Detecting Surfactant-producing Microorganisms by the Drop-collapse Test

Summary A rapid method, ’drop-collapse’, was used for screening biosurfactant production by Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Phanerochaete chrysosporium liquid cultures. Before measuring the total biosurfactant, the drop-collapse method was used in order to detect rhamnolipid presence in the culture broths. The method was performed in a microwell plate; the polystyrene platform with small wells. If the culture broth contained biosurfactant, the droplets of the broth in the oil-coated wells collapsed. If not, there was no change in the shape of the droplets. Pseudomonas aeruginosa and Bacillus subtilis culture supernatants showed spreading movement, meaning that they produced biosurfactants. However, Candida albicans and Phanerochaete chrysosporium supernatants remained beaded, meaning they did not produce any type of microbial surfactant.     

Cloning of the gene <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis> chitinase <Emphasis Type="Italic">Lpchi1</Emphasis> and identification of its potential role in the biocontrol of root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

The complete genome sequence of Bacillus subtilis reveals that sequences encoding several hemicellulases are co-localised with a gene (xynD) encoding a putative family 43 glycoside hydrolase that has not yet been characterised. In this work, xynD has been isolated from genomic DNA of B. subtilis subsp. subtilis ATCC 6051 and cloned for cytoplasmatic expression in Escherichia coli. Recombinant XynD (rXynD) was purified using ion-exchange chromatography and gel permeation chromatography. The enzyme had a molecular mass of approximately 52 kDa, a pI above 9.0 and releases α-l-arabinose from arabinoxylo-oligosaccharides as well as arabinoxylan polymers with varying degree of substitution. Using para-nitrophenyl-α-l-arabinofuranoside as substrate, maximum activity was observed at pH 5.6 and 45°C. The enzyme retained its activity over a large pH range, while activity was lost after pre-incubation above 50°C. Gas–liquid chromatography and proton nuclear magnetic resonance spectrometry analysis indicated that rXynD specifically releases arabinofuranosyl groups from mono-substituted C-(O)-2 and C-(O)-3 xylopyranosyl residues on the xylan backbone. As rXynD did not display endoxylanase, xylosidase or arabinanase activity and was inactive on arabinan, we conclude that this enzyme is best described as an arabinoxylan arabinofuranohydrolase.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

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.     

Protein Profile of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Spore

Natural wild-type strains of Bacillus subtilis spore is regarded as a non-pathogenic for both human and animal, and has been classified as a novel food which is currently being used as probiotics added in the consumption. To identify B. subtilis spore proteins, we have accomplished a preliminary proteomic analysis of B. subtilis spore, with a combination of two-dimensional electrophoretic separations and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI–TOF–MS). In this article, we presented a reference map of 158 B. subtilis spore proteins with an isoelectric point (pI) between 4 and 7. Followed by mass spectrometry (MS) analysis, we identified 71 B. subtilis spore proteins with high level of confidence. Database searches, combined with hydropathy analysis and GO analysis revealed that most of the B. subtilis spore proteins were hydrophilic proteins related to catalytic function. These results should accelerate efforts to understand the resistance of spore to harsh conditions.     

NMR assignment of the N-terminal repeat domain of Bacillus subtilis ClpC

The HSP100/AAA+ superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. We present here the backbone and side-chain assignments of the N-terminal repeat domain (residues 1–145) of ClpC from Bacillus subtilis. Douglas J. Kojetin and Patrick D. McLaughlin have equally contributed.     

Phenotypic diversity and technological properties of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> species isolated from <Emphasis Type="Italic">okpehe</Emphasis>, a traditional fermented condiment

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.     

Modeling the inactivation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> spores by ethylene oxide processing

Ethylene oxide is currently a dominant agent in medical device sterilization. This work intends to study the main effects and interactions of temperature, ethylene oxide concentration, and relative humidity on commercial spore strips of Bacillus subtilis, var. niger (ATCC 9372) inactivation, the most common microorganism used in controlling the efficacy of the process. Experiments were carried out using a full factorial experimental design at two levels (2³ factorial design). Limit target exposure conditions for ethylene oxide concentration, temperature, and relative humidity were 250–1,000 mg EO/l, 40–60°C, and 50–90%, respectively. Adopting a different approach from the first-order kinetics, a Gompertz model was successfully applied in data fitting of the inactivation curves. Bacillus subtilis kinetic behavior presented a sigmoidal inactivation with an initial shoulder (λ), followed by a maximum inactivation rate (k_max), these being model parameters. It was concluded that temperature and ethylene oxide concentration were the most significant factors and consequently, additional experiments were carried out aiming at describing the parameters' dependence on these process factors. Mathematical relations describing such dependences were successfully developed and included in the Gompertz kinetic model. The predictive ability of this integrated model was assessed, and its adequacy in predicting B. subtilis inactivation was proven.     

碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达

【背景】碱性蛋白酶是工业用酶中占比最大的酶类,广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力,这将进一步拓宽其在保健食品领域中的应用。【目的】利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。【方法】通过筛选3种枯草芽孢杆菌宿主菌株(Bacillus subtilis 1A751、MA07、MA08)和6种信号肽(AmyE、AprE、NprE、Pel、YddT、YoqM),同时优化诱导剂浓度、发酵培养基和发酵时长,最终得到最优重组菌株MA08-AmyE-subCopt。【结果】重组菌株MA08-AmyE-subCopt的胞外酶活力为3.33×103 AU/mL,胞外蛋白分泌量为胞内可溶蛋白表达量的4倍,与携带野生型信号肽的对照组菌株WT相比,酶活提高了73.4%。【结论】异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达,为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。     

Overexpression,purification and characterization of a recombinant secretary catalase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

A recombinant Bacillus subtilis strain (KN25) was generated for the large-scale preparation of catalase. The B. subtilis katA gene encoding for catalase was cloned into the shuttle vector PRB374, downstream of the constitutively active vegII promoter, followed by transformation of the B. subtilis strain WB600 with the plasmid. The transformant strain, KN25 secretes high levels (3,500 U/ml) of catalase, which facilitates its purification. Three simple purification steps yielded nearly homogeneous catalase, with ∼70% recovery. The purified recombinant catalase has a specific activity of 34,600 U/mg under optimal conditions, and is more resistant to acidic conditions than bovine liver catalase.     

Differentiated pellicle organization and lipopeptide production in standing culture of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains

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.     

DNA sequence analysis of a putative primary replicon from a cryptic plasmid pNM214 of a hydrocarbon utilizing marine <Emphasis Type="Italic">Bacillus</Emphasis> strain NM21

Marine Bacillus strain NM21 isolated from hydrocarbon-contaminated site at Naval Harbour, Mumbai grows on high-speed diesel as a source of carbon and energy. This bacterium harbours four plasmids in it. The smallest plasmid, pNM214 was digested with EcoRI enzyme and cloned in pUC19 vector. The clone Om4 containing largest insert of >3.5 kb was sequenced by primer walking. DNA sequence analysis showed this fragment to be homologous to replication initiation protein (rep) gene and dso (double strand origin) of different plasmids from Bacillus subtilis and Bacillus pumilus species. The putative rep gene sequence of pNM214 showed 74.3–91.6% DNA identity to B. subtilis plasmids (pTA1015, pTA1060 and pTA1040) and 86.3% to 88.9% DNA identity to B. pumilus plasmids (pPL7065, pPL10 and pSH1452). The translated amino acid sequence of rep shows that it contains all the three conserved motifs present in the Rep protein of pC194 family of plasmids. DNA sequence comparison of putative dso of pNM214 with other bacillus plasmids belonging to pC194 group shows that it contains highly conserved nick site sequence 5′-TCTTTTCTTATCTTGATA-3′ and surrounding inverted repeats. Thus, it indicates that pNM214 to be a rolling circle replicating plasmid belonging to the pC194 group. The presence of rep and dso like sequences in the sequenced EcoRI fragment indicate that the cloned fragment contain putative primary replicon of pNM214.     

Enrichment, isolation and characterization of pentachlorophenol degrading bacterium Acinetobacter sp. ISTPCP-3 from effluent discharge site

Three pentachlorophenol (PCP) degrading bacterial strains were isolated from sediment core of pulp and paper mill effluent discharge site. The strains were continuously enriched in mineral salts medium supplemented with PCP as sole source of carbon and energy. One of the acclimated strains with relatively high PCP degradation capability was selected and characterized in this study. Based on morphology, biochemical tests, 16S rDNA sequence analysis and phylogenetic characteristics, the strains showed greatest similarity with Acinetobacter spp. The strain was identified as Acinetobacter sp. ISTPCP-3. The physiological characteristics and optimum growth conditions of the bacterial strain were investigated. The results of optimum growth temperature revealed that it was a mesophile. The optimum growth temperature for the strain was 30°C. The preferential initial pH for the strain was ranging at 6.5–7.5, the optimum pH was 7. The bacterium was able to tolerate and degrade PCP up to a concentration of 200 mg/l. Increase in PCP concentration had a negative effect on biodegradation rate and PCP concentration above 250 mg/l was inhibitory to its growth. Acinetobacter sp. ISTPCP-3 was able to utilize PCP through an oxidative route with ortho ring-cleavage with the formation of 2,3,5,6-tetrachlorohydroquinone and 2-chloro-1,4-benzenediol, identified using gas chromatograph–mass spectrometric (GC–MS) analysis. The degradation pathway followed by isolated bacterium is different from previously characterized pathway.     

Levels and localization of mechanosensitive channel proteins in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Log phase Bacillus subtilis cells lacking the mscL gene encoding the mechanosensitive (MS) channel of large conductance are sensitive to an osmotic downshock ≥0.5 M. However, B. subtilis mscL cells develop osmotic downshock resistance in late log and early stationary phase growth that is partially dependent on three likely MS channel proteins of small conductance (MscS), YfkC, YhdY, and YkuT. Bacillus subtilis MS proteins were fused with green fluorescent protein (GFP) at their C termini; at least the MscL-, YfkC-, and YkuT-GFP fusions were functional and overexpression of YkuT-GFP, or YkuT alone abolished log phase mscL cells’ osmotic downshock sensitivity. Western blot analysis found high levels of MscL-GFP in early exponential phase cells with levels subsequently decreasing greatly. MscS-GFP proteins were present in exponential phase cells, but again disappeared almost completely in stationary phase cells and these proteins were not detected in spores. Western blot analyses further showed that MS-GFP proteins were associated with the plasma membrane, as expected. Fluorescence microscopy confirmed the localization of MscL-GFP and YhdY-GFP to the plasma membrane, with non-uniform distribution of these proteins along this membrane consistent with but by no means proving that these proteins are present in a helical array.     

Expression and secretion of a single-chain sweet protein monellin in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by <Emphasis Type="Italic">sacB</Emphasis> promoter and signal peptide

The sweet protein monellin gene was expressed in Bacillus subtilis under the control of the Bacillus subtilis sacB promoter and signal peptide sequence. A 294-bp DNA fragment, coding for sweet protein monellin, was ligated into the Escherichia coli/B. subtilis shuttle vector pHPC, producing pHPMS, which was subsequently transformed into B. subtilis QB1098, DB104, and DB403. The peptide efficiently directed the secretion of monellin from the recombinant B. subtilis cells. A maximum yield of monellin of 0.29 g protein l⁻¹ was obtained from the supernatant of B. subtilis DB403 harboring pHPMS. SDS-PAGE confirmed the purity of the recombinant product.     

原油污染土壤中生物表面活性剂菌株的筛选和产物鉴定

【目的】从原油污染的土壤中分离出产表面活性剂的枯草芽孢杆菌(Bacillus subtilis)SX-20,并对其产物进行提取及结构分析。【方法】采用氯化十六烷基吡啶和溴百里酚蓝混合溶液(cetylpyridinium chloride-bromothymol blue,CPC-BTB)显色反应结合血琼脂平板简单高效的筛选得到产脂肽的枯草芽孢杆菌。通过酸沉淀、甲醇萃取和旋转蒸发提取发酵所产生的粗产物,该产物对痤疮丙酸杆菌(Propionibacterium acnes)具有良好的抑制作用。运用傅里叶红光变换光谱(Fourier transform infrared spectroscopy,FTIR)、氨基酸分析和液相质谱联用(liquid chromatography-mass spectrometry,LC-MS)对粗产物的成分进行分析。【结果】筛选所得的菌株所产物质是含C15脂肪酸链和7个氨基酸形成的环状的脂肽表面活性剂。【结论】本研究为筛选脂肽类生物表面活性剂提供了一定的理论基础和技术路线,有利于后续获得高产的低成本的生物表面活性剂。     

Formulation of stable <Emphasis Type="Italic">Bacillus subtilis</Emphasis> AH18 against temperature fluctuation with highly heat-resistant endospores and micropore inorganic carriers

To survive the commercial market and to achieve the desired effect of beneficial organisms, the strains in microbial products must be cost-effectively formulated to remain dormant and hence survive through high and low temperatures of the environment during transportation and storage. Dormancy and stability of Bacillus subtilis AH18 was achieved by producing endospores with enhanced heat resistance and using inorganic carriers. Heat stability assays, at 90°C for 1 h, showed that spores produced under a sublethal temperature of 57°C was 100 times more heat-resistant than the ones produced by food depletion at the growing temperature of 37°C. When these highly heat-resistant endospores were formulated with inorganic carriers of natural and synthetic zeolite or kaolin clay minerals having substantial amount of micropores, the dormancy of the endospores was maintained for 6 months at 15–25°C. Meanwhile, macroporous perlite carriers with average pore diameter larger than 3.7 μm stimulated the germination of the spores and rapid proliferation of the bacteria. These results indicated that a B. subtilis AH18 product that can remain dormant and survive through environmental temperature fluctuation can be formulated by producing heat-stressed endospores and incorporating inorganic carriers with micropores in the formulation step.     

Synergistic biodegradation of pentachlorophenol by <Emphasis Type="Italic">Bacillus cereus</Emphasis> (DQ002384), <Emphasis Type="Italic">Serratia marcescens</Emphasis> (AY927692) and <Emphasis Type="Italic">Serratia marcescens</Emphasis> (DQ002385)

The consortium of Bacillus cereus (DQ002384), Serratia marcescens (AY927692) and Serratia marcescens (DQ002385) were used for pentachlorophenol (PCP) degradation. The consortia showed better overall removal efficiencies than single strains by utilization of PCP as a carbon and energy source confirmed by pH dependent dye indicator bromocresol purple (BCP) in mineral salt media (MSM). Mixed culture was found to degrade up to 93% of PCP (300 mg/l) as compared to single strains (62.75–90.33%), at optimized conditions (30 ± 1°C, pH 7 ± 0.2, 120 rpm) at 168 h incubation. PCP degradation was also recorded at 20°C (62.75%) and 37°C (83.33%); pH 6 (70%) and pH 9 (75.16%); 50 rpm (73.33%) and 200 rpm (91.63%). The simultaneous release of chloride ion up to 90.8 mg/l emphasized the bacterial dechlorination in the medium. GC–MS analysis revealed the formation of low molecular weight compound, i.e., 6-chlorohydroxyquinol, 2,3,4,6-tetrachlorophenol and tetrachlorohydroquinone, from degraded sample as compared to control.     

Bactericidal Effect of ADP and Acetic Acid on Bacillus subtilis

Bacillus subtilis is a ubiquitous soil bacterium used for measuring the β-lysin activity and in other bioassays. We observed a complete bactericidal effect of ADP on B. subtilis at concentrations of 50–100 μM at pH values <5.5, which disappeared at pH values above 6. The effect was also found for acetic acid at concentrations >17.4 μM and similar pH values. ATP, adenosine, and HCl were not bactericidal. We used BCECF-AM, a pH-sensitive probe, and found that the killing of B. subtilis was due to a change in the intracellular pH caused by the passage across the cell membrane of these weak organic acids when incubated with B. subtilis at pH values near the pK. More experiments are needed to determine the biological meaning of these in vitro findings. Received: 14 June 1996 / Accepted: 19 July 1996     

Construction of a new food-grade expression system for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> based on theta replication plasmids and auxotrophic complementation

A new food-grade expression system was constructed for Bacillus subtilis based on replicative food-grade expression plasmids and auxotrophic complementation. The food-grade B. subtilis host FG01 was created by knockout of the dal locus from the chromosome of B. subtilis 168. Two food-grade expression plasmids pXFGT03 and pXFGT05 were constructed by combining a novel theta-type Bacillus replicon with the B. subtilis endogenous gene dal and P43 promoter; while pXFGT05 was derived from pXFGT03 by deletion of two open reading frames (ORFs) from the original replicon. Upon transformation of FG01 with pXFGT03 or pXFGT05, the host phenotype was complemented on Luria–Bertani agar plates by the plasmid-coded dal gene, which served as a food-grade selection marker for recombinants. Results showed that deletion of the two ORFs had no impact on plasmid replication. A reporter gene bgaB was cloned into pXFGT03 and pXFGT05, respectively, under control of the P43 promoter, and it was successfully expressed in this food-grade expression system. Segregational stabilities of two recombinant plasmids were investigated, and they were fully stable.     

Antioxidant and antimicrobial actions of the clubmoss <Emphasis Type="Italic">Lycopodium clavatum</Emphasis> L.

Purpose of the present study was to evaluate antioxidant, antibacterial, antifungal, and antiviral activities of the petroleum ether, chloroform, ethyl acetate and methanol extracts as well as the alkaloid fraction of Lycopodium clavatum L. (LC) from Lycopodiaceae growing in Turkey. Antioxidant activity of the LC extracts was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method at 0.2 mg/ml using microplate-reader assay. Antiviral assessment of LC extracts was evaluated towards the DNA virus Herpes simplex (HSV) and the RNA virus Parainfluenza (PI-3) using Madin-Darby Bovine Kidney (MDBK) and Vero cell lines. Antibacterial and antifungal activities of the extracts were tested against standard and isolated strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis as well as the fungi; Candida albicans and C. parapsilosis. All of the extracts possessed noteworthy activity against ATCC strain of S. aureus (4 μg/ml), while the LC extracts showed reasonable antifungal effect. On the other hand, we found that only the chloroform extract was active against HSV (16–8 μg/ml), while petroleum ether and alkaloid extracts inhibited potently PI-3 (16–4 μg/ml and 32–4 μg/ml, respectively). However, all of the extracts had insignificant antiradical effect on DPPH. In addition, we also analyzed the content of the alkaloid fraction of the plant by capillary gas chromatography-mass spectrometry (GC-MS) and identified lycopodine as the major alkaloid.