The Accumulation of Proteins with Chitinase Activity in the Culture Liquids of the Parent and Mutant Serratia marcescens Strains Grown in the Presence of Mitomycin C

The study of the accumulation pattern of extracellular proteins with chitinase activity in the parent Serratia marcescens strain Bú 211 (ATCC 9986) grown in the presence of mitomycin C and its mutant strain with the constitutive synthesis of chitinases grown in the absence of the inducer showed that chitinase activity appeared in the culture liquids of both strains at the end of the exponential phase (4 h of growth) and reached a maximum in the stationary phase (18–20 h of growth). The analysis of the culture liquids (12 h of growth) by denaturing electrophoresis in PAAG followed by the protein renaturation step revealed the presence of four extracellular proteins with chitinase activity and molecular masses of 21, 38, 52, and 58 kDa.     

Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes

70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNA^Phe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N-AcPhe-tRNA^Phe/(U)₉/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319–322).     

Thiol-dependent serine proteinase from <Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis>: Purification and catalytic properties

An extracellular thiol-dependent serine proteinase was isolated from culture medium filtrate of the microscopic fungus Paecilomyces lilacinus with a yield of 33%. The enzyme is inactivated by specific inhibitors of serine proteinases, phenylmethylsulfonyl fluoride, as well as by chloromercuribenzoate and mercury acetate, but is resistant to chelating agents. The proteinase has broad specificity, hydrolyzes proteins and p-nitroanilides of N-acylated tripeptides, exhibiting maximal activity in hydrolysis of substrates containing long hydrophobic and aromatic residues (norleucine, leucine, phenylalanine) as well as arginine at the P1 position. The enzyme has a molecular weight of 33 kD. The enzyme is most active at pH 10.0-11.5; it is thermostable and is characterized by broad optimum temperature range (30-60 degrees C), displaying about 25% of maximal activity at 0 degrees C. The N-terminal sequence of the enzyme (Gly-Ala-Thr-Thr-Gln-Gly-Ala-Thr-Gly/Ile-Xxx-Gly) has no distinct homology with known primary structures of serine proteinases from fungi and bacilli. Based on its physicochemical and enzymatic properties, the serine proteinase from P. lilacinus can be classified as a thiol-dependent subtilisin-like enzyme.     

A glimpse on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> translation machinery and its control

Staphylococcus aureus is a major opportunistic and versatile pathogen. Because the bacteria rapidly evolve multi-resistances towards antibiotics, there is an urgent need to find novel targets and alternative strategies to cure bacterial infections. Here, we provide a brief overview on the knowledge acquired on S. aureus ribosomes, which is one of the major antibiotic targets. We will show that subtle differences exist between the translation at the initiation step of Gram-negative and Gram-positive bacteria although their ribosomes display a remarkable degree of resemblance. In addition, we will illustrate using specific examples the diversity of mechanisms controlling translation initiation in S. aureus that contribute to shape the expression of the virulence factors in a temporal and dynamic manner.     

The multiple flavors of GoU pairs in RNA

Wobble GU pairs (or GoU) occur frequently within double‐stranded RNA helices interspersed within the standard G═C and A─U Watson‐Crick pairs. However, other types of GoU pairs interacting on their Watson‐Crick edges have been observed. The structural and functional roles of such alternative GoU pairs are surprisingly diverse and reflect the various pairings G and U can form by exploiting all the subtleties of their electronic configurations. Here, the structural characteristics of the GoU pairs are updated following the recent crystallographic structures of functional ribosomal complexes and the development in our understanding of ribosomal translation.     

The effect of chitooligosaccharides on hydrogen peroxide production and anionic peroxidase activity in wheat coleoptiles

We studied the effects of chitooligosaccharides (ChOS) with a mol wt of 5 kD, the degree of acetylation of 65%, and the concentrations from 0.01 to 100 mg/l on the content of hydrogen peroxide in incubation medium and the activity of anionic peroxidase (pI 3.5) in the segments of wheat (Triticum aestivum) coleoptiles. H₂O₂ production and peroxidase activity were found to be dependent on the ChOS concentration. After 3 h of incubation, the highest H₂O₂ level in medium was observed at 0.01 mg/l ChOS, whereas after 6h, at 1 mg/l. After 3 h of incubation, ChOS suppressed peroxidase activity. After 6 h of incubation, high ChOS concentrations enhanced peroxidase activity. IAA favored H₂O₂ accumulation in medium and suppressed anionic peroxidase. The involvement of ChOS in the control of the level of reactive oxygen species and anionic peroxidase activity in plant cells is suggested.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 238–242.Original Russian Text Copyright © 2005 by Yusupova, Akhmetova, Khairullin, Maksimov.This revised version was published online in April 2005 with a corrected cover date.     

In vitro refolding of carboxypeptidase T precursor from Thermoactinomyces vulgaris obtained in Escherichia coli as cytoplasmic inclusion bodies

Carboxypeptidase T precursor from Thermoactinomyces vulgaris, which fails to contain its own leader peptide, has been expressed in Escherichia coli as insoluble cytoplasmic inclusion bodies. The yield of a washed recombinant protein from 1 L of culture liquid was about 60 mg. The obtained inclusion bodies were denatured in 6 M guanidine-HCl and then renatured by a rapid dilution. The important role of calcium for the complete stabilization of the refolded carboxypeptidase T precursor was established. After removal of minor admixture proteins by gel-filtration through Superdex 75, an electrophoretically homogeneous preparation of the native precursor of carboxypeptidase T was obtained. Processing of the resulting protein by subtilisin led to the formation of the mature carboxypeptidase T in which N-terminal sequence, molecular size, thermal stability, and catalytic properties were comparable to those of the natural enzyme.     

VPg unlinkase,the phosphodiesterase that hydrolyzes the bond between VPg and picornavirus RNA: a minimal nucleic moiety of the substrate

VPg unlinkase is an unusual eukaryotic enzyme that catalyzes hydrolysis of the phosphodiester bond between residues of the unique tyrosine of VPg (viral protein genome-linked) and the 5"-terminal uridylic acid of picornavirus RNA. Cellular targets of the VPg unlinking enzyme are yet unknown. To determine an essential nucleic part of the covalent linkage unit that is necessary for the VPg unlinkase reaction, the following derivatives of the encephalomyocarditis virus (EMCV) VPg–RNA complex were used: [¹²⁵I]Kp–pUpUpGp, [¹²⁵I]Kp–pUp, and [¹²⁵I]Kp–pU (Kp is residual peptides bound to RNA after proteinase K treatment of VPg–RNA). [¹²⁵I]K-peptides were unlinked from [¹²⁵I]Kp–pUpUpGp and [¹²⁵I]Kp–RNA with similar velocity, but [¹²⁵I]Kp–pUp was split much slower. Under the same conditions [¹²⁵I]Kp–pU was not dissociated at all. Thus, pUp is a minimal part of picornavirus RNA that is necessary for VPg unlinkase. We speculate that cellular substrates of the enzyme are phosphodiesters of oligo(poly)ribonucleotides and tyrosine or tyrosine peptides. In no case [¹²⁵I]VPg–pU, [¹²⁵I]VPg–pUp, and [¹²⁵I]VPg–pUpUpGp were hydrolyzed by VPg unlinkase, in contrast with [¹²⁵I]VPg–RNA and [¹²⁵I]VPg–pUpUpGpApApApGp. We conclude that the whole VPg, when bound to trinucleotide (but not to heptanucleotide), protects the inter-polymeric phosphodiester bond against hydrolysis of the covalent linkage unit. We speculate that VPg unlinkase might repair covalent complexes of RNA and topoisomerases and trigger degradation process of the picornavirus RNA.     

The path of messenger RNA through the ribosome

Using X-ray crystallography, we have directly observed the path of mRNA in the 70S ribosome in Fourier difference maps at 7 A resolution. About 30 nucleotides of the mRNA are wrapped in a groove that encircles the neck of the 30S subunit. The Shine-Dalgarno helix is bound in a large cleft between the head and the back of the platform. At the interface, only about eight nucleotides (-1 to +7), centered on the junction between the A and P codons, are exposed, and bond almost exclusively to 16S rRNA. The mRNA enters the ribosome around position +13 to +15, the location of downstream pseudoknots that stimulate -1 translational frame shifting.     

Biosynthesis of Proteus mirabilis Nuclease

The culture liquid and periplasm of Proteus mirabilis contained nuclease, an enzyme with DNase and RNase activities. The nuclease was most actively synthesized in the early exponential and stationary growth phases. Nuclease synthesis was regulated by nucleic acids (induction by substrate) and inorganic phosphate (end-product inhibition). The synthesis and secretion of nuclease by P. mirabilis was induced by mitomycin C, an inducer of the SOS functions of cells. This suggests the involvement of SOS-response proteins in the regulation of nuclease synthesis.