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Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains
FEMS Microbiology Letters 180 (1999) 39^44
Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains Philip E. Hammer a , Wassily Burd b , D. Steven Hill a , James M. Ligon a , Karl-Heinz van Pe¨e c; * a
Novartis Agribusiness Biotechnology Research, P.O. Box 12257, Research Triangle Park, NC 27709, USA b Kupala Grodno State University, 22 Ozkeshko Street, Grodno 230023, Belarus c Institut fu«r Biochemie, TU Dresden, D-01062 Dresden, Germany Received 8 July 1999; received in revised form 2 September 1999; accepted 3 September 1999
Abstract The prnABCD gene cluster from Pseudomonas fluorescens encodes the biosynthetic pathway for pyrrolnitrin, a secondary metabolite derived from tryptophan which has strong anti-fungal activity. We used the prn genes from P. fluorescens strain BL915 as a probe to clone and sequence homologous genes from three other Pseudomonas strains, Burkholderia cepacia and Myxococcus fulvus. With the exception of the prnA gene from M. fulvus, the deduced amino acid sequences were s 59% similar among the strains, indicating that the biochemical pathway for pyrrolnitrin biosynthesis is highly conserved. The prnA gene from M. fulvus is about 45% similar to prnA from the other strains and contains regions which are highly conserved among all six strains. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Pyrrolnitrin ; prnABCD gene; Halogenase; Antibiotic
1. Introduction Pyrrolnitrin (3-chloro-4-(2P-nitro-3P-chlorophenyl)pyrrole) is an antibiotic with broad-spectrum antifungal activity which was ¢rst isolated from Pseudomonas pyrrocinia [1]. Subsequently, pyrrolnitrin has been isolated from several isolates of Pseudomonas and Burkholderia and has been implicated as an important mechanism of biological control of fungal plant pathogens by these strains [2^4]. Pyrrolnitrin * Corresponding author. Tel.: +49 (351) 463-4494; Fax: +49 (351) 463-5508; E-mail: [email protected]
production has also been documented for strains of Enterobacter agglomerans [5], Myxococcus fulvus [6], Corallococcus exiguus [6], Cystobacter ferrugineus [6] and Serratia sp. [7]. In 1997, we reported the cloning and sequencing of the prnABCD gene cluster from Pseudomonas £uorescens BL915 [8]. This gene cluster confers the ability to produce pyrrolnitrin to Escherichia coli [8] and other non-producing strains and contains four genes which encode the four biochemical steps to produce pyrrolnitrin from tryptophan [9]. The prnA gene product catalyzes the chlorination of L-tryptophan to form 7-chloro-L-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxyla-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 5 2 - 8
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tion to convert 7-chloro-L-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The two chlorinating enzymes, encoded by prnA and prnC, are NADH-dependent, distinct from previously described haloperoxidases and represent a new class of halogenating enzymes [10]. In the present work, we used the prnABCD cluster from P. £uorescens BL915 to clone homologous gene clusters from P. pyrrocinia, Burkholderia cepacia LT4-12-W and M. fulvus Mx f147 and the prnA gene fragment from strain BL915 to clone prnA gene homologs from P. £uorescens CHA0 and Pseudomonas aureofaciens ACN. We sequenced the genes and aligned the sequences to study conservation of the pathway among the strains.
2. Materials and methods 2.1. Bacterial strains and growth conditions P. £uorescens BL915 is a biological control strain described by Hill et al. [1] and is the strain from which the pyrrolnitrin biosynthetic genes were ¢rst cloned [8]. P. pyrrocinia, described by Arima et al. [1], is the strain from which pyrrolnitrin was ¢rst isolated. B. cepacia (originally Pseudomonas cepacia) was described by Janisiewicz and Roitman [4,12], P. £uorescens CHA0 was described by Keel et al. [13], P. aureofaciens ACN was described by Salcher and Lingens [14] and M. fulvus Mx f147 was described by Gerth et al. [6]. Pseudomonas and Burkholderia strains were cultured at 28³C in Luria-Bertani (LB) [15] medium or on LB supplemented with 15 g agar l31 . M. fulvus was cultured at 28³C in MD1 liquid medium [6]. E. coli strains were cultured at 37³C in liquid Terri¢c broth [15], LB or on LB agar. When necessary, media for E. coli were supplemented with the following antibiotics: 100 mg l31 ampicillin, 50 mg l31 kanamycin or 15 mg l31 tetracycline. 2.2. Recombinant DNA methods General recombinant DNA techniques were per-
formed essentially as described by Sambrook et al. [15]. The prnABCD clusters in P. pyrrocinia, B. cepacia and M. fulvus were identi¢ed by Southern hybridization. 32 P-labelled DNA of the 5.8-kb XbaI to NotI DNA fragment containing the prnABCD gene cluster from P. £uorescens strain BL915 [8] was used as a probe. Hybridization and washing were carried out as described by Church and Gilbert [16] at 65³C for P. pyrrocinia and B. cepacia and at 55³C for M. fulvus. For detection of the prnA genes from P. £uorescens CHA0 and P. aureofaciens, digoxigenin-labelled prnA from P. £uorescens BL915 was used as the probe. Hybridization was performed under conditions allowing for the detection of DNA fragments with a homology of at least 75%. Based on results of the Southern hybridizations, the hybridizing fragments were excised from agarose gels, ligated into pBluescriptII (Stratagene) and used to transform E. coli to produce plasmid libraries. The libraries from P. pyrrocinia and B. cepacia were screened by colony hybridization using the prn gene probe described above. The libraries from P. £uorescens CHA0, P. aureofaciens and M. fulvus were screened by Southern analysis of puri¢ed plasmid DNA. The nucleotide sequences of both strands were determined using a Model 373A automated DNA sequencer and cycle sequencing kits from Applied Biosystems. Sequence data were assembled and edited using the Sequencher1 software package (Gene Codes Corporation, Ann Arbor, MI, USA). DNA fragments were subcloned from the pBluescriptII clones into the broad host range vector pRK290 and the resulting plasmids were mobilized into P. £uorescens 134vORF1-4 by triparental mating as described previously [11]. Pseudomonas strains containing plasmids were cultured in media containing 30 mg l31 tetracycline. Pyrrolnitrin production was detected by extraction and thin layer chromatography (TLC) essentially as described by Kirner et al. [9]. 2.3. Nucleotide sequence accession numbers The GenBank accession numbers for the DNA sequences reported here are as follows. The prn gene region from P. £uorescens BL915, U74493; the prn gene region from P. pyrrocinia, AF161186; the prn gene region from B. cepacia LT4-12-W,
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Fig. 1. Southern blot analysis of the prn gene clusters in four strains using the prn gene probe from P. £uorescens BL915. Arrows indicate the positions of molecular size markers (kb) for each blot.
AF161183; the prn gene region from M. fulvus Mx f147, AF161185; the prnA gene from P. £uorescens CHA0, AF161184 and the prnA gene from P. aureofaciens ACN, AF161182.
3. Results and discussion 3.1. Molecular cloning of prn gene homologs from P. pyrrocinia, B. cepacia and M. fulvus Southern blot analysis demonstrated that homologs of the prnABCD genes were present in P. pyrrocinia, B. cepacia and M. fulvus (Fig. 1). The prn gene
homologs from P. pyrrocinia were cloned on an approximately 20-kb BamHI fragment in plasmid pPEH80 and those from B. cepacia were cloned on an approximately 9.5-kb KpnI fragment in plasmid pPEH66. The prn gene homologs from M. fulvus were cloned on two BamHI fragments of approximately 8 and 5 kb in plasmids pPEH76 and pPEH78, respectively. Each plasmid clone was compared to genomic DNA from the corresponding source strain by Southern analysis using several restriction enzymes and the prn gene probe. In each case, the internal fragments from the plasmid clone which hybridized to the probe were identical in size to hybridizing fragments from the source strain (data
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Fig. 2. Heterologous expression of the prn gene clusters from P. pyrrocinia, B. cepacia and M. fulvus. The gene cluster from each strain was subcloned into the broad host range vector pRK290 and mobilized into P. £uorescens BL915vORF1-4, a mutant from which the entire prnABCD cluster has been deleted. The 5.8-kb prnABCD gene cluster from P. £uorescens BL915 was included as a positive control. Metabolites were extracted from the cultures and concentrated and then separated by TLC on silica plates using toluene as the mobile phase. Pyrrolnitrin was visualized by spraying with van Urk's reagent. The position of the pyrrolnitrin spot is indicated.
not shown), con¢rming that the plasmid contains DNA, cloned from the desired strain, which is highly
homologous to prnABCD from P. £uorescens BL915. A PCR approach was used to determine the native orientation of the 5- and 8-kb BamHI fragments in the M. fulvus genome. The ends of the cloned DNA fragments were sequenced and oligonucleotide primers were designed to anneal within the fragments and initiate PCR extension towards the proximal BamHI cloning site. Four PCR reactions were performed using M. fulvus genomic DNA as template and four di¡erent primer combinations. For each possible orientation of the two fragments, only one primer combination would amplify a PCR product. Furthermore, the distance from each priming site to the proximal BamHI site was known, so the length of the PCR products could be calculated. Only one primer combination produced a PCR product of the expected size. This experiment veri¢ed that the 5- and 8-kb fragments are adjacent to each other in the M. fulvus genome and revealed the native orientation of the two fragments relative to each other. The PCR product was sequenced to verify that the two fragments were indeed contiguous in M. fulvus genomic DNA and the BamHI site separating the 5- and 8-kb BamHI fragments was found to be located in the middle of the prnC gene.
Table 1 Similarity among pyrrolnitrin biosynthetic genes from six bacterial strains
prnA M. fulvus B. cepacia P. pyrrocinia P. £uorescens CHA0 P. aureofaciens ACN prnB M. fulvus B. cepacia P. pyrrocinia prnC M. fulvus B. cepacia P. pyrrocinia prnD M. fulvus B. cepacia P. pyrrocinia
P. £uorescens BL915
P. aureofaciens CAN
P. £uorescens CHA0
P. pyrrocinia
B. cepacia
44.7 89.4 94.4 94.8 95.0
45.5 90.1 93.1 93.1
45.1 88.5 92.9
44.5 92.2
45.2
61.6 80.9 86.2
61.9 85.6
59.4
79.3 93.7 95.2
79.5 94.2
79.2
62.1 87.4 91.2
62.0 87.9
61.2
Predicted amino acid sequences were compared among the strains using the Clustal alignment method. Values are the percentage similarity.
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Fig. 3. Organization of the pyrrolnitrin biosynthetic gene clusters in P. £uorescens BL915, B. cepacia and M. fulvus. Genomic clones were sequenced and the prn gene coding regions were identi¢ed by DNA sequence homology to those from P. £uorescens BL915. Arrows indicate the direction of transcription and numbers indicate the length of each open reading frame in bp. In P. £uorescens and B. cepacia strains, the prnA and prnB genes are translationally coupled.
3.2. Heterologous expression of prn gene homologs P. pyrrocinia, B. cepacia and M. fulvus The 20-kb BamHI fragment from pPEH80 and the 9.4-kb KpnI fragment from pPEH66 were subcloned into the broad host range vector pRK290 to produce the plasmids pRK(PEH80) and pRK(PEH66), respectively. The 5- and 8-kb BamHI fragments from pPEH76 and pPEH78 were ligated together into pRK290 and a clone was selected which contained both fragments in the native orientation by using restriction analysis and the PCR method described above. This plasmid construct was designated pRK(PEH76 and 78) The plasmids pRK(PEH80), pRK(PEH66) and pRK(PEH76 and 78) containing the gene clusters cloned from P. pyrrocinia, B. cepacia and M. fulvus, respectively, were mobilized into P. £uorescens BL915vORF1-4, a mutant from which the entire prn gene cluster has been deleted [8]. TLC assays revealed that each of these three plasmids restored the ability of the mutant strain to produce pyrrolnitrin (Fig. 2). These results demonstrated that the DNA cloned from each strain contains a functional pyrrolnitrin biosynthetic gene cluster. 3.3. Comparison of DNA and protein sequences The prn gene open reading frames in each strain were identi¢ed by DNA sequence homology to prnABCD from P. £uorescens BL915. The deduced amino acid sequences of the four genes were compared among the four strains using the Clustal align-
ment method and found to be very highly similar (Table 1). With the exception of prnA from M. fulvus, the deduced amino acid sequences were s 59% similar, indicating that the biochemical pathway for pyrrolnitrin production is highly conserved. The prnA gene from M. fulvus was 6 46% similar to prnA from the other strains at the amino acid level. While this level of similarity is lower than observed for the other genes, it represents signi¢cant homology. The prnA gene from M. fulvus contains regions which are highly conserved among all six strains (data not presented), including a nucleotide binding domain near the amino-terminus described by Hammer et al. [8]. In P. £uorescens, P. pyrrocinia and B. cepacia, the prn genes are arranged identically (Fig. 3) and in a linear relationship to the order of the biochemical reactions for pyrrolnitrin synthesis proposed by Kirner et al. [9]. In P. £uorescens BL915, the four genes are contained in a single transcriptional unit [8] and this appears probable for P. pyrrocinia and B. cepacia as well. In P. £uorescens BL915, P. £uorescens CHA0, P. pyrrocinia and B. cepacia, the prnB coding sequence begins with the unusual translation initiation codon GTG and is apparently translationally coupled to the prnA open reading frame, since the GTG translation initiation codon of prnB overlaps one base with the TAG stop codon of prnA [8]. This translational coupling may be a mechanism to regulate the amount of PrnB protein present in the cell and thus to prevent the diversion of tryptophan to aminophenylpyrrole [9] and/or to prevent the accumulation of 7-chlorotryptophan.
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In M. fulvus, prnB begins with ATG and is the ¢rst gene in the cluster. The prnA gene is located 3P to the other three coding regions and is transcribed in the opposite direction (Fig. 3). The distinct organization of the prn gene cluster in M. fulvus, requiring a separate promoter for prnA, indicates a di¡erent strategy for the regulation of the pyrrolnitrin biosynthesis pathway in this strain.
Acknowledgements Portions of this work were funded by the Environment and Climate Research and Technology Development Program of the European Union and the Sa«chsische Staatsministerium fu«r Umwelt und Landesentwicklung. B. cepacia strain LT-4-12W was a gift from Wojceich Janisiewicz, USDA-ARS Appalachian Fruit Research Station, Kearneysville, WV, USA, M. fulvus was a gift from Hans Reichenbach, GBF Braunschweig, Germany, and P. £uorescens CHA0 was a gift from Dieter Haas, Laboratoire de Biologie, Microbienne, Universite¨ de Lausanne, Switzerland.
References [1] Arima, K., Imanaka, H., Kousaka, M., Fukuda, A. and Tamura, G. (1964) Pyrrolnitrin, a new antibiotic substance, produced by Pseudomonas. Agric. Biol. Chem. 28, 575^576. [2] Homma, Y., Sato, Z., Hirayama, F., Konno, K., Shirahama, H. and Suzui, T. (1989) Production of antibiotics by Pseudomonas cepacia as an agent for biological control of soilborne plant pathogens. Soil Biol. Biochem. 21, 723^728. [3] Howell, C.R. and Stipanovic, R.D. (1979) Control of Rhizoctonia solani on cotton seedlings with Pseudomonas £uorescens and with an antibiotic produced by the bacterium. Phytopathology 77, 480^482. [4] Janisiewicz, W.J. and Roitman, J. (1988) Biological control of blue mold and grey mold on apple and pear with Pseudomonas cepacia. Phytopathology 78, 1697^1700.
[5] Chernin, L., Brandis, A., Ismailov, Z. and Chet, I. (1996) Pyrrolnitrin production by an Enterobacter agglomerans strain with a broad spectrum of antagonistic activity towards fungal and bacterial phytopathogens. Curr. Microbiol. 32, 208^212. [6] Gerth, K., Trowitzsch, W., Wray, V., Ho«£e, G., Irschik, H. and Reichenbach, H. (1982) Pyrrolnitrin from Myxococcus fulvus (myxobacterales). J. Antibiot. 35, 1101^1103. [7] Kalbe, C., Marten, P. and Berg, G. (1996) Strains of the genus Serratia as bene¢cial rhizobacteria of oilseed rape with antifungal properties. Microbiol. Res. 151, 433^439. [8] Hammer, P.E., Hill, D.S., Lam, S.T., van Pe¨e, K.-H. and Ligon, J.M. (1997) Four genes from Pseudomonas £uorescens that encode the biosynthesis of pyrrolnitrin. Appl. Environ. Microbiol. 63, 2147^2154. [9] Kirner, S., Hammer, P.E., Hill, D.S., Altmann, A., Fischer, I., Weislo, L.J., Lanahan, M., van Pe¨e, K.-H. and Ligon, J.M. (1998) Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas £uorescens. J. Bacteriol. 180, 1939^1943. [10] Hohaus, K., Altmann, A., Burd, W., Fischer, I., Hammer, P.E., Hill, D.S., Ligon, J.M. and van Pe¨e, K.-H. (1997) NADH-dependent halogenases are more likely to be involved in halometabolite biosynthesis than haloperoxidases. Angew. Chem. Int. Ed. Engl. 36, 2012^2013. [11] Hill, D.S., Stein, J.I., Torkewitz, N.R., Morse, A.M., Howell, C.R., Pachlatko, J.P., Becker, J.O. and Ligon, J.M. (1994) Cloning of genes involved in the synthesis of pyrrolnitrin from Pseudomonas £uorescens and the role of pyrrolnitrin synthesis in biological control of plant disease. Appl. Environ. Microbiol. 60, 78^85. [12] Roitman, J.N., Mahoney, N.E. and Janisiewicz, W.J. (1990) Production and composition of phenylpyrrole metabolites produced by Pseudomonas cepacia. Appl. Microbiol. Biotechnol. 34, 381^386. [13] Keel, C., Voisard, C., Berling, C.H., Kahr, G. and Defago, G. (1989) Iron su¤ciency, a prerequisite for the suppression of tobacco black root rot by Pseudomonas £uorescens strain CHA0 under gnotobiotic conditions. Phytopathology 79, 584^589. [14] Salcher, O. and Lingens, F. (1980) Isolation and characterization of a mutant of Pseudomonas aureofaciens ATCC 15926 with an increased capacity for synthesis of pyrrolnitrin. J. Gen. Microbiol. 118, 828^829. [15] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning : A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY. [16] Church, G.M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991^1995.
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Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains Philip E. Hammer a , Wassily Burd b , D. Steven Hill a , James M. Ligon a , Karl-Heinz van Pe¨e c; * a
Novartis Agribusiness Biotechnology Research, P.O. Box 12257, Research Triangle Park, NC 27709, USA b Kupala Grodno State University, 22 Ozkeshko Street, Grodno 230023, Belarus c Institut fu«r Biochemie, TU Dresden, D-01062 Dresden, Germany Received 8 July 1999; received in revised form 2 September 1999; accepted 3 September 1999
Abstract The prnABCD gene cluster from Pseudomonas fluorescens encodes the biosynthetic pathway for pyrrolnitrin, a secondary metabolite derived from tryptophan which has strong anti-fungal activity. We used the prn genes from P. fluorescens strain BL915 as a probe to clone and sequence homologous genes from three other Pseudomonas strains, Burkholderia cepacia and Myxococcus fulvus. With the exception of the prnA gene from M. fulvus, the deduced amino acid sequences were s 59% similar among the strains, indicating that the biochemical pathway for pyrrolnitrin biosynthesis is highly conserved. The prnA gene from M. fulvus is about 45% similar to prnA from the other strains and contains regions which are highly conserved among all six strains. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Pyrrolnitrin ; prnABCD gene; Halogenase; Antibiotic
1. Introduction Pyrrolnitrin (3-chloro-4-(2P-nitro-3P-chlorophenyl)pyrrole) is an antibiotic with broad-spectrum antifungal activity which was ¢rst isolated from Pseudomonas pyrrocinia [1]. Subsequently, pyrrolnitrin has been isolated from several isolates of Pseudomonas and Burkholderia and has been implicated as an important mechanism of biological control of fungal plant pathogens by these strains [2^4]. Pyrrolnitrin * Corresponding author. Tel.: +49 (351) 463-4494; Fax: +49 (351) 463-5508; E-mail: [email protected]
production has also been documented for strains of Enterobacter agglomerans [5], Myxococcus fulvus [6], Corallococcus exiguus [6], Cystobacter ferrugineus [6] and Serratia sp. [7]. In 1997, we reported the cloning and sequencing of the prnABCD gene cluster from Pseudomonas £uorescens BL915 [8]. This gene cluster confers the ability to produce pyrrolnitrin to Escherichia coli [8] and other non-producing strains and contains four genes which encode the four biochemical steps to produce pyrrolnitrin from tryptophan [9]. The prnA gene product catalyzes the chlorination of L-tryptophan to form 7-chloro-L-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxyla-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 5 2 - 8
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tion to convert 7-chloro-L-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The two chlorinating enzymes, encoded by prnA and prnC, are NADH-dependent, distinct from previously described haloperoxidases and represent a new class of halogenating enzymes [10]. In the present work, we used the prnABCD cluster from P. £uorescens BL915 to clone homologous gene clusters from P. pyrrocinia, Burkholderia cepacia LT4-12-W and M. fulvus Mx f147 and the prnA gene fragment from strain BL915 to clone prnA gene homologs from P. £uorescens CHA0 and Pseudomonas aureofaciens ACN. We sequenced the genes and aligned the sequences to study conservation of the pathway among the strains.
2. Materials and methods 2.1. Bacterial strains and growth conditions P. £uorescens BL915 is a biological control strain described by Hill et al. [1] and is the strain from which the pyrrolnitrin biosynthetic genes were ¢rst cloned [8]. P. pyrrocinia, described by Arima et al. [1], is the strain from which pyrrolnitrin was ¢rst isolated. B. cepacia (originally Pseudomonas cepacia) was described by Janisiewicz and Roitman [4,12], P. £uorescens CHA0 was described by Keel et al. [13], P. aureofaciens ACN was described by Salcher and Lingens [14] and M. fulvus Mx f147 was described by Gerth et al. [6]. Pseudomonas and Burkholderia strains were cultured at 28³C in Luria-Bertani (LB) [15] medium or on LB supplemented with 15 g agar l31 . M. fulvus was cultured at 28³C in MD1 liquid medium [6]. E. coli strains were cultured at 37³C in liquid Terri¢c broth [15], LB or on LB agar. When necessary, media for E. coli were supplemented with the following antibiotics: 100 mg l31 ampicillin, 50 mg l31 kanamycin or 15 mg l31 tetracycline. 2.2. Recombinant DNA methods General recombinant DNA techniques were per-
formed essentially as described by Sambrook et al. [15]. The prnABCD clusters in P. pyrrocinia, B. cepacia and M. fulvus were identi¢ed by Southern hybridization. 32 P-labelled DNA of the 5.8-kb XbaI to NotI DNA fragment containing the prnABCD gene cluster from P. £uorescens strain BL915 [8] was used as a probe. Hybridization and washing were carried out as described by Church and Gilbert [16] at 65³C for P. pyrrocinia and B. cepacia and at 55³C for M. fulvus. For detection of the prnA genes from P. £uorescens CHA0 and P. aureofaciens, digoxigenin-labelled prnA from P. £uorescens BL915 was used as the probe. Hybridization was performed under conditions allowing for the detection of DNA fragments with a homology of at least 75%. Based on results of the Southern hybridizations, the hybridizing fragments were excised from agarose gels, ligated into pBluescriptII (Stratagene) and used to transform E. coli to produce plasmid libraries. The libraries from P. pyrrocinia and B. cepacia were screened by colony hybridization using the prn gene probe described above. The libraries from P. £uorescens CHA0, P. aureofaciens and M. fulvus were screened by Southern analysis of puri¢ed plasmid DNA. The nucleotide sequences of both strands were determined using a Model 373A automated DNA sequencer and cycle sequencing kits from Applied Biosystems. Sequence data were assembled and edited using the Sequencher1 software package (Gene Codes Corporation, Ann Arbor, MI, USA). DNA fragments were subcloned from the pBluescriptII clones into the broad host range vector pRK290 and the resulting plasmids were mobilized into P. £uorescens 134vORF1-4 by triparental mating as described previously [11]. Pseudomonas strains containing plasmids were cultured in media containing 30 mg l31 tetracycline. Pyrrolnitrin production was detected by extraction and thin layer chromatography (TLC) essentially as described by Kirner et al. [9]. 2.3. Nucleotide sequence accession numbers The GenBank accession numbers for the DNA sequences reported here are as follows. The prn gene region from P. £uorescens BL915, U74493; the prn gene region from P. pyrrocinia, AF161186; the prn gene region from B. cepacia LT4-12-W,
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41
Fig. 1. Southern blot analysis of the prn gene clusters in four strains using the prn gene probe from P. £uorescens BL915. Arrows indicate the positions of molecular size markers (kb) for each blot.
AF161183; the prn gene region from M. fulvus Mx f147, AF161185; the prnA gene from P. £uorescens CHA0, AF161184 and the prnA gene from P. aureofaciens ACN, AF161182.
3. Results and discussion 3.1. Molecular cloning of prn gene homologs from P. pyrrocinia, B. cepacia and M. fulvus Southern blot analysis demonstrated that homologs of the prnABCD genes were present in P. pyrrocinia, B. cepacia and M. fulvus (Fig. 1). The prn gene
homologs from P. pyrrocinia were cloned on an approximately 20-kb BamHI fragment in plasmid pPEH80 and those from B. cepacia were cloned on an approximately 9.5-kb KpnI fragment in plasmid pPEH66. The prn gene homologs from M. fulvus were cloned on two BamHI fragments of approximately 8 and 5 kb in plasmids pPEH76 and pPEH78, respectively. Each plasmid clone was compared to genomic DNA from the corresponding source strain by Southern analysis using several restriction enzymes and the prn gene probe. In each case, the internal fragments from the plasmid clone which hybridized to the probe were identical in size to hybridizing fragments from the source strain (data
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Fig. 2. Heterologous expression of the prn gene clusters from P. pyrrocinia, B. cepacia and M. fulvus. The gene cluster from each strain was subcloned into the broad host range vector pRK290 and mobilized into P. £uorescens BL915vORF1-4, a mutant from which the entire prnABCD cluster has been deleted. The 5.8-kb prnABCD gene cluster from P. £uorescens BL915 was included as a positive control. Metabolites were extracted from the cultures and concentrated and then separated by TLC on silica plates using toluene as the mobile phase. Pyrrolnitrin was visualized by spraying with van Urk's reagent. The position of the pyrrolnitrin spot is indicated.
not shown), con¢rming that the plasmid contains DNA, cloned from the desired strain, which is highly
homologous to prnABCD from P. £uorescens BL915. A PCR approach was used to determine the native orientation of the 5- and 8-kb BamHI fragments in the M. fulvus genome. The ends of the cloned DNA fragments were sequenced and oligonucleotide primers were designed to anneal within the fragments and initiate PCR extension towards the proximal BamHI cloning site. Four PCR reactions were performed using M. fulvus genomic DNA as template and four di¡erent primer combinations. For each possible orientation of the two fragments, only one primer combination would amplify a PCR product. Furthermore, the distance from each priming site to the proximal BamHI site was known, so the length of the PCR products could be calculated. Only one primer combination produced a PCR product of the expected size. This experiment veri¢ed that the 5- and 8-kb fragments are adjacent to each other in the M. fulvus genome and revealed the native orientation of the two fragments relative to each other. The PCR product was sequenced to verify that the two fragments were indeed contiguous in M. fulvus genomic DNA and the BamHI site separating the 5- and 8-kb BamHI fragments was found to be located in the middle of the prnC gene.
Table 1 Similarity among pyrrolnitrin biosynthetic genes from six bacterial strains
prnA M. fulvus B. cepacia P. pyrrocinia P. £uorescens CHA0 P. aureofaciens ACN prnB M. fulvus B. cepacia P. pyrrocinia prnC M. fulvus B. cepacia P. pyrrocinia prnD M. fulvus B. cepacia P. pyrrocinia
P. £uorescens BL915
P. aureofaciens CAN
P. £uorescens CHA0
P. pyrrocinia
B. cepacia
44.7 89.4 94.4 94.8 95.0
45.5 90.1 93.1 93.1
45.1 88.5 92.9
44.5 92.2
45.2
61.6 80.9 86.2
61.9 85.6
59.4
79.3 93.7 95.2
79.5 94.2
79.2
62.1 87.4 91.2
62.0 87.9
61.2
Predicted amino acid sequences were compared among the strains using the Clustal alignment method. Values are the percentage similarity.
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43
Fig. 3. Organization of the pyrrolnitrin biosynthetic gene clusters in P. £uorescens BL915, B. cepacia and M. fulvus. Genomic clones were sequenced and the prn gene coding regions were identi¢ed by DNA sequence homology to those from P. £uorescens BL915. Arrows indicate the direction of transcription and numbers indicate the length of each open reading frame in bp. In P. £uorescens and B. cepacia strains, the prnA and prnB genes are translationally coupled.
3.2. Heterologous expression of prn gene homologs P. pyrrocinia, B. cepacia and M. fulvus The 20-kb BamHI fragment from pPEH80 and the 9.4-kb KpnI fragment from pPEH66 were subcloned into the broad host range vector pRK290 to produce the plasmids pRK(PEH80) and pRK(PEH66), respectively. The 5- and 8-kb BamHI fragments from pPEH76 and pPEH78 were ligated together into pRK290 and a clone was selected which contained both fragments in the native orientation by using restriction analysis and the PCR method described above. This plasmid construct was designated pRK(PEH76 and 78) The plasmids pRK(PEH80), pRK(PEH66) and pRK(PEH76 and 78) containing the gene clusters cloned from P. pyrrocinia, B. cepacia and M. fulvus, respectively, were mobilized into P. £uorescens BL915vORF1-4, a mutant from which the entire prn gene cluster has been deleted [8]. TLC assays revealed that each of these three plasmids restored the ability of the mutant strain to produce pyrrolnitrin (Fig. 2). These results demonstrated that the DNA cloned from each strain contains a functional pyrrolnitrin biosynthetic gene cluster. 3.3. Comparison of DNA and protein sequences The prn gene open reading frames in each strain were identi¢ed by DNA sequence homology to prnABCD from P. £uorescens BL915. The deduced amino acid sequences of the four genes were compared among the four strains using the Clustal align-
ment method and found to be very highly similar (Table 1). With the exception of prnA from M. fulvus, the deduced amino acid sequences were s 59% similar, indicating that the biochemical pathway for pyrrolnitrin production is highly conserved. The prnA gene from M. fulvus was 6 46% similar to prnA from the other strains at the amino acid level. While this level of similarity is lower than observed for the other genes, it represents signi¢cant homology. The prnA gene from M. fulvus contains regions which are highly conserved among all six strains (data not presented), including a nucleotide binding domain near the amino-terminus described by Hammer et al. [8]. In P. £uorescens, P. pyrrocinia and B. cepacia, the prn genes are arranged identically (Fig. 3) and in a linear relationship to the order of the biochemical reactions for pyrrolnitrin synthesis proposed by Kirner et al. [9]. In P. £uorescens BL915, the four genes are contained in a single transcriptional unit [8] and this appears probable for P. pyrrocinia and B. cepacia as well. In P. £uorescens BL915, P. £uorescens CHA0, P. pyrrocinia and B. cepacia, the prnB coding sequence begins with the unusual translation initiation codon GTG and is apparently translationally coupled to the prnA open reading frame, since the GTG translation initiation codon of prnB overlaps one base with the TAG stop codon of prnA [8]. This translational coupling may be a mechanism to regulate the amount of PrnB protein present in the cell and thus to prevent the diversion of tryptophan to aminophenylpyrrole [9] and/or to prevent the accumulation of 7-chlorotryptophan.
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In M. fulvus, prnB begins with ATG and is the ¢rst gene in the cluster. The prnA gene is located 3P to the other three coding regions and is transcribed in the opposite direction (Fig. 3). The distinct organization of the prn gene cluster in M. fulvus, requiring a separate promoter for prnA, indicates a di¡erent strategy for the regulation of the pyrrolnitrin biosynthesis pathway in this strain.
Acknowledgements Portions of this work were funded by the Environment and Climate Research and Technology Development Program of the European Union and the Sa«chsische Staatsministerium fu«r Umwelt und Landesentwicklung. B. cepacia strain LT-4-12W was a gift from Wojceich Janisiewicz, USDA-ARS Appalachian Fruit Research Station, Kearneysville, WV, USA, M. fulvus was a gift from Hans Reichenbach, GBF Braunschweig, Germany, and P. £uorescens CHA0 was a gift from Dieter Haas, Laboratoire de Biologie, Microbienne, Universite¨ de Lausanne, Switzerland.
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