Cloning, sequencing, and characterization of the genetic region relevant to biosynthesis of the lipopeptides iturin A and surfactin in Bacillus subtilis

Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea. A 3642-bp genomic region of B. subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B. subtilis 168 was cloned, sequenced, and characterized. Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B. subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B. subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system. The genetic regions between the srfD gene and lpa gene from B. subtilis B3 and B. subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B. subtilis 168 and B. subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins. There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.     

Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8

Bacillus subtilis YB8 was found to produce the lipopeptide antibiotics surfactin and plipastatin B1. A gene, lpa-8, required for the production of both lipopeptides was cloned from strain YB8. When this gene was inactivated in strain YB8, neither surfactin nor plipastatin B1 was produced. However, the defective strain transformed with an intact lpa-8 gene had restored ability to produce both peptides. Nucleotide sequence analysis of the region essential for the production of the peptides revealed the presence of a large open reading frame. The deduced amino acid sequence of lpa-8 (224 amino acid residues) showed sequence similarity to that of sfp (from surfactin-producing B. subtilis), lpa-14 (from iturin A- and surfactin-producing B. subtilis), psf-1 (from surfactin-producing Bacillus pumilus), gsp (from gramicidin-S-producing Bacillus brevis), and entD (from siderophore-enterobactin-producing Escherichia coli), which are able to complement a defect in the sfp gene and promote production of the lipopeptide antibiotic surfactin. The sequence similarity among these proteins and the product similarity of cyclic peptides suggests that they might be involved in the biosynthesis or secretion of the peptides. Received: 14 July 1995 / Accepted: 22 December 1995     

Effect of pps disruption and constitutive expression of srfA on surfactin productivity,spreading and antagonistic properties of Bacillus subtilis 168 derivatives

Aims: To analyse the effects of plipastatin operon disruption and constitutive expression of surfactin operon in Bacillus subtilis 168 on surfactin productivity, in vitro invasive growth and antagonism against fungi. Methods and Results: The srfA native promoter was replaced by the constitutive promoter P_repU in B. subtilis 168 after integration of a functional sfp gene. Moreover, the plipastatin synthesis was further disrupted in the B. subtilis 168 derivatives. In liquid media, an earlier and higher expression of P_repU, than that found with P_srfA, led to a specific surfactin production fivefold higher after 6 h of culture. On solid media, not only the invasive growth and the haemolytic activity but also the antifungal activity of the constitutive strains were improved when compared to the parental strain BBG111. As expected, the disruption of the plipastatin operon strongly reduced in vitro antifungal properties but, interestingly, enhanced specific surfactin production (1·47 g g^?1 of biomass), spreading behaviour and haemolytic activity of the strains. Conclusions: This work demonstrates for the first time the interdependency of surfactin and plipastatin regarding their biosynthesis as well as their influence on the biological activities of the producing strain. Significance and Impact of the Study: The constitutive overproduction of surfactin enhances the invasive growth and the in vitro antagonistic activity of the mutant strain. Both properties are known to play an important role in the biocontrol of plant diseases. Plipastatin operon disruption increases the surfactin productivity of mutant strains. These mutants are interesting for use in continuous bioprocesses for surfactin production or in bioremediation.     

Isolation of new variants of surfactin by a recombinant Bacillus subtilis

A recombinant Bacillus subtilis MI113(pC115), carrying a gene responsible for the production of surfactin and iturin A cloned from B. subtilis RB14C, produced new surfactin variants, in addition to the already reported surfactin, when MI113(pC115) was cultured in solid-state fermentation of soybean curd residue (okara) as a substrate. All variants isolated by HPLC were characterized. Received: 18 December 1996 / Received revision: 20 February 1997 / Accepted: 28 February 1997     

Characterization of the surfactin synthetase isolated from the Bacillus subtilis strain producing iturin

Summary The lipopeptides, surfactin and iturin, are co-produced by B. subtilis. In this work, the three subunits of surfactin synthetase have been characterized by affinity chromatography on affigel columns where the ligand is one of the amino acid components of surfactin.     

Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42

The environmental strain Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses plant pathogenic organisms present in the rhizosphere. We sampled sequenced the genome of FZB42 and identified 2,947 genes with >50% identity on the amino acid level to the corresponding genes of Bacillus subtilis 168. Six large gene clusters encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) occupied 7.5% of the whole genome. Two of the PKS and one of the NRPS encoding gene clusters were unique insertions in the FZB42 genome and are not present in B. subtilis 168. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis revealed expression of the antibiotic lipopeptide products surfactin, fengycin, and bacillomycin D. The fengycin (fen) and the surfactin (srf) operons were organized and located as in B. subtilis 168. A large 37.2-kb antibiotic DNA island containing the bmy gene cluster was attributed to the biosynthesis of bacillomycin D. The bmy island was found inserted close to the fen operon. The responsibility of the bmy, fen, and srf gene clusters for the production of the corresponding secondary metabolites was demonstrated by cassette mutagenesis, which led to the loss of the ability to produce these peptides. Although these single mutants still largely retained their ability to control fungal spread, a double mutant lacking both bacillomycin D and fengycin was heavily impaired in its ability to inhibit growth of phytopathogenic fungi, suggesting that both lipopeptides act in a synergistic manner.     

Chromosomal integration of sfp gene in Bacillus subtilis to enhance bioavailability of hydrophobic liquids

Bacillus subtilis C9 effectively degrades aliphatic hydrocarbons up to a chain length of C₁₉ and produces a lipopeptide-type biosurfactant, surfactin, yet it has no genetic competency. Therefore, to obtain a transformable surfactin producer, the sfp gene cloned from B. subtilis C9 was integrated into the chromosome of B. subtilis 168, a non-surfactin producer, by homologous recombination. The transformants reduced the surface tension of the culture broth from 70.0 mN/m to 28.0 mN/m, plus the surface-active compound produced by the transformants exhibited the same R_f value as that from B. subtilis C9 and authentic surfactin in a thin-layer chromatographic analysis. The integration of the sfp gene into the chromosome of B. subtilis 168 was confirmed by Southern hybridization. Like B. subtilis C9, the transformants readily degraded n-hexadecane, although the original strain did not. It was also statistically confirmed that the hydrocarbon degradation of the transformants was highly correlated to their surfactin production by the determination of the correlation coefficient (r²=0.997, P<0.01). Therefore, these results indicate that the surfactin produced from B. subtilis enhances the bioavailability of hydrophobic liquids.     

Molecular and Biochemical Approaches for Characterization of Antifungal Trait of a Potent Biocontrol Agent Bacillus subtilis RP24

Bacillus subtilis strain RP24, isolated from rhizoplane of field grown pigeon pea, exhibited in vitro antagonism against a wide range of phytopathogenic fungi. An attempt was made to partially purify and characterize the diffusible antifungal metabolite/s produced by the strain RP24 and its negative mutant (NM) in potato dextrose medium. High performance liquid chromatography (HPLC) of partially purified extract of RP24 showed the presence of lipopeptide antibiotic iturin as a major peak that was comparable to that of standard iturin A (5.230 min) from Sigma–Aldrich whereas the corresponding peak was absent in extract of NM. The structure was further confirmed by liquid chromatographic mass spectrometric (LCMS) analysis as iturin A. LCMS analysis also showed the presence of surfactin and fengycin besides iturin A. Amplification of the lpa-14 (encodes the 4′-phosphopantetheinyl transferase required for the maturation of template enzyme of iturin A) and ituD (encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production) genes of iturin operon of strain RP24 was carried out and the sequences obtained were compared with the existing database of NCBI. The sequences of lpa-14 and ituD gene of RP24 showed 98% and 97% homology with lpa-14 and ituD genes of B. subtilis in the existing database. The results indicated that strain RP24 harbors iturin operon in its genome and a chemical mutation in this operon might have resulted in loss of antifungal activity in the negative mutant.     

Biosynthesis of iturin and surfactin byBacillus subtilis: Evidence for amino acid activating enzymes

Summary Enzymes catalyzing ATP-PPi exchange reactions mediated by specific amino acids were found inB. subtilis lysates partially purified by gel permeation. The nature of these amino acids varied according to the stage of cell growth: activation of surfactin amino acids was observed during surfactin synthesis; activation of iturin amino acids was not detected at the begining of surfactin synthesis but appeared during iturin synthesis.     

Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.     

Cyclic lipopeptide profile of three <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains; antagonists of <Emphasis Type="Italic">Fusarium</Emphasis> head blight

The objective of the study was to identify the lipopetides associated with three Bacillus subtilis strains. The strains are antagonists of Gibberella zeae, and have been shown to be effective in reducing Fusarium head blight in wheat. The lipopeptide profile of three B. subtilis strains (AS43.3, AS43.4, and OH131.1) was determined using mass spectroscopy. Strains AS43.3 and AS43.4 produced the anti-fungal lipopeptides from the iturin and fengycin family during the stationary growth phase. All three strains produced the lipopeptide surfactin at different growth times. Strain OH131.1 only produced surfactin under these conditions. The antifungal activity of the culture supernatant and individual lipopeptides was determined by the inhibition of G. zeae. Cell-free supernatant from strains AS43.3 and AS43.4 demonstrated strong antibiosis of G. zeae, while strain OH131.1 had no antibiosis activity. These results suggest a different mechanism of antagonism for strain OH131.1, relative to AS43.3 and AS43.4.     

Cloning of <Emphasis Type="Italic">srfA</Emphasis> operon from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C9 and its expression in <Emphasis Type="Italic">E. coli</Emphasis>

The srfA operon is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. The srfA operon is composed of the four genes, srfAA, srfAB, srfAC, and srfAD, encoding the surfactin synthetase subunits, plus the sfp gene that encodes phosphopantetheinyl transferase. In the present study, 32 kb of the srfA operon was amplified from Bacillus subtilis C9 using a long and accurate PCR (LA-PCR), and ligated into a pIndigoBAC536 vector. The ligated plasmid was then transformed into Escherichia coli DH10B. The transformant ET2 showed positive signals to all the probes for each open reading frame (ORF) region of the srfA operon in southern hybridization, and a reduced surface tension in a culture broth. Even though the surface-active compound extracted from the E. coli transformant exhibited a different R _f value of 0.52 from B. subtilis C9 or authentic surfactin (R _f = 0.63) in a thin layer chromatography (TLC) analysis, the transformant exhibited a much higher surface-tension-reducing activity than the wild-type strain E. coli DH10B. Thus, it would appear that an intermediate metabolite of surfactin was expressed in the E. coli transformant harboring the srfA operon.     

Importance ofeps genes fromBacillus subtilis in biofilm formation and swarming

Unicellular organisms naturally form multicellular communities, differentiate into specialized cells, and synchronize their behaviour under certain conditions. Swarming, defined as a movement of a large mass of bacteria on solid surfaces, is recognized as a preliminary step in the formation of biofilms. The main aim of this work was to study the role of a group of genes involved in exopolysaccharide biosynthesis during pellicle formation and swarming inBacillus subtilis strain 168. To assess the role of particular proteins encoded by the group ofepsI-epsO genes that form theeps operon, we constructed a series of insertional mutants. The results obtained showed that mutations inepsJ-epsN, but not in the last gene of theeps operon (epsO), have a severe effect on pellicle formation under all tested conditions. Moreover, the inactivation of 5 out of the 6 genes analysed caused total inhibition of swarming in strain 168 (that does not produce surfactin) on LB medium. Following restoration of thesfp gene (required for production of surfactin, which is essential for swarming of the wild-type bacteria), thesfp ⁺ strains defective ineps genes (exceptepsO) generated significantly different patterns during swarming on synthetic B medium, as compared to the parental strain 168sfp ⁺.     

Rapid Detection and Characterization of Surfactin-Producing <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and Closely Related Species Based on PCR

The surfactin production genetic locus (sfp) is responsible for the ability of Bacillus subtilis to produce the lipopeptide biosurfactant, surfactin. This report demonstrates the utility of using PCR of the sfp gene as a means of identifying Bacillus species that produce surfactin. We carried out a hemolysis zone assay, quantitative HPLC and NMR in parallel to ensure that the PCR provided correct results. PCR analyses were performed for the sfp gene on 15 standard strains and 20 field-collected Bacillus spp. isolates native to Taiwan. Among the 15 standard strains, surfactin was produced by seven strains of B. subtilis and two closely related species, B. amyloliquefaciens B128 and B. circulans ATCC 4513. Of the 20 field-collected Bacillus spp. isolates; 16 strains yielded surfactin- positive results with PCR and HPLC. A good correlation was observed. Within the 16 field isolates, B. amyloliquefaciens S13 (452.5 mg/L) and B. subtilis S15 (125.6 mg/L) had high productivity of surfactin. The technique is valuable for finding out potential good yields of surfactin-producing strains. The PCR method we used could also be used to find different species or genera containing homologous genes. This is the first report of the detection of surfactin production by B. amyloliquefaciens and B. circulans based on PCR screening.     

Characterisation of antagonistic Bacillus and Pseudomonas strains for biocontrol potential and suppression of damping‐off and root rot diseases

Novel strains of rhizobacteria, Pseudomonas fluorescens Pf 9A‐14, Pseudomonas sp. Psp. 8D‐45 and Bacillus subtilis Bs 8B‐1, showed broad‐spectrum antagonistic activity and provided suppression of Pythium damping‐off and root rot of cucumber. Their biocontrol potential was further investigated for suppression of additional seedling diseases of cucumber (Phytophthora capsici) and radish (Rhizoctonia solani). Bacterial strains were also characterised for production of antibiotics, metabolites, volatiles, phytohormones and lytic enzymes. Seed and pre‐plant applications of all three antagonistic bacteria as cell suspension and talc or irradiated peat formulations to the infested potting mix provided overall high level of suppression of Phytophthora damping‐off and root rot of cucumber (66–85% healthy seedlings) and relatively low level of suppression of Rhizoctonia damping‐off of radish (18–38% healthy seedlings). Bacterial treatments also resulted in higher plant fresh masses. Seed coating with irradiated peat formulation of a mixture of three bacteria resulted in superior control of Phytophthora damping‐off and root rot of cucumber and much higher plant fresh masses. The presence of genes for biosynthesis of phenazine‐1‐carboxylic acid, 2,4‐diacetylphloroglucinol, pyrrolnitrin and pyoluteorin was confirmed in Pseudomonas strains, and that of fengycin, bacillomycin, bacilysin, surfactin and iturin A in B. subtilis Bs 8B‐1. All three strains produced HCN, salicylic acid, indole‐3‐acetic acid, protease and β‐1,3‐glucanase. Both Pseudomonas strains produced siderophores and only P. fluorescens Pf 9A‐14 showed phosphate solubilisation and chitinase activity. All three strains inhibited pathogen growth by producing volatiles, and gas chromatography–mass spectrometry analysis revealed eight compounds in Pf 9A‐14, 10 in Bs 8B‐1 and 4 in Psp 8D‐45, some with known antifungal activity. The antagonistic and plant‐growth promotion activities of these strains might be due to production of antibiotics, metabolites, lytic enzymes or phytohormones.     

The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties

The colonizing behaviour and the pellicle formation of Bacillus subtilis strains producing different families of lipopeptides were evaluated under several cultural conditions. The pattern of lipopeptides produced determined the architecture of the colony on a swarming medium as well as the flotation and the thickness of the pellicle formed at the air/liquid interface. The overproduction of mycosubtilin, a lipopeptide of the iturin family, led to increased spreading but had no effect on pellicle formation. A physico-chemical approach was developed to gain an insight into the mode of action of the biosurfactants facilitating the colonization. A relationship between surface tension of the culture medium and spreading of a lipopeptide non-producing strain, B. subtilis 168, was established. Goniometry was used to highlight the modification of the in situ wettability in the area where spreading was enhanced. On a solid medium, co-cultures of a surfactin producing with other strains showed a diffusion ring of the surfactin around the colony. This ring characterized by a higher wettability favoured the propagation of other colonies.     

Genome annotation and comparative genomic analysis of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> MJ01, a new bio-degradation strain isolated from oil-contaminated soil

One of the main challenges in elimination of oil contamination from polluted environments is improvement of biodegradation by highly efficient microorganisms. Bacillus subtilis MJ01 has been evaluated as a new resource for producing biosurfactant compounds. This bacterium, which produces surfactin, is able to enhance bio-accessibility to oil hydrocarbons in contaminated soils. The genome of B. subtilis MJ01 was sequenced and assembled by PacBio RS sequencing technology. One big contig with a length of 4,108,293 bp without any gap was assembled. Genome annotation and prediction of gene showed that MJ01 genome is very similar to B. subtilis spizizenii TU-B-10 (95% similarity). The comparison and analysis of orthologous genes carried out between B. subtilis MJ01, reference strain B. subtilis subsp. subtilis str. 168, and close relative spizizenii TU-B-10 by microscope platform and various bioinformatics tools. More than 88% of 4269 predicted coding sequences in MJ01 had at least one similar sequence in genome of reference strain and spizizenii TU-B-10. Despite this high similarity, some differences were detected among encoding sequences of non-ribosome protein and bacteriocins in MJ01 and spizizenii TU-B-10. MJ01 has unique nucleotide sequences and a novel predicted lasso-peptide bacteriocin; it also has not any similar nucleotide sequence in non-redundant nucleotide data base.     

Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis

The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilo-bases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.