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The pqqC gene is essential for antifungal activity of Pseudomonas kilonensis JX22 against Fusarium oxysporum f. sp. lycopersici

Jianhong Xu , Peng Deng , Kurt C. Showmaker , Hui Wang , Sonya M. Baird , Shi-En Lu
DOI: http://dx.doi.org/10.1111/1574-6968.12411 98-105 First published online: 1 April 2014

Abstract

Strain JX22, exhibiting a broad range of antimicrobial activities to fungal pathogens, was isolated and classified as representing Pseudomonas kilonensis. In this study, the mutant JX22MT1 was obtained by the EZ-Tn5 transposon mutation and showed no antifungal activity against Fusarium oxysporum f. sp. lycopersici as compared with wild-type strain JX22. The pqqC gene was disrupted in the mutant. Antifungal activity at the wild-type level was restored from the mutant JX22MT1 with the introduction of the functional pqqC gene, which encodes pyrroloquinoline–quinone synthesis protein C. The results suggest that pqqC is essential for antifungal activity of P. kilonensis JX22 against F. oxysporum f. sp. lycopersici.

Keywords
  • bacteria
  • antimicrobial activity
  • fungi
  • mutagenesis

Introduction

Bacteria of the genus Pseudomonas are frequently isolated from the rhizosphere of crops and many are found to have antimicrobial activity to plant pathogens (Manuel et al., 2012; Rella et al., 2012). More than 100 species have been identified in the genus and many species have interesting biotechnological applications (Peix et al., 2009; Yang et al., 2011). Pseudomonas kilonensis was described by Sikorski et al. (2001) and there is no report of it having antimicrobial activity to plant pathogens. Antimicrobial compounds are very important in managing diseases of both plants and humans (Park et al., 2011; Dharni et al., 2012). With the emergence of increasing numbers of antimicrobial-resistant pathogens, new antagonistic substances are urgently needed (Lutgen et al., 2009; Hunter et al., 2010).

Pyrroloquinoline quinone (PQQ) is a co-factor of dehydrogenases used to form various quinoproteins in bacteria and promotes bacterial growth (Ameyama et al., 1988). Recently, it was demonstrated that PQQ is associated with the production of antimicrobial substances and plant growth-promoting properties of the bacteria Pseudomonas fluorescens (Schnider et al., 1995; Choi et al., 2008; DeWerra et al., 2009) and Rahnella aquatilis (Guo et al., 2009). In addition, PQQ serves as a co-factor in the biosynthesis and excretion of gluconic acid; consequently, it promotes phosphate solubilization in soil. Genes for the biosynthesis of PQQ have been extensively studied in several bacteria (Guo et al., 2009). In P. fluorescens, the genes pqqA, B, C, D, E, F, H, I, J and M are organized as an operon. However, genetic variation has been identified between different bacteria (Choi et al., 2008).

Understanding the molecular mechanisms behind the antifungal activity of P. kilonensis is essential for the development of biological fungicides and new antibiotics. Strain JX22 was isolated in the Mississippi Delta from a soybean charcoal rot disease patch showing disease-suppressive phenomenon, and showed a broad range of antifungal and antibacterial activities to plant pathogens. In this study, JX22 was classified as belonging to P. kilonensis, and the pqqC gene, which encodes pyrroloquinoline–quinone synthesis protein C, was determined to be essential for antifungal activity of P. kilonensis against Fusarium oxysporum f. sp. lycopersici, which is one of the most important pathogens of tomato.

Materials and methods

Bacterial strains, plasmids and culture conditions

The bacterial strains and plasmids used in this study are listed in Table 1. Potato dextrose agar (Difco PDA, BD Biosciences, Detroit, MI) and nutrient broth yeast extract agar medium (NBY; Gross & DeVay, 1977) were used for plate bioassays of P. kilonensis JX22 to evaluate antifungal and antibiotic activities. Strain JX22 was cultured at 28 °C on NBY medium. Antibiotics (Sigma Chemical Co., St. Louis, MO) were added to media at the following concentrations: kanamycin, 100 μg mL−1; tetracycline, 25 μg mL−1.

View this table:

Bacterial strains and plasmids

Strain or plasmidRelevant characteristicsSource
Escherichia coli
JM109RecA1,endA1,gyrA96,thi,hsdR17,supE44,relA1,Δ (lac- proAB)/F'[traD36,proAB+,laclq, laczΔM15]Promega
EC100DTransforMax™ EC100D™ pir+Epicentre
Pseudomonas kilonensis
JX22Wild-type strainThis study
JX22MT1PqqC::Tn5 derivative of JX22; KmrThis study
Plasmid
pUCP26Expression vector for Pseudomonas; TetrWest et al. (1994)
pGEM-T EasyCloning vector; AmprPromega
pUCP1pUCP26 carrying the pqqC gene, TetrThis study
pMT1E1EZ-Tn5 carrying the 6 kb genomic DNA of JX22MT1; KmrThis study
pMT1E2EZ-Tn5 carrying the 10 kb genomic DNA of JX22MT1; KmrThis study
  • Kmr, kanamycin resistance; Tetr, tetracycline resistance; Ampr, ampicillin resistance.

Bioassays for antifungal activity

Pseudomonas kilonensis JX22 and its mutants used in this study were evaluated for antifungal activities using the PDA plate bioassays described previously (Gu et al., 2011). The fungi F. oxysporum f. sp. lycopersici ATCC 9848, Geotrichum candidum Km (Gross & DeVay, 1977) and Rhizoctonia oryzae MS120 were used as indicators for the assays (Michielse & Rep, 2009). Three replicates for the plate bioassays were performed independently.

Cloning and sequence analysis of the 16S rRNA gene

Extraction of bacterial genomic DNA was performed using the cetyltrimethylammonium bromide (CTAB) protocol (Ausubel, 1988). Primers 27F and 1492R were used to amplify the 16S rRNA gene of strain JX22 using a routine PCR procedure (Chelius & Triplett, 2001). The PCR product was purified using a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI). After purification, the 16S rRNA PCR product was ligated into pGEM T-Easy vector as recommended by the manufacturer (Promega). Sequencing reactions were run by Eurofins MWG Operon (Huntsville, AL). The nearly full-length 16S rRNA and pqqC gene sequences of strain JX22 were deposited in GenBank with accession numbers KF840730 and KF840731, respectively. Phylogenetic trees based on 16S rRNA, pqqC and rpoB gene sequences were reconstructed using mega5 software (Tamura et al., 2011). The evolutionary history was inferred using the neighbour-joining method. Evolutionary distances were computed using the maximum composite likelihood method, and were in units of the number of base substitutions per site.

Random mutagenesis analysis

To characterize the genes dedicated to antifungal activity, strain JX22 was randomly mutated using an EZ-Tn5 < R6Kγori/KAN-2 > Tnp Transposome Kit (Epicentre Biotechnologies, Madison, WI). The transposome insertion clones were screened on NBY plates with 100 μg mL−1 kanamycin. Those mutants that exhibited reduced or no antifungal activity against F. oxysporum f. sp. lycopersici were subcultured on NBY plates for further analysis. To confirm that the mutants contained the transposon, a portion of the Tn5 transposon sequence was amplified by PCR with primers EzTn5F (5′-TTACGAAACACGGAAACC-3′) and EzTn5R (5′-TCTAATACCTGGAATGCTGTT-3′). To confirm that the resulting mutants were derivatives of strain JX22, the 16S rRNA gene was cloned and sequenced as described above.

DNA cloning, Illumina sequencing and sequence analysis of the targeted genes

To characterize the gene dedicated to antifungal activity, the bacterial genomic DNA of mutant JX22MT1 (Table 1) was extracted by using the CTAB protocol described above. The bacterial genomic DNA was then digested by EcoRI, self-ligated and then transformed into TransforMax EC100D pir+ electrocompetent Escherichia coli as recommended by the manufacturer of the EZ-Tn5 kit. Sequencing reactions were performed using primers KAN-2FP-1 and KAN-2RP-1 supplied with the kit. The DNA sequences interrupted by the Tn5 transposome were obtained by the plasmid rescue protocol described by the kit manufacturer. The DNA sequence flanking the Tn5 transposome was sequenced and analysed using the blast algorithm in GenBank to identify nucleic acid and protein homologies. Promoter prediction was conducted using the web-based software bprom in the Softberry package (Solovyev & Salamov, 2011).

Illumina sequencing was used to determine the pqq gene cluster of P. kilonensis JX22, as described previously (Jalan et al., 2011). In brief, genomic DNA was extracted from a bacterial culture grown overnight at 28 °C in NBY broth by using the CTAB method. Genome sequencing was performed using Paired-end Illumina (San Diego, CA) sequencing by synthesis on an Illumina MiSeq platform. A total of 4 797 068 high-quality sequence pairs with an average read length of 150 bp, representing more than 200-fold genome coverage, were obtained and these sequence reads were assembled into contigs and scaffolds using the de novo assembler velvet 1.2 (Zerbino & Birney, 2008). This yielded 93 contigs (length weighted median N50 = 162 953; maximum length = 442 384; minimum length = 207; sum = 6 604 180) and the pqq gene cluster was located in contig 73, which is 274 kb in size. The sequence of the pqq gene cluster was deposited in GenBank under accession number KF840731.

Complementation of the mutants JX22MT1

To obtain the intact wild-type gene pqqC, which was disrupted in JX22MT1, primers with restriction enzyme gene sequences of EcoRI and HindIII were designed (JX22MT1-FP: 5′-GGAATTCGGAGACCGAAATGACTGACA-3′; JX22MT1-RP: 5′-GCTCTAGATCAT AAGGTGATCCCTTTGT-3′). The intact ORF of pqqC was amplified with these two primers using Platinum PCR SuperMix High Fidelity as recommended by the manufacturer (Invitrogen, Carlsbad, CA). The PCR product harbouring the intact pqqC gene was inserted into Pseudomonas expression vector pUCP26, to generate plasmid pUCP1 which was then electroporated into cells of strain JX22MT1; the empty vector pUCP26 was used as a control. Colonies that were able to grow on NBY plates with 25 μg mL−1 tetracycline were used for further confirmation and complementation. To confirm that the pqqC gene had been ligated to pUCP26 and introduced to JX22MT1 successfully, plasmids were extracted from JX22MT1, PCR was performed with primers JX22MT1-FP and JX22MT1-RP, and appropriately sized bands were confirmed by gel electrophoresis. Complementation experiments were conducted with plate bioassays to evaluate antifungal activity against F. oxysporum f. sp. lycopersici.

Results

Characterization of strain JX22

Cells of strain JX22 are Gram-negative, aerobic and rod-shaped. The nearly full-length 16S rRNA gene sequence of strain JX22 shared highest similarities (99–98%) with those of Pseudomonas species that were archived in GenBank. The phylogenetic tree based on 16S rRNA gene sequences indicates that strain JX22 belongs to P. kilonensis (Fig. 1). Phylogenetic analysis of the pqqC gene of species of the genus Pseudomonas revealed that strain JX22 differs significantly from those species with sequenced pqqC genes (Fig. 2). No other strains of P. kilonensis were included in the analysis due unavailability of pqqC gene sequences. Further phylogenetic analysis of the rpoB gene also showed the close relationship between strain JX22 and other strains of P. kilonensis (see supporting information, Fig. S1). At this point, we propose that JX22 strain is a member of P. kilonensis. In addition, it was noted that strains of P. fluorescens are scattered in the phylogenetic trees, suggesting the heterogeneity of Pseudomonas species.

Phylogenetic tree of strain JX22 based on 16S rRNA gene sequences. The evolutionary history was inferred using the neighbour-joining method. The optimal tree with sum of branch length = 0.335 is shown. The tree is drawn to scale, with branch lengths (next to the branches) in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed using the maximum composite likelihood method and are in units of the number of base substitutions per site. Evolutionary analyses were conducted in mega5.

Phylogenetic tree of strain JX22 based on pqqC gene sequences. The evolutionary history was inferred using the neighbour-joining method. The optimal tree with sum of branch length = 0.886 is shown. The tree is drawn to scale, with branch lengths (next to the branches) in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed using the maximum composite likelihood method and are in units of the number of base substitutions per site. The analysis involved 17 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 724 positions in the final dataset. Evolutionary analyses were conducted in mega5.

Antifungal activity

Pseudomonas kilonensis JX22 significantly inhibited mycelial growth of F. oxysporum f. sp. lycopersici ATCC 9848 with an inhibition zone of 12.0 ± 1.8 mm in the plate bioassay (Fig. 3). Pseudomonas kilonensis JX22 also had the ability to suppress the growth of R. oryzae and G. candidum (Fig. S2). Repeated experiments indicated that the antagonistic ability of strain JX22 against the fungi listed above was stable and persistent. These data suggest that strain JX22 has the potential for development of biological control and production of antimicrobial compounds.

Plate bioassays of antifungal activities of Pseudomonas kilonensis JX22 with its mutants against the indicator Fusarium oxysporum f. sp. lycopersici ATCC 9848. PDA plates were inoculated with each of the strains and incubated for 2 days at 28°C. The plates were oversprayed with the indicator fungus and further incubated for 2 days. 1, JX22 (wild-type); 2, JX22MT1; 3, JX22MT1 (pUCP26); 4, JX22MT1 (pUCP1)

Identification of the mutant JX22MT1

From two transposon insertion reactions using the EZ-Tn5 transposon system, approximately 13 000 kanamycin-resistant colonies were obtained and then tested for antifungal activity against F. oxysporum f. sp. lycoperci ATCC 9848. Twenty-nine mutants were found to show reduced or undetectable antifungal activity against strain ATCC 9848. JX22MT1 lost the antifungal activity against the fungus completely (Fig. 3). Sequence analyses of the PCR products of both the EZ-Tn5 region and the 16S rRNA gene of mutant JX22MT1 confirmed its identity as a derivative of strain JX22.

Plasmids pMT1E1 and pMT1E2 (Table 1) were obtained from the genome of JX22MT1 using the plasmid rescue method. Plasmids pMT1E1 and pMT1E2 carry 6- and 10-kb genomic DNA insertions, respectively, which include the Tn5 transposon. The genes targeted by the transposon were cloned and sequenced. blast analyses revealed that the partial gene disrupted in the mutant shares high similarity (98%) with the pqqC gene of Pseudomonas brassicacearum NFM421 (GenBank accession no. AEA71637).

Characterization of the role of the pqqC gene in production of the antifungal product

The pqqC gene targeted in the transposon mutant JX22MT1 was further characterized to determine its role in antifungal production. The PCR products amplified with primers of the pqqC gene were about 0.8 kb (pqqC only) and 2.8 kb (pqqC::EZ-Tn5) from plasmid pUCP1 and genomic DNA of JX22MT1, respectively, which are the same as predicted (Fig. S3). Plasmid extraction and restriction endonuclease digestion verified the presence of the plasmid in cells of the mutant JX22MT1 (Fig. S3). PCR analyses templated with cells of the mutant JX22MT1 (pUCP1) also reconfirmed that JX22MT1 is a true mutant of JX22 by EZ-Tn5 insertion in the pqqC gene. The plate bioassay showed that the mutant JX22MT1 containing plasmid pUCP1 produced the antifungal activity against F. oxysporum f. sp. lycopersici, with an inhibition zone (11.0 ± 2.0 mm in radius) comparable with that of the wild-type JX22 (12.0 ± 1.8 mm in radius; Fig. 3). As expected, introduction of the empty vector pUCP26 to strain JX22MT1 led to no antifungal activity against F. oxysporum f. sp. lycopersici (Fig. 3). These results show that the pqqC gene plays an important role in the production of antifungal activity of JX22.

Sequence analysis of the targeted pqqC gene and the intact pqq gene cluster

Sequence analysis revealed the gene disrupted by the transposon is the pqqC gene and starts with an ATG translation initiation codon that is preceded by a putative Shine–Dalgarno sequence (GAGA). The pqqC gene is 250 aa in length. The blast result shows that the pqqC gene shares 98% amino acid identity to the protein PqqC of P. brassicacearum NFM421. Alignment analysis of the DNA sequence previously obtained by the plasmid rescue method revealed that the transposon in the JX22MT1 genome was inserted within the pqqC gene between nucleotides 456 and 457 from its start codon.

The nucleotide sequence of the whole pqq gene cluster was obtained from the Illumina sequencing data. The pqq genes are located in a 10-kb genetic region consisting of seven ORFs (1–7), which are the genes pqqF, pqqA, pqqB, pqqC, pqqD, pqqE and pqqG, respectively (Fig. 4; Table 2). The seven genes have the same transcriptional orientation and a putative sigma 70 promoter was identified (−35 box: TTGAGA and −10 box: CGCTAGCAT) 27 nt away from the start codon of pqqF. Predicted proteins of the seven genes, except for ORF2, share 88–98% similarity with the pqq gene products of P. brassicacearum NFM421 (Ortet et al., 2011). ORF2, which was not present in the genome of strain NFM421, was predicted to be a small protein of 24 aa, having 81% identity to PqqA of Pseudomonas protegens CHA0 (Schnider et al., 1995).

View this table:

Homologues of the pqqC gene region of Pseudomonas kilonensis JX22

ORFSize (aa)HomologueIdentity (%)Accession No.StrainReference
ORF1813PqqF88 YP005210749 P. brassicacearum NFM421Ortet et al. (2011)
ORF224PqqA81 CAA60731 P. protegens CHA0Schnider et al. (1995)
ORF3303PqqB96 YP004356640 P. brassicacearum NFM421Ortet et al. (2011)
ORF4250PqqC98 AEA71637 P. brassicacearum NFM421Ortet et al. (2011)
ORF591PqqD97 YP004356642 P. brassicacearum NFM421Ortet et al. (2011)
ORF6419PqqE96 YP004353289 P. brassicacearum NFM421Ortet et al. (2011)
ORF7609PqqG91 YP004356644 P. brassicacearum NFM421Ortet et al. (2011)

Genetic organization of the pqqC gene region on the chromosome of Pseudomonas kilonensis JX22. The vertical arrow indicates insertion of the Tn5 transposon in the pqqC gene.

Discussion

Identification of Pseudomonas species relies on phylogenetic analysis of rRNA gene sequences (Palleroni, 2003). Phylogenetic analyses of additional housekeeping genes, such as rpoB and gyrB, have significantly increased the power to differentiate various species of the genus (Yamamoto et al., 2000). A recent study demonstrated that the pqqC gene can be used as a powerful marker to study diversity and evaluation of plant beneficial pseudomonads (Meyer et al., 2011). Interestingly, no matter which gene or concatenated sequences of genes are used, strains of P. fluorenscens are found scattered in the phylogenetic trees (Meyer et al., 2011), which indicates that the species is heterozygous. In this study, phylogenetic analyses of the 16S rRNA, pqqC and rpoB genes showed that strain JX22 was grouped with strains of P. kilonensis (Sikorski et al., 2001) and P. fluorenscens F113, the latter being a well-studied biological control bacterium (Fenton et al., 1992). Similarly, strain F113 grouped tightly with the type strain of P. kilonensis (520-20T) in phylogenetic analyses of rpoD, gyrB and pqqC gene sequences in a previous study (Meyer et al., 2011). Therefore, the taxonomic position of strain F113 needs to be re-evaluated.

Understanding the production of antimicrobial metabolites of bacteria provides great opportunities to develop novel biologically based fungicides or pharmaceutical drugs. Although some antimicrobial substances were identified from other organisms (Policegoudra et al., 2007; Lago et al., 2012), bacteria have received much more attention in the search for potential antimicrobial compounds from their secondary metabolites. Pseudomonas species are widely distributed in the environment and many of them can produce antimicrobial secondary metabolites such as 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, phenazine(s) and hydrogen cyanide (Ramette et al., 2011). Pseudomonas kilonensis was first described based on characterization of bacterial strains isolated from agricultural soils (Sikorski et al., 2001). We obtained strain JX22 from Mississippi soil, which possesses significant antifungal activities against a wide range of fungi. To our knowledge, this is the first report on the identification of genes associated with the antifungal activity of P. kilonensis.

The pqq genes are widely distributed in many bacteria and encode PQQ, which serves as a redox cofactor for various bacterial dehydrogenases (Puehringer et al., 2008). PQQ is associated with the production of antimicrobial substances and plant growth-promoting properties of P. fluorescens (Schnider et al., 1995; Choi et al., 2008; DeWerra et al., 2009) and R. aquatilis (Guo et al., 2009). The findings of the present study suggest that PQQ is very important in the production of antifungal activities of P. kilonensis. However, the mechanisms of PQQ's involvement in the production of antimicrobial substances and plant growth-promoting activities remain to be explored. It is expected that PQQ serves as an essential co-factor of some key enzymes that are required for biosynthesis of antimicrobial compounds of Pseudomonas strains. Direct biochemical and molecular biological evidence is needed to understand its roles in the development of antimicrobial activities.

Previous studies reported that the pqqC gene encodes the PqqC protein, which catalyses the final step in the PQQ pathway without co-factor oxidase (Puehringer et al., 2008). The pqqC gene is ubiquitous in those Pseudomonas species that have strong phosphate solubilization ability and potential as biological control agents of plant diseases (Schnider et al., 1995; Meyer et al., 2011). In this study, the crucial impact of the pqqC gene on the antifungal activity of strain JX22 was demonstrated with a series of genetic analyses including mutagenesis and complementation. This work has extended our knowledge of the impacts of PQQ on antimicrobial activity of bacteria and provided an important clue to understanding the molecular mechanisms involved. Further research will analyse antifungal compounds produced by the bacterium by using genetic and biochemical approaches, such as extraction of active compounds against the indictor fungus from agar plates and co-factor-mediated interaction analyses of PQQ with the enzymes required for antimicrobial production.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Fig. S1. Neighbour-joining phylogenetic tree of strain JX22 based on rpoB gene sequences.

Fig. S2. Plate bioassays for antifungal activities of Pseudomonas kilonensis strain JX22.

Fig. S3. Complementary confirmation with plasmids and PCR product of the pqqC gene by electrophoresis.

Acknowledgements

This research was partially supported by the National Basic Research Program of China (2013CB127805), National Natural Science Foundation of China (30901006) and Jiangsu Agriculture Science and Technology Innovation Fund of China (CX(13)3092), and partially supported by the National Institute of Food and Agriculture, US Department of Agriculture, under Project No. MIS-401170. None of the authors have a conflict of interest to declare.

Footnotes

  • Editor: Paolina Garbeva

References

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