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Makoto Shoda - One of the best experts on this subject based on the ideXlab platform.
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Horizontal Transfer of Iturin A Operon, itu, to Bacillus subtilis 168 and Conversion into an Iturin A Producer
Antimicrobial agents and chemotherapy, 2005Co-Authors: Kenji Tsuge, Takashi Ano, Satoka Inoue, Mitsuhiro Itaya, Makoto ShodaAbstract:Iturin A and its derivatives are lipopeptide antibiotics produced by Bacillus subtilis and several closely related bacteria. Three Iturin group operons (i.e., Iturin A, mycosubtilin, and bacillomycin D) of those antibiotic-producing strains have been cloned and sequenced thus far, strongly implying the horizontal transfer of these operons. To examine the nature of such horizontal transfer in terms of antibiotic production, a 42-kb region of the B. subtilis RB14 genome, which contains a complete 38-kb Iturin A operon, was transferred via competent cell transformation to the genome of a non-Iturin A producer, B. subtilis 168, using a method based on double-crossover homologous recombination with two short landing pad sequences (LPSs) in the genome. The recombinant was positively selected by confirming the elimination of the cI repressor gene, which was localized between the two LPSs and substituted by the transferred segment. The Iturin A operon-transferred strain 168 was then converted into an Iturin A producer by the introduction of an sfp gene, which encodes 4′-phosphopantetheinyl transferase and is mutated in strain 168. By inserting the pleiotropic regulator degQ, the productivity of Iturin A increased sevenfold and was restored to about half that of the donor strain RB14, without the transfer of additional genes, such as regulatory or self-resistance genes.
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Cloning, Sequencing, and Characterization of the Iturin A Operon
Journal of Bacteriology, 2001Co-Authors: Kenji Tsuge, Takanori Akiyama, Makoto ShodaAbstract:Many Bacillus subtilis strains produce a small peptide(s) with a long fatty moiety, the so-called lipopeptide antibiotics. The peptide portions of these compounds contain α-amino acids with a d configuration and are produced nonribosomally with templates of the multifunctional peptide synthetases. As in the synthesis of the peptide antibiotic gramicidin S, the peptide chain grows in a defined sequence by moving on the template of the multifunctional peptide synthetase (18, 35, 44). On the basis of the structural relationships, the lipopeptides that have been identified in B. subtilis are generally classified into three groups: the surfactin group (27), the plipastatin-fengycin group (16, 41, 42), and the Iturin group (17). The members of the surfactin and plipastatin-fengycin groups are composed of one β-hydroxy fatty acid and 7 and 10 α-amino acids, respectively, while the members of the Iturin group consist of one β-amino fatty acid and 7 α-amino acids. The presence of the β-amino fatty acid is the most striking characteristic of the Iturin A group and distinguishes this group from the other two groups. The operons that encode surfactin (3), plipastatin-fengycin (16, 37, 38, 40), and mycosubtilin (4), which is a member of the Iturin A group, have been sequenced and characterized. In particular, a study of the mycosubtilin operon of B. subtilis ATCC 6633 (4) showed that MycA, a novel template enzyme which has functional domain homology to β-ketoacyl synthetase and amino transferase and amino adenylation, was present, which implied that MycA is responsible for incorporation of the β-amino fatty acid. Recently, biological control agents for plant diseases have received considerable attention as alternatives to chemical pesticides (32). B. subtilis RB14, which has a suppressive effect against several phytopathogens, is expected to be used as a biocontrol agent (1, 5). We previously demonstrated that the biocontrol activity of RB14 can be attributed mainly to production of Iturin A (Fig. (Fig.1)1) (10, 26). Iturin A, as well as mycosubtilin, is a member of the Iturin group. The amino acid compositions of Iturin A and mycosubtilin are almost identical, except that the sixth and seventh amino acids are inverted, as shown in Fig. Fig.1.1. In our previous study, we cloned a gene, lpa-14, that encodes the 4′-phosphopantheteinyl transferase required for maturation of the template enzyme of Iturin A (6, 8, 15). For further investigation of the details of the synthesis steps and because of the interesting evolutionary relationship between Iturin A and mycosubtilin, cloning and sequencing of the complete Iturin A synthetase operon are essential. FIG. 1 Structures of Iturin A and mycosubtilin. R indicates an alkyl moiety (generally C14 to C17). The arrows represent peptide bonds in the -CO-NH- direction. The differences between the two lipopeptides are indicated by underlining. In this study, we examined the features of the Iturin A synthetase operon based on the nucleotide sequence and gene disruption. By comparing the Iturin A operon with the mycosubtilin operon, we found that the difference between the two operons may be a result of intragenic swapping of amino acid adenylation domains. We also obtained genetic evidence of probable horizontal transfer of the Iturin A operon. In addition, a promoter replacement experiment whose goal was construction of a Iturin A hyperproducer is also described below.
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cloning sequencing and characterization of the Iturin a operon
Journal of Bacteriology, 2001Co-Authors: Kenji Tsuge, Takanori Akiyama, Makoto ShodaAbstract:Bacillus subtilis RB14 is a producer of the antifungal lipopeptide Iturin A. Using a transposon, we identified and cloned the Iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The Iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in Iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as Iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser-->L-Asn, while in Iturin A these amino acids are inverted (i.e., D-Asn-->L-Ser). Comparison of the amino acid sequences encoded by the Iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the Iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the Iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of Iturin A increased threefold.
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Effect of temperature on production of lipopeptide antibiotics, Iturin A and surfactin by a dual producer, Bacillus subtilis RB14, in solid-state fermentation
Journal of Fermentation and Bioengineering, 1995Co-Authors: Akihiro Ohno, Takashi Ano, Makoto ShodaAbstract:Abstract Temperature dependency of the production of Iturin A and surfactin by a dual producer, Bacillus subtilis RB14, in the solid-state fermentation of okara was investigated. The optimal temperature for Iturin A was 25°C, while that for surfactin was 37°C, in spite of their dependency on a common gene, lpa-14 . When the effect of temperature on the relative ratios of the amount of the five homologues of Iturin A to the total Iturin A produced by RB14 was investigated, only the ratios of homologues having β-amino acid residues with normal aliphatic chains were affected and the ratio of the homologue with longer normal (C 16 -) chain increased as the temperature was increased.
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Production of the antifungal peptide antibiotic, Iturin by Bacillus subtilis NB22 in solid state fermentation
Journal of Fermentation and Bioengineering, 1993Co-Authors: Akihiro Ohno, Takashi Ano, Makoto ShodaAbstract:Abstract Production of Iturin, an antifungal peptide effective at suppressing phytopathogens, by Bacillus subtilis NB22 was investigated in solid state fermentation (SSF) using soy bean curd residue ( okara ). In scale-up from 15 g to 3 kg, aeration, temperature, and moisture content were controlling factors for the efficient production of Iturin. It was found that solid state fermentation was 6–8 times more efficient with respect to Iturin productivity than submerged fermentation on the basis of unit wet weight. Higher productivity in selective production of specific components of Iturin which are stronger inhibitors of plant pathogens was also confirmed in SSF.
Takashi Ano - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of Yield and Surface Tension-lowering Activity of Iturin A Produced by Bacillus subtilis RB14
Journal of oleo science, 2019Co-Authors: Hiroshi Habe, Toshiaki Taira, Yuya Sato, Tomohiro Imura, Takashi AnoAbstract:Bacillus subtilis RB14 produces the lipopeptide antibiotic Iturin A by submerged and biofilm fermentation. In this study, we optimized the conditions for Iturin A production in a jar fermentor. The maximum yield of Iturin A was 932 mg L-1 after 120 h. The surface tension of water decreased from 72.0 to 39.0 mN m-1 as the concentrations of C14 Iturin A increased, indicating that C14 Iturin A behaves as a surfactant in water. The critical micellar concentration obtained from the intersection of two fitted lines was 1.2 × 10-4 M. Moreover, the surface tension of water decreased as the length of the alkyl chain of Iturin A increased.
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Components of rice husk biochar in promoting the growth, sporulation and Iturin A production of Bacillus sp. strain IA.
Zeitschrift fur Naturforschung. C Journal of biosciences, 2019Co-Authors: Shohei Ebe, Tatsuya Ohike, Masahiro Okanami, Takashi AnoAbstract:In a previously study, the Bacillus sp. strain IA was successfully isolated with high sensitivity to rice husk biochar (RHB). Moreover, RHB promoted an antibiotic Iturin A production by strain IA. In order to develop the biocontrol agent, we attempted to reveal the functions of the RHB in promoting the production of Iturin A by strain IA. The promotion effects of growth, sporulation and Iturin A production of strain IA by the RHB were explained as follows. First, the manganese ion, released from RHB, promoted the sporulation and Iturin A production of strain IA. Second, the silicon dioxide contained in RHB adsorbed the metabolic inhibitor(s) and promoted the Iturin A production of strain IA. Finally, the combination of manganese ion and silicon dioxide promoted the growth, sporulation and Iturin A production of the Bacillus sp. strain IA. To culture strain IA in the medium combining manganese ion and silicon dioxide, the total cells, spore cells and Iturin A production increased 15 times, 10,000 times and 18 times higher than the control medium, respectively.
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Study of Submerged and Biofilm Fermentation of Bacillus subtilis using Fish Protein for Production of Lipopeptide Antibiotic Iturin A
Science and Technology Against Microbial Pathogens, 2011Co-Authors: Umme Salma Zohora, Masahiro Okanami, Takashi Ano, Abdul Wahab Khan, Mohammad Shahedur RahmanAbstract:Iturin A is an environmentally safe biocontrol agent produced by Bacillus subtilis as a secondary metabolite. Generally Iturin A is produced in conventional submerged fermentation. Recently, B. subtilis has received a huge interest for its nature to develop into biofilm as it shows significantly independent genetic and morphological development in biofilm compared to its planktonic culture. In this study it was attempted to compare the production of Iturin A in submerged with that in biofilm fermentation using novel marine fish protein as a medium component. When fish protein was compared with commercially available peptones, it was observed that the microbial growth and Iturin A productions were similar to those in the medium containing Polypepton S (originated from soybean) and higher than those in the medium containing Polypepton (originated from casein). Quicker cellular growth and secondary metabolite production was observed in submerged fermentation whereas slower but higher cellular growth and Iturin A production was found in biofilm fermentation.
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Production of Iturin A homologues under different culture conditions.
Journal of Environmental Sciences, 2009Co-Authors: Noriyasu Iwase, Mohammad Shahedur Rahman, Takashi AnoAbstract:Iturin A is a cyclic lipopeptide antibiotic and eight different kinds of Iturin A have been reported based on its alkyl side chains. As Iturin A is a promising biocontrol agent, total production of Iturin A was tried to enhance and comparative production of its homologues was investigated by using different nitrogen and carbon sources. When Polypepton S and defatted soybean meal were used, total production as well as the ratio of the Iturin A homologues were similar. However, production of Iturin A was relatively lower and also the ratio of the Iturin A homologues was different when Polypepton was used, where A2 was decreased and A4 was increased. Production ratio of the Iturin A homologues was similar for the carbon sources like maltose, mannitol, sucrose and starch but relative production of Iturin A2 was much enhanced compared to A3 when lactose or galactose was used. Interestingly production ratio of A4 was increased and A2 and A3 were decreased when no additional carbon source was used, and similar tendency was observed in the homologue ratio with glucose and fructose. Production of Iturin A homologue A6 was significantly increased whereas A2 and A3 were decreased when defatted rapeseed cake was used. Utilization of different amino acids did not show significant differences in their production of the Iturin A homologues. Oxygen supply found to be the factor affecting the production of Iturin A homologues when it was investigated in a varied culture volume size and shaking speed. A2 found to be increased with increased oxygen supply where the production of A3 was affected inversely.
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Biofilm formation and lipopeptide antibiotic Iturin A production in different peptone media.
Journal of environmental sciences (China), 2009Co-Authors: Umme Salma Zohora, Mohammad Shahedur Rahman, Takashi AnoAbstract:Biofilm fermentation is a newly developed promising technique in fermentation technology. In this study no.3 and no.3S media have been used for the lipopeptide antibiotic Iturin A production by Bacillus subtilis RB14. The main component of no.3 and no.3S media is Polypepton and Polypepton S, respectively. B. subtilis RB14 produces thick stable biofilm and high amount of Iturin A in no.3S medium. Whereas, impaired biofilm formation and lower Iturin A production was observed in no.3 medium. From the analytical information it was observed that the amounts of metal ions, such as K(+), Ca(2+) and Mn(2+), cysteine and cellulose are lower in Polypepton compared to the Polypepton S. To investigate their effect on biofilm formation and Iturin A production cysteine, cellulose, K(+), Ca(2+) and Mn(2+) were added respectively into the no.3 medium at similar amount that Polypepton S contains. It was observed that individual addition of K(+), Ca(2+), cysteine and cellulose had no effect on biofilm formation, cellular growth induction or Iturin A production. However, when Mn(2+) was supplemented in no.3 medium, biofilm development was restored with an improved production of Iturin A. Finally, combined addition of investigated substances into the no.3 medium resulted with highly folded, thick biofilm with high cellular growth and Iturin A production compared to the original no.3 medium.
Georges Michel - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Iturin synthetase in the wild-type Bacillus subtilis strain producing Iturin and in an Iturin deficient mutant
FEMS microbiology letters, 1996Co-Authors: Christine Feignier, Françoise Besson, Georges MichelAbstract:The multi-enzyme system responsible for the biosynthesis of Iturin, an antifungal lipopeptide of Bacillus subtilis, was partially purified by chromatography on different affigels. In the wild-type strain, two subunits of the Iturin synthetase (ITs and ITagp) were characterized: ITs activated only l-Ser, one of the Iturin amino acid components, and ITagp activated l-Asn, d-Asn, l-Gln and l-Pro, amino acids corresponding to a partial sequence of Iturin. In an Iturin deficient mutant, the activity of the ITagp subunit was modified.
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Effect of the lipopeptide antibiotic, Iturin A, on morphology and membrane ultrastructure of yeast cells
FEMS microbiology letters, 1995Co-Authors: Laurence Thimon, Françoise Peypoux, Jean Wallach, Georges MichelAbstract:The effects of Iturin A, at fungicidal concentrations, on yeast cells were studied by scanning electron microscopy and by transmission electron microscopy. A depression, observed in each Iturin A-treated cell, was the consequence of the release of electrolytes and other cytoplasmic components. Iturin A passes through the cell wall and disrupts the plasma membrane with the formation of small vesicles and the aggregation of intramembranous particles. Moreover, Iturin A passes through the plasma membrane and interacts with the nuclear membrane and probably with membranes of other cytoplasmic organelles.
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Research letterStudies on lipopeptide biosynthesis by Bacillus subtilis: Isolation and characterization of Iturin−, surfactin+ mutants
Fems Microbiology Letters, 1995Co-Authors: Christine Feignier, Françoise Besson, Georges MichelAbstract:Two mutant strains, M35 and M89, were obtained by UV irradiation from a wild-type Bacillus subtilis producing Iturin and surfactin. Sporulation and surfactin production were similar in both mutants and in the parent strain, while the Iturin production of M35 was 300-fold less than that of the wild-type strain; M89 did not produce any Iturin. The analysis of the incorporation of sodium [1-14C]acetate into cellular lipids and lipopeptides showed that M89 still synthesized β-amino fatty acids, the lipid moiety of Iturin.
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studies on lipopeptide biosynthesis by bacillus subtilis isolation and characterization of Iturin surfactin mutants
Fems Microbiology Letters, 1995Co-Authors: Christine Feignier, Frana Oise Besson, Georges MichelAbstract:Two mutant strains, M35 and M89, were obtained by UV irradiation from a wild-type Bacillus subtilis producing Iturin and surfactin. Sporulation and surfactin production were similar in both mutants and in the parent strain, while the Iturin production of M35 was 300-fold less than that of the wild-type strain; M89 did not produce any Iturin. The analysis of the incorporation of sodium [1-14C]acetate into cellular lipids and lipopeptides showed that M89 still synthesized β-amino fatty acids, the lipid moiety of Iturin.
Marius Ptak - One of the best experts on this subject based on the ideXlab platform.
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Surfactin/Iturin A interactions may explain the synergistic effect of surfactin on the biological properties of Iturin A.
Biochimie, 1992Co-Authors: Régine Maget-dana, Françoise Peypoux, Laurence Thimon, Marius PtakAbstract:Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis. Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant. The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by Iturin A. This synergistic effect seems to be in relation with interactions between Iturin A and surfactin. Iturin A adsorbs to and penetrates into surfactin monolayers. Iturin A and surfactin are miscible and interact specifically in mixed monolayers.
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surfactin Iturin a interactions may explain the synergistic effect of surfactin on the biological properties of Iturin a
Biochimie, 1992Co-Authors: Regine Magetdana, Laurence Thimon, F Peypoux, Marius PtakAbstract:Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis. Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant. The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by Iturin A. This synergistic effect seems to be in relation with interactions between Iturin A and surfactin. Iturin A adsorbs to and penetrates into surfactin monolayers. Iturin A and surfactin are miscible and interact specifically in mixed monolayers.
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Interfacial properties of the antifungal Iturins on various electrolyte solutions
Journal of Colloid and Interface Science, 1992Co-Authors: Régine Maget-dana, Laurence Thimon, F Peypoux, Marius PtakAbstract:Abstract We have studied the interfacial behavior of antifungal Iturins (principally bacillomycin F, mycosubtilin, and Iturin A) in solution and spread as monomolecular films on aqueous phases containing various chloride salts. The presence of electrolytes lowers the interfacial concentration (surface excess) of Iturin solutions. The compression isotherm curves are not superimposable, and for all the compounds the stability of the monolayer is higher on KCl as attested by the transition pressure (for the liquid-expanded part) and by the collapse pressure values. The reduction of the molecular free energy of spreading ΔGs, that measures the affinity of the polar cycle of the lipopeptide for the aqueous phase components, is maximum on KCl in the case of mycosubtilin. The energy of compression ΔGc, that measures the intermolecular forces between the film forming molecules, is generally higher on KCl. The compression leads to more ordered structures when mycosubtilin and Iturin A are spread on KCl. When spread on CaCl2, Iturin A and bacillomycin F molecules can adopt only two kinds of arrangements, as shown by the convergence of the LE parts of their isotherms. These data are interpreted as interactions between Iturins and ions and, more precisely, as discriminating interactions between Iturins and the various cations with a special role for K+.
Marc Ongena - One of the best experts on this subject based on the ideXlab platform.
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impact of rhizosphere factors on cyclic lipopeptide signature from the plant beneficial strain bacillus amyloliquefaciens s499
FEMS Microbiology Ecology, 2012Co-Authors: Venant Nihorimbere, Philippe Thonart, Hélène Cawoy, Alexandre Seyer, Alain Brunelle, Marc OngenaAbstract:Cyclic lipopeptides (cLPs) of the surfactin, Iturin and fengycin families synthesized by plant-associated Bacilli represent an important class of antibiotics as they may be tightly involved in the protective effect of selected strains against phytopathogens. However, their production by Bacillus cells developing on roots under rhizosphere conditions is still poorly understood. In this work, we combined electrospray and imaging mass spectrometry-based approaches to determine the detailed pattern of surfactins, Iturins and fengycins produced in planta by Bacillus amyloliquefaciens S499. Very different production rates were observed for the three cLPs families. Whereas surfactin accumulated in significant amounts, much lower quantities of Iturins and fengycins were detected in the environment of colonized roots in comparison with laboratory medium. In addition, the surfactin pattern produced by strain S499 evolving on roots is enriched in homologues with long fatty acid chains (C15) compared with the chains typically secreted under in vitro conditions. Additional experiments revealed that lipopeptide production by root-associated S499 cells is qualitatively and quantitatively dictated by the specific nutritional context of the rhizosphere (exudates enriched in organic acids, oxygen limitation) but also by the formation of biofilm-related structures around root hairs. As surfactins, Iturins and fengycins retain specific functions and bioactivities, the biological relevance of their differential production observed in planta is discussed in the context of biocontrol of plant diseases.
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Impact of rhizosphere factors on cyclic lipopeptide signature from the plant beneficial strain Bacillus amyloliquefaciens S499.
FEMS Microbiology Ecology, 2011Co-Authors: Venant Nihorimbere, Philippe Thonart, Hélène Cawoy, Alexandre Seyer, Alain Brunelle, Marc OngenaAbstract:Cyclic lipopeptides of the surfactin, Iturin and fengycin families synthesized by plant-associated Bacilli represent an important class of antibiotics since they may be tightly involved in the protective effect of selected strains against phytopathogens. However, their production by Bacillus cells developing on roots under rhizosphere conditions is still poorly understood. In this work, we combined electrospray and imaging mass spectrometry-based approaches to determine the detailed pattern of surfactins, Iturins and fengycins produced in planta by B. amyloliquefaciens S499. Very different production rates were observed for the three cLP families. While surfactin accumulated in significant amounts, much lower quantities of Iturins and fengycins were detected in the environment of colonized roots in comparison with laboratory medium. In addition, the surfactin pattern produced by strain S499 evolving on roots is enriched in homologues with long fatty acid chains (C15) compared to the one typically secreted under in vitro conditions. Additional experiments revealed that lipopeptide production by root-associated S499 cells is qualitatively and quantitatively dictated by the specific nutritional context of the rhizosphere (exudates enriched in organic acids, oxygen limitation) but also by the formation of biofilm-related structures around root hairs. As surfactins, Iturins and fengycins retain specific functions and bioactivities, the biological relevance of their differential production observed in planta is discussed in the context of biocontrol of plant diseases.
