Co-degradation with glucose of four surfactants,CTAB, Triton X-100, SDS and Rhamnolipid,in liquid culture media and compost matrix

Strengthened biodegradation is one of the key means to treat surfactant pollution in environment, and microorganism and surfactant have significant effects on degradation. In this paper, co-degradation of CTAB, Triton X-100, SDS and rhamnolipid with glucose by Pseudomonas aeruginosa, Bacillus subtilis and compost microorganisms in liquid culture media, as well as the degradation of rhamnolipid in compost were investigated. The results showed that CTAB was recalcitrant to degrade by the three microorganisms and it also inhibited microorganisms from utilizing readily degradable carbon source. Non-ionic surfactant Triton X-100 could also hardly be degraded, but it was not toxic to microorganisms and would not inhibit the growth of the microorganisms. Anion surfactant SDS had no toxicity to microorganisms and could be co-degraded as carbon source with glucose. Biosurfactant rhamnolipid was a kind of particular surfactant, which had no toxicity and could be degraded by Bacillus subtilis and compost microorganisms, while it could not be utilized by its producing bacterium Pseudomonas aeruginosa. Among these three bacteria, the compost consortium had the strongest degradation capacity on the tested surfactants due to their microorganisms’ diversity. In compost matrix rhamnolipid could be degraded during composting, but not preferentially utilized.     

Cloning and characterization of an ftsZ homologue from a bacterial symbiont of Drosophila melanogaster

A 1194 by open reading frame that codes for a 398 amino acid peptide was cloned from a gt11 library of Drosophila melanogaster genomic DNA. The predicted peptide sequence is very similar to three previously characterized protein sequences that are encoded by the ftsZ genes in Escherichia coli, Bacillus subtilis and Rhizobium meliloti. The FtsZ protein has a major role in the initiation of cell division in prokaryotic cells. Using a tetracycline treatment that eradicates bacterial parasites from insects, the ftsZ homologue has been found to be derived from a bacterium that lives within the strain. However, polymerase chain reaction (PCR) amplification of the gene from treated embryos suggests that it is not derived from a gut bacterium. Nevertheless, by amplifying and characterizing part of the 16S rRNA from this bacterium we have been able to demonstrate that it is a member of the genus Wolbachia, a parasitic organism that infects, and disturbs the sexual cycle of various strains of Drosophila simulans. We suggest that this ftsZ homologue is implicated in the cell division of Wolbachia, an organism that fails to grow outside the host organism. Sequence and alignment analysis of this ftsZ homologue show the presence of a potential GTP-binding motif indicating that it may function as a GTPase. The consequences of this function particularly with respect to its role in cell division are discussed.     

Cloning and expression of the PHA synthase genes phaC1 and phaC1AB into Bacillus subtilis

Summary Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1 and pBE2C1AB. The two plasmids were inserted into Bacillus subtilis DB104 and generated Bacillus subtilis/pBE2C1 and Bacillus subtilis/pBE2C1AB. The two recombinant strains were subjected to fermentation and showed PHA accumulation, the first reported example of medium-chain-length-PHA production in Bacillus subtilis. GC analysis identified the compound produced by Bacillus subtilis/pBE2C1 was a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer while that produced by Bacillus subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxydecanoate-co-hydroxydodecanoate (HB-HD-HDD) polymer. The results also showed that the recombinant B. subtilis could utilize the malt waste in the medium as a carbon source better than that of glucose and thus could substantially lower the cost of production of PHA.     

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.     

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.     

Endophytic and Biological Control Potential of Bacillus mojavensis and Related Species

The identity of a patented endophytic bacterium was established by 16S rRNA sequence analysis as a strain of Bacillus mojavensis, a recently erected species within one of the B. subtilis subgroups. This strain of B. mojavensis is antagonistic to the fungus Fusarium moniliforme, an endophytic mycotoxin-producing pathogen of maize and other plants. There are five other species within this subgroup: Bacillus amyloliquefaciens, B. atrophaeus, B. licheniformis, Brevibacterium halotolerans, Paenibacillus lentimorbus, and P. popilliae. The objectives of this research were to screen other isolates of B. mojavensis, B. subtilis, and the other closely related Bacillus species for endophytic colonizing capacity and to determine the in vitro antagonism to F. moniliforme in an effort to survey the distribution of these traits, which are desirable biological control qualities within the Bacillaceae. Antagonism was determined on nutrient agar, and endophytic colonization was established with maize plants following recovery of rifampin-resistant mutants generated from all strains used in the study. The study established that all 13 strains of B. mojavensis, isolated from major deserts of the world, endophytically colonized maize and were antagonists to F. moniliforme. The endophytic colonization of maize by B. subtilis and other species within this subgroup of the Bacillaceae varied, as did antagonism, to F. moniliforme. Thus, this study suggests that endophytic colonization is another characteristic of the species B. mojavensis. The endophytic habit and demonstrated antagonism to the test fungus indicate that isolates of this species might prove to be important biological control organisms where the endophytic habit is desired.     

Identification of lipopeptide antibiotics of a <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolate and their control of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> diseases in maize and wheat

Substances produced by Bacillus subtilis D1/2, a bacterium isolated from cultivated soil, were found to inhibit Fusarium graminearum. The antifungal activity of the bacterium was attributable to major extracellular lipopeptides isolated and identified as fengycins. Their synthesis was enhanced by casamino acids added to the culture medium. The unpurified cell-free spent medium elicited hemolysis with increasing concentration. Its application to field-cultivated maize and chamber-grown wheat suppressed gibberella ear rot and Fusarium head blight, respectively, when the plants were inoculated with F. graminearum macroconidia. The treatment of maize ears consistently arrested ear-rot development, while the treatment of wheat spikes retarded the progress of Fusarium head blight. Although the deoxynivalenol and ergosterol contents of treated maize kernels were halved, they remained high because of the experimental requirement to inoculate with a high number (1.5 × 10⁴) of macroconidia. As a potential antifungal agent for controlling Fusarium diseases, B. subtilis D1/2 can be further developed as a useful component of integrated pest management. Handling Editor: Reijo Karjalainen.     

Seasonal variation in allelopathic potential of<Emphasis Type="Italic">Artemisia princeps</Emphasis> var.<Emphasis Type="Italic">orientalis</Emphasis> on plants and microbes

We investigated seasonal variations in allelopathic potential ofArtemisia princeps var.orientalis. Aqueous and meth-anol extracts and volatile substances were prepared in the laboratory from samples collected monthly (April through October). Their impacts were then assessed on the germination and seedling growth ofLactuca sativa andAchyranthes japonica. The allelopathic potential varied with the time of sample collection and the concentration tested. For example, germination ofL. sativa was not inhibited by the aqueous extract but seedling growth (shoots and roots) was, with its seasonal effect being significant. ForA. japonica, seed germination was not inhibited at lower concentrations (except for August samples). However, at higher concentrations and in certain months (especially July), germination was more negatively affected. The degree of seedling growth inhibition also differed by month and by extract concentration, with roots being impacted more than shoots. Volatile substances also had a time-dependent influence on the germination and seedling elongation ofA. japonica. In a separate experiment, the ethyl-acetate and water fractions of a crude methanol extract were prepared monthly fromA. princeps var.orientalis. Here, we examined their antimicrobial activities against three gram-positive bacteria (Bacillus cereus, Bacillus subtilis, andStaphylococcus aureus), two gramnegative bacteria (Escherichia coli andPseudomonas fluorescens), and one lactic acid bacterium,Lactobacillus plantar urn. The ethyl-acetate fraction that was sampled in September was remarkably potent againstB. cereus andB. subtilis, whereas the water fraction collected in August and September showed great antimicrobial activity against the grampositive and -negative bacteria. In contrast,L. plantarum was not inhibited by the water fraction, regardless of the sampling month. Likewise, the ethyl-acetate and water fractions collected in April and October had the lowest levels of antimicrobial activity.     

椿根皮的化学成分及抑菌活性研究

为探寻椿根皮抑菌的物质基础，该研究采用硅胶、Sephadex LH-20等方法对椿根皮甲醇提取物进行分离和纯化，通过理化性质和波谱数据分析单体化合物的结构，并以卡那霉素为对照组采用流式细胞法测试化合物的抑菌活性。结果表明：从椿根皮中得到22个化合物，分别鉴定为pleuchiol (1)、withastramonolide (2)、7-ketositosterol (3)、白桦酯醇(4)、桦木酸甲酯(5)、1, 2, 4-trimethoxybenzene (6)、顺丁烯二酸二甲酯(7)、sonderianol (8)、dibutyl phthalate (9)、pinoresinol (10)、对羟基苯甲酸乙酯(11)、avenalumic acid methyl ester (12)、5,3′-dihydroxy-3,7,4′-trimethoxy-flavone (13)、spathulenol (14)、2-甲基-5-丙基酮-7-羟基色原酮(15)、 7,4′-dihydroxyflavone (16)、annphenone (17)、3-羟基-4-甲氧基苯甲酸(18)、5,3′...     

Production of a lipopeptide antibiotic surfactin with recombinantBacillus subtilis

Summary Production of a lipopeptide antibiotic surfactin was carried out using a recombinantBacillus subtilis. Surfactin yield of the recombinant strain was about one and half times as much as that ofBacillus subtilis RB 14, the strain in which the surfactin gene was originated. This system is especially noteworthy because a recombinant strain surpassed the original strain in the production of a bacterial antibiotic as a secondary metabolite of the bacterium.     

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。     

Enhancement of γ-aminobutyric acid production in Chungkukjang by applying a Bacillus subtilis strain expressing glutamate decarboxylase from Lactobacillus brevis

For a foreign glutamate decarboxylase (GAD) to be expressed in Bacillus host system, a recombinant DNA (pLip/LbGAD) was constructed by ligating an LbGAD gene from Lactobacillus brevis OPK-3 into Escherichia coli–Bacillus shuttle vector, pLip. The pLip/LbGAD construct was then transformed into Bacillus subtilis. The culture of the transformed Bacillus strain with the pLip/LbGAD construct had higher GAD activity and γ-aminobutyric acid (GABA) concentration than those of untransformed Bacillus counterpart. In addition, Chungkukjang, a traditional Korean fermented soybean product prepared by the transformed Bacillus subtilis, contained a significantly higher level of GABA than conventional ones. Thus, by introducing a foreign GAD gene, Bacillus strains have been genetically engineered to produce high levels of GAD and GABA.     

Changes in the antibiotic production by co-culture of Rhizopus peka P8 and Bacillus subtilis IFO3335

The co-culture of Bacillus subtilis IFO 3335 with Rhizopus peka P8 or Rhizopus oligosporus P12 in liquid medium was found to increase production of antibiotic activity and to alter the spectrum of activity relative to the pure cultures. However, a mixed culture of Rhizopus arrhizus P7 and Rhizopus oryzae P17 did not produce antibiotic activity. The concentration, ratio, and time of addition of B. subtilis to the R. peka culture was found to influence antibiotic yields. Solid-state fermentations using mixed cultures of R. peka and B. subtilis were investigated. The growth of Escherichia coli IFO 3792 as a target bacterium was inhibited by the mixed culture. These results suggest the possibility of biopreservation of fermented foods by novel co-culture systems.     

The effects of Bacillus subtilis on Caenorhabditis elegans fitness after heat stress

Microbes can provide their hosts with protection from biotic and abiotic factors. While many studies have examined how certain bacteria can increase host lifespan, fewer studies have examined how host reproduction can be altered. The nematode Caenorhabditis elegans has been a particularly useful model system to examine how bacteria affect the fitness of their hosts under different contexts. Here, we examine how the bacterium Bacillus subtilis, compared to the standard C. elegans lab diet, Escherichia coli, affects C. elegans survival and reproduction after experiencing a period of intense heat stress. We find that under standard conditions, nematodes reared on B. subtilis produce fewer offspring than when reared on E. coli.However, despite greater mortality rates on B. subtilis after heat shock, young adult nematodes produced more offspring after heat shock when fed B. subtilis compared to E. coli. Because offspring production is necessary for host population growth and evolution, the reproductive advantage conferred by B. subtilis supersedes the survival advantage of E. coli. Furthermore, we found that nematodes must be reared on B. subtilis (particularly at the early stages of development) and not merely be exposed to the bacterium during heat shock, to obtain the reproductive benefits provided by B. subtilis. Taken together, our findings lend insight into the importance of environmental context and interaction timing in shaping the protective benefits conferred by a microbe toward its host.     

Construction of a bifunctional genetically labelled plasmid for Bacillus thuringiensis subsp. israelensis

A small cryptic plasmid of Bacillus thuringiensis subsp. israelensis was labelled in vitro with two genetic markers. One of the recombinant plasmids was mapped and transformed in Escherichia coli, Bacillus subtilis and Bacillus thuringiensis. This and similar shuttle plasmids could be very useful as vectors for the investigation of the toxin genes in their own host.Abbreviations BTI Bacillus thuringiensis subsp. israelensis - MDal megadaltons     

Isolation and partial purification of a novel type II restriction endonuclease Bsu121 I,from Bacillus subtilis – Bsu121I,a type II restriction endonuclease from Bacillus subtilis

A new type II restriction endonuclease which we designated as Bsu121I has been isolated from gram-positive bacterium Bacillus subtilis strain 121 and partially purified. The restriction endonuclease was isolated from cell extracts using step-wise purification through ammonium sulfate precipitation, followed by phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights (23 and 67 kDa) of the endonuclease. The partially purified enzyme restricted pBR322 DNA into two fragments of 3200 and 1700 bp. The endonuclease activity required Mg⁺² as cofactor like other type II endonucleases.     

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.     

The roles of signal peptide and mature protein in RNase (barnase) export from Bacillus subtilis

Barnase, an extracellular RNAse from Bacillus amyloliquefaciens is secreted post-translationally from B. subtilis. The rate of secretion of barnase from B. subtilis was improved by replacement of the barnase signal peptide with a heterologous signal peptide. However, the barnase signal peptide exported Escherichia coli alkaline phosphatase faster than mature barnase. Heat shock of B. subtilis cells did not significantly alter the export of barnase using the barnase signal peptide. The slow rate of export of barnase from B. subtilis is due to both the signal peptide and the mature protein sequence rather than either alone.     

Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis

Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis. They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns. Protease I was found to be similar to an already characterized B. subtilis protease. Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors. While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times. Distinct functions for these proteases in post exponential B. subtilis are likely.     

On the Problem of Genome Redundancy in Viruses and Prokaryotes

A specific index of nucleotide sequence redundancy, the specific restriction length of a finite frequency dictionary, was determined for a complete set of genes in some viral genomes and a genome of a bacterium, Bacillus subtilis. The distribution of the gene number over the specific restriction length was shown to be bimodal for viral genomes and unimodal for the Bac. subtilis genome. These results agree with earlier data.