Molecular biology of Bacillus subtilis cytochromes

Bacillus subtilis cells must have cytochromes for growth and can synthesize cytochromes of a-, b-, c-, d-, and o-types. After a long lag, our knowledge of the structure, genetics and specific role for these cytochromes is now growing exponentially as the result of recent research. This progress is reviewed here and includes, for example, the discovery of two different cytochrome a systems and genes required for their biogenesis.     

Spectral analysis of cytochromes inAquaspirillum magnetotacticum

The respiratory chain ofAquaspirillum magnetotacticum strain MS-1 cells denitrifying microaerobically included a-, a₁-, b-, c-, cd₁-, and o-type hemes. More than 85% of the total cytochromes detected were of the c type. Virtually all of the a and b types were detected in cell membranes, whereas 70% of the c-type hemes were soluble. Large quantities of soluble c-type hemes were released with periplasm by freezing and thawing cells. Soluble c₅₅₁ occurred in two forms: as a single compound of apparent molecular weight of 17,000 daltons, which bound CO, and, together with d₁ heme, as a component of nitrite reductase. Both a₁-type hemes (which usually comprise part of the low aeration cytochrome oxidase) and o types (usually part of the high aeration oxidase) were simultaneously expressed in microaerobically grown denitrifying cells ofA. magnetotacticum; this indicated branching of the respiratory chain.     

Spectral properties of cytochromes from Staphylococcus aureus

Two cytochrome b with peaks at 554 and 558 nm and cytochrome a with alpha-peak at 603 nm were found in intact cells and membranes of Staphylococcus aureus using low-temperature spectrophotometry and registration of second- and fourth-order finite difference spectra of cytochromes. Analysis of the cytochrome functioning in membranes isolated from the cells at the exponential and stationary growth phases revealed no difference in the set of these carriers. Analysis of cytochrome reduction with different substrates demonstrated identity of the cytochrome composition in the respiratory chain, reduced with NADH, lactate, alpha-glycerophosphate, malate and succinate. Cytochrome omicron with gamma-peak at 416 nm in the CO-spectra was found to be involved in oxidation of all the substrates tested both in intact cells and membranes of Staphylococcus aureus.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

Method for blot-hybridization analysis of mRNA molecules from Bacillus subtilis

By modifying hybridization techniques which are currently available to analyze RNA molecules we have developed a sensitive and reproducible method for 'Northern' analysis of RNA from Bacillus subtilis. The use of a thin (1 mm) vertical 2% agarose-6% formaldehyde gel seems to allow more efficient transfer and higher resolution of RNA upon hybridization analysis than does the use of thicker horizontal slab gels. Our improved hybridization method results in greatly reduced background upon autoradiography regardless of whether or not 32P-labelled nick-translated probes or probes synthesized on M13 vectors were purified from the unincorporated radionucleotides.     

Calmodulin-like protein from Bacillus subtilis

The first example of a calmodulin-like activity in a Gram-positive bacterium, Bacillus subtilis, is reported. A calcium ion-dependent, 3', 5' cyclic-AMP phosphodiesterase-stimulating activity was found in the soluble fraction of cell-free extracts of cells sporulating in a chemically-defined medium; activation was reversed by trifluoperazine. The activity was heat stable, bound to phenothiazine-agarose in a calcium ion-dependent manner and was eluted therefrom with buffer containing EGTA, and displaced authentic beef brain calmodulin from its antibody in a radioimmunoassay.     

DNA-methylases from different strains of Bacillus subtilis and Bacillus natto

DNA-methyltransferase activity has been detected in some of Bacillus subtilis and Bacillus natto strains. Two strains of Bacillus subtilis exhibited DNA-cytosine methyltransferase activity, and the strains of Bacillus natto exhibited DNA-adenine methyltransferase activity. A possible effect of DNA-methyltransferase specificity on transformation efficiency is discussed.     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

枯草芽胞杆菌L-天冬氨酸a脱羧酶的酶学性质

b-丙氨酸是一种重要的医药化工原料,目前主要依靠化学法进行生产。探寻更为环保和高效的生物生产法是未来研究的一个方向。L-天冬氨酸a脱羧酶 (PanD) 能特异地脱去L-天冬氨酸的a羧基,生成b-丙氨酸。本文比较了3种分别来源于大肠杆菌、谷氨酸棒状杆菌及枯草芽胞杆菌的PanD比酶活 (分别为0.98、7.52和8.4 U/mg)。后两者的最适pH均为6.5,最适反应温度分别为65 ℃及60 ℃。与目前研究最多的来源于大肠杆菌和谷氨酸棒状杆菌的PanD相比,来源于枯草芽胞杆菌的PanD具有更好的活性和热稳定性,具有更强的工业应用潜力。同时,本文对该酶特有的翻译后自剪切及机理性失活现象进行了分析和讨论。     

Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis

The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat.     

Isolation of laccase gene from Bacillus subtilis and analysis of its physicochemical properties

The present study reports the cloning and sequencing of lac2 from Bacillus subtilis. The gene is composed of 1542 bp and encodes a 514-amino acid protein. The gene has 86% homology with a published laccase with GeneID 936023. The lac2 gene was deposited in GenBank as a new nucleotide sequence. This new sequence was cloned into the multiple cloning site of pPIC9K to generate pPIC9K-lac2, which was then transformed into Pichia pastoris GS115 via electroporation. The recombinant GS115 (pPIC9K-lac2) was grown initially in BMGY medium and transferred to BMMY to induce gene expression for 48 h. The recombinant Lac2 protein shows laccase activity with α-naphthol and guaiacol as substrates. The optimal pH is between 3.2 and 4.7, and the optimal temperature is 25 °C for enzyme reaction.