Assay of the pyruvate dehydrogenase complex by coupling with recombinant chicken liver arylamine N-acetyltransferase

The activity of the pyruvate dehydrogenase complex has long been determined in some laboratories by coupling the production of acetyl-coenzyme A (acetyl-CoA) to the acetylation of 4-aminoazobenzene-4'-sulfonic acid by arylamine N-acetyltransferase. The assay has some advantages, but its use has been limited by the need for large amounts of arylamine N-acetyltransferase. Here we report production of recombinant chicken liver arylamine N-acetyltransferase and optimization of its use in miniaturized assays for the pyruvate dehydrogenase complex and its kinase.     

蜡状芽孢杆菌S458-1对水产养殖系统中水质调节的作用

【目的】水产养殖过程中蜡状芽孢杆菌(Bacillus cereus)作为益生菌被广泛应用。本研究旨在深入了解分离于养殖水体中的一株蜡状芽孢杆菌S458-1对养殖水体以及养殖对象的影响,以期为菌株S458-1在水产养殖生产上的应用提供理论依据。【方法】根据控制变量法,各试验组鲫鱼养殖模式设置相同温度、盐度、溶氧和pH,利用分光光度法测定水体的水化学指标;利用试剂盒法测定鲫鱼的血清生理生化指标。【结果】添加菌株S458-1处理组(终浓度分别为10~6、10~7、10~8CFU/mL)与对照组(未加S458-1菌)相比:水体活性磷浓度显著性增加(P0.05);同时具有把NH_4~+-N和NO_2~–-N转化为NO_3~–-N的趋势,但无显著性差异(P0.05);养殖鲫鱼血清检测结果显示谷草转氨酶(GOT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)以及丙二醛(MDA)含量均显著降低(P0.05)。【结论】蜡状芽孢杆菌S458-1具有脱氮解磷、调节水质的作用,并可增强养殖对象的生理健康状态,可作为益生菌应用于水产养殖中,具有较高的应用价值。     

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.     

Mercury resistance determined by a self-transmissible plasmid inBacillus cereus 5

Summary Inducible mercuric reductase activity inBacillus cereus 5 was plasmid-encoded. Plasmid analysis revealed three plasmids with molecular masses of 2.6, 5.2 and 130 MDa. A mating system permitted transfer of the resistance determinant among strains ofB. cereus andB. thuringiensis. Transfer of mercury resistance fromB. cereus 5 toB. cereus 569 andB. thuringiensis occurred during mixed culture incubation on agar surfaces. The 130-MDa plasmid (pGB130) was responsible for transfer; frequencies ranged from 10^–5 to 10^–4.B. cereus 569 transconjugants inheriting pGB130 were also effective donors. High transfer frequencies and the finding that cell-free filtrates of donor cultures were ineffective in mediating transfer suggested mercury-resistance transfer was not phage-mediated. Transfer was also insensitive to DNase activity. Further evidence that pGB130 DNA carried the mercury-resistance determinant was transformation ofB. cereus 569 by electroporation with pGB130 DNA isolated fromB. cereus 5 and a mercury-resistantB. cereus 569 transconjugant. Mercury-resistant transconjugants and transformants exhibited mercuric reductase activity. Plasmid pGB130 also conferred resistance to phenylmercuric acetate.     

Specificity of translation of spore polypeptides in bacillus in vitro systems

Cell free extracts prepared from exponentially growing Escherichia coli and Bacillus cereus as well as from Bacillus cereus at the end of exponential growth were optimized for various factors required for amino acid incorporation when programmed with Bacillus ribonucleic acid. All three preparations synthesized glutamine synthetase antigen when ribonucleic acid from a Bacillus subtilis strain that overproduces glutamine synthetase was added. The post exponential Bacillus cereus extract, however, was most active for the synthesis of Bacillus cereus spore coat antigen when supplemented with the appropriate ribonucleic acid. There appears to be some specificity in the translation of at least this sporulation messenger RNA.Non-Standard Abbreviations PMSF phenyl methyl sulfonylfluoride - GS glutamine synthetase - UDS 8 M urea, 1% (W/V) sodium dodecyl sulfate, 50 mM dithioerythritol, 2 mM PMSF, 5 mM cyclohexylaminoethane sulfonic acid, pH 9.6     

Bacillus manliponensis sp. nov., a new member of the Bacillus cereus group isolated from foreshore tidal flat sediment

A Gram-positive, endospore-forming, new Bacillus species, strain BL4-6^T, was isolated from tidal flat sediment of the Yellow Sea. Strain BL4-6^T is a straight rod, with motility by peritrichate flagella. The cell wall contains meso-diaminopimelic acid, and the major respiratory quinone is menaquinone-7. The major fatty acids are iso-C_15:0 and summed feature 3 (containing C_16:1 ω7c/iso-C_15:0 2OH, and/or iso-C_15:0 2OH/C_16:1 ω7c). Cells are catalase-positive and oxidase-negative. The G+C content of the genomic DNA is 38.0 mol%. Based on a comparative 16S rRNA gene sequence analysis, the isolate belongs to the genus Bacillus, forms a clade with the Bacillus cereus group, and is closely related to Bacillus mycoides (98.5%), Bacillus cereus (98.5%), Bacillus anthracis (98.4%), Bacillus thuringiensis (98.4%), Bacillus weihenstephanensis (98.1%), and Bacillus pseudomycoides (97.5%). The isolate showed less than 85% similarity of the gyrA gene sequence and below 95% similarity of the rpoB gene sequence to the members of this group. DNA-DNA relatedness between strain BL4-6^T and B. cereus group was found to be in a range of 22.8–42.3%, and thus BL4-6^T represents a unique species. On the basis of these studies, strain BL4-6^T (=KCTC 13319^T =JCM 15802^T) is proposed to represent the type strain of a novel species, Bacillus manliponensis sp. nov.     

Untersuchungen über das Transportsystem für Thiamin bei Bacillus cereus

Zusammenfassung Bei dem untersuchten Transportsystem für Thiamin bei Bacillus cereus wechseln rhythmisch 1–2 h andauernde Aufnahme-und Abgabephasen miteinander ab. Diese großen Phasen sind in kleinere von durchschnittlich 45 s Dauer unterteilt. Die Geschwindigkeit der Thiaminaufnahme wird von pH-Wert, Temperatur, Alter der Zellen, Energie-und Nährstoffversorgung sowie Thiaminkonzentration des Mediums beeinflußt. Sie folgt der Michaelis-Menten-Kinetik: K _m =1,98x10^-8 M; V _max=1,19x10^-6 mol/g TGxmin. Gefördert wird die Aufnahmerate durch K⁺, Ca²⁺ und Mg²⁺, gehemmt wird sie durch Pyrithiamin, EDTA, H⁺-Ionen, Wasserstoffacceptoren und-donatoren; OH^--Ionen bewirken eine Umkehr der Transportrichtung. Als erklärende Theorie wird eine Kopplung der Thiaminpermeation mit Protonenverschiebungen in der Membran diskutiert.

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Studies on the thiamine transport system in Bacillus cereus

The thiamine transport system in Bacillus cereus exhibits rhythmical changes of resorption-and excretion-phases lasting 1–2 h. These main phases are subdivided in shorter ones with an average duration of 45 s. The velocity of the thiamine uptake is influenced by pH, temperature, age of cells, energy and substrate supply and thiamine concentration of the medium. The Michaelis-Menten-Kinetic can be used to describe the uptake: K _m =1.98x10^-8 M; V _max=1.19x10^-6 mol/g dry weightxmin. The rate is enhanced by K⁺, Ca²⁺ and Mg²⁺, and inhibited by Pyrithiamin, EDTA, H⁺-ions, proton donors and proton acceptors; OH^--ions cause a change in the direction of transport. A theoretical explanation can be given by assuming a coupling of the thiamine permeation with proton movements in the membrane.

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Liste der Abkürzungen AMP Adenosinmonophosphat - ADP Adenosindiphosphat - ATP Adenosintriphosphat - DNP Dinitrophenol - EDTA Athylendiamintetraessigsäure - FAD Flavinadenindinucleotid - NAD Nicotinamidadenindinucleotid - NADP Nicotinamidadenindinucleotidphosphat - TPP Thiaminpyrophosphat     

Biosorption of cadmium by a Cd2+-hyperresistant Bacillus cereus strain HQ-1 newly isolated from a lead and zinc mine

A Cd²⁺-hyperresistant bacterial strain HQ-1 was isolated from a lead–zinc mine. The strain was characterized and identified as Bacillus cereus based on morphology, physiological tests and 16S rRNA gene analysis. The minimal inhibitory concentration of Cd²⁺ for the bacterium was 0.012 mol/l. Isotherms for cadmium (Cd) biosorption by cells of B. cereus strain HQ-1 were investigated. The equilibrium data could be fitted by a Langmuir isotherm equation. The possible functional sites that might be influenced by the sorption were determined. The results indicate that this B. cereus strain has excellent potential for biosorption of Cd. Physiological characterization of the isolate also indicates possible application of this strain for bioremediation of sites with Cd contamination.     

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.     

Bacillus gaemokensis sp. nov., isolated from foreshore tidal flat sediment from the Yellow Sea

A Gram-positive, rod-shaped, endospore-forming organism, strain BL3-6^T, was isolated from tidal flat sediments of the Yellow Sea in the region of Tae-An. A 16S rRNA gene sequence analysis demonstrated that this isolate belongs to the Bacillus cereus group, and is closely related to Bacillus mycoides (99.0% similarity), Bacillus thuringiensis (99.0%), Bacillus weihenstephanensis (99.0%), Bacillus cereus (98.9%), Bacillus anthracis (98.8%), and Bacillus pseudomycoides (98.1%). The phylogenetic distance from any validly described Bacillus species outside the Bacillus cereus group was less than 95.6%. The DNA G+C content of the strain was 39.4 mol% and the major respiratory quinone was menaquinone-7. The major cellular fatty acids were iso-C_14:0 (17.8%), iso-C_16:0 (15.8%), and iso-C_12:0 (11.3%). The diagnostic amino acid of the cell wall was meso-diaminopimelic acid and the major cell wall sugar was galactose. The results of DNA-DNA hybridization (<55.6%) and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain BL3-6^T from the published Bacillus species. BL3-6^T therefore represents a new species, for which the name Bacillus gaemokensis sp. nov. is proposed, with the type strain BL3-6^T (=KCTC 13318^T =JCM 15801^T).     

Distribution of toxigenic Bacillus cereus in rice samples marketed in Hong Kong

Eleven of 16 samples of rice on sale in rice shops and supermarkets in Hong Kong contained Bacillus cereus. Although B. cereus counts did not exceed 100 bacteria/g in most of the positive samples, a sample of Thai red rice and a poor quality rice originating from China contained between 300 to 1000 cells/g and 10⁴ to 2×10⁵ cells/g, respectively. Nine strains produced an enterotoxin responsible for the diarrhoeal-type B. cereus food poisoning and seven of these strains also produced a haemolysin (haemolysin BL), a dermonecrotic vascular permeability factor which may be a virulence determinant in diarrhoeal illness caused by this bacterium.P.K. Lee and J.A. Buswell are with the Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; K. Shinagawa is with the Department of Veterinary Medicine, Iwate University, Iwate, Japan.     

Expression of parasporal crystal protein (δ—endotoxin) gene(s) ofBacillus thuringiensis var.israelensis in sporogenic and asporogenic mutant strains ofBacillus cereus

The expression of parasporal crystal protein (δ-endotoxin) coding gene(s) ofBacillus thuringlensis var.israelensis and its association, if any, with sporulation was studied in sporogenicBacillus cereus and its asporogenic mutant strains. Five asporogenous mutants ofBacillus cereus blocked at different stages of sporulation, were isolated from a streptomycin-resistant strain, The transconjugants isolated from the plasmid transfer experiments betweenBacillus thuringiensis var.israelensis and streptomycin resistantBacillus cereus and its asporogenous mutants, showed larvicidal activity. The crystal protein gene(s) are, therefore, expressed both in sporulating and in non-sporulating mutant strains ofBacillus cereus suggesting that the expression of crystal protein gene(s), is independent of sporulation specific functions inBacillus cereus. Part of the work was carried out at Biotechnology Programme, Jadavpur University, Calcutta 700 032, India.     

A predictive model for the growth rate of Bacillus cereus in broth by response surface methodology

A response surface methodology (RSM) was developed for predicting the growth rate of Bacillus cereus in a tryptic soy broth medium as a function of temperature (10 to 40°C), pH (5.5 to 8.5), and the NaCl concentration (0 to 8%). The primary model showed a good fit (r² = 0.920 to 0.999) to a Gompertz equation to obtain growth rates each condition. The quadratic polynomial model was found to be significant (p < 0.0001) and predicted values were found to be in good agreement with experimental values (R² value of 0.9486). The evaluation of RSM for describing the growth rate of B. cereus used the bias factor (B_f) and the accuracy factor (A_f). Both the B_f value (1.11) and the A_f value (1.50) were within acceptable ranges. This model was provided an efficient and accurate method for predicting the growth of B. cereus as a function of the controlling factors.     

Evidence for an arene oxide-NIH shift pathway in the transformation of naphthalene to 1-naphthol by Bacillus cereus

Bacillus cereus ATCC 14579 transformed naphthalene predominately to 1-naphthol. Experiments with [¹⁴C]naphthalene showed that over a 24 h period, B. cereus oxidized 5.2% of the added naphthalene. 1-Naphthol accounted for approximately 80% of the total metabolites. B. cereus incubated with naphthalene under the presence of ¹⁸O₂ led to the isolation of 1-naphthol that contained 94% ¹⁸O. The metabolism of [1-²H]-and [2-²H]-naphthalene by B. cereus yielded 1-naphthol which retained 95% and 94% deuterium, respectively, as determined by mass spectral analysis. NMR spectroscopic analysis of the deuterated 1-naphthol formed from [1-²H]-naphthalene indicated an NIH shift mechanism in which 19% of the deuterium migrated from the C-1 to the C-2 position. The ¹⁸O₂ and NIH shift experiments implicate naphthalene-1,2-oxide as an intermediate in the formation of 1-naphthol from naphthalene by B. cereus.Abbreviations HPLC High performance liquid chromatography - NMR nuclear magnetic resonance     

Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method

Bacillus subtilis fmbj can produce a lipopeptide antimicrobial substance, the main components of which are surfactin and fengycin. In this paper, the sensitivity of Bacillus cereus to antimicrobial lipopeptides from B. subtilis fmbj was observed, and the effect of the microstructure of antimicrobial lipopeptide on spores of B. cereus was investigated. At the same time, the optimization of the inactivation of antimicrobial lipopeptides to spores of B. cereus by a response surface methodology was studied. Results showed that B. cereus had high sensitivity to it, whose minimal inhibitory concentration was 156.25 μg/ml. It could result in the death of spores by destroying the structure of resting spores and sprouting spores, as was observed by transmission electron microscopy. The optimization result indicated that spores of B. cereus could be inactivated by 2 orders of magnitude when the temperature was 29.6°C, the action time was 7.6 h, and the concentration was 3.46 mg·ml⁻¹.     

Isolation and characterization of phages infecting Bacillus cereus

Aim: To isolate and characterize bacteriophages (phages) that infect the foodborne pathogen Bacillus cereus. Methods and Results: Two phages were isolated from soil based on their ability to form plaques on four indicator hosts including Bacillus thuringiensis subsp. israelensis, and three isolates of B. cereus. The purified phages were characterized by morphology, host range, single‐step growth curves and restriction enzyme digestion profiles. The phages appeared to be of the Myoviridae family based on their structure in electron micrographs. The phages lysed bacteria of several species, produced average burst sizes of 322 and 300 phages per infected cell, and both had genomes over 90 kb. The phages were chloroform‐resistant and stable at 4°C. They reduced the concentration of B. cereus in mashed potatoes by >6 log₁₀ CFU ml^?1 within 24 h at room temperature, when applied at a high concentration. Conclusions: The relatively narrow host range within B. cereus might mean that these phages need to be used as part of a ‘cocktail’ of phages for biocontrol, but their efficacy for the control of their host in food was demonstrated. Significance and Impact of the Study: This is the first report of biocontrol by phages of B. cereus in food.     

Isolation of spore-forming bacilli from mosquitoes in natural breeding habitats in Iraq

New isolates of spore-forming bacilli from larvae and pupae of 3 species of mosquitoes are recorded in central Iraq.Bacillus sphaericus Meyer and Neide was isolated fromCulex pipiens (L.) larva.Bacillus carotarum Koch andBacillus cereus Frankland & Frankland were isolated fromTheobaldia longiareolata (Macquart) pupae.Bacillus laterosporus Laubach andBacillus thuringiensis (H 18) were isolated fromAedes caspius (Pallas) larvae. In addition, unidentifiedBacillus spp. were isolated fromCx. pipiens, T. longiareolata andAe. caspius larvae. Examination of soil samples collected from mosquito natural breeding habitats revealed isolates ofB. cereus, Bacillus thuringiensis H4a 4b; H 12 and H 16 and an unidentifiedBacillus sp.

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Résumé Des souches bactériennes sporogènes sont isolées de moustiques qui se trouvent dans la région centrale de l'Irak. Les résultats obtenus sont les suivants:Bacillus sphaericus Meyer & Neide [Culex pipiens (L.), larve],Bacillus carotarum Koch etBacillus cereus Frankland & Frankland [Theobaldia longiareolata (Macquart), nymphe],Bacillus laterosporus Laubach etBacillus thuringiensis (H 18) [Aedes caspius (Pallas), larvel]. L'examen des larves deCulex pipiens. T. longiareolata etAe. caspius, ainsi que l'analyse des échantillons du sol prélevés dans la région montrent la présence deBacillus cereus, Bacillus thuringiensis H4a 4b; H12 plus H16 et d'autresBacillus non identifiés.

     

Occurrence and pathogenic potential of Bacillus cereus group bacteria in a sandy loam

The major part (94%) of the Bacillus cereus-like isolates from a Danish sandy loam are psychrotolerant Bacillus weihenstephanensis according to their ability to grow at temperatures below 7 °C and/or two PCR-based methods, while the remaining 6% are B. cereus. The Bacillus mycoides-like isolates could also be␣divided into psychrotolerant and mesophilic isolates. The psychrotolerant isolates of B. mycoides could␣be discriminated from the mesophilic by the two PCR-based methods used to characterize B.␣weihenstephanensis. It is likely that the mesophilic B. mycoides strains are synonymous with Bacillus pseudomycoides, while psychrotolerant B. weihenstephanensis, like B. mycoides, are B. mycoides senso stricto. B. cereus is known to produce a number of factors, which are involved in its ability to cause gastrointestinal and somatic diseases. All the B. cereus-like and B. mycoides like isolates from the sandy loam were investigated by PCR for the presence of 12 genes encoding toxins. Genes for the enterotoxins (hemolysin BL and nonhemolytic enterotoxin) and the two of the enzymes (cereolysin AB) were present in the major part of the isolates, while genes for phospolipase C and hemolysin III were present in fewer isolates, especially among B. mycoides like isolates. Genes for cytotoxin K and the hemolysin II were only present in isolates affiliated to B. cereus. Most of the mesophilic B. mycoides isolates did not possess the genes for the nonhemolytic enterotoxin and the cereolysin AB. The presence of multiple genes coding for virulence factors in all the isolates from the B. cereus group suggests that all the isolates from the sandy loam are potential pathogens.     

Identification of three Zwittermicin A biosynthesis-related genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> strain YBT-1520

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.