Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in<Emphasis Type="Italic"> Bacillus megaterium</Emphasis>

Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of Act_Bm, a ⁵⁴ regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium.     

Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium

The aim of this study was to investigate environmental conditions affecting chicken feather degradation and keratinolytic enzyme production by Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. B. megaterium F7-1 degraded whole chicken feather completely within 7 days. The bacterium grew with an optimum at pH 7.0–11.0 and 25–40 °C, where maximum keratinolytic activity was also observed. The production of keratinolytic enzyme by B. megaterium F7-1 was inducible with feather. Keratinolytic enzyme production by B. megaterium F7-1 at 0.6% (w/v) skim milk was 468 U/ml, which was about 9.4-fold higher than that without skim milk. The amount of keratinolytic enzyme production depended on feather concentrations. The degradation rate of autoclaved chicken feathers by cell-free culture supernatant was 26% after 24 h of incubation, but the degradation of untreated chicken feathers was unsuccessful. B. megaterium F7-1 effectively degraded feather meal, duck feather and human nail, whereas human hair and sheep wool showed relatively low degradation rates. B. megaterium F7-1 presented high keratinolytic activity and was very effective in feather degradation, providing potential use for biotechnological processes of keratin hydrolysis.     

Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources

Poly(hydroxybutyric acid) (PHB) and other biodegradable polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of cane molasses and corn steep liquor, two of the cheapest substrates available in Egypt, may help to reduce the cost of producing such biopolyesters. In this work, the effect of different carbon sources was studied. Maximum production of PHB was obtained with cane molasses and glucose as sole carbon sources (40.8, 39.9 per mg cell dry matter, respectively). The best growth was obtained with 3% molasses, while maximum yield of PHB (46.2% per mg cell dry matter) was obtained with 2% molasses. Corn steep liquor was the best nitrogen source for PHB synthesis (32.7 mg per cell dry matter), on the other hand, best growth was observed when ammonium chloride, ammonium sulphate, ammonium oxalate or ammonium phosphate were used as nitrogen sources.     

Distribution of Bacillus megaterium QM B1551 plasmids among other B. megaterium strains and Bacillus species

Bacillus megaterium QM B1551 contains seven plasmids. Two are small rolling circle plasmids and five are theta-replicating plasmids with cross-hybridizing replicons that define a new family of very homologous yet compatible theta replicons. Previous sequencing of several of the plasmids has shown genes with high similarity to those on the genomes and plasmids of other Gram-positive bacteria. To test the possible distribution of these plasmids, nine other B. megaterium strains and 20 other Bacillus or related species were tested for the presence of similar replicons, and specific flanking DNA by both hybridization and PCR. The theta replicons were widespread among the B. megaterium strains, and two had one or more of the rolling circle plasmids, but none of the plasmid replicon regions were observed in the other Bacillus or related species. It appears from the data that even though some plasmids carry genes suggesting horizontal transfer, their replicons seem to be unique to B. megaterium, or rarely present in related species.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

一株高产胞外多糖巨大芽孢杆菌的筛选鉴定、发酵条件优化及其对土壤团聚体的影响

【目的】筛选具有高产胞外多糖(exopolysaccharides,EPS)特性的微生物菌株,为功能性土壤改良菌剂的研制提供菌种资源。【方法】采用LB-苯胺蓝平板结合菌丝拉丝法定性筛选获得具有产EPS特性的植物根际促生细菌(plant growth promoting rhizobacteria,PGPR)功能菌株;采用低温醇沉分离、硫酸-蒽酮比色法测定PGPR功能菌株经4种培养基发酵后发酵液中EPS含量,获得具有高产EPS特性的PGPR功能菌株;以发酵液中EPS含量为衡量指标,采用正交试验对高产EPS菌株发酵条件进行优化;采用培养皿土壤培养试验,分析F1发酵液对砂壤土土壤大团聚体含量的影响。【结果】初选获得8株具有产EPS特性的PGPR功能菌株;经复选确定菌株F1具有高产EPS特性,PDA是F1产EPS的最优培养基,EPS含量为867.54 μg/mL。基于形态学、生理生化特性测定,以及16S rRNA基因序列系统发育分析、全基因组平均核苷酸相似度分析,确定F1为一株巨大芽孢杆菌(Bacillus megaterium)。F1产EPS的最优培养条件：28℃、180 r/min培养24 h,在该条件下EPS产量为1 123.39μg/mL。F1应用于砂壤土进行土壤培养40 d后,粒径>0.25 mm土壤水稳性大团聚体含量比对照提高了4.44倍。【结论】F1菌株具有较强的产EPS能力,能够促进砂壤土水稳性大团聚体的形成;F1产EPS最优培养基为PDA,最优发酵条件为28℃、180 r/min培养24 h。     

耐盐碱解磷菌的溶磷效果及其对黄豆萌发的影响

【背景】土壤中能够供植物直接吸收利用的磷含量较少,而传统施用磷肥的方式会导致环境污染,并破环土壤结构和功能等问题。【目的】研究巨大芽孢杆菌(Bacillus megaterium)和雷氏普罗威登斯菌(Providencia rettgeri)的耐盐性、溶磷机制及其对黄豆种子萌发的影响,为植物根际盐碱土壤接种定殖及促生效果奠定坚实的基础,以期通过根际土壤解磷菌的解磷作用来解决作物缺磷问题。【方法】结合接种针点接法、平板法、液体培养法及发芽盒发芽等试验,剖析了2株解磷菌的耐盐碱性、溶磷效果及其对黄豆萌发的促进效应。【结果】巨大芽孢杆菌和雷氏普罗威登斯菌分别在盐浓度为0−10%、0−6%的范围及pH值为7.0−12.0的环境下均可以生长;巨大芽孢杆菌和雷氏普罗威登斯菌在平板上的D/d值分别达到2.17和2.05,第4天于液体培养基中溶磷量达到峰值,分别为355.53 mg/L和272.17 mg/L,培养介质pH值由7.5分别降至4.61和4.81。巨大芽孢杆菌和雷氏普罗威登斯菌溶磷效果随NaCl浓度的增大均呈现“先略增强后持续减弱”的趋势,pH值均呈现“先略减小后持续增大”的趋势,NaCl浓度分别为0.4 mol/L和0.2 mol/L时溶磷效果最好,溶磷量达到峰值364.35 mg/L和285.58 mg/L,培养介质的pH值降至最小值4.28和4.73。巨大芽孢杆菌和雷氏普罗威登斯菌对种子发芽率及胚轴、胚根的伸长均有提高,在正常环境和NaCl处理环境下,与对照组相比,经2种菌液浸泡后黄豆的平均发芽率、胚轴及胚根平均增长率分别为2.70%、10.10%、9.00%和5.40%、19.40%、20.30%。【结论】2株菌均具有较强的耐盐性和溶磷效果,能促进种子萌发,可以进一步为解磷菌的接种及应用提供理论依据。     

Evaluation of microbial strains for linoleic acid hydroxylation and reclassification of strain ALA2

In previous studies, a new microbial strain ALA2 was isolated which produced many new products from linoleic acid [Gardner H.W., Hou C.T., Weisleder D. and Brown W. 2000. Lipids 35: 1055–1060; Hou C.T. 1998. 12,13,17-Trihydroxy-9(Z)-Octodecenoic acid and derivatives and microbial isolate for production of the acid. US Patent No. 5, 852, 196]. Strain ALA2 was preliminary identified as Clavibacter sp. based on its physiological and fatty acid profiles. To determine if strain ALA2 is the optimal strain for industrial applications, other related strains were screened for their abilities to convert linoleic acids. Two strains from Clavibacter and 20 type strains from the phylogenetically related genus Microbacterium were studied. Surprisingly, all of these strains tested showed very little or no activity in converting linoleic acid. On reexamination of the identification of strain ALA2, the sequence of the 16S ribosomal RNA gene of ALA2 was found to be 99% identical to that of Bacillus megaterium and the strain was also found to have 76.3% DNA homology to the B. megaterium type strain. Therefore, strain ALA2 is now reclassified as B. megaterium. Screening of 56 strains of B megaterium strains showed that many of them were able to produce reasonable amounts of hydroxyl fatty acids from linoleic acid, although strain ALA2 possessed the greatest activity.     

Characterization of the primary structural homology between the 16 S ribosomal RNAs ofEscherichia coli andBacillus megaterium by oligomer cataloging

Summary The 16 S ribosomal RNAs of twoProcaryotes, Escherichia coli andBacillus megaterium were characterized by oligomer cataloging (oligomers produced by T1 nuclease digestion), in an attempt to detect their primary structural homology and as an initial step in characterizing this homology. Oligomer sequence coincidence between the two catalogs far in excess of the random expected levels was observed. Statistically significant coincidence was most pronounced for the hexamers and pentamers, suggesting that the overall structure of 16 S ribosomal RNA may be such that conservation of large stretches of its primary structure (e.g. over eight nucleotides in length) is not in general essential.     

A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium

Gene expression systems based on the RNA polymerase of the bacteriophage T7 are often the ultimate choice for the high level production of recombinant proteins. During the last decade, the Gram-positive bacterium Bacillus megaterium was established as a useful host for the intra- and extracellular production of heterologous proteins. In this paper, we report on the development of a T7 RNA polymerase-dependent expression system for B. megaterium. The system was evaluated for cytosolic and secretory protein production with green fluorescent protein (GFP) from Aequoria victoria as intracellular and Lactobacillus reuteri levansucrase as extracellular model protein. GFP accumulated rapidly at high levels up to 50 mg/l shake flask culture intracellularly after induction of T7 RNA polymerase gene expression. The addition of rifampicin for the inhibition of B. megaterium RNA polymerase led to an increased stability of GFP. L. reuteri levansucrase was also successfully produced and secreted (up to 20 U/l) into the culture supernatant. However, parallel intracellular accumulation of the protein indicated limitations affiliated with the Sec-dependent protein translocation process.     

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。     

Export,purification, and activities of affinity tagged <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> levansucrase produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His₆- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins. After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His₆-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates d-Gal-Fru, d-Xyl-Fru, d-Man-Fru, and d-Fuc-Fru. R. Biedendieck and R. Beine contributed equally to this work.     

Effect of lacZY-marking of the 2,4-diacetyl-phloroglucinol producing Pseudomonas fluorescens-strain 5-2/4 on its physiological performance and root colonization ability

Transgenic Pseudomonas fluorescens 5-2/4 with reinforced 2,4-diacetyl phloroglucinol (phl) production had shown increased biocontrol ability towards Pythium ultimum (Pu), but inferior root colonization ability compared to its wild type 5.014. Therefore, enhanced root colonization ability of the transgenic strain by repeated inoculation and reisolation on tomato plants was suggested. As a preparation for repeated inoculation and reisolation cycles, the construction of a negative control of the transgenic strain 5-2/4 by marking with lacZY and screening for a mutant possessing qualities comparable to 5-2/4 was performed. Morphologically, colonies of all of the 11 selected mutants were similar on MLXgal medium. The root colonization ability of two of the lacZY-marked strains (mutants 1 and 10) was comparable to the parental strain. These were also able to compete with the resident microflora of tomato seedlings to the same extent as the parental strain. Five mutants were excluded due to lower growth rates on Yeast Malt, King's B Medium (KB) and 0.1 Tryptic Soy Agar (mutant 4, 5 and 8), excessive growth and higher siderophore production on KB (mutant 10) and increased protease production (mutant 2). With respect to in vitro-antagonism of Pu, no differences could be found between the target strain and mutants 1, 3, 6, 7 and 9. Examination of sole carbon source utilization of these five lacZY-marked strains revealed a significantly higher utilization of alpha-D-lactose and lactulose compared to 5-2/4. However, significant differences could be found for 51% of the utilized carbon sources. Cluster analysis showed a high degree of similarity between 5-2/4 and mutant 1 both when analyzed with and without alpha-D-lactose. As mutant 1 also represented the colonization pattern most similar to the parental strain 5-2/4, it presents a presumptive subject for a negative control in the following inoculation and reisolation studies on tomato.     

Isolation and characterisation of catechol-like siderophore from cowpea Rhizobium RA-1

Cowpea Rhizobium RA-1 produced a catechol-like siderophore. Secondary hydroxamic acids were not detected. Bioassay of the siderophore exhibited a distinct zone of growth of cowpea rhizobia. One litre of culture filtrate gave 6.2 mg of catechol-like siderophore. Glycine and threonine were detected in the siderophore. Maximum production of siderophore was found at 36 h of growth of cowpea Rhizobium RA-1.Abbreviations 2,3-DHBA 2,3-dihydroxy benzoic acid - EDTA ethylenediamine tetraacetic acid     

Survival of a strain of Bacillus megaterium carrying a lepidopteran-specific gene of Bacillus thuringiensis in the phyllospheres of various economically important plants

The colonizing ability of a transcipient strain of Bacillus megaterium carrying a lepidopteran-specific cryIA (a) gene of Bacillus thuringiensis in the phyllospheres of various economically important plants was studied. Similar experiments were also carried out using the parental B. thuringiensis var. kurstaki strain HD1 for a comparison. While the transcipient remained on the leaves of cotton and okra for more than 28 days, its survival in phyllospheres of mulberry, peanut, chickpea, tomato and rice was rather limited to about 3 – 5 days. The persistence of B. thuringiensis, on the other hand, was extremely short (i.e. less than 4 days) on all the crop plants tested.     

Acht neue Arten der GattungAnthemis (Compositae) aus Persien

The following species are described as new:Anthemis mazandaranica in N. and NW. Iran is allied toA. coelopoda; A. moghanica in NW. Iran is close toA. candidissima andA. sintenisii; A. atropatana also in NW. Iran is similar toA. hyalina; A. gracilis in W. Iran is close toA. plebeia; A. bushehrica in SW. Iran is similar toA. susiana; andA. rhodocentra in S. and E. Iran and in Pakistan is akin toA. austro-iranica, A. gayana, andA. kandaharica.

Anschrift des Herausgebers: Hofrat Univ.-Prof. Dr.Karl Heinz Rechinger, Beckgasse 22, A-1130 Wien, Österreich.     

Effect of complex nitrogen sources on the production of penicillin acylase byBacillus megaterium

The following complex nitrogen sources were evaluated for the production of penicillin acylase byBacillus megaterium: casein hydrolysate, corn steep liquor, stick water concentrate, blood meal and defatted sunflower meal. Experiments were run in shake flasks at 30‡C and pH 7.0. Best results were obtained with casein hydrolysate: 244 IU/I were produced with a productivity of 20.3 IU/l/h and yield of 717.6 IU/g of nitrogen. The lowest results correspond to sunflower meal with 39 IU/1.