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.     

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.     

Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences

Partial gyrA sequences were determined for twelve strains belonging to Bacillus amyloliquefaciens, B. atrophaeus, B. licheniformis, B. mojavensis,B. subtilis subsp. subtilis, B. subtilissubsp. spizizenii and B. vallismortis. The average nucleotide and translated amino acid similarities for the seven type strains were 83.7 and 95.1%, respectively, whereas the corresponding value for the 16S rRNA sequences was 99.1%. All of the type strains were sharply separated; the closest relationship was found between B. atrophaeus and B. mojavensis which shared a nucleotide similarity of 95.8%. Phylogenetic trees were inferred from gyrA nucleotide sequences using the neighbor-joining, Fitch–Margoliash and maximum parsimony algorithms. The test strains were divided into four groups, which generally reflected results previously reported in restriction digest and DNA-DNA hybridization studies. It is concluded from the comparative sequence analysis that the gyrA sequences provide a firm framework for the rapid and accurate classification and identification of Bacillus subtilis and related taxa.     

枯草芽孢杆菌中高效响应N-乙酰神经氨酸生物传感器的构建

枯草芽孢杆菌(Bacillus subtilis)是公认的食品安全菌株,目前已被用于多种高附加值产品的生物合成,包括被广泛用作营养化学品和药物中间体的N-乙酰神经氨酸(N-acetylneuraminic acid, NeuAc)。响应目标产物的生物传感器被广泛用于代谢工程中的动态调控和高通量筛选等方面,以提高生物合成效率。但是,枯草芽孢杆菌中缺乏可高效响应NeuAc的生物传感器。因此,本文首先测试和优化了能将胞外NeuAc转运进胞内的转运蛋白,获得了一系列具有不同转运能力的菌株,以用于后续响应NeuAc的生物传感器的验证;随后将响应NeuAc的转录因子Bbr_NanR的结合位点插入枯草芽孢杆菌组成型启动子的不同位置,筛选具有活性的杂合启动子;接下来,通过在具有NeuAc转运能力的枯草芽孢杆菌中表达Bbr_NanR,选择能响应NeuAc的杂合启动子,并进一步通过优化Bbr_NanR表达量获得了一系列动态范围广、激活倍数高的生物传感器,其中生物传感器P535-N2能灵敏地响应胞内NeuAc浓度的变化,具有最大的动态范围,为(180–20 245) AU/OD;P566-N2则具有最高的激活倍数,为122倍,是已报道的枯草芽孢杆菌中响应N-乙酰神经氨酸的生物传感器的2倍。本文构建的响应NeuAc的生物传感器可用于高产NeuAc的酶突变体和枯草芽孢杆菌菌株的筛选,为枯草芽孢杆菌生物合成NeuAc提供了高效、灵敏的分析和调控工具。     

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.     

Biodegradation of native feather keratin by Bacillus subtilis recombinant strains

A mixed culture containing two recombinant Bacillus subtilis strains; was used to hydrolyze 1% chicken feather; both were previously transformed with late-expressed and early expressed alkaline protease (aprE) carrying plasmids pS1 and p5.2, respectively. Proteolytic and keratinolytic activities of the mixed culture increased in parallel with those of the culture of B. subtilis DB100 (p5.2), and both were higher than that of B. subtilis (pS1) cultures. On the other hand, data indicated that degradation of feather by the recombinant strains B. subtilis DB100 (p5.2), was greatly enhanced when using a previously optimized medium. High levels of free amino groups as well as soluble proteins were also obtained. The concentration of amino acids was considerably increased during the fermentation process. It was found that, the amino acids Phe, Gly and Tyr were the major amino acids liberated in the cultures initiated by both strains. Results render these recombinant strains suitable for application in feather biodegradation large scale processes.     

Discrimination of riboflavin producing Bacillus subtilis strains based on their fed-batch process performances on a millilitre scale

Forty-eight single-use stirred tank bioreactors on a 10-mL scale operated in a magnetically inductive driven bioreaction block and automated with a liquid handler were applied for discrimination of different riboflavin producing Bacillus subtilis strains based on their performances in the parallel fed-batch processes. It was shown that a discrimination of the B. subtilis riboflavin producer strains can efficiently be achieved within one parallel fermentation run based on the integral riboflavin yield after 48 h. The possibility to perform replicates within the parallel fermentation run allows for a robust statistical analysis and is a prerequisite for the discrimination of producer strains under fed-batch process conditions. Within the estimation error, all of the riboflavin producing B. subtilis strains under study showed the same fed-batch process performances on the litre scale compared to the millilitre scale.     

The Structure of the Transposable Genetic Element ISBsu2 from the Cryptic Plasmid p1516 of a Soil Bacillus subtilis Strain and the Presence of Homologues of This Element in the Chromosomes of Various Bacillus subtilis Strains

A cryptic plasmid from a soil strain of Bacillus subtiliswas found to contain a sequence having features of an IS element. Homologous sequences were also found in the chromosome of this strain and in the chromosomes of some other B. subtilis strains.     

Effect of Carbon Source on the Antimicrobial Activity of the Air Flora

Summary In an attempt to screen for air flora producing new potent antimicrobial substances, Bacillus megaterium NB-3, Bacillus cereus NB-4, Bacillus cereus NB-5, Bacillus subtilis NB-6 and Bacillus circulans NB-7, were isolated and were found to be antagonistic to bacteria and/or fungi. Production of antimicrobial substances by the bacterial strains was greatly influenced by variation of carbon sources. Glycerol strongly enhanced the antimicrobial activity of strains NB-3 and NB-6, whereas glucose increased the antimicrobial activity of strains NB-4 and NB-5. The maximum antibiotic yield of NB-7 was achieved with fructose as a carbon source. Starch (Bacillus megaterium NB-3), maltose (Bacillus cereus NB-5), glycerol (Bacillus circulans NB-7), arabinose, ribose (Bacillus cereus NB-4) and arabinose, fructose, glucose, ribose and sucrose (Bacillus subtilis NB-6) repressed the production of antimicrobial substances by the respective bacterial strains.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Determination of poly-β-hydroxybutyrate (PHB) production by some <Emphasis Type="Italic">Bacillus</Emphasis> spp.

In this study, 29 strains of the genus Bacillus were isolated from different soil samples which were taken from grasslands of Ankara, Turkey and were identified as B. brevis, B. sphaericus, B. cereus, B. megaterium, B. circulans, B. subtilis, B. licheniformis and B. coagulans. Two strains, B. sphaericus ATCC 14577 and B. subtilis ATCC 6633 were also included in this study. Poly-β-hydroxybutyrate (PHB) production by these strains was determined by the spectrophotometric method, and it was found that PHB production ranged from 1.06–41.67% (w/v) depending on the dry cell weight. The highest PHB production and productivity percentage was found in B. brevis M6 (41.67% w/v).     

Secretory expression in Escherichia coli and Bacillus subtilis of human interferon alpha genes directed by staphylokinase signals

Summary A DNA segment covering the signal sequence coding region, the ribosome binding site, and the promoter of the staphylokinase (sak) 42D gene (Behnke and Gerlach 1987) was cloned into pUC19 to form a portable expression-secretion unit (ESU). Fusion of human interferon α₁ (hIFNα₁) and hybrid hIFNα_1/2 genes to thissak ESU resulted in secretory expression of the two gene products in bothEscherichia coli andBacillus subtilis. While most of the IFNα was exported to the periplasmic space ofE. coli, about 99% was secreted to the culture medium by recombinantB. subtilis strains. The total yield inE. coli was 1.2×10⁵ IU/ml. This level of expression and export led to instability of the recombinant strains that was spontaneously relieved in vivo by inactivation of thesak ESU through insertion of an IS1 element. No such instability was observed withB. subtilis although expression and secretion levels reached even 3×10⁶ IU/ml. Proteolytic degradation of IFNα by extracellular proteases was avoided by a combination of constitutive expression and secretion during the logarithmic growth phase and the use of exoprotease-reduced host strains. The IFNα₁ protein purified fromB. subtilis culture supernatant was correctly processed, carried the expected 11 amino acid N-terminal elongation that resulted from DNA manipulations and proved to be homogenous in Western blotting experiments. The same recombinant plasmid that directed efficient secretion of hIFNα₁ inB. subtilis gave poor yields when introduced intoStreptococcus sanguis.     

Desulfurization of dibenzothiophene by Bacillus subtilis recombinants carrying dszABC and dszD genes

The desulfurization (dsz) genes from Rhodococcus erythropolis DS-3 were successfully integrated into the chromosomes of Bacillus subtilis ATCC 21332 and UV1 using an integration vector pDGSDN, yielding two recombinant strains, B. subtilis M29 and M28 in which the integrated dsz genes were expressed efficiently under the promoter, Pspac. The dibenzothiophene (DBT) desulfurization efficiency of M29 was 16.2 mg DBT l⁻¹ h⁻¹ at 36 h, significantly higher than that of R. erythropolis DS−3 and B. subtilis M28 and also showed no product inhibition. The interfacial tension of the supernatant fermented by M29 varied from 48 mN m⁻¹ to 4.2 mN m⁻¹, lower than that of the recombinant strain, M28, reveals that the biosurfactant secreted from M29 may have an important function in the DBT desulfurization process.     

Cellulose Decomposition under Nitrogen Deficiency by Bacteria Isolated from the Intestines of Phytophagous Vertebrates

A nitrogen-fixing strain identified as Klebsiella pneumonia 402-2 and two endoglucanase-synthesizing Bacillus strains were isolated from the intestines of phytophagous animals. One of the Bacillus strains was identified as Bacillus subtilis GL. Klebsiella pneumoniae 402-2 increased the endoglucanase activity of both Bacillusstrains in mixed cultures.The data on the taxonomic position of strains 402-2 and GL and on the nitrogen-fixing capacity of strain 402-2 were confirmed by sequencing and analyzing their 16S rRNA genes and by amplifying the nitrogenase gene nifH.     

Impact of Bacillus subtilis JA, a biocontrol strain of fungal plant pathogens, on arbuscular mycorrhiza formation in Zea mays

Summary  In the present study, the influence of Bacillus subtilis JA on arbuscular mycorrhizal fungi (AMF) was evaluated by either pot culture or in vitro conditions, respectively. Under the pot culture conditions, the inoculation of B. subtilis JA decreased the frequency (% F) of the root colonization by indigenous arbuscular mycorrhizal fungi and the shoot dry weight of maize (Zea mays L.), but had no apparent effect on the intensity (% I) of AM fungal root colonization. The unknown volatile emitted from the B. subtilis JA in vitro significantly inhibited spore germination and the hyphal growth in the dual-compartment experiments. Moreover, the data from the direct interaction between B. subtilis JA and Glomus etunicatum showed that soluble antifungal lipopeptides influenced the development of AMF. Therefore, the application of antifungal Bacillus strains should take the compatibility with the indigenous beneficial fungi into consideration.     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.     

Phenotypic and biological properties of two antagonist <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains

Bacillus subtilis KB-1111 and KB-1122 were studied to illustrate their phenotypic and biological properties. Comparison of KB-1111 with KB-1122 in morphology was carried out by microscopy and agar plate assays. Biological assay of the test strains showed that they may possess different physiological pathways from those of reference strain ATCC6501. The assessment of antagonism against the indicator fungi showed that both test strains had broad antifungal characteristics against eight phytopathogenic fungi. Of those fungal species, Magnaporthe grisea P131, Sclerotinia sclerotiorum, and F. oxysporium exhibited high sensitivity to the test strains.     

Detailed identification of desert-originated bacteria carried by Asian dust storms to Japan

Several halotolerant bacteria were isolated from dust allowed to settle passively on saline medium in Higashi-Hiroshima, Japan during Asia dust events in 2005–2006. The primary identification, based on the sequence similarity of the 16S rRNA gene, revealed that these isolates were strains of Bacillus subtilis, B. licheniformis, Staphylococcus epidermidis, Gracillibacillus sp., and Halomonas venusta. A parallel investigation carried out on desert sand collected directly from sand dunes in Dunhuang, Gobi Desert, China resulted in the revivification of seven bacterial strains that were highly identical to the B. subtilis and B. licheniformis strains obtained in Higashi-Hiroshima (99.7 and 100% of 16S rDNA sequence similarity, respectively). A subsequent genetic analysis on the group of B. licheniformis isolates based on the universally house-keeping genes, gyrB and parE, revealed high sequence similarities in both genes among the strains of both locations (99.0–99.4%), which clustered them in a monophyletic line. Phenotype characterized by numerical taxonomy for 150 physiological tests confirmed the close relatedness between strains (similarity coefficient S _SM = 96.0%). The remarkable agreement between phenotype and genotype of the bacterial isolates allows us to conclude that there may have been an aerosolized dispersion of a Gobi Desert B. licheniformis by dust storms to Japan. This study provides evidence of microbial transport by yellow dust events in North-East Asia.     

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.