</td><td>416734</td></tr><tr><td>70</td><td>snp_nonsens</td><td>T</td><td>3260203</td><td>3260203</td><td>3260203</td></tr><tr><td>5035</td><td>FJOH_RS150</td><td>small_indel</td><td>3440151</td><td>3440164</td><td>3440151</td></tr><tr><td>30</td><td>small_indel</td><td></td><td>1442876</td><td>1442908</td><td>1442876</td></tr><tr><td></td><td>small_indel</td><td>CTAA</td><td>4493481</td><td>4493481</td><td>4493481</td></tr></table>
repeat_lengt repeat_new repeat_ref_c repeat_seq seq_id size SNP_type

<table><tbody><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>33</td><td></td></tr><tr><td>1</td><td>7</td><td>6 T</td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td>1</td><td>8</td><td>7 T</td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>33</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>12</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>synonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td>1</td><td>6</td><td>7 T</td><td></td><td>NC_009441</td><td>1</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>1</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>33</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>290</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>1</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>14</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>51</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td>1</td><td>8</td><td>9 A</td><td></td><td>NC_009441</td><td>1</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsense</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>3</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>1</td><td></td></tr><tr><td>1</td><td>8</td><td>9 T</td><td></td><td>NC_009441</td><td>1</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td></td><td>nonsynonymous</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>273</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>NC_009441</td><td>3</td><td></td></tr></tbody></table>
<table><tr><td></td><td></td><td></td><td>NC_009441</td><td>nonsynonym</td></tr><tr><td></td><td></td><td></td><td>NC_009441</td><td>45</td></tr><tr><td></td><td></td><td></td><td>NC_009441</td><td>nonsynonym</td></tr><tr><td></td><td></td><td></td><td>NC_009441</td><td>nonsynonym</td></tr><tr><td></td><td></td><td></td><td>NC_009441</td><td>nonsynonym</td></tr><tr><td></td><td></td><td></td><td>NC_009441</td><td>93</td></tr><tr><td></td><td></td><td></td><td>NC_009441</td><td>nonsense</td></tr><tr><td></td><td></td><td></td><td>NC_009441</td><td>14</td></tr><tr><td></td><td></td><td></td><td>NC_009441</td><td>33</td></tr><tr><td>4</td><td>17</td><td>16</td><td>CTAA</td><td>NC_009441</td></tr></table>
Type

<table><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr></table>
<table><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>INS</td><td></td></tr></table>
<table><caption>Summary of results from breseq</caption><thead><tr><th>Organism</th><th>Evolved line and replicate</th><th>gene_name</th></tr></thead><tbody><tr><td>B. thailandensis</td><td>Coevolved replicate 1</td><td>BTH_RS35420</td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 2</td><td>tssH</td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 2</td><td>BTH_RS04590</td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 2</td><td>BTH_RS06525/BTH_RS065</td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 2</td><td>tssF/BTH_RS15400</td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 3</td><td>BTH_RS04590</td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 3</td><td>fliH</td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 3</td><td>BTH_RS13245/BTH_RS132</td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 4</td><td>BTH_RS06340</td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 5</td><td>vgrG</td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 5</td><td>BTH_RS06600/BTH_RS066</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 1</td><td>BTH_RS06600/BTH_RS066</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 1</td><td>BTH_RS10750/BTH_RS107</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 1</td><td>BTH_RS35120</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 1</td><td>arsC/BTH_RS12180</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 1</td><td>BTH_RS17640/nuoE</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 2</td><td>BTH_RS10275/BTH_RS304</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 2</td><td>BTH_RS11790</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 2</td><td>tssM</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td>BTH_RS04585/BTH_RS045</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td>BTH_RS04590</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td>BTH_RS12945/gatC</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td>BTH_RS33635</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td>waaF/BTH_RS15555</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td>waaF/BTH_RS15555</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 4</td><td>BTH_RS10750/BTH_RS107</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 4</td><td>mnmE/BTH_RS28660</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 5</td><td>BTH_RS06600/BTH_RS066</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 5</td><td>fliH</td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 5</td><td>BTH_RS18425/BTH_RS184</td></tr></tbody></table>
<table><tr><td>gene_position</td></tr><tr><td>pseudogene (127-133/135 nt)</td></tr><tr><td>coding (2789-2797/2877 nt)</td></tr><tr><td>coding (595/912 nt)</td></tr><tr><td>intergenic (+87/+147)</td></tr><tr><td>intergenic (-714/-269)</td></tr><tr><td>coding (595/912 nt)</td></tr><tr><td>coding (77-122/681 nt)</td></tr><tr><td>intergenic (+337/-191)</td></tr><tr><td>coding (326/1593 nt)</td></tr><tr><td>2475</td></tr><tr><td>intergenic (-453/-72)</td></tr><tr><td>intergenic (-453/-72)</td></tr><tr><td>intergenic (-143/-13)</td></tr><tr><td>coding (221-229/477 nt)</td></tr><tr><td>intergenic (-222/-297)</td></tr><tr><td>intergenic (+143/-19)</td></tr><tr><td>intergenic (-41/-276)</td></tr><tr><td>coding (33-58/276 nt)</td></tr><tr><td>coding (1364-1369/3906 nt)</td></tr><tr><td>intergenic (-79/-33)</td></tr><tr><td>coding (595/912 nt)</td></tr><tr><td>intergenic (-176/-188)</td></tr><tr><td>coding (105/207 nt)</td></tr><tr><td>intergenic (-84/+44)</td></tr><tr><td>intergenic (-88/+40)</td></tr><tr><td>intergenic (-143/-13)</td></tr><tr><td>intergenic (-281/-58)</td></tr><tr><td>intergenic (-453/-72)</td></tr><tr><td>coding (77-122/681 nt)</td></tr><tr><td>intergenic (-213/-254)</td></tr></table>
<table><tr><td>gene_product</td></tr><tr><td>6-phosphofructokinase</td></tr><tr><td>type VI secretion system ATPase TssH</td></tr><tr><td>TetR/AcrR family transcriptional regulator</td></tr><tr><td>helix-turn-helix transcriptional regulator/hypothetical protein</td></tr><tr><td>type VI secretion system baseplate subunit TssF/penicillin acylase family protein</td></tr><tr><td>TetR/AcrR family transcriptional regulator</td></tr><tr><td>flagellar assembly protein FliH</td></tr><tr><td>EscU/YscU/HrcU family type III secretion system export apparatus switch protein/PepSY-asso</td></tr><tr><td>MBL fold metallo-hydrolase</td></tr><tr><td>type VI secretion system tip protein VgrG</td></tr><tr><td>amidohydrolase family protein/efflux RND transporter periplasmic adaptor subunit</td></tr><tr><td>amidohydrolase family protein/efflux RND transporter periplasmic adaptor subunit</td></tr><tr><td>GNAT family N-acetyltransferase/GntR family transcriptional regulator</td></tr><tr><td>hypothetical protein</td></tr><tr><td>arsenate reductase (glutaredoxin)/AAA family ATPase</td></tr><tr><td>NADH-quinone oxidoreductase subunit D/NADH-quinone oxidoreductase subunit NuoE</td></tr><tr><td>helix-turn-helix domain-containing protein/H-NS histone family protein</td></tr><tr><td>hypothetical protein</td></tr><tr><td>type VI secretion system membrane subunit TssM</td></tr><tr><td>Tm-1-like ATP-binding domain-containing protein/TetR/AcrR family transcriptional regulator</td></tr><tr><td>TetR/AcrR family transcriptional regulator</td></tr><tr><td>rod shape-determining protein/Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase subunit Gat</td></tr><tr><td>hypothetical protein</td></tr><tr><td>lipopolysaccharide heptosyltransferase II/zinc-finger domain-containing protein</td></tr><tr><td>lipopolysaccharide heptosyltransferase II/zinc-finger domain-containing protein</td></tr><tr><td>GNAT family N-acetyltransferase/GntR family transcriptional regulator</td></tr><tr><td>tRNA uridine-5-carboxymethylaminomethyl(34) synthesis GTPase MnmE/transcriptional regu</td></tr><tr><td>amidohydrolase family protein/efflux RND transporter periplasmic adaptor subunit</td></tr><tr><td>flagellar assembly protein FliH</td></tr><tr><td>MFS transporter/LysR substrate-binding domain-containing protein</td></tr></table>
<table><thead><tr><th>mutation_category</th><th>new_seq</th><th>position</th><th>position_end</th><th>position_start</th><th>size</th><th>snp_type</th></tr></thead><tbody><tr><td>small_indel</td><td></td><td>3545222</td><td>3545228</td><td>3545222</td><td>7</td><td></td></tr><tr><td>small_indel</td><td></td><td>1012569</td><td>1012577</td><td>1012569</td><td>9</td><td></td></tr><tr><td>small_indel</td><td>TGCAGGC</td><td>1039294</td><td>1039294</td><td>1039294</td><td></td><td></td></tr><tr><td>small_indel</td><td></td><td>1519708</td><td>1519714</td><td>1519708</td><td>7</td><td></td></tr><tr><td>small_indel</td><td>ACAGGCG</td><td>725350</td><td>725350</td><td>725350</td><td></td><td></td></tr><tr><td>small_indel</td><td>TGCAGGC</td><td>1039294</td><td>1039294</td><td>1039294</td><td></td><td></td></tr><tr><td>small_indel</td><td></td><td>232548</td><td>232593</td><td>232548</td><td>46</td><td></td></tr><tr><td>small_indel</td><td></td><td>239205</td><td>239212</td><td>239205</td><td>8</td><td></td></tr><tr><td>small_indel</td><td>TCGGCGC</td><td>1476934</td><td>1476934</td><td>1476934</td><td></td><td></td></tr><tr><td>snp_synonymous</td><td>G</td><td>153747</td><td>153747</td><td>153747</td><td>NC_00765</td><td>synonymous</td></tr><tr><td>snp_intergenic</td><td>A</td><td>1539855</td><td>1539855</td><td>1539855</td><td>NC_00765</td><td>intergenic</td></tr><tr><td>snp_intergenic</td><td>A</td><td>1539855</td><td>1539855</td><td>1539855</td><td></td><td>intergenic</td></tr><tr><td>snp_intergenic</td><td>A</td><td>2581752</td><td>2581752</td><td>2581752</td><td></td><td>intergenic</td></tr><tr><td>small_indel</td><td></td><td>2713058</td><td>2713066</td><td>2713058</td><td>9</td><td></td></tr><tr><td>small_indel</td><td>GTTGGC</td><td>2908216</td><td>2908216</td><td>2908216</td><td></td><td></td></tr><tr><td>snp_intergenic</td><td>T</td><td>1206554</td><td>1206554</td><td>1206554</td><td></td><td>intergenic</td></tr><tr><td>small_indel</td><td></td><td>2458559</td><td>2458559</td><td>2458559</td><td>1</td><td></td></tr><tr><td>small_indel</td><td></td><td>2815276</td><td>2815301</td><td>2815276</td><td>26</td><td></td></tr><tr><td>small_indel</td><td></td><td>3397661</td><td>3397666</td><td>3397661</td><td>6</td><td></td></tr><tr><td>snp_intergenic</td><td>G</td><td>1038667</td><td>1038667</td><td>1038667</td><td></td><td>intergenic</td></tr><tr><td>small_indel</td><td>TGCAGGC</td><td>1039294</td><td>1039294</td><td>1039294</td><td></td><td></td></tr><tr><td>small_indel</td><td></td><td>177802</td><td>177804</td><td>177802</td><td>3</td><td></td></tr><tr><td>small_indel</td><td>ATCGGC</td><td>728113</td><td>728113</td><td>728113</td><td></td><td></td></tr><tr><td>snp_intergenic</td><td>G</td><td>757464</td><td>757464</td><td>757464</td><td></td><td>intergenic</td></tr><tr><td>snp_intergenic</td><td>G</td><td>757468</td><td>757468</td><td>757468</td><td></td><td>intergenic</td></tr><tr><td>snp_intergenic</td><td>A</td><td>2581752</td><td>2581752</td><td>2581752</td><td></td><td>intergenic</td></tr><tr><td>snp_intergenic</td><td>A</td><td>3686634</td><td>3686634</td><td>3686634</td><td></td><td>intergenic</td></tr><tr><td>snp_intergenic</td><td>A</td><td>1539855</td><td>1539855</td><td>1539855</td><td></td><td>intergenic</td></tr><tr><td>small_indel</td><td></td><td>232548</td><td>232593</td><td>232548</td><td>46</td><td></td></tr><tr><td>large_deletion</td><td></td><td>1371337</td><td>1371387</td><td>1371337</td><td>51</td><td></td></tr></tbody></table>
<table><thead><tr><th></th><th></th></tr></thead><tbody><tr><td>type</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>INS</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>SNP</td><td></td></tr><tr><td>DEL</td><td></td></tr><tr><td>DEL</td><td></td></tr></tbody></table>
<table><caption>Summary of results from breseq</caption><thead><tr><th>Organism</th><th>Evolved line and replicate</th><th>aa_new_s</th><th>aa_positio</th><th>aa_ref_seq</th><th>clone</th></tr></thead><tbody><tr><td>B. thailandensis</td><td>Coevolved replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 4</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 5</td><td>P</td><td>825</td><td>P</td><td></td></tr><tr><td>B. thailandensis</td><td>Coevolved replicate 5</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 1</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 2</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 3</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 4</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 4</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 5</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 5</td><td></td><td></td><td></td><td></td></tr><tr><td>B. thailandensis</td><td>Monoculture replicate 5</td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th>codon_new</th><th>codon_nur</th><th>codon_pos</th><th>codon_pos</th><th>codon_ref</th><th>gene_name</th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td>BTH_RS35420</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>tssH</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>BTH_RS04590</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>BTH_RS06525/BTH</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>tssF/BTH_RS15400</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>BTH_RS04590</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>fliH</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>BTH_RS13245/BTH</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>BTH_RS06340</td></tr><tr><td>CCG</td><td>825</td><td>3</td><td>CCA</td><td>vgrG</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS06600/BTH</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS06600/BTH</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS10750/BTH</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS35120</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>arsC/BTH_RS12180</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS17640/nuoE</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS10275/BTH</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS11790</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>tssM</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS04585/BTH</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS04590</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS12945/gatC</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS33635</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>waaF/BTH_RS1555</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>waaF/BTH_RS1555</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS10750/BTH</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>mnmE/BTH_RS2860</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS06600/BTH</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>fliH</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>BTH_RS18425/BTH</td><td></td></tr></tbody></table>
<table><thead><tr><th>gene_position</th><th>gene_product</th><th>gene_strain</th><th>genes_inactive</th></tr></thead><tbody><tr><td>pseudogene (127-133/135 nt)</td><td>6-phosphofructokinase</td><td></td><td></td></tr><tr><td>coding (2789-2797/2877 nt)</td><td>type VI secretion system ATPase</td><td></td><td></td></tr><tr><td>coding (595/912 nt)</td><td>TetR/AcrR family transcriptional</td><td></td><td>BTH_RS04590</td></tr><tr><td>intergenic (+87/+147)</td><td>helix-turn-helix transcriptional</td><td></td><td></td></tr><tr><td>intergenic (-714/-269)</td><td>type VI secretion system basep</td><td></td><td></td></tr><tr><td>coding (595/912 nt)</td><td>TetR/AcrR family transcriptional</td><td></td><td>BTH_RS04590</td></tr><tr><td>coding (77-122/681 nt)</td><td>flagellar assembly protein FliH</td><td></td><td>fliH</td></tr><tr><td>intergenic (+337/-191)</td><td>EscU/YscU/HrcU family type III</td><td></td><td></td></tr><tr><td>coding (326/1593 nt)</td><td>MBL fold metallo-hydrolase</td><td></td><td>BTH_RS06340</td></tr><tr><td>2475</td><td>type VI secretion system tip pro</td><td></td><td></td></tr><tr><td>intergenic (-453/-72)</td><td>amidohydrolase family protein</td><td></td><td></td></tr><tr><td>intergenic (-453/-72)</td><td>amidohydrolase family protein</td><td></td><td></td></tr><tr><td>intergenic (-143/-13)</td><td>GNAT family N-acetyltransferase</td><td></td><td></td></tr><tr><td>coding (221-229/477 nt)</td><td>hypothetical protein</td><td></td><td>BTH_RS35120</td></tr><tr><td>intergenic (-222/-297)</td><td>arsenate reductase (glutaredoxin)</td><td></td><td></td></tr><tr><td>intergenic (+143/-19)</td><td>NADH-quinone oxidoreductase</td><td></td><td></td></tr><tr><td>intergenic (-41/-276)</td><td>helix-turn-helix domain-contain</td><td></td><td></td></tr><tr><td>coding (33-58/276 nt)</td><td>hypothetical protein</td><td></td><td>BTH_RS11790</td></tr><tr><td>coding (1364-1369/3906 nt)</td><td>type VI secretion system memb</td><td></td><td>tssM</td></tr><tr><td>intergenic (-79/-33)</td><td>Tm-1-like ATP-binding domain</td><td></td><td></td></tr><tr><td>coding (595/912 nt)</td><td>TetR/AcrR family transcriptional</td><td></td><td>BTH_RS04590</td></tr><tr><td>intergenic (-176/-188)</td><td>rod shape-determining protein</td><td></td><td></td></tr><tr><td>coding (105/207 nt)</td><td>hypothetical protein</td><td></td><td>BTH_RS33635</td></tr><tr><td>intergenic (-84/+44)</td><td>lipopolysaccharide heptosyltransferase</td><td></td><td></td></tr><tr><td>intergenic (-88/+40)</td><td>lipopolysaccharide heptosyltransferase</td><td></td><td></td></tr><tr><td>intergenic (-143/-13)</td><td>GNAT family N-acetyltransferase</td><td></td><td></td></tr><tr><td>intergenic (-281/-58)</td><td>tRNA uridine-5-carboxymethylase</td><td></td><td></td></tr><tr><td>intergenic (-453/-72)</td><td>amidohydrolase family protein</td><td></td><td></td></tr><tr><td>coding (77-122/681 nt)</td><td>flagellar assembly protein FliH</td><td></td><td>fliH</td></tr><tr><td>intergenic (-213/-254)</td><td>MFS transporter/LysR substrate</td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th>genes_overlapping</th><th>genesPromoter</th><th>insert_position</th><th>locus_tag</th><th>locuselo tags</th><th>locuselo tags</th></tr></thead><tbody><tr><td>BTH_RS35420</td><td></td><td></td><td>BTH_RS35420</td><td></td><td>BTH_RS35</td></tr><tr><td>tssH</td><td></td><td></td><td>BTH_RS04485</td><td></td><td>BTH_RS04</td></tr><tr><td></td><td></td><td>1</td><td>BTH_RS04</td><td>BTH_RS04590</td><td></td></tr><tr><td></td><td>BTH_RS06530</td><td></td><td>BTH_RS06525/BTH_RS06530</td><td></td><td></td></tr><tr><td></td><td></td><td>1</td><td>BTH_RS15395/BTH_RS15400</td><td></td><td></td></tr><tr><td></td><td></td><td>1</td><td>BTH_RS04</td><td>BTH_RS04590</td><td></td></tr><tr><td></td><td></td><td></td><td>BTH_RS13</td><td>BTH_RS13210</td><td></td></tr><tr><td></td><td>BTH_RS13250</td><td></td><td>BTH_RS13245/BTH_RS13250</td><td></td><td></td></tr><tr><td></td><td></td><td>1</td><td>BTH_RS06</td><td>BTH_RS06340</td><td></td></tr><tr><td>vgrG</td><td></td><td></td><td>BTH_RS00660</td><td></td><td>BTH_RS00</td></tr><tr><td></td><td></td><td></td><td>BTH_RS06600/BTH_RS06605</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>BTH_RS06600/BTH_RS06605</td><td></td><td></td></tr><tr><td></td><td>BTH_RS10750</td><td></td><td>BTH_RS10750/BTH_RS10755</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>BTH_RS35</td><td>BTH_RS35120</td><td></td></tr><tr><td></td><td></td><td>1</td><td>BTH_RS12175/BTH_RS12180</td><td></td><td></td></tr><tr><td></td><td>nuoE</td><td></td><td>BTH_RS17640/BTH_RS17645</td><td></td><td></td></tr><tr><td></td><td>BTH_RS10275</td><td></td><td>BTH_RS10275/BTH_RS30445</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>BTH_RS11</td><td>BTH_RS11790</td><td></td></tr><tr><td></td><td></td><td></td><td>BTH_RS27</td><td>BTH_RS27260</td><td></td></tr><tr><td></td><td>BTH_RS04585</td><td></td><td>BTH_RS04585/BTH_RS04590</td><td></td><td></td></tr><tr><td></td><td></td><td>1</td><td>BTH_RS04</td><td>BTH_RS04590</td><td></td></tr><tr><td></td><td></td><td></td><td>BTH_RS12945/BTH_RS12950</td><td></td><td></td></tr><tr><td></td><td></td><td>1</td><td>BTH_RS33</td><td>BTH_RS33635</td><td></td></tr><tr><td></td><td>waaF</td><td></td><td>BTH_RS15550/BTH_RS15555</td><td></td><td></td></tr><tr><td></td><td>waaF</td><td></td><td>BTH_RS15550/BTH_RS15555</td><td></td><td></td></tr><tr><td></td><td>BTH_RS10750</td><td></td><td>BTH_RS10750/BTH_RS10755</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>BTH_RS28655/BTH_RS28660</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>BTH_RS06600/BTH_RS06605</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>BTH_RS13</td><td>BTH_RS13210</td><td></td></tr><tr><td></td><td></td><td></td><td>BTH_RS18425/BTH_RS18430</td><td></td><td></td></tr></tbody></table>
<table><thead><tr><th>locus Shell tags</th><th>mutation</th><th>mutator</th><th>new Shell seq</th><th>population</th><th>position</th><th>position_e</th><th>position_s</th><th>ref Shell seq</th></tr></thead><tbody><tr><td>420</td><td>small_indel</td><td></td><td></td><td></td><td>3545222</td><td>3545228</td><td>3545222</td><td>GCGGCAA</td></tr><tr><td>485</td><td>small_indel</td><td></td><td></td><td></td><td>1012569</td><td>1012577</td><td>1012569</td><td>CGTTGGG</td></tr><tr><td></td><td>small_indel</td><td></td><td>TGCAGGCGGCGCTCG</td><td></td><td>1039294</td><td>1039294</td><td>1039294</td><td>G</td></tr><tr><td>BTH_RS06</td><td>small_indel</td><td></td><td></td><td></td><td>1519708</td><td>1519714</td><td>1519708</td><td>CCATCCG</td></tr><tr><td></td><td>small_indel</td><td></td><td>ACAGGCG</td><td></td><td>725350</td><td>725350</td><td>725350</td><td>G</td></tr><tr><td></td><td>small_indel</td><td></td><td>TGCAGGCGGCGCTCG</td><td></td><td>1039294</td><td>1039294</td><td>1039294</td><td>G</td></tr><tr><td></td><td>small_indel</td><td></td><td></td><td></td><td>232548</td><td>232593</td><td>232548</td><td>46-bp</td></tr><tr><td>BTH_RS13</td><td>small_indel</td><td></td><td></td><td></td><td>239205</td><td>239212</td><td>239205</td><td>ACCGGATC</td></tr><tr><td></td><td>small_indel</td><td></td><td>TCGGCGC</td><td></td><td>1476934</td><td>1476934</td><td>1476934</td><td>C</td></tr><tr><td>660</td><td>snp_synonymous</td><td></td><td>G</td><td></td><td>153747</td><td>153747</td><td>153747</td><td>A</td></tr><tr><td></td><td>snp_intergenic</td><td></td><td>A</td><td></td><td>1539855</td><td>1539855</td><td>1539855</td><td>T</td></tr><tr><td></td><td>snp_intergenic</td><td></td><td>A</td><td></td><td>1539855</td><td>1539855</td><td>1539855</td><td>T</td></tr><tr><td>BTH_RS10</td><td>snp_intergenic</td><td></td><td>A</td><td></td><td>2581752</td><td>2581752</td><td>2581752</td><td>G</td></tr><tr><td></td><td>small_indel</td><td></td><td></td><td></td><td>2713058</td><td>2713066</td><td>2713058</td><td>GTAATGC</td></tr><tr><td></td><td>small_indel</td><td></td><td>GTTGGC</td><td></td><td>2908216</td><td>2908216</td><td>2908216</td><td>C</td></tr><tr><td>BTH_RS17</td><td>snp_intergenic</td><td></td><td>T</td><td></td><td>1206554</td><td>1206554</td><td>1206554</td><td>G</td></tr><tr><td>BTH_RS10</td><td>small_indel</td><td></td><td></td><td></td><td>2458559</td><td>2458559</td><td>2458559</td><td>A</td></tr><tr><td></td><td>small_indel</td><td></td><td></td><td></td><td>2815276</td><td>2815301</td><td>2815276</td><td>26-bp</td></tr><tr><td></td><td>small_indel</td><td></td><td></td><td></td><td>3397661</td><td>3397666</td><td>3397661</td><td>CGCTCG</td></tr><tr><td>BTH_RS04</td><td>snp_intergenic</td><td></td><td>G</td><td></td><td>1038667</td><td>1038667</td><td>1038667</td><td>A</td></tr><tr><td></td><td>small_indel</td><td></td><td>TGCAGGCGGCGCTCG</td><td></td><td>1039294</td><td>1039294</td><td>1039294</td><td>G</td></tr><tr><td></td><td>small_indel</td><td></td><td></td><td></td><td>177802</td><td>177804</td><td>177802</td><td>GCG</td></tr><tr><td></td><td>small_indel</td><td></td><td>ATCGGC</td><td></td><td>728113</td><td>728113</td><td>728113</td><td>C</td></tr><tr><td>BTH_RS15</td><td>snp_intergenic</td><td></td><td>G</td><td></td><td>757464</td><td>757464</td><td>757464</td><td>C</td></tr><tr><td>BTH_RS15</td><td>snp_intergenic</td><td></td><td>G</td><td></td><td>757468</td><td>757468</td><td>757468</td><td>C</td></tr><tr><td>BTH_RS10</td><td>snp_intergenic</td><td></td><td>A</td><td></td><td>2581752</td><td>2581752</td><td>2581752</td><td>A</td></tr><tr><td></td><td>snp_intergenic</td><td></td><td>A</td><td></td><td>3686634</td><td>3686634</td><td>3686634</td><td>A</td></tr><tr><td></td><td>snp_intergenic</td><td></td><td>A</td><td></td><td>1539855</td><td>1539855</td><td>1539855</td><td>T</td></tr><tr><td></td><td>small_indel</td><td></td><td></td><td></td><td>232548</td><td>232593</td><td>232548</td><td>46-bp</td></tr><tr><td></td><td>large_deletion</td><td></td><td></td><td></td><td>1371337</td><td>1371387</td><td>1371337</td><td>51-bp</td></tr></tbody></table>
<table><thead><tr><th>repeat_ler</th><th>repeat_ne</th><th>repeat_ref_c</th><th>repeat_seq</th><th>seq_id</th><th>size</th><th>snp_type</th><th>time</th></tr></thead><tbody><tr><td>7</td><td>13</td><td>14</td><td>GCGGCAA</td><td>NC_00765</td><td>7</td><td></td><td>-1</td></tr><tr><td>9</td><td>8</td><td>9</td><td>CGTTGGGCG</td><td>NC_00765</td><td>9</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_007650</td><td></td><td></td><td>-1</td></tr><tr><td>7</td><td>8</td><td>9</td><td>CCATCCG</td><td>NC_00765</td><td>7</td><td></td><td>-1</td></tr><tr><td>7</td><td>8</td><td>7</td><td>ACAGGCG</td><td>NC_007651</td><td></td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_007650</td><td></td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_00765</td><td>46</td><td></td><td>-1</td></tr><tr><td>8</td><td>5</td><td>6</td><td>ACCGGATC</td><td>NC_00765</td><td>8</td><td></td><td>-1</td></tr><tr><td>7</td><td>2</td><td>1</td><td>TCGGCGC</td><td>NC_007650</td><td></td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_00765</td><td>synonymic</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_00765</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_007650</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_007650</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td>9</td><td>6</td><td>7</td><td>GTAATGCGC</td><td>NC_00765</td><td>9</td><td></td><td>-1</td></tr><tr><td>6</td><td>4</td><td>3</td><td>GTTGGC</td><td>NC_007650</td><td></td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_007651</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_00765</td><td>1</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_00765</td><td>26</td><td></td><td>-1</td></tr><tr><td>6</td><td>2</td><td>3</td><td>CGCTCG</td><td>NC_00765</td><td>6</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_007650</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_007650</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td>3</td><td>3</td><td>4</td><td>GCG</td><td>NC_00765</td><td>3</td><td></td><td>-1</td></tr><tr><td>6</td><td>9</td><td>8</td><td>ATCGGC</td><td>NC_007651</td><td></td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_007651</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_007651</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td>G</td><td>NC_007650</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td>G</td><td>NC_007651</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_007650</td><td>intergenic</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_00765</td><td>46</td><td></td><td>-1</td></tr><tr><td></td><td></td><td></td><td></td><td>NC_00765</td><td>51</td><td></td><td>-1</td></tr></tbody></table>
<table><thead><tr><th>title</th><th>transl_tab</th><th>treatment</th><th>type</th></tr></thead><tbody><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>INS</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>INS</td></tr><tr><td>output</td><td></td><td></td><td>INS</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>INS</td></tr><tr><td>output</td><td>11</td><td></td><td>SNP</td></tr><tr><td>output</td><td></td><td></td><td>SNP</td></tr><tr><td>output</td><td></td><td></td><td>SNP</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>INS</td></tr><tr><td>output</td><td></td><td></td><td>SNP</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>SNP</td></tr><tr><td>output</td><td></td><td></td><td>INS</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>INS</td></tr><tr><td>output</td><td></td><td></td><td>SNP</td></tr><tr><td>output</td><td></td><td></td><td>SNP</td></tr><tr><td>output</td><td></td><td></td><td>SNP</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr><tr><td>output</td><td></td><td></td><td>DEL</td></tr></tbody></table>
<table><thead><tr><th>Organism</th><th>Strain</th><th>Replicate</th></tr></thead><tbody><tr><td>Burkholderia thailandensis</td><td>Ancestor</td><td>0</td></tr><tr><td>Burkholderia thailandensis</td><td>Ancestor</td><td>0</td></tr><tr><td>Burkholderia thailandensis</td><td>Ancestor</td><td>0</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>1</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>1</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>1</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>2</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>2</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>2</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>3</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>3</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>3</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>4</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>4</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>4</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>5</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>5</td></tr><tr><td>Burkholderia thailandensis</td><td>Coevolved</td><td>5</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>1</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>1</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>1</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>2</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>2</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>2</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>3</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>3</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>3</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>4</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>4</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>4</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>5</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>5</td></tr><tr><td>Burkholderia thailandensis</td><td>Monoculture evolved</td><td>5</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Ancestor</td><td>0</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Ancestor</td><td>0</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Ancestor</td><td>0</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>1</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>1</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>1</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>2</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>2</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>2</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>3</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>3</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>3</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>4</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>4</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>4</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>5</td></tr><tr><td>Flavobacterium johnsoniae</td><td>Coevolved</td><td>5</td></tr></tbody></table>