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1.
Pellicle formation and lipopeptide production was analysed in standing cultures of different Bacillus subtilis strains producing two or three families of lipopeptides. Despite its ability to produce surfactin, B. Subtilis ATCC 6633 was unable to form stable pellicle at air–water interface. For the ATTC 21332 and ATCC 9943 strains, it was shown
for the first time that the lipopeptides were also produced in standing cultures at productivities similar or lower than those
obtained when the culture medium is agitated. A differentiated behaviour was observed between these strains in repetitive
batch cultures. B. subtilis 9943 formed a wrinkled, thinner and more resistant pellicle than B. subtilis 21332. The structure of the pellicle determined by electron microscopy observations showed that cells of B. subtilis 9943 formed microcolonies whereas those of B. subtilis 21332 rapidly died. Under these conditions, surfactin production by strain 21332 decreased after 2 days whereas it remained
stable for B. subtilis 9943 during the 6 days of the cultures. These data indicate that cells of B. subtilis strains growing in pellicle can produce lipopeptides differently depending on their cellular organisation.
M. Chollet-Imbert and F. Gancel have contributed equally to the scientific work. 相似文献
2.
DC Sabaté MJ Gonzaléz MP Porrini MJ Eguaras MC Audisio JM Marioli 《World journal of microbiology & biotechnology》2012,28(4):1415-1422
The aim of this work was to determine the in vitro effect of the mixture between the lipopeptide surfactin, synthesized by
Bacillus subtilis C4 (strain isolated from honey) and the most active vegetal extract from Achyrocline satureioides, a traditional medicinal plant, on local strains of Paenibacillus larvae, the agent of American Foulbrood in honeybees. Five P. larvae strains isolated in Córdoba, Argentina, were phenotypically characterized. These and 12 other P. larvae strains from different regions of Argentina were analysed. The antimicrobial activities of the essential oil, hexane (HE)
and benzene extracts from A. satureioides were assessed against P. larvae and the HE showed the highest anti-P. larvae activity. A combination of the biosurfactant surfactin, produced by B. subtilis C4, and the HE of A. satureioides revealed a synergistic action on P. larvae. The effective surfactin concentration in the mixture decreased from 32 to 1 μg ml−1 and the HE concentration from 32 to 4 μg ml−1, values similar or equal to minimal inhibitory concentrations observed for oxytetracycline. The fractional inhibitory concentration
index confirmed synergism in 4 strains and partial synergism in one strain. The combination of surfactin synthesized by B. subtilis C4 and the HE from A. satureioides could be a natural alternative to help beekeepers to combat the American foulbrood agent P. larvae. 相似文献
3.
Etchegaray A de Castro Bueno C de Melo IS Tsai SM Fiore MF Silva-Stenico ME de Moraes LA Teschke O 《Archives of microbiology》2008,190(6):611-622
Lipopeptides produced by Bacillus subtilis are known for their high antifungal activity. The aim of this paper is to show that at high concentration they can damage
the surface ultra-structure of bacterial cells. A lipopeptide extract containing iturin and surfactin (5 mg mL−1) was prepared after isolation from B. subtilis (strain OG) by solid phase extraction. Analysis by atomic force microscope (AFM) showed that upon evaporation, lipopeptides
form large aggregates (0.1–0.2 μm2) on the substrates silicon and mica. When the same solution is incubated with fungi and bacteria and the system is allowed
to evaporate, dramatic changes are observed on the cells. AFM micrographs show disintegration of the hyphae of Phomopsis phaseoli and the cell walls of Xanthomonas campestris and X. axonopodis. Collapses to fungal and bacterial cells may be a result of formation of pores triggered by micelles and lamellar structures,
which are formed above the critical micelar concentration of lipopeptides. As observed for P.
phaseoli, the process involves binding, solubilization, and formation of novel structures in which cell wall components are solubilized
within lipopeptide vesicles. This is the first report presenting evidences that vesicles of uncharged and negatively charged
lipopeptides can alter the morphology of gram-negative bacteria. 相似文献
4.
Lee YK Yoon BD Yoon JH Lee SG Song JJ Kim JG Oh HM Kim HS 《Applied microbiology and biotechnology》2007,75(3):567-572
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. 相似文献
5.
Sarah P. Canova Tânia Petta Luciana F. Reyes Tiago D. Zucchi Luiz A. B. Moraes Itamar S. Melo 《World journal of microbiology & biotechnology》2010,26(12):2241-2247
The main aim was to identify the active compound against Rhizoctonia solani produced by the cassava endophyte Paenibacillus sp. IIRAC-30. The compounds produced were extracted with ethyl acetate and purified by Sephadex column prior to analysis
by Q-TOF mass spectrometry. A C15-lipopeptide with an estimated molecular weight of 1036 Da and homologues were identified. The lipopeptide had a cyclic structure,
which was deduced by interpreting the ESI–MS/MS spectra of main protonated homologues containing 15:0 FA, and the amino acid
composition was Glu-Leu-Leu-Val-Asp-Leu-Leu. Therefore, the lipopeptides produced by isolate IIRAC-30 was characterized as
a surfactin series. Thus, the main mechanism used by Paenibacillus sp. IIRAC-30 to suppress R. solani was elucidated. Furthermore, because lipopeptides active against phytopathogens generally show low toxicity to humans and
the environment, the positive findings presented here suggest that the isolate IIRAC-30 could be a possible candidate for
biocontrol of R. solani. 相似文献
6.
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. 相似文献
7.
Screening of high-yielding biocontrol bacterium Bs<Emphasis Type="Italic">-</Emphasis>916 mutant by ion implantation 总被引:1,自引:0,他引:1
Bacillus subtilis 916 was an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. To further improve its antagonistic ability, low-energy ion implantation was applied in Bs-916. We studied the effects of
different doses of N+ implantation. The optimum dose of ion implantation for the Bs-916 was from 15 × 2.6 × 1014 N+/cm2 to 25 × 2.6 × 1014 N+/cm2. The mutant strain designated as Bs-H74 was obtained, which showed higher inhibition activity in the screening plate. Its
inhibition zone against the indicator organism increased by 30.7% compared to the parental strain. The control effect of rice
sheath blight was improved by 14.6% over that of Bs-916. Thin-layer chromatography and high-performance liquid chromatography
analysis indicated that lipopeptides produced by Bs-916 and the mutant strains belonged to the surfactin family. Bs-H74 produced
approximately 3.0-fold surfactin compared to that of Bs-916. To determine the role of surfactin in biocontrol by Bs-916, we
tested another mutant strain, Bs-M49, which produced lower levels of surfactin significantly, and found that Bs-M49 had no
obvious effects against R. solani. These results suggested that the surfactin produced by Bs-916 plays an important role in the suppression of sheath blight.
These observations also showed that the Bs-H74 mutant strain is a better biocontrol agent than the parental strain. 相似文献
8.
Bacillus subtilis strain IB exhibiting inhibitory activity against the Fusarium head blight disease fungus Fusarium graminearum was isolated and identified. The major inhibitory compound was purified from the culture broth through anion exchange, hydrophobic
interaction, and reverse phase high-performance liquid chromatography (RP-HPLC) steps. It was a 1,463-Da lipopeptide and had
an amino acid composition consisting of Ala, Glx, Ile, Orn, Pro, Thr, and Tyr at a molar ratio of 1:3:1:1:1:1:2. Electrospray
ionization mass spectrometry/mass spectrometry (ESI MS/MS) analyses of the natural and the ring-opened peptides showed the
antagonist was fengycin, a kind of macrolactone molecule with antifungal activity produced by several Bacillus strains. Fluorescence microscopic analysis indicated this peptide permeabilized and disrupted F. graminearum hyphae. 相似文献
9.
Background
Bacillus spp. have prominent ability to suppress plant pathogens and corresponding diseases. Previous analyses of Bacillus spp. revealed numerous gene clusters involved in nonribosomal synthesis of cyclic lipopeptides with distinct antimicrobial action. The 4′-phosphopantetheinyl transferase (PPTase) encoded by sfp gene is a key factor in lipopeptide synthesis in Bacillus spp. In previous study, B. amyloliquefaciens strain HAB-2 was found to inhibit a broad range of plant pathogens, which was attributed to its secondary metabolite lipopeptide.Results
A sfp homologue lpaH2 which encoded phosphopantetheinyl transferase but shared 71% sequence similarity was detected in strain HAB-2. Disruption of lpaH2 gene resulted in losing the ability of strain HAB-2 to produce lipopeptide, as well as antifungal and hemolytic activities. When lpaH2 replaced sfp gene of B. subtilis strain 168, a non-lipopeptide producer, the genetically engineered strain 168 could produced lipopeptides and recovered antifungal activity. Quantitative PCR assays indicated that, the expression level of lpaH2 in B. subtilis 168 strain decrease to 0.27-fold compared that of the wild type B. amyloliquefaciens strain HAB-2.Conclusion
Few studies have reported about lpa gene which can replace sfp gene in the different species. Taken together, our study showed for the first time that lpaH2 from B. amyloliquefaciens could replace sfp gene.10.
F. A. Oguntoyinbo A. I. Sanni C. M. A. P. Franz W. H. Holzapfel 《World journal of microbiology & biotechnology》2007,23(3):401-410
The Bacillus subtilis wild strains isolated from okpehe, a traditional fermented condiment used as seasoning in Nigeria, the reference and typed strains were investigated for their
phenotypic diversity and their technological parameters with a view to obtain adequate data that would enable selection of
appropriated starter cultures for vegetable protein fermentation in West Africa. All the 7 strains studied demonstrated diverse
phenotypic characteristics and they were identified as Bacillus subtilis, based on the API 50 CHB combined with API 20E profile. Specific sugars that indicated a good hydrolytic potential of the
wild strains were fermented. The highest proteinase activity of 90 AU/ml determined quantitatively was observed in the strain
Bacillus subtilis BFE 5372, the proteinase was identified by the APIZYM gallery as chymotrypsin. Highest amylase activity of 13 AU/ml was noticed
in strain Bacillus subtilis DSM 347 while only 4 strains produced polyglutamic acid with the strain Bacillus subtilis BFE 5359 producing the highest polyglutamate activity of 2.5 mm. Although strain Bacillus subtilis BFE 5301 did not release detectable polyglutamate, the strain demonstrated antagonism against different bacteria and the
antimicrobial substance produced by strain Bacillus subtilis BFE 5301 was confirmed as a bacteriocin since its activities were lost after treatment with chymotrypsin and pepsin. The
data generated showed the technological parameters that can aid selection of wild strains such as Bacillus subtilis BFE 5301, BFE 5359 and BFE 5372 for optimization of condiment production. 相似文献
11.
Roongsawang N Thaniyavarn J Thaniyavarn S Kameyama T Haruki M Imanaka T Morikawa M Kanaya S 《Extremophiles : life under extreme conditions》2002,6(6):499-506
Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin. 相似文献
12.
The biological activity and the presence of genes sfp and ituD (surfactin and iturin A) among Bacillus strains isolated from the Amazon basin were determined. Bacillus spp. were tested for hemolytic activity and inhibition of fungal growth by agar plate assays in parallel with PCR for identification
of sfp and ituD genes. All strains tested produced surface-active compounds, giving evidence by lysis of erythrocytes and emulsifying activity
on mineral oil and soybean oil. These strains of Bacillus caused growth inhibition of several phytopathogenic fungi, including Fusarium spp., Aspergillus spp., and Bipolaris sorokiniana. The presence of genes ituD and sfp was confirmed by PCR and sequence analysis. The only exception was Bacillus sp. P34 that lacks sfp gene. Lipopeptides were isolated from culture supernatants and analyzed by mass spectrometry. Characteristic m/z peaks for surfactin and iturin were observed, and some strains also produced fengycin and bacillomycin. The remarkable antifungal
activity showed by the strains could be associated with the co-production of three or more lipopeptide antibiotics. Screening
for novel bacteria producing useful biosurfactants or biocontrol agents for agriculture is a topic of greatest importance
to eliminate chemical pollutants. 相似文献
13.
Isolation and characterization of a biosurfactant producing strain, <Emphasis Type="Italic">Brevibacilis brevis</Emphasis> HOB1 总被引:1,自引:1,他引:0
Biosurfactant-producing bacteria were isolated from the production water of an oil field. Isolates were screened for biosurfactant
production using surface tension test. The highest reduction of surface tension was achieved with a bacterial strain which
was identified by 16S rRNA gene sequencing as Brevibacilis brevis HOB1. It has been investigated using different carbon and nitrogen sources. It showed that the strain was able to grow and
reduce the surface tension of the broth to 29 mN/m on commercial sugar and maltose, and to 32 mN/m on glucose after 72 h of
growth. The maximum amount of biosurfactant was obtained when nitrate ions were supplied as nitrogen source. Biosurfactant
produced by Brevibacilis brevis HOB1 was confirmed as a lipopeptide class of biosurfactant using TLC test and mass spectra. Lipopeptide isoforms were isolated
from cell-free supernatants by acid-precipitation followed by one step of chromatographic separation on solid-phase ODS C18
column. The separation was confirmed by HPLC and ESI Q-TOF MS spectroscopy. Comparing the mass data obtained and the mass
numbers reported for the lipopeptide complexes from other strains, it can be concluded that the major lipopeptide product
of Brevibacilis brevis HOB1 is the surfactin isoform. This lipopeptide showed strong antibacterial and antifungal activity. It is a candidate for
the biocontrol of pathogens in agriculture and other industries. 相似文献
14.
Yi-Ming Li Namir I. A. Haddad Shi-Zhong Yang Bo-Zhong Mu 《International journal of peptide research and therapeutics》2008,14(3):229-235
A lipopeptide producing strain was isolated from an oil field and identified as Bacillus licheniformis HSN221. Nine different substrates were used to cultivate the strain under the same incubation conditions. Using a rapid method,
Electrospray Ionization Mass Spectrometry (ESI-MS) combined with Thin Layer Chromatography (TLC), nine different lipopeptide
homologues were found and identified. The strain produced four [Leu]surfactin homologues, surfactin C13, surfactin C14, surfactin
C15 and surfactin C16, when cultivated in the medium with glucose, yeast extract and ammonium chloride, but it produced five
lichenysin homologues, lichenysin C12, lichenysin C13, lichenysin C14, lichenysin C15 and lichenysin C16, when cultivated
in the remaining eight media. Additionally, it showed that the type and relative content of each homologue were consistent
with in each medium which is helpful for optimizing the medium components to cultivate the similar species. 相似文献
15.
Pabel CT Vater J Wilde C Franke P Hofemeister J Adler B Bringmann G Hacker J Hentschel U 《Marine biotechnology (New York, N.Y.)》2003,5(5):424-434
The aim of this study was to isolate bacteria that are resistant to the strong antimicrobial metabolites
characteristic of Aplysina aerophoba. For this purpose, bacterial isolation was performed on agar plates to which
sponge tissue extract had been added. Following screening for antifungal and antimicrobial activities, 5 strains
were chosen for more detailed analyses. 16S ribosomal DNA sequencing revealed that all isolates belonged to
the genus Bacillus, specifically B. subtilis and B. pumilus. Using a combination of matrix-assisted laser desorption/
ionization mass spectrometry typing of whole cells and antimicrobial bioassays against selected reference
strains, the bioactive metabolites were identified as lipopeptides. 相似文献
16.
Novel rhamnolipid-producing strains of three thermophilic bacteria, Thermus sp., T. aquaticus and Meiothermus ruber were identified that have not been previously described as rhamnolipid producers. Rhamnolipids were extracted from supernatant
and further purified by thin-layer chromatography. Mass spectrometry with negative electrospray ionization revealed 77 rhamnolipid
homologues varying in chain length and unsaturation. Tandem mass spectrometry identified mono-rhamnolipid and di-rhamnolipid
homologues containing one or two 3-hydroxy-fatty acids, saturated, monounsaturated or diunsaturated, even- or odd-chain, up
to unusual long chains with 24 carbon atoms. The stereochemistry of rhamnose was L and that of 3-hydroxy-fatty acids was R, the position of double bonds in monoenoic acids was cis ω-9. All three strains produced a rhamnolipid that differs in structure from Pseudomonas aeruginosa rhamnolipids and exhibits excellent surfactant properties. Importantly, in comparison to P. aeruginosa both strains, i.e., Thermus and Meiothermus, are Biosafety level 1 microorganisms and are not pathogenic to humans. 相似文献
17.
The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through
cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the
assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the
application of genetically engineered micro-organisms. 相似文献
18.
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. 相似文献
19.
The bacterial strain Bacillus subtilis UTM 126 produced antimicrobial activity against pathogenic Vibrio species, including V. alginolyticus, V. parahaemolyticus, and V. harveyi. The probiotic effect of B. subtilis was tested by feeding juvenile shrimp (Litopenaeus vannamei) food supplemented with B. subtilis (105 CFU/g) for 28 days before an immersion challenge with V. harveyi at 105 CFU/mL for 24 h. The treatment with B. subtilis UTM 126 decreased final mortality to 18.25%, compared with 51.75% in the control group. Bacillus subtilis UTM 126 has potential applications for controlling pathogenic V. harveyi in shrimp aquaculture. 相似文献
20.
Durban MA Silbersack J Schweder T Schauer F Bornscheuer UT 《Applied microbiology and biotechnology》2007,74(3):634-639
Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for
cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into
B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed
by an acetoin-controlled expression system. For PLC516, 13.7 U g−1 wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted
in pure PLC516 with a specific activity of 13,190 U mg−1 protein. 相似文献