Predatory selection of mucoid, antibiotic resistant Pseudomonas putida phenotype by myxobacterium Cystobacter ferrugineus

Predation contributes to the structure and diversity of microbial communities. Predatory myxobacteria are ubiquitous to a variety of microbial habitats and capably consume a broad diversity of microbial prey. Predator-prey experiments utilizing myxobacteria have provided details into predatory mechanisms and features that facilitate consumption of prey. However, prey resistance to myxobacterial predation remains underexplored, and prey resistances have been observed exclusively from predator-prey experiments that included the model myxobacterium Myxococcus xanthus. Utilizing a predator-prey pairing that instead included the myxobacterium, Cystobacter ferrugineus, with Pseudomonas putida as prey, we infrequently observed surviving phenotypes capable of eluding predation. Comparative transcriptomics between P. putida unexposed to C. ferrugineus and the survivor phenotype suggested that increased expression of efflux pumps, genes associated with mucoid conversion, and various membrane features contribute to predator avoidance. The P. putida survivor phenotype was confirmed to be resistant to the antibiotics kanamycin, gentamicin, and tetracycline and to produce more alginate than predator-unexposed P. putida. Unique features observed from the survivor phenotype including small colony variation, efflux-mediated antibiotic resistance, and increased mucoid conversion overlap with traits associated with Pseudomonas aeruginosa predator avoidance and pathogenicity. The survivor phenotype also benefited from increased predator resistance during subsequent predation assays. These results demonstrate the utility of myxobacterial predator-prey models and provide insight into prey resistances in response to predatory stress might contribute to the phenotypic diversity and structure of bacterial communities.

to C. ferrugineus and the survivor phenotype suggested that increased expression of 23 efflux pumps, genes associated with mucoid conversion, and various membrane features 24 contribute to predator avoidance. The P. putida survivor phenotype was confirmed to be 25 resistant to the antibiotics kanamycin, gentamicin, and tetracycline and to produce more 26 alginate than predator-unexposed P. putida. Unique features observed from the survivor 27 phenotype including small colony variation, efflux-mediated antibiotic resistance, and 28 increased mucoid conversion overlap with traits associated with Pseudomonas 29 aeruginosa predator avoidance and pathogenicity. The survivor phenotype also benefited 30 from increased predator resistance during subsequent predation assays. These results 31 demonstrate the utility of myxobacterial predator-prey models and provide insight into 32 { PAGE } exposed to grazing amoeba (Matz et al., 2004). Also associated with iron limitation, 128 regulatory and biosynthetic genes contributing to production of the siderophore 129 pyoverdine were significantly up-regulated in the survivor phenotype. These included 130 PvdA an L-ornithine monooxygenase (WP_016499102.1) and PvdH a diaminobutyrate-  (Figure 2A). While not a membrane associated feature, we also found 144 the overexpression of a formaldehyde dehydrogenase (WP_016497631.1) by the 145 survivor phenotype to be of note due to the recent observation that suggests 146 formaldehyde secretion to be a predation-resistance trait of P. aeruginosa (Sutton et al.,147 2019). The abundance of up-regulated genes that encode membrane-associated 148 products in the survivor phenotype including those responsible for increased mucoid 149 production provides insight into features involved in predation avoidance. Numerous { PAGE } efflux and transport proteins were also among the most up-regulated genes observed 151 from the survivor phenotype ( Figure 2D). Upregulated efflux genes included inner 152 (WP_016498130 and WP_016499477.1) and outer (WP_016498128.1) membrane 153 components highly homologous to the AcrAD-TolC-type multidrug resistance-nodulation- to multidrug efflux pumps contributing to virulence of human pathogens were also up-158 regulated in the survivor phenotype including a P-type ATPase (WP_016502100) and an 159 additional RND/MmpL (Mycobacterial membrane protein Large) protein ( Figure 2D). 160 Provided theses observed differences in gene expression, we sought to determine if the 161 survivor phenotype demonstrated antibiotic resistance and increased mucoidy, which 162 have both been previously associated with predator avoidance strategies. 163

Antibiotic resistance of survivor phenotype 164
Provided the observation that numerous transport proteins were overexpressed in the 165 survivor phenotype ( Figure 2D) including RND-type efflux pumps known to contribute to 166 P. aeruginosa antibiotic resistances, we were interested to determine if the survivor 167 phenotype exhibited antibiotic resistance. The survivor phenotype was capable of initially 168 forming colonies on LB agar supplemented with the antibiotics gentamicin (10 µg/ml), 169 kanamycin (50 µg/ml), and tetracycline (10 µg/ml), with no colonies from predation 170 sensitive P. putida observed on identical medias. Subsequent growth curve assays 171 comparing predation sensitive P. putida with the survivor phenotype when grown in LB 172 and LB supplemented with antibiotics ( Figure 3A and 3B) confirmed that the survivor { PAGE } phenotype was uniquely resistant to gentamicin, kanamycin, and tetracycline. These 174 results combined with the overexpression of efflux-associated proteins observed from the 175 survivor phenotype confirms that C. ferrugineus selection provides prey that benefit from 176 antibiotic resistance and suggests that efflux-mediated antibiotic resistance is a P. putida 177 predation avoidance feature. 178

Increased production of alginate by survivor phenotype 179
Alginate-based formation of mucoid biofilms has previously been associated with P. we sought to determine if the increased transcription of genes known to impact alginate 184 production observed from the survivor phenotype resulted in increased production of 185 alginate when compared to predator unexposed P. putida. Comparing isolated alginate 186 from predation sensitive P. putida with the survivor phenotype, we observed that the 187 survivor phenotype indeed produced more alginate when compared to P. putida not 188 exposed to C. ferrugineus ( Figure 4). 189

Predation resistance of survivor phenotype in subsequent predation assays 190
Additional subsequent predation assays were done to assess the predator avoidance trait 191 of the P. putida survivor phenotype with P. putida not exposed to C. ferrugineus included 192 as a control. These predation assays included an assay as previously described with assays the survivor phenotype demonstrated increased predator avoidance when 206 compared to control P. putida ( Figure 5B and 5C). However, no differences between 207 swarming diameters of C. ferrugineus grown on lawns of control P. putida and the P. 208 putida survivor phenotype were observed ( Figure 5D). These data suggest the P. putida 209 survivor phenotype benefits from a predator avoidance trait when subjected to 2 out of 3 210 subsequent predation assays. 211

Discussion 212
Building upon previously reported resistance strategies to avoid predatory myxobacteria 213 The survivor phenotype observed to be resistant to C. ferrugineus predation was 296 assessed with subsequent predation assays to determine the resiliency of the predator 297 avoidance trait. While comparative transcriptomic data provided insight into key up-298 regulated features potentially involved in prey survival, down-regulation of 1,178 genes in 299 the survivor phenotype when compared to predator unexposed P. putida suggests 300 potentially diminished survivor fitness. Despite this, the survivor phenotype maintained 301 the predator avoidance trait in 2 out of 3 subsequent predation assays. While it is unclear 302 why no significant predator avoidance was observed from lawn culture predation assays, 303 we suspect the introduction of predator to the center of prey lawns as opposed to 304 consistent introduction to the peripheral interface of prey potentially reduce any predator 305 avoidance mechanisms. Predator avoidance observed from direct and indirect contact 306 predation assays and the heterogeneity associated with differences between the growth 307 and metabolism of peripheral cells and central cells of bacterial colonies supports this 308 suspicion (Reyrolle and Letellier, 1979;Jeanson et al., 2015). We anticipate that P. putida 309 phenotypes resistant to myxobacterial predation continually subjected to such predatory 310 selection will afford adapted phenotypes for further scrutiny of any resulting mutations { PAGE } and genotypes. Ultimately, these results demonstrate the utility of predator-prey 312 experiments using lesser studied myxobacteria and provide insight into how predatory 313 stress might contribute to adaptive phenotypic diversity of bacterial communities. 314

Bacterial Strains and Cultivation 316
The myxobacterium Cystobacter ferrugineus strain Cbfe23, DSM 52764 was employed 317 as the predator. Pseudomonas putida type strain, ATCC 12633, and the discussed P. 318 putida survivor phenotype resulted from predation assays with this P. putida type strain 319 were included as prey. C. ferrugineus was grown on VY/2 solid (1.4% w/v agar, 0.1% w/v 320 CaCl2 * 2 H2O, 0.5% w/v Baker's yeast, 500 μM vitamin B12) media for 5-7 days. Luria-321 Bertani (LB) solid (1.5% agar) and liquid media were utilized for the cultivation of P. replicates. Any prey spot with visible biomass of P. putida after day 14 was considered 335 as survivor phenotype of P. putida, and five observed P. putida survivor stocks were 336 acquired that remained visible after day 14 of the assay. 337 For subsequent predation assays, P. putida predation sensitive (control) and the survivor 338 phenotypes were employed as prey. The direct spot assay was performed as mentioned 339 above. Statistical significance of recorded time to predation reported in was calculated 340 using an unpaired t test with Welch's correction in Prism 7.0d. The subsequent directed 341 contact predation assay was also performed on VY/2 (1.4% w/v agar, 0.1% w/v CaCl2 * 342 2 H2O, 0.5% w/v Baker's yeast, 500 μM vitamin B12) media as well, considered as 343 nutrient-rich media. For indirect contact spot assay, C. ferrugineus was inoculated at a 344 distance of 5-7 mm from the established P. putida control and P. putida survivor prey 345 spots. Next, time C. ferrugineus took to reach the prey spots and the time of its swarming 346 over the spots was recorded. Indirect predation assays were performed with 8 replicates 347 per phenotype. Statistical significance of recorded time to predation reported in days and 348 recorded time to initial contact between C. ferrugineus and prey reported in days was 349 calculated using an unpaired t test with Welch's correction in Prism 7.0d.  Technologies). The library was diluted (to 6.5 pM) and sequenced paired end for 500 389 cycles using the MiSeq system (Illumina). with P. putida control). All raw RNAseq data is publicly available at the NCBI Sequence 416 Read Archive (PRJNA577468). 417

Antibiotic Susceptibility Assays. 418
The standard growth curve assay was employed to determine the sensitivity of the 419 unexposed P. putida and the survivor phenotype towards kanamycin (50 mg/mL), 420 gentamicin (10 mg/mL), and tetracycline (10 mg/mL) antibiotics. The Lauria Bertani (LB) 421 broth without any supplemented antibiotics was used as a control. The test was 422 performed in a 96-well microtiter plate. In a 96-well plate, each well having 200 µL of LB 423 or LB supplemented with an antibiotic was inoculated with a colony of P. putida. Next, the 424 absorbance at OD620 was recorded using a plate reader every 30 min for 10 hr to examine 425 the growth dynamics of each phenotype. The test was performed in 12 replicates per { PAGE } condition for both phenotypes. Recorded OD620 values were plotted using Prism version 427 7.0d with error bars depicting standard deviation of replicate data. 428 Carbazole Assays. 429 The alginate was isolated according to Jones et al. (Jones et al., 2013). In brief, both P. 430 putida phenotype unexposed to the predatory stress and the survivor phenotype were 431 grown overnight in 2 ml LB media and adjusted to an OD600 of 1.00. Following 432 centrifugation, 1 ml of supernatant was treated with 2% cetyl pyridinium chloride to 433 precipitate the alginate. The precipitated alginate was further collected by centrifugation.  introduced 2 cm from predator (n=8; p≤0.01) and time to predator-prey contact recorded 656 (C). Swarming assays with C. ferrugineus plated on prey lawns and swarming diameters 657 recorded after 7 days (n=8). Statistical significance calculated using an unpaired t test 658 with Welch's correction. 659