Appl Microbiol Biotechnol (2013) 97:10081–10090 DOI 10.1007/s00253-013-5247-5 APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY The highly modified microcin peptide 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 biosynthesis 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 mass spectrometry 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, Corynebacterium, 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 proteases 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 antibiotics (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