PCR amplification of hydrogen cyanide biosynthetic locus hcnAB in Pseudomonas spp.

Note

PCR amplification of hydrogen cyanide biosynthetic locus hcnAB in Pseudomonas spp.

M. Svercel

, B. Duffy

, G. Défago

^⁎

aPlant pathology, Institute of Integrative Biology, ETH-Zürich, CH-8092 Zürich, Switzerland

bAgroscope Changins-Wädenswil, Swiss Federal Research Station for Horticulture, Plant Protection Division, CH-8820 Wädenswil, Switzerland Received 8 December 2006; received in revised form 27 March 2007; accepted 27 March 2007

Available online 6 April 2007

Abstract

A PCR-based assay targeting

pseudomonads between logs 2.9 and 3.5 cells per PCR reaction tube. RFLP analysis revealed 13 allele combinations among selected 2,4- diacetylphloroglucinol-producing (Phl

)HCN

, and 13 alleles in Phl

HCN

strains from a global collection.

© 2007 Elsevier B.V. All rights reserved.

Keywords: Pseudomonas; Hydrogen cyanide (HCN); Diversity

Production of hydrogen cyanide (HCN), as well as production of 2,4-diacetylphloroglucinol (Phl), is an impor- tant biocontrol determinant (Haas and Defago, 2005). In Pseudomonas fluorescens strains (e.g., Q2-87, CHA0), the hcnABC genes encode for HCN synthetase critical for HCN production (Haas and Defago, 2005). Very little is known about the polymorphism of these genes. The phylogenies based on partial hcnBC sequences determined from a worldwide collection of Phl

HCN

biocontrol fluorescent Pseudomonas spp. and the deduced protein sequences revealed four main bacterial groups (Ramette et al., 2003).

Because the specificity and sensitivity of previously estab- lished hcnBC primers have proven insufficient for analysis of mixed pseudomonad populations from grapevine (Svercel unpublished) and to detect HCN

but Phl

pseudomonads, hcnAB specific primers were developed and validated.

Additionally, hcnAB-RFLP analysis allowed us to study the variety in our pseudomonad collection and present a basic overview about hcnAB allelic diversity in HCN

(Phl

and Phl

) strains.

Primers for hcnAB genes were designed with MultAlin (Corpet, 1988) from the consensus of the hcn sequences between P. fluorescens strain CHA0 (accession number AF053760) and P. aeruginosa strain PAO1 (AF208523). Regions of the alignment were scanned for areas with high sequence identity that could be used as priming sites for PCR amplification. Potential priming sites were selected based on the following criteria for the annealing primer: (i) ≥90% identity of primer to compared sequences, (ii) a Tm ≥55 °C, (iii) priming site ≥350 bp distant from that of nearest complementary primer and (iv) a C or G in the terminal 3′ position. Optimal amplification and specificity were obtained using forward primer PM2 (31-mer 5′- TGCGGCATGGGCGTGTGCCATTGCTGCCTGG-3′) and re- verse primer PM7-26R (26-mer 5′-CCGCTCTTGATCTG- CAATTGCAGGCC-3′) (synthesized by MWG Biotech, Basel, Switzerland). Amplifications were carried out in 12-μl reaction mixtures containing 4 μ l of lysed bacterial suspension, 1× PCR buffer (Amersham Pharmacia, Uppsala, Sweden), bovine serum albumin (0.5 g l

; Fluka, Buchs, SG, Switzerland), 5% dimethyl sulfoxide (Fluka), 100 μM each of dATP, dCTP, dGTP and dTTP (Amersham Pharmacia), 0.40 μM of each primer and 1.4 U of Taq DNA polymerase (Amersham Pharmacia). The PCR started with the initial denaturation (2 min at 94 °C) was followed by 35 cycles of 94 °C for 30 s, 67 °C for 30 s and 72 °C for 60 s and final

⁎Corresponding author. Tel.: +41 44 632 38 69; fax: +41 44 632 15 72.

E-mail address:genevieve.defago@agrl.ethz.ch(G. Défago).

0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.mimet.2007.03.018

extension at 72 °C for 10 min. Amplifications were performed with a PTC-100TM cycler (MJ Research Inc., Watertown, MA), and the resulting PCR products were separated in 1.5% agarose gels in 0.5x Tris–borate–EDTA (TBE) buffer at 160 V for 1 h.

The amplified fragment was 570 bp and included 136 bp of hcnA (312 nucleotides) and 434 bp of hcnB (1404 nucleotides) (Fig. 1).

A total of 57 Phl

HCN

strains representing each of the four previously described phylogenetic HCN groups of biocontrol pseudomonads (Ramette et al., 2003) were used to test the specificity of primers (Table 1). Additionally, the specificity of primers was tested on 41 diverse Phl

HCN

strains (Table 1). P. fluorescens strains 2–79 and P3 were included as HCN-negative controls. All pseudomonads were routinely grown at 27 °C on King's B agar (KBA, King et al., 1954) and stored at −80 °C in 40% glycerol. DNA preparation was done as described by Wang et al. (2001).

To test the specificity of the primers, all strains were adjusted to a constant concentration of approximately 10

cells per ml. To test the sensitivity of the assay for detection of hcnAB

strains in a background of hcnAB

bacteria, two different types of template mixtures were prepared. The first mixture consisted of a ten-fold serial dilution of strain CHA0 or Q2-87 (1 × 10

to 1 × 10

cells per ml) in a constant concentration of negative control strain P3 (1 × 10

cells per ml). The second mixture consisted of ten-fold serial dilution of a 24:1 mixture of strain P3 with either strain CHA0 or Q2-87 (1 × 10

to 1 × 10

cells per ml). Dilutions were initially frozen

at −80 °C for a minimum of 1 h, and then transferred to a

−20 °C freezer for storage.

A single amplicon of about 570 bp in length was obtained for all HCN

strains using our PCR method, whereas no amplicon was obtained from the two negative HCN pseudomonads.

When we varied the concentration of HCN

bacteria in samples but kept a fixed background population of HCN

bacteria (e.g., P3 at log 7.5 cells per PCR reaction tube) we were able to detect HCN

strains present at between log 2.9 cells (CHA0) and log 3.2 cells (Q2-87) per PCR reaction tube. When we tested dilutions of mixtures containing a fixed proportion of high background (i.e., 24:1 of P3:CHA0 or P3:Q2-87), a clear amplification signal was obtained with an average of log 3.1 hcnAB

per PCR tube of CHA0 and log 3.5 per PCR tube of Q2-87. This demonstrates that HCN

bacteria can be sensitively detected in samples where they represent a low percentage of the total pseudomonad community and/or where the numbers are low regardless of their relative proportion to the total community, which is an important feature for environmental biodiversity analyses.

To characterize hcnAB

alleles, 5 μ l of amplified product were used for restriction analysis with 1.5 U of HaeIII, MspI or TaqI enzymes (Boehringer, Mannheim). The combination of just these three digests was sufficient to discriminate all phlD alleles and the achieved polymorphism corresponded exactly to that defined previously by BOX-PCR genomic fingerprinting in Phl

pseudomonads (McSpadden Gardener et al., 2001). Reactions

Fig. 1. Alignment of partialhcnABsequences ofPseudomonas fluorescensCHA0 (accession number AF053760) andP. aeruginosaPAO (AF208523). Dots and dashes represent conserved bases and alignment gaps, respectively. ThehcnBstart codon is underlined and the sites annealing to the PCR primers PM2 and PM7-26R are overlined.

Table 1

Pseudomonasspp. strains tested in the study

Strain Phl HCN hcnABallele Year/origin Source

P. fluorescens

Q65c-80 + + A 1980/USA Harrison et al. (1993)

Q95-87, Q107-87, Q128-87, Q139-87 + + A 1987/USA (Harrison et al., 1993; Keel et al., 1996)

CM1’A2, TM1’A4, TM1’A5 + + A 1992/Switzerland Keel et al. (1996)

C⁎1A1, TM1’A3, TM1’B2 + + A 1992/Switzerland Fuchs and Défago (1991)

Q8r1-96 + + A 1996/USA Raaijmakers and Weller (1997)

OC4-1, W2-6 + + A 1998/USA McSpadden Gardener et al. (2000)

Delaw1R, Wood1R + + A 2001/USA McSpadden Gardener et al. (2005)

K94.31 + + B 1994/Czech rep. Wang et al. (2001)

K94.37 + + C 1994/Czech rep. Wang et al. (2001)

FFL1R18 + + C 1997/USA McSpadden Gardener et al. (2000)

F113 + + D 1992/Ireland Shanahan et al. (1992)

P12 + + E 1996/Switzerland Keel et al. (1996)

Q37-87 + + F 1987/USA (Harrison et al., 1993; Keel et al., 1996)

Q2-2 + + F 1998/USA McSpadden Gardener et al. (2000)

Q2-87 + + G 1987/USA Vincent et al. (1991)

Q1-87, Q6-87, Q7-87, Q9-87, Q12-87, Q13-87, Q86-87

+ + G 1987/USA (Harrison et al., 1993; Keel et al., 1996)

PITR2 + + H 1993/Italy Keel et al. (1996)

PILH1 + + I 1993/Italy Keel et al. (1996)

D27B1 + + I 1998/USA McSpadden Gardener et al. (2000)

F96.27 + + J 1996/Estonia Wang et al. (2001)

P97.26 + + J 1997/Bhutan Wang et al. (2001)

P97.30 + + J 1997/Czech rep. Wang et al. (2001)

P97.38 + + K 1997/Switzerland Wang et al. (2001)

Clinto1R, Wood3R + + K 2001/USA McSpadden Gardener et al. (2005)

Pf5 + + L 1979/USA Howell and Stipanovic (1979)

CHA0 + + L 1985/Switzerland Stutz et al. (1986)

PF + + L 1992/USA Levy et al. (1992)

PINR3 + + L 1993/Italy Keel et al. (1996)

PGNR1, PGNR2, PGNR3, PGNR3, PGNR4, PGNL1

+ + L 1993/Ghana Keel et al. (1996)

Pf1 + + L 1996/Switzerland Keel et al. (1996)

Cosho1R, Wayne1R , Wayne2, Wayne4 + + L 2001/USA McSpadden Gardener et al. (2005)

JMP6, JMP6R + + M 1998/USA McSpadden Gardener et al. (2000)

K92.2 − + A 1992/Switzerland Rezzonico et al. (2007)

K92.46 − + A 1992/Switzerland Rezzonico et al. (2004)

P96.36 − + A 1996/Russia Rezzonico et al. (2007)

K92.60 − + I 1992/Switzerland Rezzonico et al. (2007)

K92.3 − + L 1992/Switzerlans Rezzonico et al. (2007)

K93.35 − + O 1993/Switzerland Rezzonico et al. (2007)

K94.27 − + P 1994/India Rezzonico et al. (2007)

P96.47 − + P 1996/Mexico Rezzonico et al. (2007)

K94.34 − + Q 1994/Czech rep. Rezzonico et al. (2007)

K94.10, K94.11, K94.12 − + R 1994/Switzerland Rezzonico et al. (2007)

K94.36 − + S 1994/Czech rep. Rezzonico et al. (2007)

K93.50 − + T 1993/Ghana Rezzonico et al. (2007)

K94.20 − + U 1994/Switzerland Rezzonico et al. (2007)

K95.6 − + V 1995/Pakistan Rezzonico et al. (2007)

K95.8 − + V 1995/China Rezzonico et al. (2007)

K94.13 − + W 1994/Switzerland Rezzonico et al. (2007)

KD − + W 1995/China Rezzonico et al. (2005)

K95.14 − + W 1995/China Rezzonico et al. (2007)

K93.40, K93.41 − + X 1993/Ghana Rezzonico et al. (2007)

P97.18, P97.28 − + X 1997/Bhutan Rezzonico et al. (2007)

P97.7, P97.8, P97.9, P97.10, P97.15, P97.41, P97.42

− + X 1997/Switzerland Rezzonico et al. (2007)

P97.24, P97.29 − + X 1997/Hungary Rezzonico et al. (2007)

P97.35, P97.36 − + X 1997/India Rezzonico et al. (2007)

P97.12 − + X 1997/USA Rezzonico et al. (2007)

P96.16 − + Y 1996/China Rezzonico et al. (2007)

K94.14 − + Z 1994/Italy Rezzonico et al. (2007)

K95.31 − + Z 1995/China Rezzonico et al. (2007)

(continued on next page)

were incubated 3 h at either 37 °C (HaeIII, MspI) or 60 °C (TaqI) and then stored at −20 °C. Restriction fragments were separated by electrophoresis in ethidium bromide-stained 2.5% agarose gels, as described (Sambrook et al., 1989). A 100-bp ladder (GIBCO-BRL Life Technologies Inc., Gaithersburg, MD) was used as a molecular size marker. Two replicates were tested for each sample. RFLP digestion pattern system for hcnAB amplicons was characterized by using all 98 strains applied for hcnAB primers development (Table 1). Restriction analyses of hcnAB for strains in our worldwide collection of Phl

HCN

pseudomonads revealed 13 allele combinations, and 13 allelic combinations from Phl

HCN

strains, respectively (Fig. 2).

This study describes a protocol that can be applied to study HCN-producing pseudomonads, which may lead to the selection of specific Pseudomonas spp. strains and therefore to develop- ment of more effective and ecologically sound application of biological control of plant pathogens.

Acknowledgements

This project was funded by the National Centre of Competence in Research (NCCR) Plant Survival, research program of the Swiss National Science Foundation. We are grateful to Yvan Moënne-Loccoz (Université Claude Bernard,

Strain Phl HCN hcnABallele Year/origin Source

P. fluorescens

2–79 − − − 1979/USA Thomashow and Weller (1988)

P3 − − − 1989/Switzerland Voisard et al. (1989)

P. chlororaphis

LMG 1245 − + N BCCM^a

LMG 5004 − + N BCCM

P. aeruginosa

PAO1^b − + 1995/human wound Holloway (1955)

a Strains from Belgian Coordinated collection of Microorganisms (http://www.belspo.be/bccm/index.html).

b hcnsequence of human pathogenic strain PAO1 (accession number AF208523) was used only forhcnABspecific primers design.

Fig. 2. Genotypic determination (A–Z) of hydrogen cyanide producingPseudomonasspp. by restriction fragment length polymorphism (RFLP) assay ofhcnAB sequences amplified with PM2 and PM7-26R. The 570-bp product ofhcnABgene amplification was digested withHaeIII,TaqI orMspI. Amplicons were separated on 2.5% agarose gel and visualized by staining with ethidium bromide. A 100-bp DNA size standard (λ) indicates the size of the banding patterns. Strains used as templates for the PCR-based assay were as follows: Phl⁺HCN⁺—lines 1, C⁎1A1; 2, K94.31; 3, K94.37; 4, F113; 5, P12; 6, Q37-87; 7, Q2-87; 8, PITR2; 9, PILH1;

10, F96.27; 11, P97.38; 12, CHA0; 13, JMP6. Phl⁻HCN⁺—lines 1, LMG 1245; 2, K94.27; 3, P96.49; 4, K94.34; 5, K94.10; 6, K94.36; 7, K93.35; 8, K94.20; 9, K95.6; 10, K94.13; 11, P97.7; 12, P96.16; 13, K93.40.

Table 1 (continued)

Lyon, France) for helpful discussions, Brian B. McSpadden Gardener (Department of Plan Pathology, Ohio State Univer- sity, Wooster, OH, USA) and Linda S. Thomashow (USDA- ARS, Washington State University, WA, USA) for providing reference pseudomonad strains.

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