Spectral-kinetic analysis of recombination reaction of heme centers of <Emphasis Type="Italic">bd</Emphasis>-type quinol oxidase from <Emphasis Type="Italic">Escherichia coli</Emphasis> with carbon monoxide

Recombination of the isolated, fully reduced bd-type quinol oxidase from Escherichia coli with carbon monoxide was studied by pulsed absorption spectrophotometry with microsecond time resolution. Analysis of the kinetic phases of recombination was carried out using the global analysis of multiwavelength kinetic data (“Global fitting”). It was found that the unresolved photodissociation of CO is followed by a stepwise (with four phases) recombination with characteristic times (τ) of about 20 μs, 250 μs, 1.1 ms, and 24 ms. The 20-μs phase most likely reflects bimolecular recombination of CO with heme d. Two subsequent kinetic transitions, with τ ~ 250 μs and 1.1 ms, were resolved for the first time. It is assumed that the 250-μs phase is heterogeneous and includes two different processes: recombination of CO with ~7% of heme b₅₉₅ and transition of heme d from a pentacoordinate to a transient hexacoordinate state in this enzyme population. The 24-ms transition probably reflects a return of heme d to the pentacoordinate state in the same protein fraction. The 1.1-ms phase can be explained by recombination of CO with ~15% of heme b₅₅₈. Possible models of interaction of CO with different heme centers are discussed.     

The Activity and Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Wheat Plants as Affected by Water Stress and Kartolin-4

The carboxylating activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39), and other soluble proteins in young seedlings and mature leaves of Lutescens-758, a drought-sensitive cultivar of soft spring wheat Triticum aestivum L., were studied under the conditions of drought and subsequent rehydration. Seedlings and mature plants preliminarily treated with the cytokinin-like compound kartolin-4 were compared to untreated plants. Drought-induced decrease in RuBPCO activity should be attributed not only to proteolytic decomposition of the enzyme protein itself but also to a partial inhibition of its catalytic activity. The decrease in RuBPCO activity was larger than that in RuBPCO content. Water stress induced a marked decrease in the soluble protein content. Kartolin-4 increased the resistance to drought.     

Activity of carbon metabolism enzymes in wheat plants treated with kartolin-4 and exposed to water stress]

Enzymatic activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39), phospho(enol)pyruvate carboxylase (EC 4.1.1.31), NAD malate dehydrogenase (EC 1.1.1.37), and NADP glyceraldehydephosphate dehydrogenase complex including phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehydephosphate dehydrogenase (EC 1.2.1.13) were comparatively assayed in wheat seedlings of the cultivar Lyutestsens 758 grown under normal conditions, water deficiency conditions, and subsequent rehydration. Water stress was found to decrease the activity of all enzymes tested, the effect being most pronounced in case of Rubisco. The content of Rubisco in wheat plants exposed to water deficiency was reduced less significantly than the activity of the enzyme. Preliminary treatment of plant seeds with kartolin-4 (o-isopropyl-N-2-hydroxyethyl carbamate), a preparation with cytokinin activity, reduced the dehydration-induced inhibition of enzymatic activity. Upon a subsequent rehydration, kartolin-4 facilitated rapid recovery of the photosynthetic activity, the process being based on the kartolin-induced stimulation of reparation reactions. Under conditions of water stress, a partial decrease in the activity of carbon metabolism enzymes in vitro was accompanied by complete inhibition of photosynthesis in vivo, perhaps, as a result of an abrupt increase in the stomatal resistance.     

Analysis of toxin genetic determinants of Vibrio cholerae virulence cassette and neuraminidase with DNA probes

V. cholerae strains of different origin have been studied for the presence of cholera toxin genes (vct), the proximal part of the virulence cassette including genes zot, ace and orfU, as well as neuraminidase genes (neu), in their genomes with the use of molecular DNA probes. The possibility, in principle, for some strains to lose only a part of their virulence cassette (gene vct), while retaining its proximal part has been shown. In most cases such strains are isolated from patients with diarrhea of different severity and may probably play some etiological role, provided that the expression of the genes of additional toxins of the virulence cassette occurs. The gene expressing neuraminidase which facilitates the penetration of cholera toxin into the epithelial cells of the intestine is always present in vct+ strains and may be absent in vct- strains. The absence of genetic relationship between neuraminidases in V. cholerae O139 and V. cholerae O1 and non-O1 (non-O139) has been confirmed. The problems in connection with the integration and deletion of genetic determinants of V. cholerae virulence factors are discussed.     

Point mutation of the Virbrio cholerae O139 cef (CHO cell elongating factor) gene alters the substrate specificity of its product

Sequencing of the cef (CHO cell elongating factor) of Vibrio cholerae serogroup O139 revealed one nucleotide substitution (C for T in position 2015) in comparison with classical V. cholerae O1 and two substitutions (AC for GT in positions 2014-2015) in comparison with V. cholerae O1 E1 Tor. A comparative bioinformatic analysis showed that the substitution determines a threonine residue in position 672 of the Cefprotein, while the position is occupied by an isoleucine residue in the classical strains and a valine residue in the El Tor group. The last two amino acids are hydrophobic, while threonine is hydrophilic, having a polar R group. The non- synonymous substitution affects the predicted secondary and, probably, tertiary structures of the Cef-O139 protein and explained our previous finding that the protein fails to degrade tributyrin, while retaining the tweenase activity spectrum and all other characteristics. It cannot be excluded that the inability of Cef-O139 to cleave triglycerides, along with other genetic specifics, contribute to the fact that the O139 serogroup has been displaced from a dominating position in etiology of cholera by the El Tor genotype. The nucleotide sequence of the V. cholerae O139 cefgene and the deduced amino acid sequence of its product are reported for the first time and were deposited in GenBank under accession nos. JF499787 and AEC04822.1, respectively.     

Efficiency of Vibrio parahaemolyticus tdh gene expression depends upon two point mutations in its promoter region

A molecular study of Vibrio parahaemolyticus clinical isolates containing the thermostable direct hemolysin (Tdh) gene and the Tdh-related hemolysin (Trh) gene have been conducted. Southern blot hybridization revealed that in the genomes of strains carrying the determinants of both hemolysins (tdh ⁺ trh ⁺) the tdh gene is presented by a single copy while tdh ⁺ trh ^? strains have two copies (tdh1 and tdh2). All investigated tdh ⁺ trh ⁺ and some tdh ⁺ trh ^? strains did not express the tdh gene (Kanagawa-negative, KP^?) or expressed it weakly and inconstantly (Kanagawa-intermediate, KP^±) in contrast to several Kanagawa-positive (KP⁺) strains. To establish the reasons for the KP^?/± phenotypes we sequenced tdh, tdh1 and tdh2 genes of 13 strains isolated in Russia and neighbouring foreign countries followed by bioinformatics analysis of the obtained sequences in comparison with those of a number of strains presented in GenBank. The results revealed that poor expression of the tdh gene depends not only on a single point mutation in the promoter region (substitution of A to G in the ?35 sequence) as it was believed earlier, but to the same extent on a second substitution (G to A at ?3 nucleotide position from the t-10 sequence) which appeared to be sufficient in the absence of the first one. Therefore, a reversion of KP^-/± strains to KP⁺ actually may occur as a result of a single point back mutation, and such strains are to be considered as potentially dangerous. Those bearing both substitutions may reverse with less probability as this process requires two simultaneous mutations.     

Cholera caused by Vibrio cholerae O1 ctxAB- tcpA+

Results of analysis of cholera outbreak during which V. cholerae O1 biovar El-Tor ctxAB- tcpA+ was isolated from 2 patients and 30 carriers are presented. Epidemic was caused by contamination of water source and water route of transmission. Strains identical to ones detected in humans were isolated from water of surface well in zone of water intake. Genome and VNTR-analysis of ctxAB- tcpA+ vibrios that caused outbreak in Rostov region in 2005 showed that they differed from ctxAB- tcpA- and ctxAB- tcpA+ vibrios isolated previously during and beyond of outbreaks from patients, carriers and environment and formed separate group with certain genotype. These results confirms conclusions of epidemiological analysis about imported cause of recent outbreak.