Appl Microbiol Biotechnol (2013) 97:10081–10090 DOI 10.1007/s00253-013-5247-5

APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY

The highly modified microcin plantazolicin is associated with nematicidal activity of Bacillus amyloliquefaciens FZB42

Zhongzhong Liu & Anto Budiharjo & Pengfei Wang & Hui Shi & Juan Fang & Rainer Borriss & Keqin Zhang & Xiaowei Huang

Received: 9 May 2013 /Revised: 19 August 2013 /Accepted: 22 August 2013 /Published online: 2 October 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Bacillus amyloliquefaciens FZB42 has been shown plantazolicin bearing a molecular weight of 1,354 Da was to stimulate plant growth and to suppress the growth of plant present in wild-type B. amyloliquefaciens FZB42, but absent pathogenic organisms including nematodes. However, the in the ΔRABM_007470 mutant. Furthermore, bioassay of the mechanism underlying its effect against nematodes remains organic extract containing plantazolicin also showed a mod- unknown. In this study, we screened a random mutant library erate nematicidal activity. We conclude that a novel gene of B. amyloliquefaciens FZB42 generated by the mariner RBAM_007470 and its related metabolite are involved in the transposon TnYLB-1 and identified a mutant strain F5 with antagonistic effect exerted by B. amyloliquefaciens FZB42 attenuated nematicidal activity. Reversible polymerase chain against nematodes. reaction revealed that three candidate genes RAMB_007470, yhdY,andprkA that were disrupted by the transposon in strain Keywords Bacillus amyloliquefaciens FZB42 . Nematicidal F5 potentially contributed to its decreased nematicidal activ- activity . RBAM_007470 gene . Plantazolicin . Himar1 ity. Bioassay of mutants impaired in the three candidate genes transposon library . ESI-TOF-MS demonstrated that directed deletion of gene RBAM_007470 resulted in loss of nematicidal activity comparable with that of the F5 triple mutant. RBAM_007470 hasbeenreportedas Introduction being involved in of plantazolicin, a thiazole/ oxazole-modified microcin with hitherto unknown function. Plant-parasitic nematodes cause serious losses to a variety of Electrospray ionization time-of-flight agricultural crops worldwide. However, because traditional (ESI-TOF-MS) analyses of surface extracts revealed that nematicides are associated with major environmental and health concerns, developing safe and effective nematicides is urgently needed. Among the recent developments, biocontrol : : : : : * Z. Liu P. Wang H. Shi J. Fang K. Zhang X. Huang ( ) measures have shown significant promises and attracted much Laboratory for Conservation and Utilization of Bio-Resources, and Key Laboratory for Microbial Resources of the Ministry of attention (Duncon 1991; Schneider et al. 2003). Education, Yunnan University, Kunming, China Several successful biocontrol agents have been devel- e-mail: [email protected] oped and put into use over the years. These agents include nematophagous fungi and bacteria (Åhman 2000; Z. Liu Department of Dermatology, Xijing Hospital, Fourth Military Tikhonov et al. 2002;Tianetal.2007). Among them, the Medical University, Xi′an, China bacterial genera. such as Pasteuria, Pseudomonas, Bacillus, Actinomycetes, Agrobacterium, Arthrobacter, Alcaligenes, A. Budiharjo Aureobacterium, Azotobacter, Beijerinckia, Chromobacterium, Biology Department, Faculty of Sciences and Mathematics, Diponegoro University, Semarang, Indonesia Clavibacter, Clostridium, Comamonas, , Curtobacterium, Desulforibtio, Enterobacter, Flavobacterium, R. Borriss Gluconobacter, Hydrogenophaga, Klebsiella, Methylobacte- Institute for Biology, Humboldt University Berlin, Berlin, Germany rium, Phyllobacterium, Phingobacterium, Rhizobium, R. Borriss Stenotrotrophomonas,andVariovorax, have shown great poten- ABiTEP, Berlin, Germany tials for controlling nematode infections (Tian et al. 2007). 10082 Appl Microbiol Biotechnol (2013) 97:10081–10090

Additionally, several human pathogens, such as Burkholderia, et al. 2007). However, despite its obvious abilities to reduce Serratia, Enterococcuss, Streptococcus,andStaphylococcus, nematode eggs in roots, juvenile worms in soil, and plant galls have also been reported to have antagonistic effects against on tomato, the specific nematicide-related genes as well as the nematodes (O'Quinn et al. 2001; Kurz and Ewbank 2000; molecular mechanisms have remained completely unknown in Garsin et al. 2001; Qin et al. 2000; Sifri et al. 2002). Studies B. amyloliquefaciens FZB42 (Burkett-Cadena et al. 2008). In have revealed that different bacterial genera employ different this study, after we screened a random mutant library of B. mechanisms in pathogenesis against nematodes. For example, amyloliquefaciens FZB42 prepared with the mariner transposon four Pasteuria species (Pasteuria ramosa, Pasteuria penetrans, TnYLB-1 (Le Breton et al. 2006), the RABM_007470 gene, Pasteuria thornei,andPasteuria nishizawae) can parasitize which is located in a cluster of 12 genes that covers 10 kb, was root-knot nematodes Meloidogyne spp as well as cyst nematodes demonstrated to be involved in the capability of killing nema- Heterodera and Globodera, respectively (Ebert et al. 1996; todes. Because this gene cluster had been described to be respon- Atibalentja et al. 2000). During their pathogenesis, the spores sible for the biosynthesis, modification, export, and self- of Pasteuria first attach to the cuticles of the second-stage immunity of plantazolicin, a type of newly reported athiazole/ juveniles and germinate after the worms enter plant roots and oxazole-modified microcin (TOMM) (Scholz et al. 2011), we begin feeding. Bacillus thuringiensis produces toxic crystal pro- further compared the extracellular metabolites formed by the teins and six Cry proteins (Cry5, Cry6, Cry12, Cry13, Cry14, ΔRABM_007470 mutant and the wild-type strain. LC-TOF- and Cry21) are known to be toxic to larvae of some free-living or MS assay demonstrated that a component with molecular weight + parasitic nematodes (Bravo et al. 1998; Marroquin et al. 2000; of 1354 Da [M+H+H2O] was absent due to the disruption of Wei et al. 2003; Kotze et al. 2005). After ingestion of toxin by RABM_007470 gene, and this compound displayed a moderate target nematode larvae, the crystals dissolve within the gut of the nematicidal activity. To our knowledge, this is the first report on a nematode, followed by forming lytic pores in the cell membrane metabolic product and its encoded gene in B. amyloliquefaciens of gut epithelial cells and the subsequent proteolytic activation FZB42 that serves as a pathogenic factor against nematodes. Our (Crickmore 2005; Marroquin et al. 2000). For three strains in study represents an important step for understanding the mech- three different genera (Pseudomonas fluorescens CHA0, anism of nematicidal activity in biological control. Brevibacillus laterosporus, and Bacillus nematocida B16 strains), their mechanism of pathogenesis is to secrete virulent extracellular which are targeting on cuticle or digestive Materials and methods tract, and biocontrol plant parasitic nematodes Meloidogyne incognita and Bursaphelenchus xylophilus (Niu et al. 2006, Bacterial strains and growth conditions 2007; Huang et al. 2005). The human pathogens Burkholderia pseudomallei, Serratia marcescens, Enterococcus faecalis, B. amyloliquefaciens and Escherichia coli strains were grown Streptococcus pyogenes,andStaphylococcus aureus kill nema- at 37 °C in Luria–Bertani broth (LB) medium solidified with todes via secreting a neuromuscular endotoxin, a cytolysin, and 2 % agar, supplemented when necessary with the appropriate two extracellular proteases (O'Quinn et al. 2001;Kurzand (ampicillin at 100 mg/ml, kanamycin at 25 mg/ml, Ewbank 2000;Garsinetal.2001;Qinetal.2000; Sifri et al. and erythromycin at 1 mg/ml). A medium for producing the 2002). 1354 Da compound contained 40 g soy peptone, 40 g dextrin,

Although great achievements have been made to under- 1.8 g KH2PO4, 4.5 g K2HPO4, 0.3 g MgSO4⋅7H2O, and stand the mechanisms underlying bacterial pathogenesis 0.2 ml KellyT trace metal solution (25 mg EDTA [ethylene- against nematodes, there are only few reports on metabolites diamine-tetra-acetic acid] disodium salt dihydrate, 0.5 g as well as genes related to their biosynthesis that contribute to ZnSO4⋅7H2O,3.67gCaCl2⋅2H2O,1.25gMnCl2⋅4H2O, their virulence. Bacillus amyloliquefaciens FZB42 is a Gram- 0.25 g CoCl2⋅6H2O, 0.25 g ammonium molybdate, 2.5 g positive bacterium and is distinguished from the model organ- FeSO4⋅7H2O,and0.1gCuSO4⋅5H2O, 500 ml H2O) per liter ism Bacillus subtilis by its abilities to stimulate plant growth (Scholz et al. 2011). and antagonize plant root pathogens such as bacteria, fungi, The bacterial strains and plasmid used in this study are and even root-knot nematodes. A variety of secondary metab- listed in Table 1, and the polymerase chain reaction (PCR) olites produced by B. amyloliquefaciens FZB42 have been primers are listed in Table 2. suggested to be involved in its impressive ability to control plant pathogens and to stimulate plant growth. In B. amyloliquefaciens Transposon library FZB42, more than 340 kb, corresponding to 8.5 % of its total genetic capacity, are devoted to non-ribosomal synthesis of The mariner based transposon TnYLB-1 plasmid was used to secondary metabolites including antibacterial polyketides generate a transposon library according to Haldenwang (Le (bacillaene, difficidin, and macrolactin), lipopeptides (surfactin, Breton et al. 2006;Dieteletal.2013). Plasmids pMarA and fengycin, and bacillomycin D), siderophores (bacillibactin; Chen pMarB differ in the promoters that drive the expression of the Appl Microbiol Biotechnol (2013) 97:10081–10090 10083

Table 1 Bacterial strains and plasmid used in this study

Strain or plasmid Description Source or reference

Strains Bacillus amyloliquefaciens FZB42 Wild-type, deposited as strain 10A6 in the culture collection Bacillus Genetic Stock Center (BGSC), of the Bacillus Genetic Stock Center Columbus, OH, USA F5 Insertion of pMarA in FZB42:RBAM_007470::kan This work ΔBAM_007470 FZB42 RBAM_007470::erm (pNC),does not produce PZN; This work Escherichia coli OP50 Uracil auxotroph Escherichia coli B Caenorhabditis Genetics Center (CGC), Saint Paul, MN, USA DH5α hsdR17 recA1 gyrA endA1 relA1 Takara Bio, Dalian, China Plasmids PMUTinNC Present from Dr.Shu Ishikawa of Nara University, Nara, Japan pMD18-T, pMD19-T Apr lacZ′ Takara PMarA Plasmid containing mariner transposon TnYLB-1 Breton et al. (2006) pNC-007470 pMUTinNC with 464-bp of RBAM_007470 This work

Himar1 transposase gene. pMarA has Himar1 under the tran- electrophoresis to verify that the transformants contained the scriptional control of housekeeping σ factor σA of B. subtilis, correct plasmid. For inducing transposition, isolated clones while pMarB B uses general stress response σ factor σB for were grown overnight in liquid LB medium at 37 °C, and then transposase expression. Plasmid pMarC has no transposase portions of each culture were plated on either LB, LB and Kan gene as well as its promoter and is used as a control (Le (5 mg/L), or and Erm (1 mg/L) and incubated at non-permissive Breton et al. 2006). In brief, plasmids pMarA, pMarB, and temperature for plasmid replication (48 °C) to select for pMarC were transformed into B. amyloliquefaciens FZB42. transposants as described previously (Le Breton et al. 2006). Competent cells of B. amyloliquefaciens were obtained by modifying the two-step protocol (Kunst and Rapoport 1995) Bioassay of nematicidal activity according to Idris et al. (2007). Transformants were screened for plasmid-associated properties, i.e. Kanr and Ermr at permis- Culturing and the synchronization of nematodes were sive temperature for plasmid replication (30 °C) and Kanr and performed according to the modified methods from previous Erms at the restrictive temperature (48 °C). To verify that these reports (Brenner 1974; Lewis and Fleming 1995). Briefly, after transformants contained the original intact plasmid, the plasmid an E. coli OP50, overnight culture was seeded on nematode was extracted from the transformants and transformed into growth medium (NGM) agar plates [50 mM NaCl, 0.25 % (w/v)

E. coli DH5α. Next, plasmid DNA was extracted from E. coli peptone, 1 mM CaCl2,5μg/ml cholesterol, 25 mM KH2PO4, DH5α and subjected to restriction endonuclease analysis with 1mMMgSO4, and 1.7 % (w/v) agar], the Bristol N2 strain of C. EcoRI. The restriction was then analyzed through agarose gel elegans (the present from Kunming Animal Research Institute

Table 2 Primers used in this study

Primer name Sequencea (5′–3′) Function and source oIPCRf GCTTGTAAATTCTATCATAATTG IPCR, from Breton et al. (2006) oIPCRv AGGGAATCATTTGAAGGTTGG IPCR, from Breton et al. (2006) Hp-pTf AAGCTTCGCGATGTAGATGACGTTTG RBAM_007470 homologous amplification Hp-pTv GGATCCCCGCAAGCAAGACCATTACT RBAM_007470 homologous amplification Erm UP AACGACGAAACTGGCTAA erm gene amplification, this study Erm DW GGAACATCTGTGGTATGGC erm gene amplification, this study RBAM_0074700 gene UP ATCGATGATGAAGGTCAAGCCAGTC PCR amplifying the gene knock-out of RBAM_007470 RBAM_007470 gene DW GGCGCCCGAAGCCTCAAACCAGTT PCR amplifying the gene knock-out of RBAM_007470 a The nucleotides underlined represent sequences recognized by restriction 10084 Appl Microbiol Biotechnol (2013) 97:10081–10090 of Chinese Academy of Sciences) were cultivated for 2–3days ESI-TOF-MS at 20 °C on the bacterial lawn. When enough worm eggs (20– 50/cm2) were observed by optical microscopy, they were After the tested bacterial strains were grown in the production washed using 4 ml sterile M9 medium (5.8 g Na2HPO4⋅7H2O, medium containing 1.5 % agar at 37 °C for 24 h, the bacterial 3gKH2PO4,5gNaCl,0.25gMgSO4⋅7H2O) from a NGM agar lawn from four growth plates were collected into a 50 ml plate, followed by frequent washing in M9 medium to reduce centrifuge tube with 30 ml mixture of 70 % acetonitrile and bacterial concentration. After removing the liquid supernatant, 30 % water containing the final concentration of 0.1 % formic an alkaline hypochlorite treatment was performed to isolate the acid. After thorough vortexing and then centrifugation at eggs with 1–1.5 ml bleach lysis solution (5 M NaOH 0.3 ml, 8,000 rpm for 20 min, the supernatant was removed and purified

8%NaClO1.2ml,ddH2O 3.5 ml). The eggs were washed with a 0.45 μm filter and successively dried by a rotary evapo- againwithM9mediumandtheeggswerethenincubatedin4– rator. The samples were dissolved in 2 ml mixture of 90 % 5mlM9mediumat22°Cfor18htogetlarvalstage1(L1) formic acid and 10 % acetonitrile for electrospray ionization nematodes. These L1 nematodes were then transferred to fresh time-of-flight mass spectrometry (ESI-TOF-MS) analysis. NGM agar plates containing E. coli OP50 and incubated at ESI-TOF-MS analysis was applied in LC/TOF-MS with ESI 22 °C for 2 to 3 days until L4 stage. These L4 stage nematodes operating in positive mode, and achieved using an HPLC system were used for bioassays. (an autosampler, and a binary pump; Agilent Series 1100, Agilent In our bioassays, both the slow killing assay and liquid fast Technologies, Santa Clara, CA). The following operation param- killing assay were employed (Garsin et al. 2001;Kurzetal. eters were used in our analysis: mobile phase was composed of 2003; Garvis et al. 2009). In the slow killing assay, 40–60 L4 solvent A (60 % sterile water), solvent B (20 % acetonitril) and C. elegans worms were added to a NGM plates containing a solvent C (20 % isopropyl alcohol) at a constant flow of 100 μl/ lawn of bacteria to be tested. The nematodes were incubated at min; capillary voltage 3000 V; nebulizer pressure 40 psig; drying 25 °C and examined every 24 h for 3–5 days. In the liquid fast gas flow 7 L/min; drying gas temperature 300 °C; fragmentor killing assay, the bacterial strains to be tested were grown voltage 210 V; skimmer voltage 60 V. LC/TOF-MS accurate overnight at 37 °C with 200 rpm shaking in 3 ml liquid assay mass spectra were recorded across the range 50–3100 m/z. media (4.0 g NaCl, 2.5 g peptone, 5.0 g tryptone, 2.5 g yeast Data processing was carried out with Applied Biosystems/ extract, 5 mg cholesterol, 7.5 ml glycerol, dH2Oto1liter).Then MDS-SCIEX Analyst QS software (Frankfurt, Germany) with each 100 μl bacterial culture was diluted with M9 medium to accurate mass application-specific additions from Agilent MSD 600 μl and transferred into 16 well plates. Each well was seeded TOF software (Valverde et al. 2010; Arraez-Roman et al. 2010). with 40–60 L4 stage hermaphrodite N2 nematodes and the infection assay was performed at 25 °C for 24 h. Bioassay of nematocidal activity to surface extraction In these bioassays, the nematodes were considered dead when no movement was observed under a dissecting micro- The extraction of metabolites from the cell surface was pre- scope and when the gentle tapping of nematodes by a needle pared using the same method described above, except that the did not result in any response. Mortalities of nematodes were dried cell surface extraction was dissolved in methanol yield- defined as the ratio of dead nematodes over the tested nema- ing the concentration of 40 mg/ml. Then 20 μlliquorprepared todes. All experiments were performed in triplicates and were above was mixed with 380 μl sterile water and added into 16- repeated at least three times. well plates, achieving the end concentration of cell surface extraction as 2 mg/ml. After seeding 40–60 L4 stage hermaph- IPCR to identify of the candidate virulence genes rodite N2 nematodes in each well, the assay was incubated at 25 °C for 24 h. The mixture containing 5 % methanol and Inverse PCR (IPCR) was performed according to standard 95 % sterile water was used as negative control. procedures as follows (Le Breton et al. 2006). Genomic DNA samples isolated from mutants derived from transposon mutagenesis were first digested with the Taq I and Result then the digested linear DNAs were circularized in a ligation reaction using T4 ligase (Takara, Dalian, China) at a DNA Screening the transposon library for mutants attenuated concentration of 5 ng/μl. IPCR was performed with the tem- in virulence plate of 100 ng ligated DNA using the primers of oIPCR1 and oIPCR2, which face outward from the transposon sequence. Before screening the transposon-insertion mutagenesis library of IPCR products were purified using a recovery kit (Biotek, B. amyloliquefaciens FZB42 to identify mutants with attenuated Beijing, China), and the products were then linked ligated to nematicidal activities, auxotrophic mutants were first identified the plasmid pMD-18 T (TakaRa) and sequenced with the and these mutants were excluded from further analyses. This pMD-18 T universal primer. exclusion was necessary because auxotrophy alone could Appl Microbiol Biotechnol (2013) 97:10081–10090 10085 contribute to their decreased virulence against nematodes. After the amplicons were sequenced, we selected the DNA fragments the initial screening 400 mutants in the library, only 75 % of the that contained the partial sequence of Tc1/himar1 mariner random mutants were then assayed for their nematicidal activi- transposon vector at the two termini. This was because the ties. Finally, our assay identified 46 mutants that showed de- TnYLB-1 plasmid transposes by a ‘cut-and-paste’ mechanism creased virulence compared to the parental strain. Among them, (Le Breton et al. 2006;Fig.2b). Our analyses revealed that three F5 mutant strain was one of the candidates. In the liquid fast genes in the F5 mutant were disrupted by the transposon: killing assay, less than 10 % mortality was obtained within 4 h in RBAM_007470, yhdY,andprkA. RAMB_007470 was located F5 mutant when 31.3±4.7 % nematodes were killed by the in a cluster of 12 genes, about 10 kb in length, and that this parent strain FZB42. If the infection time was extended to 16 h, cluster encoded a docking/scaffolding protein in the protein 53.2±2.6 % mortality was obtained in F5 mutant treatment while complex for the biosynthesis of a TOMM compound (Garsin less than 10 % nematodes survived in the treatment of wild-type et al. 2001;Fig.2a). Two other genes (yhdY and prkA)were FZB42 (Fig. 1a). In the slow killing assay, nematodes treated also disrupted in F5 and these two genes respectively encoded with the F5 mutant had a 10 % higher survival than the wild-type an uncharacterized MscS family protein that likely functioned strain throughout the whole assay period. Additionally, F5 mu- in the transmembrane transport and a protein kinase (Earl tant needed at least one additional day to kill 50 % of the exposed et al. 2012; Fischer et al. 1996). nematodes in this assay (Fig. 1b). Thus, the above results dem- onstrated that the transposon-insertion mutant F5 contained mu- Disruption of RBAM_007470 gene caused attenuated tations that negatively affected its nematicidal activity. nematicidal activity

Identification of candidate genes in F5 mutant To determine which of the three transposon-disrupted genes resulted in the loss of virulence within the F5, we employed the Taking Taq I-digested genomic DNA as the template, IPCR integration vector pMUTinNC to construct suicide plasmids with a pair of primers oIPCR1 and oIPCR2 (Le Breton et al. containing internal DNA fragments of the three genes to mutate 2006), which faced outward from the transposon sequence, was each of the three genes separately. An internal DNA fragment of performed to identify the gene(s) disrupted in F5 mutant. After RBAM_007470 was PCR amplified. The amplicon of 464 bp was linked into pMD18-T, cleaved with HindIII and BamHI, cloned into pMUTinNC, and yielded the recombinant plasmid pNC-007470 for the gene locus-directed mutagenesis. The plasmid construct was introduced into B. amyloliquefaciens FZB42 strains by chemotransformation. Successful transforma- tion and chromosomal integration event of the plasmid into the RBAM_007470 gene were confirmed by PCR through ampli- fying the knock-out of our target gene RBAM_007470 as well as the knock-in of anti-erythromycin gene. The other two locus- directed mutagenesis of yhdY and prkA were constructed with the similar method (data not shown). After bioassaying the three specific mutants and then com- paring their nematicidal activities to the parent wild-type strain FZB42 as well as to mutant F5, we found that the disruption of gene RBAM_007470 led to a similar reduction of nematicidal activity as that of mutant F5. In contrast, no reduction in nematocidal activity was observed for mutations at the two other genes yhdY and prkA. In the liquid fast killing assay, mostnematodeswerekilledwithin4handallweredeadwithin 16 h after co-incubation with the wild-type strain FZB42. In contrast, the ΔRBAM_007470 mutant killed only 50.6±3.3 % Fig. 1 Nematicidal activity of the wild-type strain B. amyloliquefaciens nematodes after 16 h and it took 28 h to kill all the nematodes FZB42, F5 mutant from the transposon-insert library, and the locus-specific Δ ΔRBAM_007470 mutant. a. Liquid fast killing assays using the three (Fig. 1a). In the slow killing assay, the RBAM_007470 mutant bacterial strains. 100 μl bacterial culture was diluted with M9 medium to required at least one additional day to kill 50 % of exposed 600 μl and transferred into 16 well plates, and 40–60 L4 stage C. elegans nematodes, similar to that of the F5 mutant. While all the tested worms were added to be tested at 25 °C. b. Slow killing assays using the wormswerekilledafter168hby the wild-type strain FZB42, three bacterial strains. Similarly, 40–60 L4 C. elegans worms were added to be tested. The Y axis represents the percentage of nematode survival, and about 20.0±4.2 % of the nematodes survived the treatment of the X axis represents the time (in hours) after tests began ΔRBAM_007470 at that time (Fig. 1b). 10086 Appl Microbiol Biotechnol (2013) 97:10081–10090

Fig. 2 Alignments of nucleotide sequences of the target gene amplified from IPCR. Sequence alignment of the disrupted gene showed 99 % identity to the gene RBAM_007470, which encoded a docking/scaffolding protein, in a cluster involved in synthesizing plantazolicin (a) and the two flanking sequences from the product of IPCR showed 99 % identity to the partial sequence from Tc1/himar1 mariner transposon vector Appl Microbiol Biotechnol (2013) 97:10081–10090 10087

Fig. 3 ESI-TOF-MS detecting metabolites from organic extracts of B. anm/zrangeof900to1600Da.(b) The corresponding metabolite amyloliquefaciens FZB42 and ΔRBAM_007470 strains. (a)ESI- cpd1354 [M+H]+ could not be detected by ESI-TOF-MS in the TOF-MS of cpd1354 [M+H]+ from B. amyloliquefaciens FZB 42 in ΔRBAM_007470 strain

The metabolite plantazolicin serves as a virulence factor elimination of plantazolicin, which subsequently led to the attenuated nematicidal activity in B. amyloliquefaciens FZB42. The gene cluster containing the RABM_007470 gene has been In order to identify differences in their metabolites, the found essential for the biosynthesis of plantazolicin (Scholz surface extract from B. amyloliquefaciens FZB42 and the et al. 2011). Within this cluster, gene RABM_007470 was ΔRABM_007470 mutant were assayed by ESI-TOF-MS, re- + proposed to function as a docking/scaffolding protein in the spectively. A compound ([M+H+H2O] ) with the molecular protein complex consisting of a cyclodehydratase (C), a dehy- mass of 1354 Da was detected in the wild-type strain but drogenase (B), and a docking/scaffolding protein (D). We hy- absent in the ΔRBAM_007470 mutant (Fig. 3). The features pothesize that disruption of the RABM_007470 gene led to the of this compound are consistent with those of plantazolicin

Fig. 4 Nematicidal activity of surface extracts of B. amyloliquefaciens respectively. 5 % methanol and 95 % sterile water was used as negative FZB42 and ΔRBAM_007470 strains. 40–60 L4 stage C. elegans worms control. The Y axis represents the percentage of nematode survival, and and 2 mg/ml cell surface extraction were seeded in 16 well plates at 25 °C, the X axis represents the time (in hours) after tests began 10088 Appl Microbiol Biotechnol (2013) 97:10081–10090 previously reported (Scholz et al. 2011). The results suggest B, have been recently elucidated (Kalyon et al. 2011; Molohon that the gene ΔRABM_007470 as well as the whole gene et al. 2011). Due to their genetic and chemical structure conser- cluster is responsible for the biosynthesis of plantazolicin. vation, plantazolicin is classified into the TOMM group of To further confirm if the decrease of nematicidal activity metabolites (Haft et al. 2010). Plantazolicin A and its desmethyl resulted from the absence of plantazolicin, we determined the analogue plantazolicin B represent an unusual type of thiazole/ virulence of extracellular metabolites from both the wild-type oxazole-containing peptide antibiotics (TOMM). A unique struc- strain and the ΔRABM_007470 mutant against nematodes. Our tural feature of plantazolicin A is the adjacency of two results showed that there are no obvious differences in nema- pentaheterocyclic moieties that mainly confer a planar structure tode mortalities within the initial 2 h. Up to 24 h, extracellular to the peptide and are reminiscent of telomerase inhibitor metabolites from the ΔRABM_007470 mutant showed 10- telomestatin (Shin-ya et al. 2001). In the plantazolicin synthesis 20 % lower virulence than those of the wild-type strain process, the trimeric “BCD” complex functions in two distinct (Fig. 4). After 24 h, the nematicidal activities decreased dra- chemical transformations. The first is catalyzed by the matically in both the wild-type strain and the ΔRABM_007470 cyclodehydratase (C), which converts Cys and Ser/Thr residues mutant, probably due to the lower concentration or activity loss into the corresponding thiazoline and (methyl) oxazoline with of surface metabolites. Thus, our results here demonstrated that loss of water from the amide backbone. In the second, the the metabolic products containing the compound plantazolicin dehydrogenase (B) removes two electrons and two protons to had higher nematicidal activity, suggesting a potential virulence afford the aromatic thiazole and (methyl) oxazole (Li et al. 1996; factor in B. amyloliquefaciens FZB42. Milne et al. 1999). The docking scaffold protein (D) appears to play a role in trimer assembly and the regulation of enzymatic activity. This mechanism is similarly utilized in the biosynthetic Discussion pathways for streptolysin S (Datta et al. 2005), and microcin B17 (Li et al. 1996). Our previous analysis revealed that disruption of Defined mutant libraries have allowed efficient genome-scale one of the members of the BCD protein complex leads to a screening and provided a convenient collection of mutations for complete loss of plantazolicin production in FZB42 (Scholz et al almost any nonessential gene of interest (Cameron et al. 2008). 2011). Here, we corroborated that finding specifically for PznD: On the other hand, the genome sequence and associated anno- transposon insertion into gene RBAM_007470 (pznD)ledtoa tation information have also facilitated high-throughput gener- complete loss in plantazolicin biosynthesis. ation of comprehensive mutant libraries. Thus, this approach At present, the functions of this type of natural products have has been successfully used to identify virulence-related genes in not been completely elucidated yet. Streptolysin S secreted by various pathogens against nematodes such as Pseudomonas the human pathogen S. pyogenes is a highly cytolytic toxin and aeruginosa and B. subtilis (Xia et al. 2011). Similar to the contributes to the hemolytic phenotype of this bacterium. two successful cases, here we identified the nematotoxic factors Plantazolicin from B. amyloliquefaciens FZB42 has been shown and obtained insights into the pathogenesis mechanisms of B. as a narrow spectrum antibacterial compound (Molohon et al amyloliquefacien FZB42 using random transposon-insertion 2011). It can inhibit the growth of closely related Gram-positive mutagenesis, in which nearly all of the ORFs in the FZB42 bacteria, e.g. Bacillus anthracis, but showed no activity against genome have been disrupted at least once. Gram-negative bacteria. However, those activities were only Excluding the influences of mutation on impaired growth and observed when high concentrations of the metabolite have been viability, we found that disruption of the gene RBMA_007470 in applied. The data presented here explain the activity to B. amyloliquefaciens FZB42 decreased its nematicidal activity in antagonise root-knot nematodes that had been observed previ- both the random transposon-insertion mutant F5 and the locus- ously (Burkett-Cadena et al. 2008) and suggests a novel role of directed ΔRABM_007470 mutant. Though the other two genes plantazolicin in killing root-knot nematodes. Such an activity yhdY and prkA were also disrupted in the F5 mutant, subsequent could lead to its development as an agent for biological control. experiments for the two locus-directed mutagenesis strain showed Our study is the first report to demonstrate that plantazolicin similar nematicidal activities to the wild type, suggesting no produced by B. amyloliquefaciens FZB42 contributes to its obvious contribution of these two genes to nematotoxic activity. nematicidal activity. The detailed mode of action of plantazolicin RBAM_007470 (pznD) is part of a 12-gene cluster (pzn against nematodes awaits further investigation, but its structural cluster) covering about 10 kb and involved in synthesis of similarity to telomerase inhibitor telomestatin might serve as a plantazolicin (PZN). It has been reported that this gene cluster first starting point for further studies. is coding for the prepropeptide PznA, and the trimeric PznBCD protein complex (cyclodehydratase [C], dehydrogenase [B] and Acknowledgments We thank Prof. Jianping Xu, from McMaster Uni- docking/scaffolding protein [D]) encoding posttranslational versity in Canada, for his constructive criticism for this manuscript. This work is supported by the National Basic Research Program of China modification (Scholz et al. 2011). The structures of the (grant nos. 2011AA10A203 and 2013CB127500), the National Natural ribosomally synthesized peptide antibiotics, plantazolicin A and Science Foundation Program of China (grant nos. 30970065 and Appl Microbiol Biotechnol (2013) 97:10081–10090 10089

U1036602), and the Department of Science and Technology of Yunnan automated liquid screen reveals a specialized role for the chemotaxis Province (grant no. 2010GA012). gene cheB2 in Pseudomonas aeruginosa virulence. PLoS Pathog 5(8):1–13 Haft DH, Basu MK, Mitchell DA (2010) Expansion of ribosomally produced natural products: a nitrile hydratase- and Nif11-related References precursor family. BMC Biol 8:70 Huang XW, Tian BY, Niu QH, Yang JK, Zhang LM, Zhang KQ (2005) An extracellular from Brevibacillus laterosporus G4 with- Åhman J (2000) Extracellular serine proteases as virulence factors in out parasporal crystal can serve as a pathogenic factor in infection of nematophagous fungi: molecular characterization and functional nematodes. Res Microbiol 156:719–727 analysis of the PII protease in Arthrobotrys oligospora. PhD thesis, Idris ESE, Iglesias DJ, Talon M, Borriss R (2007) Tryptophan-dependent University of Lund, Lund, Sweden production of indole-3-acetic acid (IAA) affects level of plant Arraez-Roman D, Fu SQ, Sawalha SMS, Segura-Carretero A, Fernández- growth promotion by Bacillus amyloliquefaciens FZB42. Mol Gutiérrez A (2010) HPLC/CE-ESI-TOF-MS methods for the char- Plant Microbe Interact 20:619–626 acterization of polyphenols in almond-skin extracts. Electrophoresis Kalyon B, Helaly SE, Scholz R, Nachtigall J, Vater J, Borriss R, 31:2289–2296 Süssmuth RD (2011) Plantazolicin A and B: structure elucidation Atibalentja N, Noel GR, Domier LL (2000) Phylogenetic position of the of ribosomally synthesized thiazole/oxazole from Bacillus North American isolates of Pasteuria that parasitizes the soybean amyloliquefaciens FZB42. Org Lett 13:2996–2999 cyst nematodes, Heterodera glycines, as inferred from 16S rDNA Kotze AC, O'Grady J, Gough JM, Pearson R, Bagnall NH, Kemp DH, sequence analysis. Int J Syst Evol Microbiol 50:605–613 Akhurst RJ (2005) Toxicity of Bacillus thuringiensis to parasitic and Bravo A, Sarabia S, Lopez L, Ontiveros H, Abarca C, Ortiz A, Ortiz M, Lina free-living life stages of nematodes parasites of livestock. Int J Parasitol L, Villalobos FJ, Peña G, Nuñez-Valdez ME, Soberón M, Quintero R 35:1013–1022 (1998) Characterization of cry genes in a Mexican Bacillus Kunst F, Rapoport G (1995) Salt stress is an environmental signal thuringiensis strain collection. Appl Environ Microbiol 64:4965–4972 affecting degradative enzyme synthesis in Bacillus subtilis.J Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71–94 Bacteriol 177:2403–2407 Burkett-Cadena M, Kokalis-Burelle N, Lawrence KS, van Santen E, Kurz CL, Ewbank JJ (2000) Caenorhabditis elegans for the study of Kloepper JW (2008) Suppressiveness of root-knot nematodes me- host–pathogen interactions. Trends Microbiol 8:142–144 diated by rhizobacteria. Biol Control 47:55–59 Kurz CL, Chauvet S, Andres E, Aurouze M, Vallet I, Michel GPF, Uh M, Cameron DE, Urbach JM, Mekalanos JJ (2008) A defined transposon Celli J, Filloux A, Bentzmann SD, Steinmetz I, Hoffmann JA, Finlay mutant library and its use in identifying motility genes in Vibrio BB, Gorvel JP, Ferrandon D, Ewbank JJ (2003) Virulence factors of cholera. Proc Natl Acad Sci U S A 105:8736–8741 the human opportunistic pathogen Serratia marcescens identified by Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, in vivo screening. EMBO J 22:1451–1460 Heinemeyer I, Morgenstern B, Voss B, Hess WR, Reva O, Junge Le Breton YL, Mohapatra NP, Haldenwang WG (2006) In vivo random H, Voigt B, Jungblut PR, Vater J, Süssmuth R, Liesegang H, mutagenesis of Bacillus subtilis by sse of TnYLB-1, a mariner-based Strittmatter A, Gottschalk G, Borriss R (2007) Comparative analysis transposon. Appl Environ Microbiol 72:327–333 of the complete genome sequence of the plant growth-promoting Lewis JA, Fleming JT (1995) Basic culture methods. In: Epstein HF and bacterium Bacillus amyloliquefaciens FZB42. Nat Biotechnol 25: Shakes DC (eds) Caenorhabditis elegans: modern biological anal- 1007–1014 ysis of an organism. Academic, San Diego, CA, pp 3–29 Crickmore N (2005) Using worms to better understand how Bacillus Li YM, Milne JC, Madison LL, Kolter R, Walsh CT (1996) From peptide thuringiensis kills insects. Trends Microbiol 13:347–350 precursors to oxazole and thiazole-containing peptide antibiotics: Datta V, Myskowski SM, Kwinn LA, Chiem DN, Varki N, Kansal RG, microcin B17 synthase. Science 274:1188–1193 Kotb M, Nizet V (2005) Mutational analysis of the group A strep- Marroquin LD, Elyassnia D, Griffitts JS, Feitelson JS, Aroian RV (2000) tococcal operon encoding streptolysin S and its virulence role in Bacillus thuringiensis (Bt) toxin susceptibility and isolation of invasive infection. Mol Microbiol 56:681–695 resistance mutants in the nematode Caenorhabditis elegans. Dietel K, Beator B, Budiharjo A, Fan B, Borriss R (2013) Bacterial traits Genetics 155:1693–1699 involved in colonization of Arabidopsis thaliana roots by Bacillus Milne JC, Roy RS, Eliot AC, Kelleher NL, Wokhlu A, Nickels B, Walsh amyloliquefaciens FZB42. Plant Pathol J 29:59–66 CT (1999) Cofactor requirements and reconstitution of microcin Duncon LW (1991) Current options for nematode management. Annu B17 synthetase: a multienzyme complex that catalyzes the forma- Rev Phytopathol 29:469–490 tion of oxazoles and thiazoles in the microcin B17. Earl AM, Eppinger M, Fricke WF, Rosovitz MJ, Rasko DA, Daugherty S, Biochemistry 38:4768–4781 Losick R, Kolter R, Ravel J (2012) Whole-genome sequences of Molohon K, Melby JO, Lee J, Evans BS, Dunbar KL, Bumpus SB, Bacillus subtilis and close relatives. J Bacteriol 194:2378–2379 Kelleher NL, Mitchell DA (2011) Structure determination and in- Ebert D, Rainey P, Embley TM, Scholz D (1996) Development, life terception of biosynthetic intermediates for the plantazolicin cycle, ultrastructure and phylogenetic position of Pasteuria class of highly discriminating antibiotics. ACS Chem Biol ranosa Metchnikoff 1888: rediscovery of an obligate endopara- 16:1307–1313 site of Daphnia magna Straus. Philos Trans R Soc Lond B 351: Niu QH, Huang XW, Zhang L, Yang JK, Zhang KQ (2006) A neutral 1689–1701 protease from Bacillus nematocida, another potential virulence Fischer C, Geourjon C, Bourson C, Deutscher J (1996) Cloning and factor in the infection against nematodes. Arch Microbiol 185: characterization of the Bacillus subtilis prkA gene encoding a novel 439–448 serine protein kinase. Gene 168:55–60 Niu QH, Huang XW, Zhang L, Lian LH, Li YX, Li J, Yang JK, Zhang Garsin DA, Sifri CD, Mylonakis E, Qin X, Singh KV, Murray BE, KQ (2007) Functional identification of the gene bace16 from Calderwood SB, Ausubel FM (2001) A simple model host for identi- nematophagous bacterium Bacillus nematocida. Appl Microbiol fying Gram-positive virulence factors. Proc Natl Acad Sci U S A 98: Biotechnol 75:141–148 10892–10897 O'Quinn AL, Wiegand EM, Jeddeloh JA (2001) Burkholderia Garvis S, Munder A, Ball G, Bentzmann SD, Wiehlmann L, Ewbank JJ, pseudomallei kills the nematode Caenorhabditis elegans using an Tümmler B, Filloux A (2009) Caenorhabditis elegans semi- endotoxin-mediated paralysis. Cell Microbiol 3:381–393 10090 Appl Microbiol Biotechnol (2013) 97:10081–10090

Qin X, Singh KV, Weinstock GM, Murray BE (2000) Effects of Tian BY, Yang JK, Zhang KQ (2007) Bacteria used in the biological Enterococcus faecalis fsr genes on production of gelatinase and a control of plant-parasitic nematodes: populations, mechanisms of serine protease and virulence. Infect Immun 68:2579–2586 action, and future prospects. FEMS Microbiol Ecol 61:197–213 Schneider SM, Rosskopf EN, Leesch JG, Chellemi DO, Bull CT, Tikhonov VE, Lopez-Llorca LV, Salinas J, Jansson HB (2002) Purification Mazzola M (2003) Research on alternatives to methyl bromide: and characterization of chitinases from the nematophagous fungi pre-plant and post-harvest. Pest Manag Sci 59:814–826 Verticillium chlamydosporium and V. suchlasporium. Fungal Genet Scholz R, Molohon KJ, Nachtigall J, Vater J, Markley AL, Süssmuth RD, Biol 35:67–78 Mitchell DA, Borriss R (2011) Plantazolicin, a novel microcin B17/ Valverde A, Aguilera A, Ferrer CA, Camacho F, Cammarano A (2010) streptolysin S-like from Bacillus amyloliquefaciens Analysis of forchlorfenuron in vegetables by LC/TOF-MS after FZB42. J Bacteriol 193:215–224 extraction with the buffered QuEChERS method. J Agric Food Shin-ya K, Wierzba K, Matsuo K, Ohtani T, Yamada Y, Furihata K, Chem 58:2818–2823 Hayakawa Y, Seto H (2001) Telomestatin, a novel telomerase inhib- Wei JZ, Hale K, Carta L, Platzer E, Wong C, Fang SC, Aroian RV (2003) itor from Streptomyces anulatus. J Am Chem Soc 131:1262–1263 Bacillus thuringiensis crystal proteins that target nematodes. Proc Sifri CD, Mylonakis E, Singh KV, Qin X, Garsin DA, Murray BE, Ausubel Natl Acad Sci U S A 100:2760–2765 FM, Calderwood SB (2002) Virulence effect of Enterococcus faecalis Xia YF, Xie SS, Ma X, Wu HJ, Wang X, Gao XW (2011) The purL gene protease genes and the quorum-sensing locus fsr in Caenorhabditis of Bacillus subtilus is associated with nematicidal activity. FEMS elegans and mice. Infect Immun 70:5647–5650 Microbiol Lett 322:99–107