A novel toxin-antitoxin module SlvT–SlvA governs megaplasmid stability and incites solvent tolerance in Pseudomonas putida S12

Pseudomonas putida S12 is highly tolerant towards organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies reveal that P. putida S12 contains a single-copy 583 kbp megaplasmid pTTS12. This pTTS12 encodes several important operons and gene clusters facilitating P. putida S12 to survive and grow in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid from P. putida S12 and conclusively confirmed that the SrpABC efflux pump is the major contributor of solvent tolerance on the megaplasmid pTTS12. Importantly, we identified a novel toxin-antitoxin module (proposed gene names slvT and slvA respectively) encoded on pTTS12 which contributes to the solvent tolerant phenotype and is essential in conferring genetic stability to the megaplasmid. Chromosomal introduction of the srp operon in combination with slvAT gene pair created a solvent tolerance phenotype in non-solvent tolerant strains such as P. putida KT2440, E. coli TG1, and E. coli BL21(DE3). Importance Sustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in bacterial biocatalysis. However, during bioproduction of such chemicals and biopolymers, aromatic compounds that function as products, substrates or intermediates in the production process may exert toxicity to microbial host cells and limit the production yield. Therefore, solvent-tolerance is a highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerant P. putida S12 for molecular adaptation to organic solvent. In addition to the RND efflux pump (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to tolerance in low solvent concentration as well as to genetic stability of pTTS12. These two gene clusters were successfully transferred to non-solvent tolerant strains of P. putida and to E. coli strains to confer and enhance solvent tolerance.

16 grow in the presence of toxic compounds or other environmental stresses. We wished to revisit and 17 further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the 18 megaplasmid from P. putida S12 and conclusively confirmed that the SrpABC efflux pump is the major 19 contributor of solvent tolerance on the megaplasmid pTTS12. Importantly, we identified a novel toxin-  Importance 26 Sustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in 27 bacterial biocatalysis. However, during bioproduction of such chemicals and biopolymers, aromatic 28 compounds that function as products, substrates or intermediates in the production process may 29 exert toxicity to microbial host cells and limit the production yield. Therefore, solvent-tolerance is a 30 highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and 31 biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerant 32 P. putida S12 for molecular adaptation to organic solvent. In addition to the RND efflux pump 33 (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to tolerance in low 34 solvent concentration as well as to genetic stability of pTTS12. These two gene clusters were Introduction 39 One of the main problems in the production of aromatic compounds is chemical stress caused by the 40 added substrates, pathway intermediates, or products. These chemicals, often exhibiting 41 characteristics of organic solvents, are toxic to microbial hosts and may negatively impact product 42 yields. They adhere to the cell membranes, alter membrane permeability, and cause membrane 43 damage (1, 2). Pseudomonas putida S12 exhibits exceptional solvent tolerance characteristics, 44 enabling this strain to withstand toxic organic solvents in saturating concentrations (3, 4).

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Consequently, a growing list of valuable compounds has successfully been produced using P. putida 46 S12 as a biocatalyst by exploiting its solvent tolerance (5-9).

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Following the completion of its full genome sequence and subsequent transcriptome and 48 proteome analyses, several genes have been identified that may play important roles in controlling 49 and maintaining solvent tolerance of P. putida S12 (10-12). As previously reported, an important 50 solvent tolerance trait of P. putida S12 is conferred through the RND-family efflux pump, SrpABC, 51 which actively removes organic solvent molecules from the cells (13,14). Initial attempts to 52 heterologously express the SrpABC efflux pump in E. coli enabled instigation of solvent-tolerance and 53 production of 1-naphtol (15, 16). Importantly, the SrpABC efflux pump is encoded on the megaplasmid 54 pTTS12 of P. putida S12 (12).

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It encodes several important operons and gene clusters enabling P. putida S12 to tolerate, resist and 57 survive the presence of various toxic compounds or otherwise harsh environmental conditions.

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Interesting examples are the presence of a complete styrene degradation pathway gene cluster, the 59 RND efflux pump specialized for organic solvents (SrpABC) and several gene clusters conferring heavy 60 metal resistance (12,17,18). In addition, through analysis using TADB2.0 (19, 20) pTTS12 is predicted 61 to contain three toxin-antitoxin modules. Toxin-antitoxin modules recently have been recognized as 62 important determinants of resistance towards various stress conditions (21, 22). Toxin-antitoxin 63 modules identified in pTTS12 consist of an uncharacterized RPPX_26255 -RPPX_26260 system and 4 two identical copies of a VapBC system (23). RPPX_26255 and RPPX_26260 belong to a newly 65 characterized type II toxin-antitoxin pair COG5654-COG5642. While toxin-antitoxin systems are 66 known to preserve plasmid stability through post-segregational killing of plasmid-free daughter cells 67 (24), RPPX_26255-RPPX_26260 was also previously shown to be upregulated during organic solvent 68 exposure indicating its role in solvent tolerance (11).

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In this paper, we further address the role of pTTS12 in conferring solvent tolerance of P. putida 70 S12. Curing pTTS12 from its host strain caused a significant reduction in solvent tolerance, while 71 complementation of the cured strain with the srp operon significantly restored solvent tolerance, 72 underscoring the importance of the SrpABC solvent pump in conferring solvent tolerance in P. putida 73 S12. In addition, we showed that the novel toxin-antitoxin pair slvAT (RPPX_26260 and RPPX_26255) 74 is essential for maintaining genetic stability of megaplasmid pTSS12. We further modelled SlvT and

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Megaplasmid pTTS12 is essential for solvent tolerance in P. putida S12

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To further analyze the role of the megaplasmid of P. putida S12 in solvent tolerance, pTTS12 was 84 removed from P. putida S12 using mitomycin C. This method was selected due to its reported 85 effectivity in removing plasmids from Pseudomonas sp. (28), although previous attempts regarded 86 plasmids that were significantly smaller in size than pTTS12 (29). After treatment with mitomycin C 87 (10-50 mg L -1 ), liquid cultures were plated on M9 minimal media supplemented with indole to select 88 for plasmid-cured colonies. Megaplasmid pTTS12 encodes two key enzymes: Styrene monooxygenase 89 (SMO) and Styrene oxide isomerase (SOI), that are responsible for the formation of indigo coloration 90 from indole. This conversion results in indigo coloration in spot assays for wildtype P. putida S12 91 whereas white colonies are formed in the absence of megaplasmid pTTS12. With the removal of 92 pTTS12, loss of indigo coloration and hence, of indigo conversion was observed in all three plasmid-93 cured strains and the negative control P. putida KT2440 ( Figure 1A).

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With mitomycin C concentration of 30 mg L -1 , 3 out of 122 obtained colonies appeared to be 95 completely cured from the megaplasmid, underscoring the high genetic stability of the plasmid. No 96 colonies survived the addition of 40 and 50 mg L -1 of mitomycin C, whereas all the colonies that 97 survived the addition of 10 and 20 mg L -1 of mitomycin C retained the megaplasmid. All three 98 independent colonies cured from the megaplasmid were isolated as P. putida S12-6, P. putida S12-10, 99 and P. putida S12-22. Complete loss of the megaplasmid was further confirmed by phenotypic analysis 100 (Figure 1), and by full genome sequencing. Several operons involved in heavy metal resistance were 101 previously reported in the pTTS12 (12). The terZABCD operon contributes to tellurite resistance in 102 wildtype P. putida S12 with minimum inhibitory concentration (MIC) as high as 200 mg L -1 ( Figure 1B).

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Genomic DNA sequencing confirmed complete loss of pTTS12 from P. putida strains S12-6, 106 S12-10, and S12-22 without any plasmid-derived fragment putatively being inserted within the 6 chromosome. Complementation of pTTS12 into the plasmid-cured P. putida S12 strains restored the 108 indole-indigo transformation and high tellurite resistance to the similar level with wildtype strain 109 (Figure S1). Repeated megaplasmid curing experiments indicated that P. putida S12 can survive the 110 addition of 30 mg L -1 Mitomycin C with the frequency of 2.48 (± 0.58) x 10 -8 . Among these survivors, 111 only 2% colony population lost the megaplasmid, confirming the genetic stability of pTSS12. In 112 addition, other plasmid-curing attempt by introducing double strand break as described by Wynands 113 and colleagues (30) was not successful due to the pTTS12 stability.

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Growth comparison in solid and liquid culture in the presence of toluene was performed to 115 analyze the effect of megaplasmid curing in constituting solvent tolerance trait of P. putida S12. In 116 contrast with wildtype P. putida S12, the plasmid-cured strains were unable to grow under toluene 117 atmosphere. In liquid LB medium, plasmid-cured P. putida S12 strains were able to tolerate a 118 maximum of 0.15% v/v toluene, whereas the wildtype P. putida S12 can grow in the presence of 0.30% 119 v/v toluene (Figure 2). In the megaplasmid-complemented P. putida S12-C strains, solvent tolerance 120 was restored to the wildtype level ( Figure S1-D). Hence, absence of megaplasmid pTTS12 caused a 121 significant reduction of solvent tolerance in P. putida S12. We chose P. putida S12-6 for further 122 experiments representing megaplasmid-cured P. putida S12.

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The significant reduction of solvent-tolerance in plasmid-cured P. putida S12 underscored the 127 important role of megaplasmid pTTS12 in solvent-tolerance. Besides encoding the efflux pump SrpABC 128 enabling efficient intermembrane solvent removal (12, 13), pTTS12 encodes more than 600 genes and 129 hence, may contain multiple additionally solvent-tolerance related genes. Two adjacent hypothetical 130 proteins, RPPX_26255 and RPPX_26260, encoded on the megaplasmid pTTS12 were previously 131 reported to be upregulated in the presence of toluene (11). We propose to name RPPX_26255-26260 132 gene pair as 'slv' due to its elevated expression in the presence of solvent. In a first attempt to identify 7 additional potential solvent tolerance regions of pTTS12, we deleted the srpABC genes (∆srp), 134 RPPX_26255-26260 genes (∆slv), and the combination of both gene clusters (∆srp ∆slv) from pTTS12 135 in wild-type P. putida S12.

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All strains were compared for growth under increasing toluene concentrations in liquid LB 137 medium ( Figure 2). In the presence of low concentrations of toluene (0.1% v/v), all strains showed 138 similar growth. With the addition of 0.15% v/v toluene, S12 ∆slv, S12 ∆srp and S12 ∆srp ∆slv exhibit 139 slower growth and reached a lower OD600nm compared to the wildtype S12 strain. S12 ∆slv and S12 140 ∆srp achieved a higher OD600nm in batch growth compared to S12 ∆pTTS12 and S12 ∆srp ∆slv due to 141 the presence of SrpABC efflux pump or RPPX_26255-26260 gene pair. Interestingly, S12 ∆srp ∆slv (still 142 containing pTSS12) exhibit diminished growth compared to S12 ∆pTTS12. This may be an indication of 143 megaplasmid burden in the absence of essential genes for solvent tolerance. With 0.2% and 0.3% v/v 144 toluene added to the medium, S12 ∆srp, S12 ∆srp ∆slv, and S12 ∆pTTS12 were unable to grow while 145 the wildtype S12 and S12 ∆slv were able to grow although S12 ∆slv reached a clearly lower OD600nm 146 compared to wildtype S12. Taken together, these results demonstrate an important role for both the

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Chromosomal introduction of slv into S12-6 and KT2440, improved growth of the resulting 156 strains at 0.15% v/v toluene compared to S12-6 and KT2440 (Figure 3). The introduction of srp or a 8 toluene compared to the addition of only srp ( Figure 3B). Interestingly, the growth of S12-6 srp,slv and 160 S12.6 srp are better in comparison with S12 wildtype ( Figure 3A). The observed faster growth of S12-161 6 srp,slv and S12.6 srp may be due to more efficient growth in the presence of toluene supported by 162 a chromosomally introduced srp operon, compared to its original megaplasmid localization. Indeed, 163 replication of this large megaplasmid is likely to require additional maintenance energy. To 164 corroborate this, we complemented the megaplasmid lacking the solvent pump, pTTS12 (Tc R ::srpABC) 165 into P. putida S12-6 srp resulting in the strain P. putida S12-9. Indeed, P. putida S12-9 showed further 166 reduced growth in the presence of 0.20 and 0.30 % toluene ( Figure S2), indicating the metabolic 167 burden of carrying the megaplasmid. We conclude that the SrpABC efflux pump can be regarded as 168 the major contributor to solvent tolerance from pTTS12. The slv gene pair appears to promote 169 tolerance of P. putida S12 at least under moderate solvent concentrations.

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The intrinsic solvent tolerance of E. coli strains was observed to be clearly lower than that of P. 171 putida ( Figure 4). The wild type E. coli strains were able to withstand a maximum 0.10% v/v toluene, 172 whereas plasmid-cured P. putida S12-6 and P. putida KT2440 were able to grow in the presence of  (Table S2). Interestingly, the CAI 9 values were higher for E. coli (0.664) than for P. putida (0.465) predicting a better protein translation 186 efficiency of the srp operon in E. coli. Hence, reduced translation efficiency is not likely to be the cause 187 of lower performance of srp operon in E. coli strains for generating solvent tolerance. Overall, our 188 results indicate that in addition to the solvent efflux pump, P. putida S12 and P. putida KT2440 are 189 intrinsically more robust compared to E. coli TG1 and E. coli BL21 DE3 in the presence of toluene. In addition to its role in solvent tolerance, localization of the slvAT pair on megaplasmid pTTS12 may 258 have an implications for plasmid stability. pTTS12 is a very stable megaplasmid that cannot be 259 spontaneously cured from P. putida S12 and cannot be removed by introducing double strand breaks 260 (see above). We deleted slvT and slvAT from the megaplasmid to study their impact in pTTS12 stability.

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With the deletion of slvT and slvAT, the survival rate during treatment with mitomycin C improved 262 had a survival rate of 2.48 (± 0.58) x 10 -8 .

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Among all the genes encoded on the megaplasmid pTTS12, the SrpABC efflux pump appears 293 as the major effector of solvent tolerance in P. putida S12 (Figure 9). A previous report applied SrpABC 294 in whole-cell biocatalysis while optimizing the production of 1-naphtol in E. coli TG1 (15, 16).

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Implementation of the SrpABC efflux pump increased the production of 1-naphtol from E.coli, 296 however, production was still higher using P. putida S12 as the production host. Here, we compared 297 the performance of SrpABC efflux pump in several established industrial strains. SrpABC was 298 expressed at a basal level and upregulated in the presence of 0.10 % v/v toluene in P. putida S12, P.  sequencing. Complementation of megaplasmid pTTS12 was performed using bi-parental mating 369 between P. putida S12-1 (pTTS12 Km R ) and plasmid-cured strains P. putida S12 ∆pTTS12 (Gm R :: Tn7) 370 and followed by selection on LB agar supplemented with Kanamycin and Gentamicin.
The loss of the megaplasmid band in megaplasmid-cured P. putida S12 proven by