New SINE families from rice, OsSN, with poly(A) at the 3' ends

A database search of the sequences flanking a member of rice retrotransposon RIRE7 revealed that a 298-bp sequence in the region downstream of the member is a repetitive sequence interspersed in the genome of Oryza sativa cv. Nipponbare. Most of the repetitive sequences were flanked by a direct repeat of a target-site sequence, about 14 bp in length. The consensus sequence, 293 bp in length, had no regions encoding any proteins but had sequence motifs of an internal promoter of RNA polymerase III. These indicate that the sequence is a retroposon SINE, designated OsSN1 (Oryza sativa SINE1). OsSN1 is a new rice SINE, because it has no homology with any of the three p-SINE families previously identified from rice, and because it has a stretch of A at the 3' end, unlike p-SINE and any other Gramineae SINEs which have a stretch of T at the 3' end. The Nipponbare genome was found to have many members related to OsSN1, forming two additional new SINE families (designated OsSN2 and OsSN3). OsSN2 and OsSN3 are highly homologous to the 3' and 5' regions of OsSN1, respectively. This suggests that OsSN1 has a mosaic structure, which is generated by sequence exchange (or shuffling) between ancestral OsSN2 and OsSN3. Despite the absence of homology in the 3' regions between OsSN1 (or OsSN2) and OsSN3, a sequence, 5'-TTCTC-3', is commonly present in the region preceding the A stretch at the 3' end. This sequence together with the A stretch and a stem-loop structure found in the region near the A stretch are assumed to be important for retroposition. OsSN members were present in strains of Oryza species, as were p-SINE members. Some of the members showed insertion polymorphism at the respective loci among the rice strains. p-SINE had such polymorphic members, which are useful for classification and phylogenetic analysis of various strains of Oryza species. The polymorphic members of OsSN were more frequently found than those of p-SINE, and therefore, such members are likely to be useful for extensive taxonomic and phylogenetic studies on various rice strains.     

Synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis

The synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis was studied. A significant amount of RNA puls-labelled with 3H-uridine is polyadenylated. With the beginning of RNA synthesis in outgrowing spores labelled poly(A)-containing RNA was detected. The amount of poly(A)-RNA during the outgrowth and first cell division remains constant. Besides poly(A)-RNA the synthesis of tRNA and rRNA occurs. These results indicate a simultaneous activation of synthesis of tRNA, rRNA as well as of poly(A)-containing RNA during outgrowth of B. subtilis spores.     

The yvaJ gene of Bacillus subtilis encodes a 3'-to-5' exoribonuclease and is not essential in a strain lacking polynucleotide phosphorylase

Studies of Bacillus subtilis RNases that are involved in mRNA degradation reveal a different pattern from that of Escherichia coli. A strain lacking polynucleotide phosphorylase, the major 3'-to-5' exoribonuclease activity in cell extracts, is viable. Here, we show that the B. subtilis yvaJ gene encodes a second 3'-to-5' exoribonuclease. A strain lacking both of these RNases grows slowly but is viable. The existence of another, as yet unknown, 3'-to-5' exoribonuclease in B. subtilis is suggested.     

Template-independent poly(A) x poly(U) synthesizing activity of different forms of Bacillus subtilis RNA polymerase

The steady-state kinetic parameters of the tripeptides D-Val-Leu-Lys-, Ala-Phe-Lys-, and < Glu-Phe-Lys- in which the free carboxyl group was substituted with p-nitroaniline (substrate) or chloromethane (inhibitor), towards the serine proteinases plasmin (EC 3.4.21.7), thrombin (EC 3.4.21.5), urokinase, factor Xa, and trypsin (EC 3.4.21.4) were investigated. The p-nitroanilide derives were found to be very good substrates for plasmin, 2.5--40-times less efficient towards trypsin and very poor (100--10 000-times less efficient) substrates for thrombin, factor Xa and urokinase. The chloromethyl ketone derivatives were comparably efficient inhibitors of plasmin and trypsin and in general very poor (100--10 000-times weaker) inhibitors of thrombin, factor Xa and urokinase. D-Val-Leu-Lys-pNA however was a very poor substrate but D-Val-Leu-Lys-CH2Cl a very efficient inhibitor for thrombin. The variability in susceptibility of the substrates towards the enzymes was due to differences in their Michaelis constant, in their deacylation rate constant or both. the variable efficiency of the inhibitors was mostly due to differences in their dissociation constant and much less to differences in their alkylation rate constant. Only a poor correlation (r = 0.25) was found between the efficiency of the p-nitroanilides as substrate and their homologous chloromethyl ketones as inhibitor. The most notable discrepancy was observed with the D-Val-Leu-Lys derivatives towards thrombin.     

A template independent, rifampicin sensitive poly (A).poly (U) synthesizing activity present in Bacillus subtilis.

A template independent poly (A)·poly (U) synthesizing activity has been isolated from $\text{Bacillus subtilis}$. This activity is eluted from a DNA-cellulose column along with DNA-dependent RNA polymerase. The column fractions which exhibit this activity contain RNA polymerase holoenzyme plus a polypeptide which is slightly larger than sigma factor; pure RNA polymerase holoenzyme did not synthesize poly (A)·poly (U). The activity was dependent on the presence of ATP, UTP, and Mn⁺⁺ (Mg⁺⁺ could not substitute), and was inhibited by rifampicin, streptolydigin, and Cibacron Blue. The incorporation of nucleotides was not linear with time, but appeared after a lag period. The results suggest that a modified form of DNA-dependent RNA polymerase analogous to $\text{Escherichia coli}$ holoenzyme II is catalyzing the synthesis of poly (A)·poly (U).     

Alteration of tyrosine isoaccepting transfer ribonucleic acid species in wild-type and asporogenous strains of Bacillus subtilis.

The relative amounts of two isoacceping species of tyrosine transfer ribonucleic acid, tRNATyrI and tRNATyrII, determined from reversed phase 5 profiles of tyrosyl-tRNA, prepared from Bacillus subtilis strain W168, were growth phase and medium dependent. The growth phase-dependent alterations in the relative amounts of tRNATyr species were also demonstrated in 11 asporogenous strains of B. subtilis. The proportion of tRNA-Tyr species and the extent of the alteration in their relative amounts during the transition from the exponential to the stationary phase of growth of these strains was not directly correlated with the formation of spores by strain W168 grown in various media or the stage at which the asporogenous strains are blocked in the process of sporulation.     

Polymerase C1 levels and poly(R-3-hydroxyalkanoate) synthesis in wild-type and recombinant Pseudomonas strains.

A functional antibody highly specific for polymerase C1 of Pseudomonas oleovorans GPo1 was raised and used to determine polymerase C1 levels in in vivo experiments. The polymerase C1 antibodies did not show a cross-reaction with polymerase C2 of P. oleovorans. In wild-type P. oleovorans GPo1 and Pseudomonas putida KT2442, amounts of 0.075 and 0.06% polymerase relative to total protein, respectively, were found. P. oleovorans GPo1(pGEc405), which contained additional copies of the polymerase C1-encoding gene under the control of its native promoter, contained 0.5% polymerase C1 relative to total protein. Polymerase C1 reached 10% of total cell protein when the polymerase C1-encoding gene was overexpressed through the P(alk) promoter in P. oleovorans GPo1(pET702, pGEc74). Amounts of poly(R-3-hydroxyalkanoate) (PHA) increased significantly under non-nitrogen-limiting conditions when additional polymerase C1 was expressed in P. oleovorans. Whereas P. oleovorans produced 34% (wt/wt) PHA under these conditions, a PHA level of 64% (wt/wt) could be reached for P. oleovorans GPo1(pGEc405) and a PHA level of 52% (wt/wt) could be reached for P. oleovorans GPo1(pET702, pGEc74) after induction, compared to a PHA level of 13% for the uninduced control. All recombinant Pseudomonas strains containing additional polymerase C1 showed small changes in their PHA composition. Larger amounts of 3-hydroxyhexanoate monomer and smaller amounts of 3-hydroxyoctanoate and -decanoate were found compared to those of the wild type. Two different methods were developed to quantify rates of incorporation of new monomers into preexisting PHA granules. P. oleovorans GPo1 cells grown under nitrogen-limiting conditions showed growth stage-dependent incorporation rates. The highest PHA synthesis rates of 9.5 nmol of C8/C6 monomers/mg of cell dry weight (CDW)/min were found during the mid-stationary phase, which equals a rate of production of 80 g of PHA/kg of CDW/h.     

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.     

Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.     

Chelation of calcium ions by poly(gamma-glutamic acid) from Bacillus subtilis(chungkookjang)

Many studies have clarified that poly(gamma-glutamic acid) (PGA) increases the solubility of Ca(2+), suggesting that PGA enhances calcium absorption in small intestine. However, there has been no report on the specific interaction between PGA and Ca(2+) in water. We studied the aqueous solution properties of PGA calcium salt (PGA-Ca complex). The chelating ability and binding strength of PGA for Ca(2+) were evaluated. PGA-Ca complex was soluble in water in contrast with the insolubility of poly(acrylic acid) (PAA) calcium salt and the chelating ability of PGA for Ca(2+) was almost the same than that of PAA. The globular conformation of PGA-Ca complex in water was estimated by SEC and viscosity measurements. The chelation of PGA for Ca(2+) was examined by 1H NMR. The present study showing the characteristics of PGA-Ca complex will provide useful information of the calcium absorption by PGA in vivo.     

Adsorption of rare earth ions onto the cell walls of wild-type and lipoteichoic acid-defective strains of Bacillus subtilis

The aim of this study is to investigate the potential of cell walls of wild-type and lipoteichoic acid-defective strains of Bacillus subtilis 168 to adsorb rare earth ions. Freeze-dried cell powders prepared from both strains were used for the evaluation of adsorption ability for the rare earth ions, namely, La(III), Eu(III), and Tm(III). The rare earth ions were efficiently adsorbed onto powders of both wild-type strain (WT powder) and lipoteichoic acid-defective strain (?LTA powder) at pH 3. The maximum adsorption capacities for Tm(III) by WT and ?LTA powders were 43 and 37 mg g^?1, respectively. Removal (in percent) of Tm(III), La(III), and Eu(III) from aqueous solution by WT powder was greater than by ?LTA powder. These results indicate that rare earth ions are adsorbed to functional groups, such as phosphate and carboxyl groups, of lipoteichoic acid. We observed coagulated ?LTA powder in the removal of rare earth ions (1–20 mg L^?1) from aqueous solution. In contrast, sedimentation of WT powder did not occur under the same conditions. This unique feature of ?LTA powder may be caused by the difference of the distribution between lipoteichoic acid and wall teichoic acid. It appears that ?LTA powder is useful for removal of rare earth ions by adsorption, because aggregation allows for rapid separation of the adsorbent by filtration.     

Transketolase mutation in riboflavin-synthesizing strains of Bacillus subtilis

Unlike its predecessors B. subtilis rosR and 41, riboflavin producing B. subtilis 24 strain does not utilize pentose and gluconate and poorly assimilates glucose. Simultaneous addition of glutamic and shikimic acid restored its capacity to grow and produce riboflavin in medium with pentose and gluconate. This strain lacks the activity of transketolase, the key enzyme of the pentose phosphate cycle, and possesses normal ribulose-5-phosphate-epimerase and glucose phosphate isomerase activities. Like enterobacteria, B. subtilis has two different transport systems for glucose and mannose. The data are discussed from the viewpoint of increasing riboflavin production by transketolase mutants. Probable consequences of cell wall and cytoplasmatic membrane damage in B. subtilis with this mutation are discussed.     

Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis.

Continuous cultivation in a glucose-limited chemostat was used to determine the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coefficients of 0.45 and 0.66 mmol of glucose g-1 h-1 were determined for the wild-type and recombinant strains, respectively. However, the maximum molar growth yield of 82 to 85 g (cell dry weight)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin production rate and the dilution rate was observed, revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For molecular characterization of B. subtilis, the total amino acid composition of the wild type was experimentally determined and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approximately 9% of B. subtilis biomass, three- to fivefold more than in Escherichia coli. Estimation of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle.     

Mutational analysis of the mengovirus poly(C) tract and surrounding heteropolymeric sequences.

Previously, we described three mengovirus mutants derived from cDNA plasmids, containing shortened poly(C) tracts (C8, C12, and C13UC10), that exhibited strong attenuation for virulence in mice yet grew like wild-type virus in HeLa cells. Thirteen additional mutants hav now been constructed and characterized. Five of these differ only in poly(C) length, including one with a precise deletion of the tract. The other mutants bear deletions into the regions juxtaposing poly(C). Studies with HeLa cells confirm the essential dispensability of mengovirus's poly(C) tract but reveal a subtle, measurable correlation between poly(C) length and plaque diameter. Virulence studies with mice also revealed a strong correlation between poly(C) length and virulence. For the poly(C)-flanking mutations, the 15 bases directly 5' of the tract proved dispensable for virus viability, whereas the 20 to 30 bases 3' of poly(C) were critical for growth, thus implicating this region in the basal replication of the virus.     

Optimization of cell growth and poly(glutamic acid) production in batch fermentation by Bacillus subtilis

Poly(glutamic acid) was produced maximally by Bacillus subtilis in batch fermentations at pH 7 and using glycerol at 20 g l^–1 in a glutamic acid/citric acid medium. Poly(glutamic acid) reached 23 g l^–1 after 30 h.