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

José A Gutiérrez-Barranquero; Antonio de Vicente; Víctor J Carrión; George W Sundin; Francisco M Cazorla

doi:10.1128/AEM.02644-12

. 2013 Feb;79(3):1028–1033. doi: 10.1128/AEM.02644-12

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

José A Gutiérrez-Barranquero ^a, Antonio de Vicente ^a, Víctor J Carrión ^a, George W Sundin ^b, Francisco M Cazorla ^a,^✉

PMCID: PMC3568574 PMID: 23183969

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.

TEXT

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, 12–14). 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, 19–22), 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 plasmids share the major replication gene repA (33), and phylogenetic analysis indicates that individual pPT23A family plasmids (PFPs) have been transferred between P. syringae pathovars, and individual plasmid-borne genes have been transferred among PFPs (34).

Table 1.

Bacterial strains used in this study and their relevant characteristics

Bacterial strain	Origin	Host	Plasmid size (kb)^a	Source or reference
Pseudomonas fluorescens Pf-5	United States	Soil	–	28
Pseudomonas syringae
pv. garcae 2708	Africa	Coffee	72.6	NCPPB^c
pv. syringae
3910	Greece	Lemon	–	29
6–9	United States	Sweet cherry	61.6	30
7B44	United States	Ornamental pear	72.1	29
847	Italy	Cherry	–	29
B61	United States	Wheat	–	29
B728a	United States	Bean	–	31
B86-17	United States	Bean	54^b	29
DAP11	Sweden	Willow	–	29
FF5	United States	Ornamental pear	–	7
PS270	Unknown	Apple	–	29
UMAF0081	Spain	Mango	61.6	3
UMAF0158	Spain	Mango	63.0	3
UMAF0170	Spain	Mango	64.5	3
UMAF1029	Spain	Mango	63.0	3
pv. tabaci 0893-29	Hungary	Tobacco	73.8	29

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–, no plasmid.

The plasmid of this strain is currently being closed by sequencing; 54 kb is the provisional size determined.

NCPPB, National Collection of Plant Pathogenic Bacteria (United Kingdom).

Based on a PFP sequencing project that included each plasmid listed in Table 1 (J. A. Gutiérrez-Barranquero, F. M. Cazorla, A. De Vicente, G. W. Sundin, unpublished data), the presence of copG, a putative metal transporting P-type ATPase, and cusCBA (plasmid-encoded variant of cus genes) inserted within the copABCD operon (Fig. 1A) was observed and described for the first time associated with a conjugative-conserved native plasmid of 61.6 kb in two strains of P. syringae pv. syringae from different hosts and countries (P. syringae pv. syringae UMAF0081 and P. syringae pv. syringae 6-9). This novel arrangement has been also detected in the draft genomes of P. syringae pv. tabaci ATCC 11528 (35) and P. syringae pv. tomato NCPPB 1108 (36). The novel plasmid-encoded structure encompasses 16,703 bp and includes 11 open reading frames (ORFs) showing 99% nucleotide sequence identity and similarity among the four P. syringae strains. This genetic organization was then compared with the chromosomal and plasmid variants of czcCBA and cusCBA genes from different Pseudomonas species and Cupriavidus metallidurans CH34 (Fig. 1B to F) and also with the copABCD operon present in a native plasmid from P. syringae pv. tomato PT23 (Fig. 1G). The cus system of Cupriavidus metallidurans CH34 is located in chromosome 2 (2.5 Mbp), as is the copABCDRS operon (16, 37). However, sequence similarity and synteny with the P. syringae plasmid is low (i.e., query coverage of 32%). A different structure related to copper resistance can be found in the plasmid pMOL30 (38, 39), but with low similarity and synteny to the P. syringae plasmid (i.e., query coverage of 34%).

Fig 1 — Genetic arrangement of the *czc*, *cus*, and *cop* genes located in different *Pseudomonas* and *Cupriavidus metallidurans* strains. (A) Arrangement of the *copG* and *cusCBA* genes within the *copABCDRS* operon in the 61.6-kb conjugative conserved native plasmid of *P. syringae* pv. syringae UMAF0081 and 6-9. Note that these sequences were also identified in draft genome sequences of *P. syringae* pv. tabaci ATCC 11528 and *P. syringae* pv. tomato NCPPB 1108. (B) Genetic organization of the *czc* operon in the chromosome of *P. syringae* pv. syringae B728a; (C) genetic organization of the *czc* operon in the chromosome of *Pseudomonas putida* KT2440; (D) genetic organization of the *cus* (incorrectly annotated as *czc*) and *copABRS* operons in the chromosome of *Pseudomonas putida* KT2440; (E) genetic organization of the *czc* operon in the plasmid pMOL30 of *Cupriavidus metallidurans* CH34; (F) genetic organization of the *cus* operon in chromosome 2 of *Cupriavidus metallidurans* CH34; (G) genetic organization of the *copABCD* operon from *P. syringae* pv. tomato PT23 present in the plasmid PT23D. The number inside each ORF denotes the percentage of GC content.

Thus, the presence of the novel structure led us to hypothesize that a combination of the plasmid-encoded copABCD and cusCBA genes along with the copG gene could be involved in the increase of copper resistance in the phytopathogenic bacterium P. syringae pv. syringae.

Fourteen strains of P. syringae pv. syringae from different plant hosts, including the Cu^r strains P. syringae pv. syringae UMAF0081 and 6-9, and two strains from different pathovars were studied. Pseudomonas fluorescens Pf-5 was used in this study as an external control. Strains and plasmids used in this study to obtain genetically derived strains are listed in Table 2. The bacterial strains were routinely grown in Luria-Bertani broth or agar (LB), at 28°C for all the P. syringae strains and at 37°C for the E. coli DH5α strain.

Table 2.

Bacterial strains and plasmids used in this study to obtain the transformant strains

Strain or plasmid	Relevant characteristic(s)^d	Source or reference
Bacteria
Escherichia coli DH5α	recA lacZDM15	GIBCO-BRL
P. syringae pv. syringae FF5	Sm^s Cu^s; plasmidless	7
Plasmids
pCR2.1	Vector for TA cloning of amplicons and integration mutagenesis; Ap^r Km^r	Invitrogen Corporation
pCR2.1cusCBA	pCR2.1 with cusCBA genes from strain UMAF0081; Ap^r Km^r	This study
pCR2.1copG	pCR2.1 with copG gene from strain UMAF0081; Ap^r Km^r	This study
pBBR1MCS-5	lacPOZ′ mob, broad-host-range vector; Gm^r	40
pBBR1MCS-5cusCBA	pBBR1MCS-5 with cusCBA genes from strain UMAF0081; Gm^r	This study
pBBR1MCS-5copG	pBBR1MCS-5 with copG gene from strain UMAF0081; Gm^r	This study
Transformant strains
FF5pBBR1MCS-5	FF5 transformed with the empty pBBR1MCS-5 vector; Cu^s	This study
FF5pBBR1cusCBA^a	FF5 transformed with pBBR1MCS-5cusCBA; Cu^r	This study
FF5pBBR1copG^b	FF5 transformed with pBBR1MCS-5copG; Cu^r	This study
Transconjugant strain
UMAFCB^c	Pss FF5-km (Km^r) × Pss UMAF0081 (Cu^r)	41

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Transformant strain of Pseudomonas syringae FF5 harboring the pBBR1MCS-5 plasmid with the cusCBA genes cloned.

Transformant strain of Pseudomonas syringae FF5 harboring the pBBR1MCS-5 plasmid with the copG gene cloned.

Transconjugant strain (Cu^r Km^r) selected from biparental matings of P. syringae pv. syringae FF5-km (Km^r) × P. syringae pv. syringae UMAF0081 (Cu^r). The plasmid of 61.6 kb was mobilized by conjugation.

Ap^r, ampicillin resistant; Cu^r, copper resistant; Cu^s, copper sensitive; Gm^r, gentamicin resistant; Km^r, kanamycin resistant; Sm^s, streptomycin sentitive.

To determine functionality of copG and the cusCBA genes arranged with the copABCD operon, transformation experiments were performed. Specific primers were designed with restriction sites for double digestion and directional ligation into the pBBR1MCS-5 vector (40), based on copG and cusCBA sequences from the conjugative conserved native plasmid of the strains P. syringae pv. syringae UMAF0081 and P. syringae pv. syringae 6-9. Amplification was conducted using a high-fidelity Taq polymerase (Expand Long Range, dNTPack; Roche), and the PCR product was cloned into the pCR2.1 vector (Invitrogen Corporation) and transformed into competent E. coli DH5α cells. Subsequently, the PCR product was recovered from the pCR2.1 vector by double digestion, cloned into pBBR1MCS-5, and transformed into electrocompetent cells (42) of the copper-sensitive strain P. syringae pv. syringae FF5 (7). Determination of the MIC for copper, cadmium, zinc, and cobalt was then carried out for all P. syringae strains, the two transformant strains (FF5pBBR1copG and FF5pBBR1cusCBA), and the transconjugant strain (P. syringae pv. syringae UMAFCB) harboring the conjugative conserved native plasmid (Table 2). Agar plates of MG medium (5) supplemented with different concentrations of metals were used. The doses tested for the different heavy metals were Cu₂SO₄ · 5H₂O, from 0.2 mM to 1.8 mM; CdCl₂, from 0.05 mM to 1 mM; ZnCl₂, from 0.05 mM to 6 mM; and CoCl₂, from 0.01 mM to 1 mM. The MIC values (Table 3) displayed only a resistance pattern associated with the novel genes in the case of copper, among all the different strains tested. The P. syringae pv. syringae UMAF0081 and P. syringae pv. syringae 6-9 strains and the transconjugant strain UMAFCB showed the highest resistance level to copper. The transformant Cu^s strains confirmed that copG and cusCBA genes associated with the copABCD operon were involved in copper resistance (Table 3).

Table 3.

MICs of cadmium, zinc, cobalt, and copper for the Pseudomonas syringae strains studied, including the transformant and the transconjugant strains

Bacterial strain	MIC^a (mM)
Bacterial strain	CdCl₂	ZnCl₂	CoCl₂	Cu₂SO₄ · 5H₂O^b
Pseudomonas fluorescens Pf-5	>1	6	0.25	0.6
Pseudomonas syringae
pv. garcae 2708	0.3	4	<0.01	0.4
pv. syringae
3910	0.3	4	<0.01	0.4
6-9	0.3	4	<0.01	1.8
7B44	0.5	4	0.025	1.2
847	0.5	4	0.025	0.6
B61	0.5	4	<0.01	0.6
B728a	0.5	5	0.25	1.6
B86-17	1	4	0.25	0.6
DAP11	0.3	4	<0.01	0.4
FF5	0.3	4	<0.01	0.8
PS270	0.5	1	0.05	0.6
UMAF0081	0.3	4	<0.01	1.8
UMAF0158	0.3	4	<0.01	0.4
UMAF0170	0.3	4	0.05	1.2
UMAF1029	0.3	4	0.05	0.6
pv. tabaci 0893-29	0.3	4	0.025	0.6
Transformant strains
FF5pBBR1MCS-5^c	0.3	4	<0.01	0.8
FF5pBBR1cusCBA	0.3	4	<0.01	1.2
FF5pBBR1copG	0.3	4	<0.01	1.0
Transconjugant strain
UMAFCB	0.3	4	<0.01	1.8

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The doses tested for the different heavy metals were as follows: CdCl₂, from 0 to 1; ZnCl₂, from 0.05 to 6; CoCl₂, from 0.01 to 1; and Cu₂SO₄ · 5H₂O, from 0.2 to 1.8.

Strains with MICs of ≤0.8 mM are considered sensitive to copper.

Control strain: P. syringae pv. syringae FF5 transformed with the empty pBBR1MCS-5 vector.

Next, the copper resistance of the transformant, the transconjugant (UMAFCB), and the wild-type copper-sensitive (P. syringae pv. syringae FF5) strains was also studied by examining growth in liquid MG medium supplemented with copper at 0.8 mM (Fig. 2). The transformant strains were able to grow at 0.8 mM copper. The bacterial counts of the FF5pBBR1cusCBA strain were higher than those of FF5pBBR1copG, in agreement with the previous results observed in the MIC analysis (Table 3). These experiments confirmed the role of both copG and cusCBA in the increase of copper resistance. As expected, P. syringae pv. syringae UMAFCB displayed the highest copper resistance level. All of these results seem to confirm that the novel plasmid arrangement of the copG, cusCBA, and copABCD operon is involved in increased levels of copper resistance.

Fig 2 — Growth curve of *Pseudomonas syringae* pv. syringae strains in MG liquid medium supplemented with 0.8 mM copper sulfate. *P. syringae* pv. syringae strains harboring different constructs were tested: wild-type copper-sensitive plasmidless strain (*P. syringae* pv. syringae FF5), transformant strains (*P. syringae* pv. syringae FF5pBBR1cusCBA, *P. syringae* pv. syringae FF5pBBR1copG), and the transconjugant strain (UMAFCB) harboring the 61.6-kb conserved native plasmid.

Quantitative real-time PCR (qRT-PCR) analysis was conducted in order to determine if expression of the copG and cusA genes was induced by copper. RNA was extracted from four independent cultures of P. syringae pv. syringae UMAF0081 grown in MG liquid medium, two of them with copper at 0.8 mM. qRT-PCR analysis (triplicate technical replicates) on two independent RNA isolations under each condition (with or without copper) (43, 44) was carried out, as has been previously described (45) with minor modifications. The copA gene was used as a positive control, and the rpoD gene was used as a reference gene. The expression of the cusA and copG genes was clearly increased in the presence of copper (13-fold and 100-fold increases, respectively), and the baseline expression of the cusA gene was 3-fold higher than that of copG. These results correlate with the previous MIC (Table 3) and growth (Fig. 2) results.

A phylogenetic analysis was conducted using the complete sequences from chromosomal and plasmid czcCBA and cusCBA genes. Concatenated sequences were used for each strain for the multiple alignments using ClustalW2 software (46). Phylogenetic trees were constructed by using MEGA 5.05 (47) with neighbor-joining, minimum evolution and by eliminating all positions containing gaps. Confidence levels of the branching points were determined using 10,000 bootstrap replicates. This tree was compared to that of a phylogenetic tree derived from a multilocus sequence analysis using partial sequences of gyrB and rpoD genes of P. syringae pv. syringae and other related strains (belonging to the genus Pseudomonas, Burkholderia, Cupriavidus, Ralstonia, and Xanthomonas). The phylogenetic distribution based on the housekeeping genes revealed a clear clustering of the Pseudomonas spp. as well as for P. syringae strains examined in this study (see Fig. S1 in the supplemental material). Likewise, when using czc genes for phylogenetic analysis, a similar clustering was observed. Interestingly, all the cus genes grouped together, forming a completely separate cluster, with only one exception (RsPSI07cusI) (Fig. 3). As far as we know, this is the first report of the presence of the cus system in a phytopathogenic bacterium such as P. syringae pv. syringae. In Fig. 3, some genes annotated as czc (PsA1501, Pf0-1, and PpKT2440) grouped with the plasmidic cus genes of P. syringae, but a deeper analysis of their sequences showed the typical domains of the cus genes (see Fig. S2 in the supplemental material). The incorrect annotations, as well as the scarce sequences of the cus system, led to difficulties in proper study of the phylogeny of this gene family. A correct annotation of the cus genes would be needed in order to elucidate the evolutionary history of this gene family.

Fig 3 — Phylogenetic distribution of *Pseudomonas syringae* strains and other related Gram-negative bacteria based on the sequences of the *czc* and *cus* genes from chromosomal and plasmid origin obtained from sequencing in this study or from the GenBank database. The neighbor-joining tree was constructed with combined sequences (*czcCBA* or *cusCBA* genes) using MEGA 5.05. Evolutionary distances are in units of nucleotide substitutions per site. Based on the sequences of the *cusCBA* plasmid-borne genes, strains of *P. syringae* pv. syringae of this study (UMAF0081 and 6-9) together with the strains ATCC 11528 (*P. syringae* pv. tabaci) and NCPPB 1108 (*P. syringae* pv. tomato) are grouped together and marked in green. Based on the sequences of the chromosomal *czc* genes, strains from different pathovars of *P. syringae*, including PstaATCC11528 and PstNCPPB1108, are grouped together and marked in pink. Bootstrap values (10,000 repetitions) are shown on branches. Sequences from the following strains were used in this analysis: *Burkholderia cenocepacia* (BcJ2315), *Burkholderia pseudomallei* (BpK96243), *Cupriavidus metallidurans* (CmCH34), *Pseudomonas aeruginosa* (PaPAO1), *Pseudomonas entomophila* (PeL48), *Pseudomonas fluorescens* (Pf-5, PfSBW25, and Pf0-1), *Pseudomonas putida* (PpKT2440), *Pseudomonas stutzeri* (PsA1501), *Pseudomonas syringae* pv. phaseolicola (Pph1448A), *Pseudomonas syringae* pv. syringae (PssB728a, PssUMAF0081, and Pss6-9), *Pseudomonas syringae* pv. tabaci (PstaATCC11528), *Pseudomonas syringae* pv. tomato (PstDC3000 and PstNCPPB1108), *Ralstonia solanacearum* (RsPSI07), *Xanthomonas anoxopodis* pv. citri (Xac306), *Xanthomonas campestris* pv. campestris (XccATCC33913), and *Xanthomonas campestris* pv. vesicatoria (Xcv85-10). ^a, DNA sequences belong to the *cus* system based on the conserved motif domains but are annotated as *czc* genes.

In conclusion, we have described for the first time the presence of a cus system and its relationship with copper resistance in Pseudomonas syringae. Also in this study we report a novel arrangement of the copABCD operon, including insertions of copG and cusCBA encoded on a conjugative conserved native plasmid of 61.6 kb from two strains of P. syringae pv. syringae (6-9 and UMAF0081) that were isolated from different hosts and continents. Transformation experiments, MIC analysis, and qRT-PCR confirmed the role of the copG and cusCBA in the increase of copper resistance in P. syringae.

Nucleotide sequence accession numbers.

Database searches were performed using the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov). All the accession numbers from the different DNA sequences used in this study are summarized in Table S1 in the supplemental material. The newly determined cus sequences deposited in GenBank have the accession numbers JX645720 for P. syringae pv. syringae UMAF0081 and JX645721 for P. syringae pv. syringae 6-9. The sequences of the housekeeping genes gyrB and rpoD of P. syringae pv. syringae 6-9 have the accession numbers JX867861 and JX867862, respectively.

Supplementary Material

Supplemental material

supp_79_3_1028__index.html^{(1.2KB, html)}

ACKNOWLEDGMENTS

This work has been supported by grants from CICE-Junta de Andalucía, Ayudas Grupo PAIDI AGR-169, Plan Nacional de I+D+I del Ministerio de Ciencia e Innovación (AGL2011-30354C0201), and Proyecto de Excelencia (P07-AGR-02471), cofinanced by FEDER (EU) and Michigan AgBioResearch.

Footnotes

Published ahead of print 26 November 2012

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02644-12.

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24. Cánovas D, Cases I, de Lorenzo V. 2003. Heavy metal tolerance and metal homeostasis in Pseudomonas putida as revealed by complete genome analysis. Environ. Microbiol. 5:1242–1256 [DOI] [PubMed] [Google Scholar]
25. Haritha A, Sagar KP, Tiwari A, Kiranmayi P, Rodrigue A, Mohan PM, Singh SS. 2009. MrdH, a novel metal resistance determinant of Pseudomonas putida KT2440, is flanked by metal-inducible mobile genetic elements. J. Bacteriol. 191:5976–5987 [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Leedjärv A, Ivask A, Virta M. 2008. Interplay of different transporters in the mediation of divalent heavy metal resistance in Pseudomonas putida KT2440. J. Bacteriol. 190:2680–2689 [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Goldberg M, Pribyl T, Juhnke S, Nies DH. 1999. Energetics and topology of CzcA, a cation/proton antiporter of the RND protein family. J. Biol. Chem. 274:26065–26070 [DOI] [PubMed] [Google Scholar]
28. Paulsen IT, Press CM, Ravel J, Kobayashi DY, Myers GS, Mavrodi DV, DeBoy RT, Seshadri R, Ren Q, Madupu R, Dodson RJ, Durkin AS, Brinkac LM, Daugherty SC, Sullivan SA, Rosovitz MJ, Gwinn ML, Zhou L, Schneider DJ, Cartinhour SW, Nelson WC, Weidman J, Watkins K, Tran K, Khouri H, Pierson EA, Pierson LS, III, Thomashow LS, Loper JE. 2005. Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat. Biotechnol. 23:873–878 [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Sundin GW, Murillo J. 1999. Functional analysis of the Pseudomonas syringae rulAB determinant in tolerance to UVB (290–320 nm) radiation and distribution of rulAB among P. syringae pathovars. Environ. Microbiol. 1:75–87 [DOI] [PubMed] [Google Scholar]
30. Renick LJ, Cogal AG, Sundin GW. 2008. Phenotypic and genetic analysis of epiphytic Pseudomonas syringae populations from sweet cherry in Michigan. Plant Dis. 92:372–378 [DOI] [PubMed] [Google Scholar]
31. Hirano SS, Charkowski AO, Collmer A, Willis DK, Upper CD. 1999. Role of the Hrp type III protein secretion system in growth of Pseudomonas syringae pv. syringae B728a on host plants in the field. Proc. Natl. Acad. Sci. U. S. A. 96:9851–9856 [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Sundin GW. 2007. Genomic insights into the contribution of phytopathogenic bacterial plasmids to the evolutionary history of their hosts. Annu. Rev. Phytopathol. 45:129–151 [DOI] [PubMed] [Google Scholar]
33. Sesma A, Sundin GW, Murillo J. 1998. Closely related plasmid replicons coexisting in the phytopathogen Pseudomonas syringae show a mosaic organization of the replication region and altered incompatibility behavior. Appl. Environ. Microbiol. 64:3948–3953 [DOI] [PMC free article] [PubMed] [Google Scholar]
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35. Studholme DJ, Ibanez SG, MacLean D, Dangl JL, Chang JH, Rathjen JP. 2009. A draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of Pseudomonas syringae pathovar tabaci 11528. BMC Genomics 10:395. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Cai R, Lewis J, Yan S, Liu H, Clarke CR, Campanile F, Almeida NF, Studholme DJ, Lindeberg M, Schneider D, Zaccardelli M, Setubal JC, Morales-Lizcano NP, Bernal A, Coaker G, Baker C, Bender CL, Leman S, Vinatzer BA. 2011. The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity. PLoS Pathog. 7:e1002130 doi:10.1371/journal.ppat.1002130 [DOI] [PMC free article] [PubMed] [Google Scholar]
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39. Monchy S, Benotmane MA, Wattiez R, van Aelst S, Auquier V, Borremans B, Mergeay M, Taghavi S, van der Lelie Vallaeys T. 2006. Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34. Microbiology 152:1765–1776 [DOI] [PubMed] [Google Scholar]
40. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, II, Peterson KM. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176 [DOI] [PubMed] [Google Scholar]
41. Cazorla FM, Codina JC, Abad C, Arrebola E, Torés JA, Murillo J, Pérez-García A, de Vicente A. 2008. 62-kb plasmids harboring rulAB homologues confer UV-tolerance and epiphytic fitness to Pseudomonas syringae pv. syringae mango isolates. Microb. Ecol. 56:283–291 [DOI] [PubMed] [Google Scholar]
42. Choi KH, Kumar A, Schweizer HP. 2006. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Methods 64:391–397 [DOI] [PubMed] [Google Scholar]
43. De Brujin I, de Kock MJD, de Waard P, van Beek TA, Raaijmakers JM. 2008. Massetolide A biosynthesis in Pseudomonas fluorescens. J. Bacteriol. 190:2777–2789 [DOI] [PMC free article] [PubMed] [Google Scholar]
44. De Brujin I, Raaijmakers JM. 2009. Regulation of cyclic lipopeptide biosynthesis in Pseudomonas fluorescens by the ClpP protease. J. Bacteriol. 191:1910–1923 [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Carrión VJ, Arrebola E, Cazorla FM, Murillo J, de Vicente A. 2012. The mbo operon is specific and essential for biosynthesis of mangotoxin in Pseudomonas syringae. PLoS One 7:e36709 doi:10.1371/journal.pone.0036709 [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948 [DOI] [PubMed] [Google Scholar]
47. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28:2731–2739 [DOI] [PMC free article] [PubMed] [Google Scholar]

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[B25] 25. Haritha A, Sagar KP, Tiwari A, Kiranmayi P, Rodrigue A, Mohan PM, Singh SS. 2009. MrdH, a novel metal resistance determinant of Pseudomonas putida KT2440, is flanked by metal-inducible mobile genetic elements. J. Bacteriol. 191:5976–5987 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26. Leedjärv A, Ivask A, Virta M. 2008. Interplay of different transporters in the mediation of divalent heavy metal resistance in Pseudomonas putida KT2440. J. Bacteriol. 190:2680–2689 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. Goldberg M, Pribyl T, Juhnke S, Nies DH. 1999. Energetics and topology of CzcA, a cation/proton antiporter of the RND protein family. J. Biol. Chem. 274:26065–26070 [DOI] [PubMed] [Google Scholar]

[B28] 28. Paulsen IT, Press CM, Ravel J, Kobayashi DY, Myers GS, Mavrodi DV, DeBoy RT, Seshadri R, Ren Q, Madupu R, Dodson RJ, Durkin AS, Brinkac LM, Daugherty SC, Sullivan SA, Rosovitz MJ, Gwinn ML, Zhou L, Schneider DJ, Cartinhour SW, Nelson WC, Weidman J, Watkins K, Tran K, Khouri H, Pierson EA, Pierson LS, III, Thomashow LS, Loper JE. 2005. Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat. Biotechnol. 23:873–878 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Sundin GW, Murillo J. 1999. Functional analysis of the Pseudomonas syringae rulAB determinant in tolerance to UVB (290–320 nm) radiation and distribution of rulAB among P. syringae pathovars. Environ. Microbiol. 1:75–87 [DOI] [PubMed] [Google Scholar]

[B30] 30. Renick LJ, Cogal AG, Sundin GW. 2008. Phenotypic and genetic analysis of epiphytic Pseudomonas syringae populations from sweet cherry in Michigan. Plant Dis. 92:372–378 [DOI] [PubMed] [Google Scholar]

[B31] 31. Hirano SS, Charkowski AO, Collmer A, Willis DK, Upper CD. 1999. Role of the Hrp type III protein secretion system in growth of Pseudomonas syringae pv. syringae B728a on host plants in the field. Proc. Natl. Acad. Sci. U. S. A. 96:9851–9856 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32. Sundin GW. 2007. Genomic insights into the contribution of phytopathogenic bacterial plasmids to the evolutionary history of their hosts. Annu. Rev. Phytopathol. 45:129–151 [DOI] [PubMed] [Google Scholar]

[B33] 33. Sesma A, Sundin GW, Murillo J. 1998. Closely related plasmid replicons coexisting in the phytopathogen Pseudomonas syringae show a mosaic organization of the replication region and altered incompatibility behavior. Appl. Environ. Microbiol. 64:3948–3953 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B34] 34. Ma Z, Smith JJ, Zhao Y, Jackson RW, Arnold DL, Murillo J, Sundin GW. 2007. Phylogenetic analysis of the pPT23A plasmid family of Pseudomonas syringae. Appl. Environ. Microbiol. 73:1287–1295 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35. Studholme DJ, Ibanez SG, MacLean D, Dangl JL, Chang JH, Rathjen JP. 2009. A draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of Pseudomonas syringae pathovar tabaci 11528. BMC Genomics 10:395. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Cai R, Lewis J, Yan S, Liu H, Clarke CR, Campanile F, Almeida NF, Studholme DJ, Lindeberg M, Schneider D, Zaccardelli M, Setubal JC, Morales-Lizcano NP, Bernal A, Coaker G, Baker C, Bender CL, Leman S, Vinatzer BA. 2011. The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity. PLoS Pathog. 7:e1002130 doi:10.1371/journal.ppat.1002130 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37. Jannsen PJ, Van Houdt R, Moors H, Monsieurs P, Morin N, Michaux A, Benotmane MA, Leys N, Vallaeys T, Lapidus A, Monchy S, Médigue C, Taghavi S, McCorkle S, Dunn J, van der Lelie D, Mergeay M. 2010. The complete genome sequence of Cupriavidus metallidurans strain CH34, a master survivalist in harsh and anthropogenic environments. PLoS One 5:e10433 doi:10.1371/journal.pone.0010433 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38. Monchy S, Benotmane MA, Janssen P, Vallaeys T, Taghavi S, van der Lelie D, Mergeay M. 2007. Plasmids pMOL28 and pMOL30 of Cupriavidus metallidurans are specialized in the maximal viable response to heavy metals. J. Bacteriol. 189:7417–7425 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39. Monchy S, Benotmane MA, Wattiez R, van Aelst S, Auquier V, Borremans B, Mergeay M, Taghavi S, van der Lelie Vallaeys T. 2006. Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34. Microbiology 152:1765–1776 [DOI] [PubMed] [Google Scholar]

[B40] 40. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, II, Peterson KM. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176 [DOI] [PubMed] [Google Scholar]

[B41] 41. Cazorla FM, Codina JC, Abad C, Arrebola E, Torés JA, Murillo J, Pérez-García A, de Vicente A. 2008. 62-kb plasmids harboring rulAB homologues confer UV-tolerance and epiphytic fitness to Pseudomonas syringae pv. syringae mango isolates. Microb. Ecol. 56:283–291 [DOI] [PubMed] [Google Scholar]

[B42] 42. Choi KH, Kumar A, Schweizer HP. 2006. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Methods 64:391–397 [DOI] [PubMed] [Google Scholar]

[B43] 43. De Brujin I, de Kock MJD, de Waard P, van Beek TA, Raaijmakers JM. 2008. Massetolide A biosynthesis in Pseudomonas fluorescens. J. Bacteriol. 190:2777–2789 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B44] 44. De Brujin I, Raaijmakers JM. 2009. Regulation of cyclic lipopeptide biosynthesis in Pseudomonas fluorescens by the ClpP protease. J. Bacteriol. 191:1910–1923 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B45] 45. Carrión VJ, Arrebola E, Cazorla FM, Murillo J, de Vicente A. 2012. The mbo operon is specific and essential for biosynthesis of mangotoxin in Pseudomonas syringae. PLoS One 7:e36709 doi:10.1371/journal.pone.0036709 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B46] 46. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948 [DOI] [PubMed] [Google Scholar]

[B47] 47. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28:2731–2739 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

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

José A Gutiérrez-Barranquero

Antonio de Vicente

Víctor J Carrión

George W Sundin

Francisco M Cazorla

Abstract