Anionic polymers of the cell wall of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> subsp. <Emphasis Type="Italic">subtilis</Emphasis> VKM B-501<Superscript>T</Superscript>

Teichoic acid and disaccharide-1-phosphate polymer were identified in the cell walls of Bacillus subtilis subsp. subtilis VKM B-501^T. The teichoic acid represents 1,3-poly(glycerol phosphate) 80% substituted by α-D-glucopyranose residues at O-2 of glycerol. The linear repeating unit of disaccharide-1-phosphate polymer contains the residues of β-D-glucopyranose, N-acetyl-α-D-galactosamine, and phosphate and has the following structure: -6)-β-D-Glcp-(1→3)-α-D-GalpNAc-(1-P-. The structures of two anionic polymers were determined by chemical and NMR-spectroscopic methods. The ¹H- and ¹³C-NMR spectral data on disaccharide-1-phosphate polymer are presented for the first time.     

Cloning of <Emphasis Type="Italic">rec</Emphasis>A gene of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and phenotypic complementation of <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM^®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm², while non-transformed cells tolerated only up to dose 0.08 J/cm². It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.     

D-ribose-5-phosphate isomerase B from Escherichia coli is also a functional D-allose-6-phosphate isomerase, while the Mycobacterium tuberculosis enzyme is not

Interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate is an important step in the pentose phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars d-allose-6-phosphate (All6P) and d-allulose-6-phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme's primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-d-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme's structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate ribose-5-phosphate, shed additional light on the reaction mechanism of RpiBs generally.     

Chromosomal differentiation of the sibling species <Emphasis Type="Italic">Pichia membranifaciens</Emphasis> and <Emphasis Type="Italic">Pichia manshurica</Emphasis>

The molecular karyotyping analysis of 21 strains within the taxonomic complex Pichia membranifaciens allowed the sibling species P. membranifaciens and P. manshurica, as well as P. deserticola and P. punctispora, to be differentiated. Heterogeneity of the species P. membranifaciens at the variety level is discussed.     

Antirestriction and antimodification activities of T7 Ocr: Effects of amino acid substitutions in the interface

The T7 antirestriction protein Ocr, encoded by 0.3 (ocr), specifically inhibits ATP-dependent type I restriction-modification systems. T7 0.3 (ocr) was cloned in pUC18. Ocr inhibited both restriction and modification activities of the type I restriction-modification system (EcoKI) in Escherichia coli K12. The Ocr F53D A57E mutant was obtained and proved to inhibit only restriction activity of EcoKI. The 0.3 (ocr) and Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P _ltetO-1 promoter, strongly controlled by the TetR repressor. The bioluminescence intensity and luciferase content varied up to 5000-fold in E. coli K12 MG1655Z1 tetR⁺ (pZE21-luxCDABE) cells, depending on the environmental concentration of the inductor anhydrotetracycline. The antirestriction activity of Ocr and Ocr F53D A57E was studied as a function of their concentration in the cell. The dissociation constant K _d, characterizing the binding with EcoKI, differed 1000-fold between Ocr and Ocr F53D A57E (10^?10 M versus 10^?7 M).     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Interaction of single-stranded DNA with the second DNA-binding site of the RecA nucleoprotein filament

RecA first forms a filament on single-stranded DNA (ssDNA), thereby forming the first site for ssDNA binding and, simultaneously, the second site for binding double-stranded DNA (dsDNA). Then, the nucleoprotein filament interacts with dsDNA, although it can bind ssDNA as well. The resulting complex searches for homology sites and performs strand exchange between homologous DNA molecules. The interaction of various ssDNAs with the second DNA-recognizing site of RecA was studied by gradually increasing the structural complexity of the DNA ligand. Recognizing ssDNA with the second site, the protein interacts with each nucleotide of the ligand, forming contacts with both internucleotide phosphate groups and nitrogen bases. Pyrimidine oligonucleotides d(pC) _n and d(pT) _n interacted with the second site of the RecA filament more efficiently than d(pA) _n did. This was due to a more efficient interaction of the RecA filament with the 5′-terminal nucleotide of pyrimidinic DNA and to the difference in specific conformational changes of the nucleoprotein filament in the presence of purinic and pyrimidinic DNAs. A comparison of thermodynamic characteristics of DNA recognition at the first and second DNA-binding sites of the filament showed that, at n > 10, d(pC) _n and d(pN) _n were bound at the second site less tightly than at the first site. At n > 20, the second site bound d(pA) _n more efficiently than the first site. The difference in d(pN) _n affinity for the first and second sites increased monotonically with increasing n. Possible mechanisms of a RecA-dependent search for homology and DNA strand exchange are discussed.     

Isolation,structure modeling and function characterization of a trypsin inhibitor from <Emphasis Type="Italic">Cassia obtusifolia</Emphasis>

A trypsin inhibitor gene (CoTI1) from Cassia obtusifolia was isolated and the deduced amino acid sequence was attributed to the Kunitz-type trypsin inhibitor. The recombined CoTI1, expressed in E. coli, exhibited strong inhibitory effect on bovine trypsin and trypsin-like proteases from Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura. CoTI1 thus presents insecticidal properties that may be useful for the genetic engineering of plants. Leu84, Arg86 and Thr88 were predicted as three key residues by molecular modeling in which Arg86, inserted into the substrate pocket of trypsin, interacted directly with residue Asp189 of trypsin causing the specific inhibition against trypsin. The predicted results were confirmed by site-directed mutagenesis with L84A, R86A and T88A, respectively. The substantial changing expression level of CoTI1 under salt, drought and abscisic acid treatment suggested that CoTI1 might play important role in the resistance against abiotic stress.     

Crystal structure of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> ribose-5-phosphate isomerase B reveals properties critical for fast enzyme kinetics

Ribose-5-phosphate isomerase (Rpi) catalyzes the conversion of d-ribose 5-phosphate (R5P) to d-ribulose 5-phosphate, which is an important step in the non-oxidative pathway of the pentose phosphate pathway and the Calvin cycle of photosynthesis. Recently, Rpis have been used to produce valuable rare sugars for industrial purposes. Of the Rpis, d-ribose-5-phosphate isomerase B from Clostridium thermocellum (CtRpi) has the fastest reactions kinetics. While Thermotoga maritime Rpi (TmRpi) has the same substrate specificity as CtRpi, the overall activity of CtRpi is approximately 200-fold higher than that of TmRpi. To understand the structural basis of these kinetic differences, we determined the crystal structures, at 2.1-Å resolution or higher, of CtRpi alone and bound to its substrates, R5P, d-ribose, and d-allose. Structural comparisons of CtRpi and TmRpi showed overall conservation of their structures with two notable differences. First, the volume of the CtRpi substrate binding pocket (SBP) was 20% less than that of the TmRpi SBP. Second, the residues next to the sugar-ring opening catalytic residue (His98) were different. We switched the key residues, involved in SBP shaping or catalysis, between CtRpi and TmRpi by site-directed mutagenesis, and studied the enzyme kinetics of the mutants. We found that tight interactions between the two monomers, narrow SBP width, and the residues near the catalytic residue are all critical for the fast enzyme kinetics of CtRpi.     

Development and relations of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> and <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> in soil

The development of Fusarium culmorum and Pseudomonas fluorescens in soil, and the relations between them, were studied using membrane filters containing the fungus, the bacterium, or both microorganisms; the filters were incubated in soil. F. culmorum was identified by indirect immunofluorescence; the GUS-labeled strain was used to visualize P. fluorescens. It was found that F. culmorum introduced in soil can develop as a saprotroph, with the formation of mycelium, macroconidia, and a small amount of chlamydospores. Introduction of glucose and cellulose resulted in increased density of the F. culmorum mycelium and macroconidia. P. fluorescens suppressed the development of the F. culmorum mycelium in soil, but stimulated chlamydospore formation. Decreased mycelial density in the presence of P. fluorescens was more pronounced in soil without additions and less pronounced in the case of introduction of glucose or cellulose. F. culmorum had no effect on P. fluorescens growth in soil.     

Purification and properties of recombinant DNA methyltransferase M2.<Emphasis Type="Italic">Bst</Emphasis>SE of the <Emphasis Type="Italic">Bst</Emphasis>SEI nickase-modification system

The operon for the Bacillus stearothermophilus SE-589 nickase-modification system (NM.BstSEI, recognition site 5′-GAGTC-3′) includes two DNA methyltransferase (M.) genes, bstSEIM1 and bstSEIM2. The gene encoding M2.BstSEI was cloned in pJW and expressed in Escherichia coli cells. M2.BstSEI was purified by chromatography and displayed maximal activity at 55° C and pH 7.5. The enzyme modified adenine in the nickase recognition site 5′-GAGTC-3′ and was specific for 5′-GASTC-3′ substrates. The kinetic parameters of the methylation reaction were determined. The catalytic constant was 2.2 min^?1, and the Michaelis constant was 9.8 nM on T7 DNA and 5.8 μM on SAM.     

Mapping of the Ogura fertility restorer gene <Emphasis Type="Italic">Rfo</Emphasis> and development of <Emphasis Type="Italic">Rfo</Emphasis> allele-specific markers in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader^® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.     

The role of thiol redox systems in the response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to far-UV irradiation

Escherichia coli mutants deficient in glutathione (gshA), glutaredoxin (grxA), thioredoxin (trxA), and thioredoxin reductase (trxB) synthesis were studied with respect to their resistance to far-UV (UV₂₅₄) exposure. The trxA, trxB, and grxA mutants subjected to a short-term UV exposure were found to be more resistant to UV irradiation than the parent cells. Under the same conditions, the trxA and trxB mutants demonstrated a high level of induction of the sulA gene, a component of the SOS regulon. The mutagenic effect of long-term UV exposure of all the mutants with redox deficiencies was more pronounced than in the case of the parent strain, and the trxA and trxB mutants were found to be the least viable microorganisms. Pretreatment of the cells with low concentrations of the thiol-oxidizing agent diamide enhanced the sulA gene expression; however, high concentrations of diamide inhibited sulA expression. The data obtained indicate that the thiol redox systems of E. coli are involved in its response to far-UV irradiation.     

Polymorphism of <Emphasis Type="Italic">yellow</Emphasis> locus in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> mutants from natural populations

We have studied the molecular characteristics of the yellow locus (y; 1–0.0), which determines the body color of phenotypically wild-type and mutant alleles isolated in different years from geographically distant populations of Drosophila melanogaster. According to the Southern blot, data restriction maps of the yellow locus of all examined strains differ from one another, as well as from Oregon stock. FISH analysis shows that, in the neighborhood of the yellow locus in the X chromosome, neither P nor hobo elements are found in y^1–775 stock, while only hobo is found in these region in y^1–859 and y^1–866 stocks, only the P element is found in y⁺sn⁸⁴⁹ stock, and both elements are found in y^1–719 stock. Thus, all yellow mutants studied are of independent origin. Locus yellow located on the end of X chromosome (region 1A5–8 on the cytologic map) carries significantly more transposon than retrotransposon induced mutations compared to the white locus (region 3C2). It is possible that, at the ends of Drosophila melanogaster chromosomes, transposons are more active than retrotransposons.     

Photosynthetic activity and components of the electron transport chain in the aerobic bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>-containing bacterium <Emphasis Type="Italic">Roseinatronobacter thiooxidans</Emphasis>

Bioenergetics of the aerobic bacteriochlorophyll a-containing (BCl a) bacterium (ABC bacterium) Roseinatronobacter thiooxidans is a combination of photosynthesis, oxygen respiration, and oxidation of sulfur compounds under alkaliphilic conditions. The photosynthetic activity of Rna. thiooxidans cells was established by the photoinhibition of cell respiration and reversible photobleaching discoloration of the BCl a of reaction centers (RC), connected by the chain of electron transfer with cytochrome c ₅₅₁ oxidation. The species under study, like many purple bacteria and some of the known ABC bacteria, possesses a light-harvesting pigment-protein (LHI) complex with the average number of 30 molecules of antenna BCl a per one photosynthetic RC. Under microaerobic growth conditions, the cells contained bc ₁ complex and two terminal oxidases: cbb ₃-cytochrome oxidase and the alternative cytochrome oxidase of the a ₃ type. Besides, Rna. thiooxidans was shown to have several different soluble low- and high-potential cytochromes c, probably associated with the ability of utilizing sulfur compounds as additional electron donors.     

Ammonites of Tethyan origin from the Ryazanian of the Russian platform: Genus <Emphasis Type="Italic">Riasanites</Emphasis> spath

The genus Riasanites, represented in Central Russia by two successive dimorphic species, is revised. R. swistowianus is found in the basal beds of the rjasanensis Zone. Its descendant R. rjasanensis is also found in this zone, but upwards in the section, including the beds with Surites spasskensis and Externiceras solowaticum. The representatives of Riasanites from the Crimea and Northern Caucasus are assigned to two species, R. crassicostatus and R. maikopensis, respectively. It is suggested that Riasanites evolved from Sub-Mediterranean Himalayatidae, which migrated from the Western Tethys via the Polish Passage into the Central Russian Basin, and from there to Mangyshlak, the Northern Caucasus, and the Crimea.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Histone-antihistone interactions in the <Emphasis Type="Italic">Escherichia coli-Tetrahymena pyriformis</Emphasis> association

Using the Escherichia coli-Tetrahymena pyriformis model system, we revealed the involvement of bacterial antihistone activity and protozoan histones in interactions between pro-and eukaryotic microorganisms. Antihistone activity enhanced the viability of E. coli in association with T. pyriformis, according to our data on the dynamics of E. coli cell numbers. The strain with antihistone activity induced incomplete phagocytosis in the infusorians, resulting in cytological changes and ultrastructural alterations that indicated the retention of bacterial cells in phagosomes. Bacteria with antihistone activity located in the T. pyriformis cytoplasm influenced the eukaryotic nucleus. This was accompanied by in macronucleus decompactization and a decrease in the average histone content in the population of infusorians. The data obtained suggest that protozoan histone inactivation by bacteria is one of the mechanisms involved in prokaryote persistence in associations with eukaryotic microorganisms.     

Three new species of <Emphasis Type="Italic">Passiflora</Emphasis> subgenus <Emphasis Type="Italic">Decaloba</Emphasis> (Passifloraceae) from Brazil

Three new species from Brazil are described and illustrated. Passiflora cervii, P. jiboiaensis, and P. transversalis all belong to Passiflora subg. Decaloba.