Recruitment and Rearrangement of Three Different Genetic Determinants into a Conjugative Plasmid Increase Copper Resistance in Pseudomonas syringae

Abstract

We describe the genetic organization of a copper-resistant plasmid containing copG and cusCBA genes in the plant pathogen Pseudomonas syringae. Chromosomal variants of czcCBA and a plasmid variant of cusCBA were present in different P. syringae pathovar strains. Transformation of the copper-sensitive Pseudomonas syringae pv. syringae FF5 strain with copG or cusCBA conferred copper resistance, and quantitative real-time PCR (qRT-PCR) experiments confirmed their induction by copper. Pseudomonas syringae is a common foliar bacterium and causal agent of plant diseases in many different hosts worldwide, affecting both woody trees and herbaceous plants (1).Copper bactericides have been widely used for decades to control bacterial infections in crop plants (2). However, the extensive use of copper bactericides can lead to many problems, among them the reduction of efficacy of this antimicrobial agent due to the selection of copper-resistant (Cu r ) strains (3, 4). Copper resistance determinants have been detected and described in several pathovars of P. syringae (3, 5-7) and are frequently encoded within native plasmids (3,8,9). These native Cu r plasmids contribute to the dissemination of resistance genes among P. syringae strains from different pathovars (5, 10, 11). The copABCD genes, which were first described in a plasmid of P. syringae pv. tomato PT23 (8), is an important determinant of copper resistance in P. syringae and other phytopathogenic bacteria (6,8,9,(12)(13)(14). However, additional studies have shown that P. syringae may harbor other variants of Cu r determinants (3, 7, 15). The efflux system czcCBA and the analogous system cusCBA are probably the best-characterized members of the heavy metal efflux (HME)-RND (resistance-nodulation-cell division) family. The czcCBA system functions in the detoxification of cadmiun, zinc, and cobalt (16,17), and the cusCBA system works in detoxifying monovalent cations, such as silver and copper (16,18). These efflux systems have been widely studied in Cupriavidus metallidurans (formerly Ralstonia metallidurans or Alcaligenes eutrophus) (16,(19)(20)(21)(22), Escherichia coli (18, 23), and Pseudomonas putida KT2440 (24-26), but no further studies have been carried out for the plant pathogen P. syringae pv. syringae.In this work, we detected that the majority of the cusCBA genes from different Pseudomonas species could be wrongly annotated as czcCBA genes, based on the analysis of the conserved motifs of the RND transporter domains (27). This inaccurate annotation creates problems in the database regarding czcCBA or cusCBA genes in the Pseudomonas genus.Our goal was to identify and characterize plasmid-encoded Cu r genes in the P. syringae pv. syringae UMAF0081 strain. The bacterial strains used, and sizes of native plasmids harbored by the strains, are listed in Table 1. Each native plasmid examined is a member of the pPT23A plasmid family, a group of related plasmids broadly distributed within P. syringae and the related phytopathogen Pseudomonas savastanoi (32). These...

Genome sequencing and characterization of an extensively drug-resistant sequence type 111 serotype O12 hospital outbreak strain of Pseudomonas aeruginosa

“…Other integrated conjugative elements containing Trb conjugal protein loci were also found in RGP26, RGP36, and RGP75, the latter two also containing the copG gene, which encodes a putative metal-transporting P-type AT-Pase, thought to confer copper resistance [20]. Furthermore, the element contained within RGP75 has been sequenced in P. aeruginosa strain NCGM2.S1 previously, as well as having 99% identity with the P. putida sequenced in this study.…”

Evolution of copper resistance in the kiwifruit pathogen Pseudomonas syringae pv. actinidiae through acquisition of integrative conjugative elements and plasmids

“…Partway through copS the two ICEs diverge at a recombination breakpoint with the downstream variable region from Psa NZ45 ICE_Cu being homologous to a set of copper resistance genes found on plasmid pPaCu1 from the divergent (non‐pandemic) Japanese isolate of Psa (J2) (Nakajima et al ., ). This region comprises a putative copper transporting ATPase encoded by copG (Gutiérrez‐Barranquero et al ., ) , cusABC genes involved in the detoxification of monovalent cations, including copper and silver (Mergeay et al ., ; Rensing and Grass, ) and copABCD (Fig. B).…”

Paleomicrobiology to investigate copper resistance in bacteria: isolation and description of Cupriavidus necator B9 in the soil of a medieval foundry

“…tomato (Lim and Cooksey, 1993) and on the C. metallidurans CH34 pMOL28 and pMOL30 (Monchy et al, 2006). Interestingly, the genetic organization of a copper-resistant conjugative plasmid containing copABCD, copG and cusCBA genes in the plant pathogen Pseudomonas syringae was recently described (Guti errez- Barranquero et al, 2013) and it was shown that copG and cusCBA increased copper resistance in P. syringae. Similarly, in strain B9, the cluster of cop genes on contig 195 also includes copG and features a cusCBAF operon upstream.…”