Biology subjects

de Winde, J. H.

Publications and source records attributed to de Winde, J. H..

3 recordsLinked to original sources

bioRxiv2020

Adaptive laboratory evolution restores solvent tolerance in plasmid-cured Pseudomonas putida S12; a molecular analysis

Pseudomonas putida S12 is intrinsically solvent-tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The genome of P. putida S12 consists of a 5.8 Mbp chromosome, and a 580 kbp megaplasmid pTTS12 that carries several gene clusters involved in solvent tolerance. Removal of pTTS12 caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent-tolerance of this strain. Whole genome sequencing revealed several single nucleotide polymorphisms (SNPs) and a mobile element insertion, enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% v/v). Mutations were identified in an RND efflux pump regulator arpR, resulting in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit {beta}, global two-component regulatory system (GacA/GacS) and a putative AraC-family transcriptional regulator Afr. RNA-seq analysis further revealed a constitutive down-regulation of energy consuming activities in ALE-derived strains, including flagellar assembly, F0F1 ATP synthase, and membrane transport proteins. Out results indicate that constitutive expression of an alternative solvent extrusion pump in combination with high metabolic flexibility ensures restoration of solvent-tolerance in P. putida S12 lacking its megaplasmid.

microbiology↗

bioRxiv2020

Comparative analysis reveals the modular functional build-up of megaplasmid pTTS12 of Pseudomonas putida S12: a paradigm for transferable traits, plasmid stability and inheritance?

The Pseudomonas putida S12 genome contains 583 kbp megaplasmid pTTS12 that carries over 600 genes enabling tolerance to various stress conditions, including the solvent extrusion pump SrpABC. We performed a comparative analysis of pTTS12 against 28915 plasmids from NCBI databases. We investigated putative roles of genes encoded on pTTS12 and further elaborated on its role in the establishment and maintenance of several stress conditions, specifically focusing on solvent tolerance in P. putida strains. The backbone of pTTS12 was found to be closely related to that of the carbapenem-resistance plasmid pOZ176, member of the IncP-2 incompatibility group, although remarkably the carbapenem resistance cassette is absent from pTTS12. Megaplasmid pTTS12 contains multiple transposon-flanked cassettes mediating resistance to various heavy metals such as tellurite, chromate (Tn7), and mercury (Tn5053 and Tn5563). Additionally, pTTS12 also contains a P-type, Type IV secretion system (T4SS) supporting self-transfer to other P. putida strains. This study increases our understanding in the build-up of IncP-2 plasmids and several promising exchangeable gene clusters to construct robust microbial hosts for biotechnology applications. ImportanceOriginating from various environmental niches, large numbers of bacterial plasmids have been found carrying heavy metal and antibiotic resistance genes, degradation pathways and specific transporters for organic solvents or aromatic compounds. Such genes may constitute promising candidates for novel synthetic biology applications. Our systematic analysis of gene clusters encoded on megaplasmid pTTS12 underscores that a large portion of its genes is involved in stress response increasing survival under harsh conditions like heavy metal and organic solvent resistance. We show that pTTS12 belongs to the IncP-2 plasmid family. Comparative analysis of pTTS12 provides thorough insight into the structural and functional build-up of members of the IncP-2 plasmid family. pTTS12 is highly stable and carries a complex arrangement of transposable elements containing heavy metal resistance clusters as well as distinct aromatic degradation pathways and solvent-extrusion pump. This offers interesting insight into the evolution of solvent tolerance in the P. putida family.

microbiology↗

bioRxiv2020

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). ImportanceSustainable 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.

microbiology↗