Uptake of branched-chain alpha-keto acids in Bacillus subtilis.

Bacillus subtilis has a constitutive system for the uptake of alpha-keto-beta-methylvalerate, alpha-ketoisovalerate, and (probably) alpha-ketoisocaproate. A mutation, kauA1, which blocks the uptake of alpha-keto-beta-methylvalerate and alpha-ketoisovalerate, is located between metB and citK on the B. subtilis chromosome.     

Cloning, expression, purification, and characterisation of the dUTPase encoded by the integrated Bacillus subtilis temperate bacteriophage SPbeta

The cDNA encoding dUTPase, an enzyme catalysing the hydrolysis of dUTP to dUMP and pyrophosphate, from the integrated Bacillus subtilis temperate bacteriophage SPbeta has been cloned and over-expressed at high levels in Escherichia coli. The resulting recombinant dUTPase was purified to homogeneity in one step by phosphocellulose chromatography with a final yield of 700 mg pure crystallisable protein per litre of bacterial culture. The molecular mass of the 142 amino acid polypeptide was 16 kDa as judged by electrophoretic analysis and gel filtration chromatography revealed the enzyme to exist as a homotrimer in solution. Isoelectric focusing indicated the isoelectric point to be 7. Functionality of the purified recombinant dUTPase was proven by demonstrating catalytic activity towards the substrate dUTP. The optimal activity of SPbeta dUTPase proved to be dependent on the presence of divalent metal ions, with Mg(2+) conferring the highest activity.     

Identification of the nonA and nonB loci of Bacillus subtilis Marburg permitting the growth of SP10 phage

Mutational inactivation of both nonA and nonB genes are required for the permissiveness of Bacillus subtilis Marburg cells to infection by phage SP10. By transformational analysis of the nonA strain with DNAs from gently lysed protoplasts carrying the integrative plasmid pMUTIN (em) insertions in every 20 kb along the whole chromosome, we have identified the nonA to be the cured state of endogenous prophage SPbeta. Direct DNA sequencing, on the other hand, revealed one nonsense mutation of nonB in ydiR, which is a component gene of the intrinsic restriction system BsuMR of B.subtilis Marburg. Introduction of the wild type ydiR into the nonB strain at aprE locus resulted in complementation of nonB. Furthermore, as the SP10 genome was found to possess multiple BsuM target sites, it is considered that SP10 can infect and multiply in B.subtilis cells, which are SPbeta free and possess a defective BsuMR restriction system.     

The Prophage of SPβc2DcitK1, a Defective Specialized Transducing Phage of BACILLUS SUBTILIS

The defective specialized transducing phage SP beta c2dcitK1 carries two known bacterial genes, kauA and citK, as well as SP beta hage markers including the heat-sensitive repressor allele, c2. Some phage genes (including essential ones) are missing. When SP beta c2dcitK1 transduces SP beta-sensitive cells of Bacillus subtilis, the defective prophage is inserted into sites in the homologous bacterial DNA of the attSP beta-kauA-citK region of the recipient chromosome. During the growth of these transductants, occasional excisions occur that result in the loss of the phage genes and of the heterogenotic state. These excisions increase greatly in frequency during growth at repressor-inactivating temperatures. The kinds of insertions and excisions seen suggest that a Campbell-type (CAMPBELL 1962) circular phage genome may occur transiently. If the transductants are superinfected by SP beta c2 or by the clear-plaque mutant SP beta c1, the resulting double lysogen can be heat induced to release high-frequency-of-transduction (HFT) lysates for kauA and citK.     

DNA methyltransferases affecting the sequence 5''CCGG

B. subtilis phage SPbeta and Moraxella sp. code for DNA methyltransferases which methylate both cytosines of the sequence 5'CCGG. Experiments using a B. subtilis strain whose DNA is sensitive to HpaII and resistant to MspI degradation, indicated that methylation of the outer C of this sequence provides protection against the restriction enzyme MspI.     

Determination of the chromosomal locations of four Bacillus subtilis genes which code for a family of small, acid-soluble spore proteins.

The chromosomal locations of four genes which code for small, acid-soluble spore proteins (SASP) in Bacillus subtilis have been determined. Although these four genes code for extremely homologous small, acid-soluble spore proteins (greater than 65% sequence identity), the genes are not clustered but are located at 70 degrees (adjacent to glyB [sspB gene]), 115 degrees (between metC and pyr cluster [sspD gene]), 180 degrees (between metB and kauA [sspC gene]), and 260 degrees (between ilvC and aroG [sspA gene]) on the B. subtilis genetic map.     

Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168-166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPbeta established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPbeta, plays a key role in the formation of so-called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of 'transstamping'. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X-ray-induced, rearrangement.     

Subspecies-specific distribution of intervening sequences in the Bacillus subtilis prophage ribonucleotide reductase genes

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.     

Initiation of Bacillus subtilis sporulation by the stringent response to partial amino acid deprivation

We have controlled the rates at which three different amino acids were available to auxotrophs of Bacillus subtilis by avoiding active transport of the respective substrate. The active transport of oxomethylvalerate, a precursor of isoleucine, was prevented by a kauA mutation, the uptake of L-aspartate was competed by 20 mM L-glutamate, and D-methionine was used instead of L-methionine. When in this way conditions of partial amino acid deprivation were achieved, a partial "stringent response" occurred which included the increase of ppGpp and pppGpp, and the decrease of GTP; such conditions initiated sporulation. In the corresponding relaxed (relA) mutants, the changes of guanine nucleotides were greatly reduced and no sporulation was observed at any substrate concentration; but addition of decoyinine produced a further decrease of GTP and caused sporulation.     

Identification of Bacillus subtilis sigma-dependent genes that provide intrinsic resistance to antimicrobial compounds produced by Bacilli

Bacillus subtilis produces many antibiotics of varying structures and specificity. Here we identify a prominent role for sigma(W), an extracytoplasmic function (ECF) sigma factor, in providing intrinsic resistance to antimicrobial compounds produced by other Bacilli. By using a panel of B. subtilis mutants disrupted for each of the 30 known sigma(W)-dependent operons we identified resistance genes for at least three different antimicrobial compounds. The ydbST and fosB genes contribute to resistance to antimicrobial compound(s) produced by B. amyloliquefaciens FZB42, the yqeZyqfAB operon provides resistance to the SPbeta prophage-encoded bacteriocin sublancin, and the yknWXYZ operon and yfhL provide resistance to the antimicrobial peptide SdpC. YfhL encodes a paralogue of SdpI, a membrane protein that provides immunity to SdpC. In competition experiments, we identify sigma(W) as a key factor in allowing B. subtilis to resist antibiotic killing and encroachment by competing strains. Together with the previous observation that sigma(W) provides inducible resistance against the Streptomyces antibiotic fosfomycin, these studies support the notion that sigma(W) controls an antibiosis regulon important in the microbial ecology of soil bacteria.     

Genome engineering reveals large dispensable regions in Bacillus subtilis

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.     

Attachment of the chromosomal terminus of Bacillus subtilis to a fast-sedimenting particle

After gently lysed protoplasts of exponential phase cells of Bacillus subtilis were treated with restriction endonuclease BamHI, 99% of the DNA did not sediment with the plasma membrane. This DNA was fractionated on sucrose gradients into (i) a fast-sedimenting fraction highly enriched for genes from the origin and terminus (purA and ilvA), (ii) a 50 to 100S component also enriched for purA and ilvA, and (iii) the bulk of the DNA. The fast-sedimenting fraction was dissociated by Sarkosyl; this fraction contained a substantial amount of protein and is probably a membrane subparticle. The S value of the 50 to 100S component was not greatly affected by Sarkosyl treatment, but these particles were unable to penetrate an agarose gel during electrophoresis and were retained by nitrocellulose filters. The terminus DNA in the fast-sedimenting fraction and the 50 to 100S component contained a large restriction fragment (1.5 x 10(7) to 2.0 x 10(7) daltons) encoding ilvA, thyB, and ilvD. The bulk of the SP beta prophage and metB, which lie to the right and left, respectively, of the ilvA-ilvD cluster, were not part of the complex. citK, which lies to the right of SP beta, appeared to be present in the fast-sedimenting complexes. The neighboring genes kauA and gltA were not part of the fast-sedimenting complexes. The presence of terminus DNA in the fast-sedimenting components was also demonstrated by a radiochemical method.     

Functional morphology of the hindleg in two kangaroos Macropus giganteus and Aepyprymnus rufescens

In this study the structures in the hindleg of the kangaroo which are potentially available for jumping were examined. Specimens of two species, Macropus giganteus and Aepyprymnus rufescens, were examined and are described and compared. The basic pattern of the jump of the two species is similar. This is reflected anatomically by the fact that in both species the extensors of the hip, knee and ankle as a percentage of the total weight of the hindleg are greater than the flexors of the same joints. An additional similarity is that the biceps femoris and adductor magnus have the greatest share in the weight of the hip extensors. Furthermore the estimated total force of the hip, knee and ankle extensors and total moment of the hip and ankle extensors are always greater than the flexors of the same joints. However, the percentage of the hip and knee extensors, the absolute forces and moments of both the extensors and flexors and the range of movement especially of the hip and knee are always greater in M. giganteus than in A. rufescens. As well as these differences, the long tibia and the position of the knee in view of the hip may be important factors for the longer jump achieved by M. giganteus. In comparison A. rufescens has a anatomical construction which seems to be a compromise between walking and jumping.     

马铃薯'转心乌'块茎色素的组成和含量

马铃薯转心乌块茎的内、外韧皮部和髓为淡黄色,周皮和木质部为紫色;木质部的紫色形成一个不规则的环",并向内韧皮部蔓延.系列特征颜色反应和紫外可见光谱分析表明:‘转心乌’块茎紫色素属于黄酮类化合物,可能含有酚性邻位二羟基,并被肉桂酸酰化,不含类胡萝卜素、查耳酮、噢哢、异黄酮、儿茶素;花色苷和/或其苷元花色素奠定了‘转心乌’块茎着色的基础,其它的非红色的黄酮类化合物发挥共色素的作用.块茎的皮"紫色最浓","环"其次,"肉"最淡,这与"皮"、"环"和"肉"的色价、花色苷含量和总黄酮类化合物含量的变化趋势呈正相关.     

黄土高原小叶杨与其他树种枯落叶混合分解对土壤性质的影响

将小叶杨分别与其他11个树种枯落叶粉碎混合后进行室内分解培养,分析不同树种枯落叶混合分解对土壤性质的影响及其相互作用.结果表明:12个树种枯落叶单独混土分解均明显提高了土壤脲酶、脱氢酶、磷酸酶活性和有机质、碱解N含量,但对土壤速效P含量和土壤阳离子交换量(CEC)的影响差异较大,其中柠条和紫穗槐枯落叶改善土壤性质的效果明显.小叶杨分别与油松、侧柏、刺槐、白榆枯落叶混合分解,对土壤微生物数量的影响存在相互促进作用;小叶杨分别与侧柏、柠条枯落叶混合分解对土壤有机质、速效P、速效K含量和CEC的影响存在相互促进作用,但对土壤大部分酶活性的影响却存在相互抑制作用;小叶杨与落叶松枯落叶混合分解对土壤多数酶活性和养分含量的影响存在相互促进作用,而与樟子松枯落叶混合分解时则有抑制作用.总体上,小叶杨分别与白榆、油松、落叶松和刺槐枯落叶混合分解可促进土壤性质的改善,而与侧柏、柠条、樟子松、沙棘和紫穗槐枯落叶混合分解时则相互抑制.     

Biomimetic nanosystems and novel composite nanobiomaterials

Biophysicochemical approaches to the solution of nanotechnology problems associated with the design of functional biomimetic nanosystems, hybrid and composite nanobiomaterials and study of their structure-function relationships. The results of studies concerned with physicochemical mechanisms of the formation of organized biomimetic nanostructures and bioinorganic nanomaterials in systems involving a bulky liquid phase and the interface (gas-liquid, solid-liquid, liquid-liquid)during the synthesis and structure formation with the participation of the components of colloid systems, inorganic nanoparticles of various composition and clusters of metals, surfactants, polyelectrolytes and their complexes are discussed. In the development of the methods for the formation of composite bioinorganic nanosystems containing inorganic nanocomponents, two major approaches were used: adsorption and incorporation into the biomolecular matrix or colloid system of presynthesized inorganic nanoparticles, as well as the synthesis of the inorganic nanophase immediately in the biomolecular system. The methods of obtaining biomaterials and nanosystems are based on the principles of biomimetics, biomineralization, self-assembly and self-organization, combination and integration of a number of synthetic and physicochemical methods (physical and chemical adsorption, Langmuir technique, the formation of polycomplexes, chemical linking, competitive interactions, and substitution of ligands in supramolecular and coordination complexes) and nanocomponents of different nature. In particular, a novel approach to the preparation of highly organized nanofilm materials was developed, which is based on the effect of self-assembly and self-organization of colloid nanoparticles during the formation of their complexes with polyfunctional biogenic ligands in the volume of the liquid phase in the absence of any surfaces and interfaces. The physical and chemical factors responsible for the formation of structurally ordered biomolecular and composite nanosystems including nano-sized components of different nature and the possibilities to control the composition, structure, and properties of resulting nanomaterials and nanosystems are discussed. The experimental methods and approaches developed may be useful in studies of structure-property relationships and basic mechanisms of structural organization and transformation at the nanoscales level in biological, artificial, and hybrid nanosystems. The problems of practical application of the synthetic methods and the corresponding nanomaterials are discussed.     

Lignification and cell wall thickening in nodes of Phyllostachys viridiglaucescens and Phyllostachys nigra

BACKGROUND AND AIMS: Bamboos are among the most important plants in the world. The anatomical structure and mechanical properties of the culm internode are well documented. Fewer details are available of the culm node. The aim of this study was a topochemical investigation on lignification and cell wall thickening in developing and maturing bamboo nodes. The deposition sequence and distribution of lignin structural units and cell wall thickening in different anatomical regions of the node of Phyllostachys viridiglaucescens and Phyllostachys nigra are discussed. METHODS: Cell wall thickening and lignification are investigated in the outer part of the nodal region and in the diaphragm of developing and maturing P. nigra culms and in maturing culms of P. viridiglaucescens of different age classes. The lignification during ageing was studied topochemically by means of cellular UV microspectrophotometry. A combination of light microscopy and image analysis techniques were used to measure cell wall thickness. KEY RESULTS: The fibre and parenchyma cell wall thickness does not significantly increase during ageing. In the diaphragm, the cell walls are thinner and the cell diameter is larger than in the outer part of the node. In shoots, the lignin content in the epidermis, hypodermis and in both fibre and parenchyma cells of the diaphragm is relatively low compared with older culms. The fibre and parenchyma cells of the diaphragm have higher values of p-coumaric and ferulic acids than fibre and parenchyma cells of the outer part of the node. CONCLUSIONS: It was hypothesized that the combination of more hydroxycinnamic acids and of thinner cell walls in combination with higher cell diameters (lower density and lower stiffness) in the diaphragm than in the outer part of the node may play an important role in the biomechanical function of the node by acting as a spring-like joint to support the culm by bending forces.     

Comparison of the physiological and biochemical indices of the phagocytosis of ram erythrocytes by macro- and microphages]

Morita and Perkins' method was applied to the study of the stage of ingestion and destruction of an antigen (sheep erythrocytes) in the macrophages of peritoneal exudate of rabbits and rats and in the microphages of rabbit pleural exudate. Ingestion and intracellular destruction of the antigen were accompanied by intensified respiration and glycolysis of phagocytes. Respiration of the three types of phagocytes at two stages of phagocytosis and also the digestive capacity of microphages proved to be sensitive to cyanide and colchicine. The latter failed to influence the ingestion of the antigen by the three types of phagocytes and its digestion by macrophages. The differences in the metabolism routes of macro- and microphages in intracellular destruction of the antigen was postulated. An intensification of the phagocytic activity after the immunization was characteristic of rabbit and rat macrophages.     

不同生态地理区域杉木人工林土壤微生物及生化活性的研究

 本文研究了广西龙胜县里骆林区、宜山县庆远林区、岑溪县七坪林区和田林县老山林区四种不同生态地理区域杉木人工林土壤微生物及其生化活性。研究结果表明：1．土壤微生物的总数及各类群数量均以龙胜县里骆林区和田林县老山林区杉木人工林土壤最高,岑溪县七坪林区次之,宜山县庆远林区最低。2．土壤生化活性包括土壤氮的转化强度,土壤碳的氧化代谢能力和土壤酶活性,也均以龙胜县里骆林区和田林县老山林区杉木人工林土壤最强,岑溪县七坪林区次之,宜山县庆远林区最弱。3．四种不同生态地理区域杉木人工林生长状况与土壤微生物的数量分布和生化活性表现相似规律,龙胜县里骆林区和田林县老山林区杉木人工林林木生长最好,岑溪县七坪林区次之,宜山县庆远林区最差。