Nonribosomal Peptide Synthase Gene Clusters for LipopeptideBiosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions

Chuping Luo,a,b Xuehui Liu,c Huafei Zhou,a,b Xiaoyu Wang,a Zhiyi Chena,b

College of Plant Protection, Nanjing Agriculture University, Nanjing, Chinaa; Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, Chinab;Institute of Biophysics, Chinese Academy of Sciences, Beijing, Chinac

Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research hasshown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility,biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e.,surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genomeof B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsiblefor the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutantslacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. Wedemonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak anti-fungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the im-paired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fenresulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity,swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. Inconclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in anintricate way.

As alternatives to chemical pesticides, biological agents for con-trolling crop diseases have drawn more and more attention

from microbiologists and plant pathologists. Due to the produc-tion of abundant secondary metabolites with a broad spectrum ofantimicrobial activities, many Bacillus strains are widely used inthe biocontrol of crop diseases (1–5). One extensively used andintensively studied microorganism, Bacillus subtilis, has the po-tential to produce antimicrobial compounds. Among these com-pounds, cyclic lipopeptides (LPs) generated by nonribosomalpeptide synthases (NRPSs) have the well-recognized potential tobe used in biotechnology and biopharmaceutical applications be-cause of their antimicrobial and surfactant properties (6, 7). Threefamilies of Bacillus cyclic LPs—surfactins, iturins, and fengy-cins—are well described with regard to most of the mechanismsthat account for the biocontrol effect of different Bacillus strains todate (5, 8, 9).

Surfactins, iturins, and fengycins are synthesized by large mul-tienzyme complexes and show striking product microheterogene-ity due to variations in both length and branching of fatty acidchains and in amino acids of peptide sequences (6, 7). All thebiochemical and genetic results showed that these three families ofLPs are synthesized by modular organization of NRPSs and obeythe linear rule. It is not difficult to make novel LP analogues withcertain peptide substitutions by manipulation of existing LP syn-thetases, e.g., via subunit exchange, module exchange, and com-binatorial biosynthesis (10, 11). The prospect of creating numer-ous bioactive LPs by genetic engineering has stimulated the searchfor new lipopeptide synthetases from Bacillus strains.

With the development of genome mining strategies, a largenumber of new natural products with good antimicrobial activi-ties were identified from traditional biocontrol strains (12, 13).Therefore, the opportunities to find novel LPs and synthetases willbe largely enhanced with the number of Bacillus strain genomes

sequenced recently. B. subtilis 916 was isolated from paddy soil inJurong County, Jiangsu Province, China, in 1994. The genomesequence of B. subtilis 916 was recently analyzed, and four NRPSgene clusters were identified (14). In addition to the three conven-tional gene clusters, srf, bmy, and fen, B. subtilis 916 also containsa new gene cluster, loc, which is responsible for the biosynthesis ofa new family of LP called locillomycin. In comparison to otherBacillus strains, it is unusual for B. subtilis 916 to have the potentialto coproduce four families of LPs, since the other Bacillus strainswith good control efficiency usually coproduce 2 or 3 families ofLPs (6, 7, 15).

Recent research showed that the LPs were also involved in mul-ticellular behaviors in terms of swarming motility, biofilm forma-tion, and colony morphology (3, 5, 6, 16–19). B. subtilis strains canuse swimming- and sliding-type mechanisms to swarm on sur-faces. Swarming motility enables cells to spread on plant surfacesand is an important component of the initial development of mi-crobial biofilms (17, 20). Swarming motility and biofilm forma-

Received 6 September 2014 Accepted 22 October 2014

Accepted manuscript posted online 31 October 2014

Editor: R. M. Kelly

Address correspondence to Chuping Luo, luochuping@163.com, or Zhiyi Chen,chzy84390393@163.com.

Citation Luo C, Liu X, Zhou H, Wang X, Chen Z. 2015. Nonribosomal peptidesynthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 andtheir phenotypic functions. Appl Environ Microbiol 81:422– 431.doi:10.1128/AEM.02921-14.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02921-14.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.02921-14

422 aem.asm.org January 2015 Volume 81 Number 1Applied and Environmental Microbiology

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

tion are major adaptive colonization strategies of Bacillus strainsin new environments (17, 21). While many research results sug-gested that surfactins played a major role in influencing the mul-ticellular behaviors of B. subtilis (3, 5, 22), results concerning thefunctions of the other LPs remain inconclusive.

Here, we report the identification, molecular characterization,and functional domain analysis of the four NRPS gene clusters inthe genome of B. subtilis 916. We also report the construction of aseries of single, double, and triple mutants which are disrupted indifferent NRPS gene clusters. We demonstrated that the fourNRPS gene clusters are responsible for the productions of the fourfamilies of LPs—surfactins, bacillomycin Ls, fengycins, and locil-lomycins. Function investigation of the four families of LPsshowed that they are involved in the phenotypic features of B.subtilis 916 in an intricate way.

MATERIALS AND METHODSBacterial strains, plasmids, and media. The strains and plasmids used inthis study are listed in Table 1. Luria-Bertani (LB) broth medium (10g/liter tryptone, 5 g/liter yeast extract, 5 g/liter NaCl) was used for culti-vation of Escherichia coli and B. subtilis. When necessary, antibiotics wereadded at the following concentrations: ampicillin, 100 �g/ml; spectino-mycin, 100 �g/ml; erythromycin, 1 �g/ml; neomycin, 20 �g/ml; chlor-amphenicol, 5 �g/ml. MSgg medium was used for biofilm formation andcolony morphology and contained 100 mM morpholinepropanesulfonicacid (MOPS) (pH 7), 50 �g/ml tryptophan, 50 �g/ml phenylalanine, 2

mM MgCl2, 700 �M CaCl2, 50 �M FeCl3, 50 �M MnCl2, 2 �M thiamine,1 �M ZnCl2. The fungal strains were cultured at 28°C on potato dextroseagar (PDA) medium containing (per liter) 200 g of potato infusion, 20 g ofglucose, and 20 g of agar (pH 7.0).

Genome sequence, DNA analysis, and domain structure. The draftgenome of B. subtilis 916 was sequenced with Illumina/Solexa HiSeq 2000as reported previously (14). On the basis of the draft genome, the com-plete genome of B. subtilis 916 was sequenced using the Pacbio sequencingplatform. The genome bioinformatics analysis was performed by Shang-hai Hanyubiotech company. DNA analyses and translation were con-ducted with the Vector NTI 10 and DANMAN software packages. BLASTwith annotated domains of similar nonribosomal peptide synthetases(NRPSs) was used to detect conserved active-site motifs. The amino acidsequences of discrete Bacillus adenylation domains, thiolation or peptidylcarrier domains, and condensation domains were extracted from modu-lar Bacillus NPRSs and were used for BLASTP comparison in order todetect their closest orthologs.

Construction of mutants deficient in four families of LPs synthesis.B. subtilis 916 mutants were generated according to a modified protocoloriginally developed for B. subtilis 168 (23). The bmyD gene was disruptedby insertion of a neomycin cassette via double-crossover homologousrecombination, as reported previously (24), and the mutant BSBM wasselected. Disruption of the fenA gene was achieved by insertion of a chlor-amphenicol cassette. A PCR product of 729 bp, obtained with the primersFenAF (5=-TTTCTCGAGGTCTTGATGGTGCAGTCAGA-3=) and FenAR(5=-TTTGAATTCCTGGACCTGTTTGTCTTTGT-3=) (XhoI and EcoRIrestriction sites are underlined), was cloned into pSG1164, resulting inpSGFen. pSGFen was transformed into B. subtilis 916, and the mutantBSFM was selected. The locD gene was disrupted by insertion mutagenesiswith an erythromycin cassette derived from pMUTIN4. A PCR product of812 bp, obtained with the primers LocDF (5=-TTTAAGCTTTCAGGTACCAACGATGAACA-3=) and LocDR (5=-TTTGGATCCTTGTCCATTACAGCTACGGT-3=) (HindIII and BamHI restriction sites are underlined),was cloned into pMUTIN4, resulting in pMUTINLoc. pMUTINLoc wastransformed into B. subtilis 916, and the mutant BSLM was selected. ThesrfAA gene was disrupted by insertion mutagenesis with a spectinomycincassette derived from pDG1728 and pUC19. A PCR product of 1,182 bpcontaining the spectinomycin resistance gene and promoter was ob-tained with the primers SpecF (5=-TTTGGATCCCTGCAGCCCTGGCGAATG-3=) and SpecR (5=-TTTGAATTCAGATCCCCCTATGCAAGG-3=) (BamHI and EcoRI restriction sites are underlined) from pDG1728.After digestion with BamHI and EcoRI, the spectinomycin cassette wascloned into pUC19, resulting in pUCSC. A 908-bp PCR product from thesrfAA gene region was amplified with the primers SrfA-AF (5=-TTTAAGCTTACACAGATATCAGGCAAGC-3=) and SrfA-AR (5=-TTTGGATCCGTCCCATCGTTCCTTCACA-3=) (HindIII and BamHI restriction sitesare underlined) and inserted into pUCSC, yielding the vector pUCSCSrf.pUCSCSrf was transformed into B. subtilis 916, and the mutant BSSM wasselected. To obtain double and triple mutants, the vectors constructedabove were transformed into competent B. subtilis 916 cells one by one,and the mutants BSBFM (�bmyD::Nmr �fenA::Cmr), BSBLM (�bmyD::Nmr �locA::Emr), BSFLM (�fenA::Cmr �locA::Emr), and BSBFLM(�bmyD::Nmr �fenA::Cmr �locA::Emr) were selected. All the single, dou-ble, and triple disruptions of genes described above were demonstrated inthe resistant colonies by PCR with appropriate primers and by Southernhybridization (data not shown).

Isolation and purification of LPs and HPLC-MS analysis. Surfactins,bacillomycin Ls, and locillomycins were isolated by adding concentratedhydrochloric acid to the culture broth of BSFM after the biomass wasremoved by centrifugation. Precipitates were formed at pH 2.0 whichcould be collected, dried, and extracted with methanol (MeOH). Thesolvents were removed under reduced pressure and white solids werecollected after the solvents were removed. The white solids were dissolvedin methanol followed by charcoal treatment and passed through a 0.22-�m-pore-size filter. The MeOH extractions were added to a 3-fold vol-

TABLE 1 B. subtilis strains and plasmids used

Strain orplasmid Descriptiona

Source orreference

B. subtilis strains916 Producer of surfactin, fengycin,

bacillomycin L, and locillomycinCGMCC no.

0808BSBM �bmyD::Nmr; BGG105 Laboratory stockBSFM �fenA::Cmr This studyBSLM �locD::Emr This studyBSSM �srfA::Specr This studyBSBFM �bmyD::Nmr �fenA::Cmr This studyBSBLM �bmyD::Nmr �locD::Emr This studyBSFLM �fenA::Cmr �locD::Emr This studyBSBFLM �bmyD::Nmr �fenA::Cmr �locD::Emr This study

PlasmidspUC19 Cloning vector; Apr Laboratory stockpBEST501 pGEM4 carrying the PrepU promoter

and neo gene from pUB110, Nmr

BGSC

pBAC105 pUC19 carrying the PrepU promoterand the 750-bp and 800-bpfragments from the bmyD operon

Laboratory stock

pSG1164 Integrated vector; Ampr Cmr BGSCpSGFen pSG1164 carrying a 729-bp fragment

of fenAThis study

pMUTIN4 Integrated vector; Ampr Emr BGSCpMUTINLoc pMUTIN4 carrying a 812-bp

fragment of locDThis study

pDG1728 Integrated vector; Ampr SpeCr BGSCpUCSC pUC19 carrying the spectinomycin

cassette from pDG1728This study

pUCSCSrf pUCSC carrying a 908-bp fragmentof srfAA

This study

a Apr, ampicillin resistance; Emr, erythromycin resistance; Cmr, chloramphenicolresistance; Specr, spectinomycin resistance.

LP Biosynthesis and Phenotypic Features in B. subtilis

January 2015 Volume 81 Number 1 aem.asm.org 423Applied and Environmental Microbiology

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

ume of H2O and titrated to pH 7.0 with 5 M NaOH. The extracted impu-rities were added to an Agilent amino solid-phase extraction column andwashed with 50% (vol/vol) MeOH-H2O, 100% MeOH, 1% (vol/vol) for-mic acid-MeOH, and 2% (vol/vol) formic acid-MeOH step by step. The1% (vol/vol) formic acid-MeOH elutions were concentrated by nitrogendrying to partial purification of surfactin and bacillomycin. The 2% (vol/vol) formic acid-MeOH elutions were concentrated by nitrogen drying topartial purification of locillomycin. All the partially purified samples werethen loaded on an Agilent C18 solid-phase extraction column separatelyand washed with 40%/50% (vol/vol) MeOH-H2O for locillomycin, with50%/70% (vol/vol) MeOH-H2O for bacillomycin L, and with 75%/90%(vol/vol) MeOH-H2O for surfactin. The 50%, 70%, and 90% (vol/vol)MeOH-H2O elution products were concentrated by nitrogen drying topurify locillomycins, bacillomycin Ls, and surfactins, respectively. Fengy-cin purification was the same as surfactin purification except that it startedwith BSSM culture broth. The extraction and purification products wereanalyzed by high-performance liquid chromatography (HPLC) with anacetonitrile-water-trifluoroacetic acid solvent system (40:60:0.5 [vol/vol/vol] for bacillomycin Ls and locillomycins, 50:50:0.5 [vol/vol/vol forfengycins, and 80:20:0.5 [vol/vol/vol] for surfactins) with a 0.5-ml/minflow rate. Except for the locillomycins, which were monitored at 230 nm,the LPs (i.e., the surfactins, fengycins, and bacillomycins) were monitoredat 210 nm. All the samples were further analyzed by matrix-assisted laserdesorption ionization-time of flight mass spectrometry (MS) with an Agi-lent 1100 series HPLC-MS/MS system. When necessary, tandem MS spec-trometry was also performed, in which a precursor ion was mass selectedand the parent ion was divided into the daughter ions to give structurallysignificant product ions using an Agilent 1100 series LC-MS/MS system(Agilent Technologies).

Assay of the antifungal activities against Fusarium oxysporum. Theactivities of the wild-type and mutant strains against spore germination ofF. oxysporum were assessed as follows. Spores of F. oxysporum were dilutedto 106 ml�1 and inoculated by using 500 �l to flood LB medium plates.The excess liquid was removed, and the plates were allowed to dry undera laminar flow hood for 30 min. Portions (50 �l) of culture strains weredeposited in 7-mm-diameter wells created in the solidified medium usingsterile glass tubes. The plates were inoculated at 28°C, and inhibitionzones were measured after 1 to 3 days. To further determine the antifungalactivity of the four purified LPs, five doses of each family of LPs wereadded separately to three aliquots each containing 25 ml potato dextroseagar at 45°C, mixed rapidly, and poured into three separate plates. Afterthe agar had cooled, mycelial plugs were added to the plates in equalamounts. Samples consisting of buffer only served as controls, and all theplates were inoculated at 28°C. When the mycelial colony of the controlhad grown to almost fill the plate, the area of the colony was measured,and the inhibition of fungal growth on the other plates was determined bycalculating the percent reduction of the area of the mycelial colony.

Evaluation of hemolytic activities. To evaluate the hemolytic activi-ties of wild-type B. subtilis 916 and the mutants, strains were inoculatedonto the blood agar plates with 5% defibrinated sheep blood by streaking.Hemolytic activities were visualized by development of a clear haloaround the growth of the strains after incubation at 37°C for 1 to 4 days. Inall cases, three replicate plates were used for each strain, and the experi-ment was repeated once.

Evaluation of colony architecture, swarming motility, and biofilmformation. For colony architecture, 1 �l of starting culture was spottedonto the surface of an MSgg agar plate containing 20 �g/ml Congo redand 10 �g/ml Coomassie brilliant blue and incubated at 30°C for 48 h, aspreviously described (25). To evaluate swarming motility, 1 �l of an over-night culture was used to inoculate the center of LB plates containing 20�g/ml Congo red and 10 �g/ml Coomassie brilliant blue solidified with0.7% agar. The plates were incubated at 37°C and were evaluated forcolony spread over time. For pellicle (floating biofilm) formation analysis,each strain was grown in 4 ml of LB at 37°C overnight, and 100 �l of thisstarting culture was used to inoculate 4 ml of MSgg containing 20 �g/ml

Congo red and 10 �g/ml Coomassie brilliant blue in 12-well microtiterplates, which was then incubated without agitation at 37°C for 24 h.

Nucleotide sequence accession numbers. The draft genome sequenceand complete genome sequence of B. subtilis 916 and the new gene clusterfor biosynthesis of locillomycins described here have been submitted toGenBank under accession numbers AFSU00000000.1, CP009611, andKF866134, respectively.

RESULTSIdentification and quantification analysis of four families of LPsproduced by B. subtilis 916. The LPs of B. subtilis 916 were inves-tigated by HPLC-MS. Four groups of mass peaks were detected(see Fig. S1 in the supplemental material), and their mass numbersare summarized in Table 2. Families 1, 2, and 4 of the LPs wereidentified as surfactins, bacillomycin Ls, and fengycins (Fig. 1) bycomparing their mass data with those previously obtained by MSanalysis of the LPs of numerous Bacillus strains. The novel family3 of the LPs were identified as locillomycins by evaluation of thefragment spectra obtained from electrospray ionization (ESI)-MS/MS and nuclear magetic resonance (NMR) spectra (C. Luo, Z.Chen, J. Y. Guo, X. Liu, X. Wang, Y. Liu, and Y. Liu, U.S. patentapplication 14/190,817; C. Luo and Z. Chen, unpublished data). B.subtilis 916 produces C13 to C15 surfactins, C14 to C16 bacillomycinLs, and C15 to C17 fengycins. This pattern of LPs corresponds tothe metabolite spectra found for most surfactin-, bacillomycin-,and fengycin-producing Bacillus strains. Unexpectedly, B. subtilis916 also produces a novel family of LPs called locillomycins, whichare unique nonapeptides with fatty acid side chains of 13 to 15carbon atoms (Fig. 1).

Each purified LP was further analyzed by HPLC-MS, and dif-ferent peaks representing different isomers of LPs were obtained(see Fig. S5 to S8 in the supplemental material). The differentpeaks produced from each preparation were used to quantify thefour families of LPs produced by B. subtilis 916 in the agitatedErlenmeyer flasks for 3 days. The production of surfactins wasdetected at the first 8 h of growth and revealed the early synthesis

TABLE 2 Calculated mass values of M, M�H�, and M�Na� ionscorresponding to identified isoforms of surfactins, bacillomycin Ls,locillomycins, and fengycins in culture extracts from B. subtilis 916

Lipopeptide

Mass valuea

M M�H� M�Na�

Surfactin A (C13) 1,007.6 1,008.6* 1,030.6Surfactin B (C14) 1,021.7 1,022.7* 1,044.7*Surfactin C (C15) 1,035.7 1,036.7* 1,058.7*

Bacillomycin LA (C14) 1,020.5 1,021.5* 1,043.5*Bacillomycin LB (C15) 1,034.5 1,035.5* 1,057.5*Bacillomycin LC (C16) 1,048.5 1,049.5* 1,071.5

Locillomycin A (C13) 1,145.6 1,146.6* 1,168.6Locillomycin B (C14) 1,159.6 1,160.6* 1,182.6*Locillomycin C (C15) 1,173.6 1,174.6* 1,196.6

Fengycin A (C15/Ala-6) 1,448.8 1,449.8* 1,471.9Fengycin B (C16/Ala-6) 1,462.9 1,463.9* 1,485.9Fengycin C (C17/Ala-6) 1,476.9 1,477.9* 1,499.9Fengycin D (C16/Val-6) 1,490.9 1,491.9* 1,513.9Fengycin E (C17/Val-6) 1,504.9 1,505.9* 1,527.9a The data were compiled from whole cells grown on LB agar. Peaks presented in FigureS1 in the supplemental material are indicated with asterisks.

Luo et al.

424 aem.asm.org January 2015 Volume 81 Number 1Applied and Environmental Microbiology

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

of surfactins by B. subtilis 916. In contrast to surfactins, other threeLPs were not observed during the first 12 h of growth, which wasexpected since the biosynthesis of these LPs is known to occur onlyafter the exponential growth phase. The highest rates of produc-tion of surfactins and bacillomycin Ls were 22.8 mg/liter and 19.7mg/liter, respectively, at 36 h of growth. However, the highestlevels of production of fengycins and locillomycins were only 4.8mg/liter and 3.6 mg/liter, respectively (Table 3).

Organization of four nonribosomal peptide synthetase(NRPS) gene clusters in the B. subtilis 916 genome. The B. sub-tilis 916 genome contains four nonribosomal gene clusters, srf, fen,bmy, and loc, which are responsible for the synthesis of three well-known LPs (surfactin, fengycin, and bacillomycin L) and the newlocillomycin family, respectively (see Fig. S2 in the supplementalmaterial). While the gene clusters srf, fen, and bmy in B. subtilis 916locus are closely related to the corresponding gene clusters srf, fen,and bam in Bacillus amyloliquefaciens FZB42, respectively, the locgene cluster appears to be a single-site insertion relative to B. amy-loliquefaciens FZB42. Like the fen and bmy gene clusters, srf and locare also close to each other on the chromosome of B. subtilis 916.

In addition, the locus of the bmy gene cluster in B. subtilis 916 isthe same as that of the iturin A gene cluster in B. subtilis RB14 (26).The fen locus in B. subtilis 916 is related to pps in B. subtilis 168, fenin B. subtilis F29-3, and myc in B. subtilis ATCC 6633 (15). The factthat the gene clusters fen, pps, and myc occupy essentially the samelocus on the genomic DNA suggests that different NRPS geneclusters could be integrated into these loci as an insertion or as areplacement of existing NRPS gene clusters.

Interestingly, although the genes of the loc cluster have no ho-mologs in the B. amyloliquefaciens FZB42 genome, the nucleotidesequences outside loc are approximately 98% identical and con-tain two highly conserved genes, glmS and ybbR. Near the “inser-tion site” of loc in B. subtilis 168 there is a skf gene cluster which isresponsible for the biosynthesis of sporulation killing factor, andB. subtilis ATCC 6633 contains a rhi gene cluster which is respon-sible for the biosynthesis of an antifungal phosphonate oligopep-tide of rhizocticin (27). The fact that the loc, skf, and rhi geneclusters occupy the same locus on the genomic DNA may be dueto the highly conserved genes glmS and ybbR, which can be easilyswapped among different Bacillus strains through homologousrecombination. In general, the locillomycin, sporulation killingfactor, and rhizocticin in B. subtilis 916, B. subtilis 168, and B.subtilis ATCC 6633 have been found in identical loci, and it isfurther suggested that either the NRPS or polyketide synthase(PKS) gene cluster is also interchangeable among different B. sub-tilis strains.

Schematic representation and functional domain analysis ofthe four nonribosomal peptide synthetases (NRPSs). Geneticand biochemical analyses have revealed that the arrangement ofthe modules of most LP synthetases is linear with amino acidsequences of LPs (6, 7, 28). As expected, the modular organizationof NRPSs involved in biosynthesis of surfactins, bacillomycin Ls,and fengycins in B. subtilis 916 is similar to that of their counter-parts in other Bacillus strains, and all the NRPSs obey the linearrule (Fig. 2; also, see Fig. S11 and Table S1 in the supplementalmaterial). That is to say that the order and specificity of the mod-ules within proteins encoded by srf, bmy, and fen in B. subtilis 916determine the amino acid sequence of the surfactins, bacillomycinLs, and fengycins, respectively (see Fig. S3 and Table S1 in thesupplemental material). In particular, the bmy gene cluster re-sponsible for bacillomycin L biosynthesis in B. subtilis 916 is verysimilar to the bam gene cluster responsible for bacillomycin Dbiosynthesis in B. amyloliquefaciens FZB42. Although the aminoacid sequences encoded by bmy share high similarity (�97%) withtheir counterparts encoded by bam, a low-similarity (�60%) re-gion in bmy and bam was found. Further detailed bioinformationanalysis showed that this region encoded adenylation domainswhich were responsible for activation of Ser-4 for bacillomycin L

FIG 1 Structures of four families of LPs—surfactins, bacillomycin Ls, locillo-mycins, and fengycins. Amino acid residues are in the three-letter code. The Lor D configuration is indicated with a subscript before the amino acid residuedesignation. The fatty acid moieties are shaded, and the number of carbonatoms is given. The surfactin family has a polar molecule structure, which ischaracterized by a core cyclic peptide with 7 amino acid residues and an exo-cyclic acyl group consisting of a �-hydroxyl fatty acid moiety with 13 to 15carbon atoms. The bacillomycin L family is characterized by a chemical struc-ture comprising of a core cyclic peptide with 7 amino acid residues and anexocyclic acyl group consisting of a �-amino fatty acid moiety with 14 to 17carbon atoms. The novel locillomycin family is characterized by a chemicalstructure comprising a core cyclic peptide with 9 amino acid residues and a�-hydroxyl fatty acid moiety with 13 to 15 carbon atoms. The fengycin familyis characterized by a chemical structure comprising a core cyclic peptide with10 amino acid residues and an exocyclic acyl group consisting of either asaturated or unsaturated �-hydroxyl fatty acid moiety with 16 to 17 carbonatoms.

TABLE 3 Production of four families of LPs—surfactins, bacillomycin Ls, locillomycins, and fengycins— by B. subtilis 916 grown in LB mediuma

LPs

LP production (mg liter�1)

8 h 12 h 24 h 36 h 72 h 96 h

Surfactins 5.4 (0.24) 12.9 (2.1) 19.1 (2.5) 22.8 (3.0) 21.2 (2.9) 22.6 (3.1)Bacillomycin Ls 0 0 15.9 (2.4) 19.7 (2.6) 16.3 (1.8) 15.5 (1.6)Locillomycins 0 0 1.2 (0.3) 3.6 (0.5) 3.4 (0.5) 3.2 (0.4)Fengycins 0 0 1.0 (0.3) 4.8 (0.6) 4.2 (0.5) 4.4 (0.7)a The values are means from three experiments, and the values in parentheses are standard deviations.

LP Biosynthesis and Phenotypic Features in B. subtilis

January 2015 Volume 81 Number 1 aem.asm.org 425Applied and Environmental Microbiology

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

and Pro-4 for bacillomycin D (see Fig. S4 in the supplementalmaterial).

To our surprise, the biosynthesis of locillomycins by the locgene cluster deviates from the linear mechanism and exhibits anonlinear assembly (29, 30). The loc gene cluster encodes 4proteins (LocD, LocA, LocB, and LocC) which constructed a

hexamodular NRPS, but they biosynthesize cyclic nonapep-tides, the locillomycins. Based on further analysis of the orga-nization of the function modules in loc and the structure oflocillomycins, we propose that the biosynthesis of locillomy-cins exhibits a rare nonlinear mechanism, with the middlethree domains in LocB being used iteratively and a KAS domain

FIG 2 Gene clusters of surfactin, locillomycin, bacillomycin L, and fengycin. The schematic representation of the entire gene cluster for srf, bmy, loc, and fencomprises the ORFs and the domains corresponding to NRPSs and amino acids incorporated by the different modules, which encode catalytic machineriesresponsible for the biosynthesis of surfactins, bacillomycin Ls, locillomycins, and fengycins. The order and specificity of the modules within NRPSs encoded bysrf, bmy, and fen determine the amino acid sequence of the surfactins, bacillomycin Ls, and fengycins, respectively. However, the hexamodular NRPSs encodedby loc carry out biosynthesis of locillomycin nonapeptides.

FIG 3 HPLC spectrograms of four families of LPs produced by B. subtilis 916 and its mutants. (a) HPLC spectrograms of fengycins. BSFM, which is disruptedin fenA, was deficient in production of fengycins, and BSSM, which is disrupted in srfAA, had a significant decrease in the production of fengycins. (b) HPLCspectrograms for bacillomycin Ls. BSBM, which is disrupted in bmyD, and BSSM, which is disrupted in srfAA, were deficient in production of bacillomycin Ls.(c) HPLC spectrograms for surfactins. BSSM is deficient in production of surfactins. (d) HPLC spectrograms for locillomycins. BSLM, which is disrupted in locD,was deficient in production of locillomycins.

Luo et al.

426 aem.asm.org January 2015 Volume 81 Number 1Applied and Environmental Microbiology

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

in LocD being skipped (this observation will be detailed else-where).

Consecutive disruption of bmy, fen, srf, and loc gene clustersyields series of LP-deficient phenotypes. To confirm that the srf,bmy, fen, and loc gene clusters are responsible for surfactin, bacil-lomycin L, fengycin, and locillomycin biosynthesis, we disruptedsrfAA, bamD, fenA, and locD in the B. subtilis 916 genome withhomologous recombination. Double and triple mutant strainswere also obtained. Analysis of the mutant strains by HPLC-MSconfirmed that BSBM (�bac::Nmr), BSFM (�fenA::Cmr), andBSLM (�locA::Emr) were deficient in bacillomycin L, fengycin,and locillomycin production, respectively (Fig. 3). Unexpectedly,BSSM (�srfA::Specr) was deficient in producing not only surfac-tins but also bacillomycin Ls, and significantly decreased produc-tion of fengycins was also observed (Fig. 3; also, see Fig. S9 to S12and Table S2 in the supplemental material). In contrast to BSSM,while the mutant BSBM lacked bacillomycin L biosynthesis, itproduced 43.4% more surfactins and 25.6% more fengycins thanwild-type B. subtilis 916 (see Table S2 and Fig. S10 and S12 in thesupplemental material). Like BSBM, while BSFM lacked fengycinbiosynthesis, it produced 18.5% more surfactins than the wildtype (see Table S2 and Fig. S12 in the supplemental material). Asexpected, the double and triple mutant strains failed to producetwo and three LPs, respectively. The production of the four LPs bythe double and triple mutant strains was also assessed by HPLC-MS. The results showed that while the mutant strains were defi-cient in producing some of the LPs, the production of the otherscould be enhanced significantly (see Fig. S9 to S12 and Table S2 inthe supplemental material).

Evaluation of antifungal activities and hemolytic activities ofwild-type and mutant strains. In this study, the antifungal activ-ities of mutant strains against spore germination and myceliumgrowth of F. oxysporum were also investigated (Fig. 4; also, see Fig.S13 in the supplemental material). Compared to the wild type,BSBM and BSFM had significantly decreased antifungal activity.In contrast to the single mutants BSBM and BSFM, the doublemutant BSBFM had further-decreased antifungal activity. As ex-pected, the single mutant BSSM was unable to inhibit the growthF. oxysporum in a manner similar to that of BSBFM. Compared tothe wild type, BSLM had no significant change in its antifungalactivity. For the triple mutant BSBFLM, which retained the abilityto produce surfactins, the ability to suppress the growth of F. ox-ysporum was completely abolished. The toxicities of the four LPsfor F. oxysporum were also investigated in in vitro assays. F. oxys-porum showed high sensitivity to the bacillomycin Ls and fengy-cins, and the 50% inhibitory concentrations (IC50s) for hyphalgrowth were below 2 �g/ml. F. oxysporum showed moderate sen-sitivity to the locillomycins, and the IC50 for hyphal growth was18.8 �g/ml. Further, F. oxysporum showed the lowest sensitivity tosurfactins, and the IC50 for hyphal growth was over 50.0 �g/ml.The results above strongly suggest that the bacillomycin Ls andfengycins contribute mainly to the antifungal activity of B. subtilis916 against F. oxysporum.

The hemolytic activities of B. subtilis 916 and the mutants werealso compared (Fig. 5). While the wild type, BSFM, and BSLMwere able to induce a clear halo surrounding the streak-inoculatedregion on blood agar plates at 24 h of growth, BSBM was not ableto formed a clear halo until 48 h of growth. In particular, BSSMwas unable to induce a hemolytic halo around the inoculated re-gion before 36 h of growth and formed only a vague halo at 72 h of

growth. The double and triple mutant strains have less hemolyticactivity than their parent strains. The hemolytic activities of thefour purified families of LPs were also investigated. As expect, thesurfactins and bacillomycin Ls showed strong hemolytic activities,but the fengycins and locillomycins showed weak hemolytic activ-ities. Thus, we draw the conclusion that the bacillomycin Ls andsurfactins play much more important roles in the hemolytic activ-ities of B. subtilis 916 than the locillomycins and fengycins do.

Evaluation of swarming motility, biofilm formation, and col-ony morphology. B. subtilis 916 formed colonies with dense wrin-kles and compact structure on MSgg plates, showed good swarm-ing motility on semisolid LB plates, and formed a thick andwrinkled floating biofilm in MSgg broth (Fig. 6, 7, and 8). The

FIG 4 Antifungal activities of B. subtilis 916 and its mutants against F. oxys-porum. (a) A volume of 50 �l culture broth of B. subtilis 916 and its mutantstrains was dropped into agar plates which contain spores of F. oxysporum. Theplates were incubated for 1 to 3 days at 28°C. Two inhibition zones wereformed. The inner inhibition zones are marked by red circles, and the outerinhibition zones are marked by green circles. WT, wild-type B. subtilis 916;BSBM, single mutant disrupted in bmyD; BSFM, single mutant disrupted infenA; BSLM, single mutant disrupted in locD; BSSM, single mutant disruptedin srfAA; BSBFM, double mutant disrupted in both bmyD and fenA; BSFLM,double mutant disrupted both in fenA and locD; BSBLM, double mutant dis-rupted in both bmyD and locD; BSBFLM, triple mutant disrupted in bmyD,fenA, and locD. (b) Measurement the inhibition zones of B. subtilis 916 and itsmutant strains against F. oxysporum. Whereas bacillomycin L contributedmainly to the inner inhibition zones, the fengycins contributed conclusively tothe outer inhibition zones. Data are average diameters standard deviationsfor three replicates in two independent experiments. Means in the same col-umn with different letters are significantly different (P � 0.05) according toDuncan’s multiple range tests.

LP Biosynthesis and Phenotypic Features in B. subtilis

January 2015 Volume 81 Number 1 aem.asm.org 427Applied and Environmental Microbiology

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

mutant BSSM, like other strains deficient in production of surfac-tins, formed flat colonies, had significantly less swarming motility,and formed only a very thin and fragile floating biofilm (3, 5).Interestingly, the mutant BSBM also showed changes in colonymorphology and decreased floating biofilm formation, but itsswarming motility was unexpectedly significantly enhanced. Themutant BSLM showed no difference in swarming motility andbiofilm formation, and the colony morphology of BSLM was sig-nificantly different from that of the wild type, similar to that of

BSBM (Fig. 6 to 8). However, the mutant BSFM showed no dis-tinctive change in any of the three phenotypical features.

Restoration of the multicellular behavior of mutant strains byadding exogenous LPs was also investigated (Fig. 6 to 8). Theaddition of exogenous LPs failed to restore colony morphology inany of the mutant strains. However, the swarming motility of

FIG 5 Hemolytic activities of wild-type B. subtilis 916 and its mutant strains. B. subtilis 916 and its mutant strains were inoculated on blood agar plates bystreaking. Hemolytic activities were visualized by development of a clear halo around the growth of the strains after incubation at 37°C for 1 to 3 days. WT,wild-type B. subtilis 916, which coproduces four LPs; BSBM, single mutant deficient in production of bacillomycin Ls; BSFM, single mutant deficient inproductions of fengycins; BSLM, single mutant deficient in the production of locillomycins; BSSM, single mutant deficient in production of both surfactins andbacillomycin Ls and decreased in production of fengycins; BSBFM, double mutant deficient in production of both bacillomycin Ls and fengycins; BSFLM, doublemutant deficient in production of both fengycins and locillomycins; BSBLM, double mutant deficient in production of both bacillomycin Ls and locillomycins;BSBFLM, triple mutant deficient in production of bacillomycin Ls, fengycins, and locillomycins. Surfactins and bacillomycin Ls produced by B. subtilis 916 andits mutant strains contributed mainly to their hemolytic activities.

FIG 6 Comparison of colony morphology of B. subtilis 916 and its mutantstrains on LB plates with Congo red and Coomassie brilliant blue dyes. Thewild-type (WT) strain B. subtilis 916 and single mutant BSBFM disrupted infenA formed the highly structured colonies. The single mutants BSBM, BSLM,and BSSM disrupted in bmyD, locD, and srfAA, respectively, formed less struc-tured and flatter colonies than WT. Furthermore, the double mutants BSBFM,BSFLM, and BSBLM and triple mutant BSBFLM formed flatter colonies thantheir parent strains. Unlike the disruption of fenA (encoding fengycins), thedisruptions of srfAA, bmyD, and locD all had an important influence on thecolony morphology of B. subtilis 916 and its mutant strains.

FIG 7 Floating-biofilm formation of the B. subtilis 916 and its mutant strainswith or without adding exogenous surfactins and bacillomycin Ls in MSggbroth with Congo red and Coomassie brilliant blue dyes. WT B. subtilis 916formed a thick and wrinkled floating biofilm. Like the WT, the mutants BSFM(disrupted in fenA) and BSLM (disrupted in locD) also formed thick and wrin-kled floating biofilms. However, biofilm formation was weakened significantlyin mutant BSBM (disrupted in bmyD), which formed a thin and fragile floatingbiofilm. Furthermore, BSSM (disrupted in srfAA) formed a thinner and morefragile floating biofilm than BSBM. The floating biofilm was restored signifi-cantly in BSBM with either bacillomycin Ls (BSBM�BL) or surfactins(BSBM�SRF) at 20 �g/ml. However, the floating biofilm was restored in themutant BSSM only faintly with bacillomycin Ls (BSSM�BL) and surfactins(BSSM�SRF).

Luo et al.

428 aem.asm.org January 2015 Volume 81 Number 1Applied and Environmental Microbiology

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

BSSM and BSBM was restored or even enhanced upon additionof exogenous surfactins and bacillomycin Ls, respectively. Theswarming motility of the double mutant BSBSM was also restoredsignificantly by the addition of both surfactins and bacillomycinLs. To our surprise, unlike other mutant strains disrupted in srfAAreported previously, which were able to restore biofilm formation,the mutant BSSM was unable to restore biofilm formation whensurfactins and bacillomycin Ls were added. Interestingly, BSBMwas able to restore the formation of floating biofilm upon theaddition of either surfactins or bacillomycin Ls.

DISCUSSION

The endospore-forming rhizobacterium B. subtilis and closely re-lated species are well-known candidates for developing efficientbiopesticide products mainly due to their production of twodozen antibiotics with an amazing variety of structures, enablingthem to cope with competing organisms within food crop tissues(4, 6, 7, 31). Usually, B. subtilis has an average of 4 to 5% of itsgenome devoted to antibiotic synthesis (6). Recently, the com-plete genome of B. subtilis 916 was sequenced (14). The mostdistinctive feature of this genome is that a considerable part(10%, 400 kb, organized in 9 operons) is devoted to the biosyn-thesis of polyketide and peptide antibiotics. Five gene clusters en-coding PKSs and four gene clusters involved in NRPS biosynthesisof four families of LPs have been identified. In contrast to otherbiocontrol B. subtilis and B. amyloliquefaciens strains, whose ge-nomes contain only 2 or 3 NRPS gene clusters, the genome of B.subtilis 916 contains four family NRPS gene clusters. In addition,B. subtilis 916 is naturally competent for uptake of naked DNA and

homologous recombination and ideally suitable for genetic ma-nipulation. Thus, B. subtilis 916 provides an excellent model forresearch on the biosynthesis and functions of LPs.

The disruption of bmy, fen, and loc genes blocked productionof the corresponding LPs, suggesting that these gene clusters areresponsible for their biosynthesis. To our surprise, unlike theother B. subtilis strains disrupted in srfAA, which had only theproduction of surfactins blocked, a B. subtilis 916 mutant with adisruption of srfAA had the production of bacillomycin Ls as wellas surfactins blocked and had impaired production of fengycins(3, 5, 9). In contrast to the disruption of srf, the disruption of bmyenhanced the production of the other LPs significantly. Furtherinvestigations are necessary to clarify the entangled biosynthesis ofbacillomycin Ls and fengycins. The double and triple mutantstrains further confirmed that the four NRPS gene clusters areresponsible for the four families of LPs in B. subtilis 916 and thatthey interact with and affect one another.

Intriguingly, in contrast to the other Bacillus LPs reported pre-viously, locillomycins showed a novel molecular architecture (6,7). While the length of the fatty acid chains of the locillomycins issimilar to that of other Bacillus LPs, the amino acid moiety oflocillomycins is different from moieties of the iturins, surfactins,and fengycins. Moreover, unlike other Bacillus LP biosynthesis,biosynthesis of locillomycins used a nonlinear pathway, where anonapeptide is assembled by hexamodular NRPSs. With the pos-sibility of making novel LPs by rationally manipulated NRPSs, theunusual structure and unique biosynthesis pathway of locillomy-cins could inspire novel means to construct hybrid NRPSs, whichcould be used to synthesize new LP derivatives (10, 32, 33).

Our results showed that the bacillomycin Ls, together withfengycins, mainly contribute to the antifungal activity of B. subtilis916. In addition, bacillomycin Ls and surfactins obviously con-tributed to the hemolytic activity of this strain. Though the sur-factins and locillomycins showed limited antifungal activities,they showed strong antibacterial and antiviral activities (data notshown). Particular emphasis should be placed on the observationthat locillomycins and fengycins exhibited low hemolytic activitiesand strong antimicrobial activities, which could give rise to poten-tial therapeutic applications. The antifungal and hemolytic activ-ities of double and triple mutant strains were further decreasedcompared to those of their parent strains. In summary, the four LPfamilies contributed individually, differently, and synergisticallyto the antimicrobial and hemolytic activity of B. subtilis 916.

Recently, LPs were proven to function as signal molecules inthe multicellular behaviors of B. subtilis in terms of swarmingmotility, biofilm formation, and colony morphology (9, 16, 19,34). Surfactins contributed to the swarming motility and biofilmformation by acting as wetting agents for reducing the surfacetension and acting as pheromones for quorum sensing (3, 5, 9,20). In contrast to the data on surfactins, the reports on bacillo-mycins which facilitated swarming motility and were involved inbiofilm formation are fewer and inconclusive (5, 7, 18). In thisstudy, we demonstrated unambiguously that both surfactins andbacillomycin Ls are involved in swarming motility and biofilmformation of B. subtilis 916. It is interesting that while attempts torestore biofilm formation of srfAA mutant by adding both surfac-tins and bacillomycin Ls were unsuccessful, the biofilm formationof bacD mutant was restored significantly upon the addition ofeither surfactins or bacillomycin Ls. On the basis of these results,we presume that surfactins and bacillomycin Ls trigger mature

FIG 8 Swarming motility of the B. subtilis 916 and its mutants with or withoutthe addition of exogenous surfactins and bacillomycin Ls, as assessed in 0.7%agar LB plates with Congo red. The WT strain B. subtilis 916 showed goodswarming motility on semisolid LB plates. Swarming motility of BSFM andBSLM, which are disrupted in fenA and locD, respectively, showed no differ-ence from that of the WT. The swarming motility of BSSM disrupted in srfAAdecreased significantly compared to that of the WT. The swarming motility ofBSBM increased significantly compared to that of the WT. The swarmingmotility of BSBM was further enhanced by adding exogenous bacillomycin Lsat 20 �g/ml (BSBM�BL). As expected, swarming motility was restored inBSSM upon addition of exogenous surfactins at 20 �g/ml (BSSM�SRF). Toour surprise, swarming motility of BSBM decreased significantly upon addi-tion of exogenous surfactins at 20 �g/ml (BSBM�SRF). Similarly, swarmingmotility of BSSM further decreased upon addition of exogenous bacillomycinLs (BSSM�BL).

LP Biosynthesis and Phenotypic Features in B. subtilis

January 2015 Volume 81 Number 1 aem.asm.org 429Applied and Environmental Microbiology

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

biofilm formation involved in different quorum-sensing path-ways. While the locillomycins have no significant influence on theswarming motility and biofilm formation of B. subtilis 916, thecolony morphology of the loc mutant, like that of srf and bmymutants, changed significantly compared to that of wild-type B.subtilis 916. The results strongly suggest that locillomycins, likesurfactins and bacillomycin Ls, take part in the multicellular be-haviors of B. subtilis 916. However, it seems that the fengycinscontribute only to the antifungal activity and are not involved inother phenotypical features of B. subtilis 916. In view of the differ-ent contributions of surfactins, bacillomycin Ls, and locillomycinsto multicellular behaviors of B. subtilis 916, it will be interesting tofurther decipher the detailed mechanisms of different LPs withregard to how they trigger active kinase expression and regulatethe swarming motility, biofilm formation, and colony morphol-ogy of B. subtilis 916 (21, 22, 34–39).

In conclusion, we identified and characterized four NRPS geneclusters responsible for the biosynthesis of four families of LPs—surfactins, bacillomycin Ls, fengycins, and locillomycin—in B.subtilis 916. Functions of these LPs with regard to the phenotypicfeatures were evaluated. Deficiencies in producing one or more ofthese four families of LPs led to different results, some of which arestraightforward while others are elusive. It will be interesting toknow how the genes and chemicals involved in these phenotypicfeatures are affected by the four families of LPs. An investigation atthe genomics level on this question will be our next challenge.

ACKNOWLEDGMENTS

We thank Jun Yao Guo for the linguistic revision and critical review of themanuscript.

This work was supported by the National High-tech R&D Program ofChina (2011AA10A201), National Natural Science Foundation of China(grant 30900929), and the Science Foundation of the Jiangsu Academy ofAgricultural Sciences [grant CX(12)5001].

REFERENCES1. Asaka O, Shoda M. 1996. Biocontrol of Rhizoctonia solani damping-off of

tomato with Bacillus subtilis RB14. Appl Environ Microbiol 62:4081–4085.

2. Xu Z, Zhang R, Wang D, Qiu M, Feng H, Zhang N, Shen Q. 2014.Enhanced control of cucumber wilt disease by Bacillus amyloliquefaciensSQR9 by altering the regulation of its DegU phosphorylation. Appl Envi-ron Microbiol 80:2941–2950. http://dx.doi.org/10.1128/AEM.03943-13.

3. Bais HP, Fall R, Vivanco JM. 2004. Biocontrol of Bacillus subtilis againstinfection of Arabidopsis roots by Pseudomonas syringae is facilitated bybiofilm formation and surfactin production. Plant Physiol 134:307–319.http://dx.doi.org/10.1104/pp.103.028712.

4. Ahimou F, Jacques P, Deleu M. 2000. Surfactin and iturin A effects onBacillus subtilis surface hydrophobicity. Enzyme Microb Technol 27:749 –754. http://dx.doi.org/10.1016/S0141-0229(00)00295-7.

5. Zeriouh H, de Vicente A, Pérez-García A, Romero D. 2014. Surfactintriggers biofilm formation of Bacillus subtilis in melon phylloplane andcontributes to the biocontrol activity. Environ Microbiol 16:2196 –2211.http://dx.doi.org/10.1111/1462-2920.12271.

6. Stein T. 2005. Bacillus subtilis antibiotics: structures, syntheses and spe-cific functions. Mol Microbiol 56:845– 857. http://dx.doi.org/10.1111/j.1365-2958.2005.04587.x.

7. Ongena M, Jacques P. 2008. Bacillus lipopeptides: versatile weapons forplant disease biocontrol. Trends Microbiol 16:115–125. http://dx.doi.org/10.1016/j.tim.2007.12.009.

8. Romero D, de Vicente A, Rakotoaly RH, Dufour SE, Veening J-W,Arrebola E, Cazorla FM, Kuipers OP, Paquot M, Pérez-García A. 2007.The iturin and fengycin families of lipopeptides are key factors in antago-nism of Bacillus subtilis toward Podosphaera fusca. Mol Plant MicrobeInteract 20:430 – 440. http://dx.doi.org/10.1094/MPMI-20-4-0430.

9. Ongena M, Jourdan E, Adam A, Paquot M, Brans A, Joris B, Arpigny J-L,

Thonart P. 2007. Surfactin and fengycin lipopeptides of Bacillus subtilis aselicitors of induced systemic resistance in plants. Environ Microbiol9:1084 –1090. http://dx.doi.org/10.1111/j.1462-2920.2006.01202.x.

10. Sugimoto Y, Ding L, Ishida K, Hertweck C. 2014. Rational design ofmodular polyketide synthases: morphing the aureothin pathway into aluteoreticulin assembly line. Angew Chem Int Ed Engl 53:1560 –1564.http://dx.doi.org/10.1002/anie.201308176.

11. Mootz HD, Schwarzer D, Marahiel MA. 2000. Construction of hybridpeptide synthetases by module and domain fusions. Proc Natl Acad SciU S A 97:5848 –5853. http://dx.doi.org/10.1073/pnas.100075897.

12. Challis GL. 2008. Mining microbial genomes for new natural productsand biosynthetic pathways. Microbiology 154:1555–1569. http://dx.doi.org/10.1099/mic.0.2008/018523-0.

13. Velásquez JE, van der Donk WA. 2011. Genome mining for ribosomallysynthesized natural products. Curr Opin Chem Biol. 15:11–21. http://dx.doi.org/10.1016/j.cbpa.2010.10.027.

14. Wang X, Luo C, Chen Z. 2012. Genome sequence of the plant growth-promoting rhizobacterium Bacillus sp. strain 916. J Bacteriol 194:5467–5468. http://dx.doi.org/10.1128/JB.01266-12.

15. Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H, SaengerW, Bernhard F, Reinhardt R, Schmidt M, Ullrich C, Stein T, LeendersF, Vater J. 1999. The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an aminotransferase, and a fatty acid synthase. Proc Natl Acad Sci U S A 96:13294 –13299. http://dx.doi.org/10.1073/pnas.96.23.13294.

16. Shank EA, Kolter R. 2011. Extracellular signaling and multicellularity inBacillus subtilis. Curr Opin Microbiol 14:741–747. http://dx.doi.org/10.1016/j.mib.2011.09.016.

17. Houry A, Briandet R, Aymerich S, Gohar M. 2010. Involvement ofmotility and flagella in Bacillus cereus biofilm formation. Microbiology156:1009 –10018. http://dx.doi.org/10.1099/mic.0.034827-0.

18. Xu Z, Shao J, Li B, Yan X, Shen Q, Zhang R. 2013. Contribution ofbacillomycin D in Bacillus amyloliquefaciens SQR9 to antifungal activityand biofilm formation. Appl Environ Microbiol 79:808 – 815. http://dx.doi.org/10.1128/AEM.02645-12.

19. López D, Vlamakis H, Losick R, Kolter R. 2009. Cannibalism enhancesbiofilm development in Bacillus subtilis. Mol Microbiol 74:609 – 618. http://dx.doi.org/10.1111/j.1365-2958.2009.06882.x.

20. Kinsinger RF, Shirk MC, Fall R. 2003. Rapid surface motility in Bacillus subtilisis dependent on extracellular surfactin and potassium ion. J Bacteriol.185:5627–5631. http://dx.doi.org/10.1128/JB.185.18.5627-5631.2003.

21. Vlamakis H, Chai Y, Beauregard P, Losick R, Kolter R. 2013. Stickingtogether: building a biofilm the Bacillus subtilis way. Nat Rev Microbiol11:157–168. http://dx.doi.org/10.1038/nrmicro2960.

22. López D, Fischbach Ma, Chu F, Losick R, Kolter R. 2009. Structurallydiverse natural products that cause potassium leakage trigger multicellu-larity in Bacillus subtilis. Proc Natl Acad Sci U S A 106:280 –285. http://dx.doi.org/10.1073/pnas.0810940106.

23. Kunst F, Rapoport G. 1995. Salt stress is an environmental signal affect-ing degradative enzyme synthesis in Bacillus subtilis. J Bacteriol 177:2403–2407.

24. Luo C, Chen Z, Liu Y, Zhang J, Liu Y, Wang X, Nie Y. 2009. Construc-tion and function analysis of Bac operon mutants of bio-control strainBacillus subtilis Bs-916. Wei Sheng Wu Xue Bao 49:445– 452.

25. Romero D, Aguilar C, Losick R, Kolter R. 2010. Amyloid fibers providestructural integrity to Bacillus subtilis biofilms. Proc Natl Acad Sci U S A107:2230 –2234. http://dx.doi.org/10.1073/pnas.0910560107.

26. Tsuge K, Akiyama T, Shoda M. 2001. Cloning, sequencing, and charac-terization of the iturin A operon. J Bacteriol 183:6265– 6273. http://dx.doi.org/10.1128/JB.183.21.6265-6273.2001.

27. Borisova Sa, Circello BT, Zhang JK, van der Donk WA, Metcalf WW.2010. Biosynthesis of rhizocticins, antifungal phosphonate oligopeptidesproduced by Bacillus subtilis ATCC6633. Chem Biol 17:28 –37. http://dx.doi.org/10.1016/j.chembiol.2009.11.017.

28. Kessler N, Schuhmann H, Morneweg S, Linne U, Marahiel Ma. 2004.The linear pentadecapeptide gramicidin is assembled by four multimodu-lar nonribosomal peptide synthetases that comprise 16 modules with 56catalytic domains. J Biol Chem 279:7413–7419. http://dx.doi.org/10.1074/jbc.M309658200.

29. Moss SJ, Martin CJ, Wilkinson B. 2004. Loss of co-linearity by modularpolyketide synthases: a mechanism for the evolution of chemical diversity.Nat Prod Rep 21:575–593. http://dx.doi.org/10.1039/b315020h.

30. Sieber SA, Marahiel MA. 2005. Molecular mechanisms underlying non-

Luo et al.

430 aem.asm.org January 2015 Volume 81 Number 1Applied and Environmental Microbiology

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from

ribosomal peptide synthesis: approaches to new antibiotics. Chem Rev105:715–738. http://dx.doi.org/10.1021/cr0301191.

31. Leclere V, Bechet M, Adam A, Guez J-S, Wathelet B, Ongena M,Thonart P, Gancel F, Chollet-Imbert M, Jacques P. 2005. MycosubtilinOverproduction by Bacillus subtilis BBG100 enhances the organism’s an-tagonistic and biocontrol activities. Appl Environ Microbiol 71:4577–4584. http://dx.doi.org/10.1128/AEM.71.8.4577-4584.2005.

32. Kapur S, Lowry B, Yuzawa S, Kenthirapalan S, Chen AY, Cane DE,Khosla C. 2012. Reprogramming a module of the 6-deoxyerythronolide Bsynthase for iterative chain elongation. Proc Natl Acad Sci U S A 109:4110 – 4115. http://dx.doi.org/10.1073/pnas.1118734109.

33. Eppelmann K, Doekel S, Marahiel Ma. 2001. Engineered biosynthesis of thepeptide antibiotic bacitracin in the surrogate host Bacillus subtilis. J BiolChem 276:34824 –34831. http://dx.doi.org/10.1074/jbc.M104456200.

34. Chen Y, Cao S, Chai Y, Clardy J, Kolter R, Guo J, Losick R. 2012. ABacillus subtilis sensor kinase involved in triggering biofilm formation onthe roots of tomato plants. Mol Microbiol 85:418 – 430. http://dx.doi.org/10.1111/j.1365-2958.2012.08109.x.

35. Branda SS, González-Pastor JE, Dervyn E, Ehrlich SD, Losick R, Kolter

R. 2004. Genes involved in formation of structured multicellular commu-nities by Bacillus subtilis. J Bacteriol 186:3970 –3979. http://dx.doi.org/10.1128/JB.186.12.3970-3979.2004.

36. Kearns DB, Chu F, Branda SS, Kolter R, Losick R. 2005. A masterregulator for biofilm formation by Bacillus subtilis. Mol Microbiol 55:739 –749. http://dx.doi.org/10.1111/j.1365-2958.2004.04440.x.

37. Vlamakis H, Aguilar C, Losick R, Kolter R. 2008. Control of cell fate bythe formation of an architecturally complex bacterial community. GenesDev 22:945–953. http://dx.doi.org/10.1101/gad.1645008.

38. Ogura M, Yoshikawa H, Chibazakura T. 2014. Regulation of the re-sponse regulator gene degU through the binding of SinR/SlrR and exclu-sion of SinR/SlrR by DegU in Bacillus subtilis. J Bacteriol 196:873– 881.http://dx.doi.org/10.1128/JB.01321-13.

39. Hansen H, Bjelland AM, Ronessen M, Robertsen E, Willassen NP. 2014.LitR is a repressor of syp genes and has a temperature-sensitive regulatoryeffect on biofilm formation and colony morphology in Vibrio (Aliivibrio)salmonicida. Appl Environ Microbiol 80:5530 –5541. http://dx.doi.org/10.1128/AEM.01239-14.

LP Biosynthesis and Phenotypic Features in B. subtilis

January 2015 Volume 81 Number 1 aem.asm.org 431Applied and Environmental Microbiology

on March 1, 2021 by guest

http://aem.asm

.org/D

ownloaded from