IR spectroscopic research on the impact of chemical analogues of autoregulatory d<Subscript>1</Subscript> factors of microorganisms on structural changes in DNA

Using IR spectroscopy, we investigated the impact of chemical analogues of autoregulatory d₁ factors of microorganisms (methylresorcinol, hexylresorcinol, and tyrosol) on the conformational changes in DNA in films upon altering (decreasing) the relative humidity. We analyzed the appearance/disappearance of characteristic absorption bands of A and B DNA forms and determined D ₁₀₈₈/D ₁₂₂₄, the ratio between the band intensities of symmetrical and asymmetrical oscillations in their phosphate groups. The data obtained suggest the slowing down of the B → A structural transition in DNA in the presence of methylresorcinol and its speeding up in the presence of tyrosol. We discuss the mechanisms of this phenomenon in relation to the chemical composition of d 1 factors and their biological function.     

Long-term preservation of DNA in aqueous solutions in the presence of the chemical analogues of microbial autoregulators

The fact of long-term preservation of the physicochemical properties of DNA molecules in aqueous solutions in complexes with methylresorcinol, hexylresorcinol, and tyrosol, the chemical analogues of microbial autoregulators (d1 factors) from the group of alkylhydroxybenzenes (AOB), was established. Compared to the control variants of storage of aqueous DNA solutions, the AOB influence consisted in the sum of correlating effects: the prevention of DNA degradation (according to spectrophotometric parameters) and the preservation of its viscous characteristics and electrophoretic mobility. The initial DNA properties were preserved to the greatest degree in the presence of hexylresorcinol, the compound with the longest alkyl radical. Possible mechanisms of the protective action of alkylhydroxybenzenes in relation to DNA are discussed, namely, the prevention of its hydrolysis due to isolation from the aqueous environment and maintaining DNA stability in the dormant forms of microorganisms.     

Mechanisms of interaction between DNA and chemical analogues of microbial anabiosis autoinducers

The alkylhydroxybenzene (AHB) autoregulatory factors d1 (fd1) of microorganisms have been found to directly interact with highly polymeric DNA. This circumstance results in changes, related to alterations in the topology of this macromolecule, in DNA physicochemical properties. The physicochemical properties of DNA in the presence of chemical analogues of microbial AHBs (methylresorcinol; hexylresorcinol; and 2-(4-hydroxyphenyl)ethane-1-ol, also known as tyrosol) were investigated using adsorption spectrophotometry, fluorometry, heat denaturation, viscosimetry, and electrophoresis in agarose gel. A number of concordant effects pointing to DNA-AHB interactions were revealed that manifesed themselves in the hypochromic properties of the resulting complexes, an increase in their melting temperature and viscosity, a decrease in their electrophoretic mobility, and a change in the fluorescent properties of AHBs upon complexation with DNA. Such alterations were particularly significant in the presence of hexylresorcinol, which possessed the maximum alkyl radical length among the fd1 analogues tested. Using atomic force microscopy, we visualized the micelle-like DNA nanostructures forming in the presence of AHBs. The results obtained provided the basis for developing a hypothetical model of the interaction between the biopolymer macromolecule and low-molecular-weight AHBs that takes into account the differences in the hydrophobicity of individual AHB homologues functioning as ligands. In terms of our model, we discuss AHB involvement in the stabilization of DNA and alteration of its topology, i.e., in the process related to intragenomic rearrangements, which account for the intrapopulational variability of bacteria, including dissociation processes.     

Long-term preservation of DNA in aqueous solutions in the presence of the chemical analogues of microbial autoregulators

he fact of long-term preservation of the physicochemical properties of DNA molecules in aqueous solutions in complexes with methylresorcinol, hexylresorcinol, and tyrosol, the chemical analogues of microbial autoregulators (d₁ factors) from the group of alkylhydroxybenzenes (AOB), was established. Compared to the control variants of storage of aqueous DNA solutions, the AOB influence consisted in the sum of correlating effects: the prevention of DNA degradation (according to spectrophotometric parameters) and the preservation of its viscous characteristics and electrophoretic mobility. The initial DNA properties were preserved to the greatest degree in the presence of hexylresorcinol, the compound with the longest alkyl radical. Possible mechanisms of the protective action of alkylhydroxybenzenes in relation to DNA are discussed, namely, the prevention of its hydrolysis due to isolation from the aqueous environment and maintaining DNA stability in the dormant forms of microorganisms.     

Mechanisms of Interaction between DNA and Chemical Analogues of Microbial Anabiosis Autoinducers

The alkylhydroxybenzene (AHB) autoregulatory factors d₁ (fd₁) of microorganisms have been found to directly interact with highly polymeric DNA. This circumstance results in changes, related to alterations in the topology of this macromolecule, in DNA physicochemical properties. The physicochemical properties of DNA in the presence of chemical analogues of microbial AHBs (methylresorcinol; hexylresorcinol; and 2-(4-hydroxyphenyl)ethane-1-ol, also known as tyrosol) were investigated using adsorption spectrophotometry, fluorometry, heat denaturation, viscosimetry, and electrophoresis in agarose gel. A number of concordant effects pointing to DNA-AHB interactions were revealed that manifest themselves in the hypochromic properties of the resulting complexes, an increase in their melting temperature and viscosity, a decrease in their electrophoretic mobility, and a change in the fluorescent properties of AHBs upon complexation with DNA. Such alterations were particularly significant in the presence of hexylresorcinol, which possessed the maximum alkyl radical length among the fd₁ analogues tested. Using atomic force microscopy, we visualized the micellelike DNA structures forming in the presence of AHBs. The results obtained provided the basis for developing a hypothetical model of the interaction between the biopolymer macromolecule and low-molecular-weight AHBs that takes into account the differences in the hydrophobicity of individual AHB homologues functioning as ligands. In terms of our model, we discuss AHB involvement in the stabilization of DNA and alteration of its topology, i.e., in the process related to intragenomic rearrangements, which account for the intrapopulational variability of bacteria, including dissociation processes.     

Influence of chemical analogues of microbial autoregulators on the sensitivity of DNA to UV radiation

We established that chemical analogues of alkylhydroxybenzenes (AHB), belonging to alkylresorcinols and functioning as microbial autoregulatory d₁ factors, enhance the UV resistance of various DNA molecules of different origin and conformation. These include the linear DNA of the λ phage, bovine spleen DNA, and the DNA of the pUC19 plasmid that is composed of a number of annular (supercoiled and relaxed) and linearized molecules. Irradiating DNA with UV light (λ = 254 nm) in the presence of methylresorcinol (MR) or hexylresorcinol (HR) results in comparatively insignificant DNA destruction as evidenced by our data on the electrophoretic mobility pattern in agarose gel. Using the linear HindIII restricts of the λ phage DNA, we revealed that the protective effect of AHB varies depending on their chemical structure (it is more manifest with HR than MR) and the concentration. Importantly, the effect of HR on bovine spleen DNA was based on its protective activity and manifested itself after a long incubation period. Studies using the pUC19 plasmid demonstrated that AHB, apart from increasing the resistance of linearized DNA molecules to UV irradiation, prevented both the supercoiled annular-supercoiled relaxed and the supercoiled relaxed-linearized transitions. The possible mechanisms of the UV-protective effect of AHB on DNA and their contributions to the resistance of dormant microbial forms to environmental factors are discussed.     

Effect of chemical chaperones on properties of lysozyme and the reaction center protein from Rhodobacter sphaeroides

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and the functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. Selected chemical chaperones are strongly different by the structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described within the frames of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic nuclus) moieties. This fact provides additional regulation of functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in denaturation temperature T(d), and an increase in the amplitude of equilibrium fluctuation, which allows him to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in the case of methylresorcinol. Therefore, methylresorcinol does not effect the functional properties of the RC protein. It was concluded that various chaperones at one and the same concentration and chaperones at different concentrations form diverse 3D structures of proteins, which differ by dynamic and functional characteristics.     

Influence of chemical chaperones on the properties of lysozyme and the reaction center protein from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. The chosen chemical chaperones differ strongly in their structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described in the framework of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic core) moieties; this provides for additional regulation of the functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in the denaturation temperature and an increase in the amplitude of equilibrium fluctuations, allowing it to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in this case. Therefore, methylresorcinol does not affect the functional properties of the RC protein. It is concluded that different chaperones at the same concentration as well as one and the same chaperone at different concentrations produce protein 3D structures differing in dynamic and functional characteristics.     

DNA protecting and genotoxic effects of olive oil related components in cells exposed to hydrogen peroxide

In search for compounds, able to protect nuclear DNA in cells exposed to oxidative stress, extracts from olive leaves, olive fruits, olive oil and olive mill waste water were tested by using the “single cell gel electrophoresis” methodology (comet assay). Jurkat cells in culture were exposed to continuously generated hydrogen peroxide (11.8±1.5 μM per min) by direct addition into the growth medium of the appropriate amount of the enzyme “glucose oxidase” in the presence or absence of the tested total extracts. The protective effects of the tested extracts or isolated compounds were evaluated from their ability to decrease hydrogen peroxide-induced formation of single strand breaks in the nuclear DNA, while the toxic effects were estimated from the increase of DNA damage when the extracts or isolated compounds were incubated directly with the cells. Significant protection was observed in extracts from olive oil and olive mill waste water. However, above a concentration of 100 μg/ml olive oil extracts exerted DNA damaging effects by themselves in the absence of any H₂O₂. Extracts from olive leaves and olive fruits although protective, were also able to induce DNA damage by themselves. Main compounds isolated from the above described total extracts, like oleuropein glucoside, tyrosol, hydroxytyrosol and caffeic acid, were tested in the same experimental system and found to exert cytotoxic (oleuropein glucoside), no effect (tyrosol) or protective effects (hydroxytyrosol and caffeic acid). In conclusion, cytoprotective as well as cytotoxic compounds with potential pharmaceutical properties were detected in extracts from olive oil related sources by using the comet assay methodology.     

Influence of chemical analogues of microbial autoregulators on the sensitivity of DNA to UV radiation

We established that chemical analogues of alkylhydroxybenzenes (AHB), belonging to alkylresorcinols and functioning as microbial autoregulatory d1 factors, enhance the UV resistance of various DNA molecules of different origin and conformation. These include the linear DNA of the lambda phage, bovine spleen DNA, and the DNA of the pUC19 plasmid that is composed of a number of annular (supercoiled and relaxed) and linearized molecules. Irradiating DNA with UV light (lambda = 254 nm) in the presence of methylresorcinol (MR) or hexylresorcinol (HR) results in comparatively insignificant DNA destruction as evidenced by our data on the electrophoretic mobility pattern in agarose gel. Using the linear Hind III restricts of the lambda phage DNA, we revealed that the protective effect of AHB varies depending on their chemical structure (it is more manifest with HR than MR) and concentration. Importantly, the effect of HR on bovine spleen DNA was based on its protective activity and manifested itself after a long incubation period. Studies using the pUC19 plasmid demonstrated that AHB, apart from increasing the resistance of linearized DNA molecules to UV irradiation, prevented both the supercoiled annular-supercoiled relaxed and the supercoiled relaxed-linearized transitions. The possible mechanisms of the UV-protective effect of AHB on DNA and their contributions to the resistance of dormant microbial forms to environmental factors are discussed.     

Effect of alkylhydroxybenzenes, microbial anabiosis inducers, on the structural organization of Pseudomonas aurantiaca DNA and on phenotypic dissociation induction

We revealed a relationship between alkylhydroxybenzene (AHB)-induced changes in the structural organization of supramolecular complexes (SC) of the DNA of Pseudomonas auraniaca and the phenotypic dissociation of this bacterium. The addition of 0.1-0.3 mM hexylresorcinol (C6-AHB), a chemical analogue of microbial anabiosis autoinducers, caused the formation of cystlike refractile cells (CRC) in these gram-negative, nonsporulating bacteria. Inoculating pseudomonad CRC on solid nutrient media resulted in phenotypic dissociation of the microbial population that yielded several variants with different colony structure and morphology. This manifested itself in the conversion of the original S-colony-forming phenotype into the R form and in the formation of less pigmented colonies. These transitions were possibly linked to AHB-induced structural changes in the DNA. In vitro studies revealed that AHB could interact with DNA SC, resulting in their structural modification that manifested itself in changes in their elastoviscosity. DNA supramolecular complexes isolated from proliferating, stationary-phase, and anabiotic P. aurantiaca cells differed in their elastoviscosity and capacity to interact with AHB homologues with different hydrophobicity, such as hexylresorcinol and methylresorcinol (C1-AHB). The DNA SC from actively proliferating cells were characterized by smaller elastoviscosity compared with those from stationary-phase and anabiotic cells, due to the difference in the DNA superspiralization degree and the physiological age of the bacteria involved. C6-AHB produced a pronounced relaxing effect on the DNA SC from exponential-phase P. aurantiaca cells. The less hydrophobic C1-AHB produced a similar effect on the DNA SC from stationary-phase cells. The curve of the dose-effect dependence of C6-AHB had a breaking point within the submillimolar (10(-4) M) concentration range. These concentrations induce the formation of cystlike anabiotic pseudomonad cells that are characterized by an unstable genotype and dissociate into distinct variants upon inoculation on solid media.     

Determinants of HMGB proteins required to promote RAG1/2-recombination signal sequence complex assembly and catalysis during V(D)J recombination

Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.     

Alcohol-based quorum sensing plays a role in adhesion and sliding motility of the yeast Debaryomyces hansenii

The yeast Debaryomyces hansenii was investigated for its production of alcohol-based quorum sensing (QS) molecules including the aromatic alcohols phenylethanol, tyrosol, tryptophol and the aliphatic alcohol farnesol. Debaryomyces hansenii produced phenylethanol and tyrosol, which were primarily detected from the end of exponential phase indicating that they are potential QS molecules in D.?hansenii as previously shown for other yeast species. Yields of phenylethanol and tyrosol produced by D.?hansenii were, however, lower than those produced by Candida albicans and Saccharomyces cerevisiae and varied with growth conditions such as the availability of aromatic amino acids, ammonium sulphate, NaCl, pH and temperature. Tryptophol was only produced in the presence of tryptophane, whereas farnesol in general was not detectable. Especially, the type strain of D.?hansenii (CBS767) had good adhesion and sliding motility abilities, which seemed to be related to a higher hydrophobicity of the cell surface of D.?hansenii (CBS767) rather than the ability to form pseudomycelium. Addition of phenylethanol, tyrosol, tryptophol and farnesol was found to influence both adhesion and sliding motility of D.?hansenii.     

群体感应动态调控促进大肠杆菌合成酪醇

酪醇是一种多酚类天然产物,广泛应用于化工、医药和食品等领域。目前大肠杆菌(Escherichia coli)从头合成酪醇存在发酵菌体密度低和产量低等问题。为此,本研究将前期获得苯丙酮酸脱羧酶突变体ARO10F138L/D218G与不同来源的醇脱氢酶融合表达,最优组合ARO10F138L/D218G-L-YahK酪醇产量达到1.09 g/L。为进一步提高酪醇产量,敲除了4-羟基苯乙酸竞争途径关键基因feaB,使酪醇产量提高了21.15%,达到1.26g/L。针对酪醇发酵菌体密度低的问题,通过群体感应系统动态调控酪醇合成途径,减轻酪醇对底盘细胞的毒性作用,缓解生长抑制,使其产量提高了33.82%,达到1.74 g/L。在2 L发酵罐中,群体感应动态调控工程菌TRFQ5的酪醇产量达到4.22g/L,OD600值达到42.88,分别较静态诱导表达工程菌TRF5提高了38.58%和43.62%。本研究应用基因敲除技术,阻断了酪醇合成竞争途径;同时结合群体感应动态调控策略,减轻了酪醇毒性对底盘细胞的生长抑制,从而有效地提高了酪醇产量。本研究对其他高毒性化学品的生物合成具有良好的借鉴和应用价值。     

Carcinogen-nucleic acid interactions: equilibrium binding studies of aflatoxin B1 with the oligodeoxynucleotide d(ATGCAT)2 and with plasmid pBR322 support intercalative association with the B-DNA helix

Equilibrium binding of aflatoxin B1 (AFB1) to the oligodeoxynucleotide d(ATGCAT)2 was examined by using 1H NMR. AFB1 binds to double-stranded d(ATGCAT)2 with an apparent binding constant of 3.7 x 10(3) M-1. The equilibrium is rapid on the NMR time scale; the observed 1H NMR spectrum represents the population-weighted average of the chemical shifts arising from the free and bound states of the oligodeoxynucleotide and the AFB1. The spectrum of d(ATGCAT)2 exhibits exchange broadening in the presence of AFB1, manifested as decreases in apparent T2 relaxation times for the d(ATGCAT)2 base protons. Upon binding to d(ATGCAT)2, the AFB1 signals are shifted upfield, indicative of increased shielding. The adenine H2 protons are also shifted upfield in the presence of the carcinogen. Small changes in chemical shift are observed for other d(ATGCAT)2 protons. A substantial decrease in the nonselective T1 relaxation time is observed for the adenine H2 protons in the presence of AFB1. Competition binding experiments in which the competing ligands actinomycin D, ethidium bromide, and spermidine were individually added to an AFB1-d(ATGCAT)2 equilibrium mixture showed that addition of 1 equiv of actinomycin D or 4 equiv of ethidium bromide was sufficient to displace bound AFB1 from d(ATGCAT)2. In contrast, the addition of spermidine did not result in the displacement of bound AFB1 molecules and may have slightly enhanced binding, presumably due to stabilization of the DNA duplex. 1H NOESY experiments confirmed that the overall conformation for the d(ATGCAT)2 duplex was right-handed both in the absence and in the presence of AFB1. Equilibrium binding of AFB1 to d(ATGCAT)2 is greatly diminished at higher temperatures at which the oligodeoxynucleotide is single-stranded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Requirement of Cyclin/Cdk2 and protein phosphatase 1 activity for chromatin assembly factor 1-dependent chromatin assembly during DNA synthesis

The influence of reversible protein phosphorylation on nucleosome assembly during DNA replication was analyzed in extracts from human cells. Inhibitor studies and add-back experiments indicated requirements of cyclin A/Cdk2, cyclin E/Cdk2, and protein phosphatase type 1 (PP1) activities for nucleosome assembly during DNA synthesis by chromatin assembly factor 1 (CAF-1). The p60 subunit of CAF-1 is a molecular target for reversible phosphorylation by cyclin/Cdk complexes and PP1 during nucleosome assembly and DNA synthesis in vitro. Purified p60 can be directly phosphorylated by purified cyclin A/Cdk2, cyclin E/Cdk2, and cyclin B1/Cdk1, but not by cyclin D/Cdk4 complexes in vitro. Cyclin B1/Cdk1 triggers hyperphosphorylation of p60 in the presence of additional cytosolic factors. CAF-1 containing hyperphosphorylated p60 prepared from mitotic cells is inactive in nucleosome assembly and becomes activated by dephosphorylation in vitro. These data provide functional evidence for a requirement of the cell cycle machinery for nucleosome assembly by CAF-1 during DNA replication.     

The effect of anabiosis autoinducers on the bacterial genome

The mutagenic activity of chemical analogues of microbial anabiosis autoinducers (the autoregulatory d1 factors of cell differentiation), which act to inhibit cell proliferation, to enhance cell tolerance, and to induce the transition of cells to anabiotic state, was studied using the Ames test. In the range of concentrations studied (0.1 to 100 micrograms/ml), alkyl-substituted hydroxybenzenes (AHBs) differing in hydrophobicity, i.e., methylresorcinol (C1-AHB) and hexylresorcinol (C6-AHB), as well as unsubstituted resorcinol, showed different growth-inhibiting and mutagenic effects. C6-AHB was found to inhibit the growth of Salmonella typhimurium TA100 and to induce its mutagenesis at a rate of 1.8 revertants/nmol. C1-AHB taken at low concentrations not only failed to inhibit bacterial growth but even stimulated it and exerted an antimutagenic effect. Unsubstituted resorcinol virtually did not influence bacterial growth and showed weak mutagenic activity. The growth-inhibiting effect of elevated C6-AHB concentrations correlated with the degree of the transition of the original phenotype producing S-type colonies to a phenotype producing R-type colonies. The role of AHB homologues, as microbial autoregulators with mutagenic activity, in the regulation and correlation of two processes (the phenotypic dissociation of microbial populations and the formation of resting microbial forms) is discussed. The accumulation of AHBs in senescent microbial cultures may induce adaptive mutations, change the expression of genes, and promote the development of minor cell subpopulations (phenotypes), thus providing for the adaptation of these cultures to varying environmental conditions.     

Production of Salidroside Through Biotransformation of Exogenous Tyrosol in Rhodiola sachalinensis Cell Suspension Cultures

In Rhodiola sachalinensis A. Bot. cell cultures, low yields of salidroside was supposed to be associated with the low efficiency of glucosylation reaction at the stationary phase of cell growth, when large amounts of the substrate, aglycon tyrosol, were accumulated. Considering the activity of tyrosol glucosyhransferase being the highest at the exponential growth phase, the author added exogenous tyrosol into the cultures at this time so as to produce salidroside through biotransformation. The effects of tyrosol concentration, the way of tyrosol addition as well as the cell density on the transformation rate and salidroside yield were investigated. It was found that the transformation rate attained 95 % after cells were incubated in the medium containing 1 mmol/L tyrosol for 24 h. Excess high concentrations of tyrosol in medium ( > 3 mmol/L) caused inhibition of transformation rate and cell growth. By 3 repeated additions of tyrosol in low concentrations, the salidroside yields of 1 320 mg/L, 1 740 mg/L and 1 980 mg/L to the cell densities of 6 g DW/L, 12 g DW/L and 18 g DW/L were obtained respectively.     

Sequence-dependent variations in the 31P NMR spectra and backbone torsional angles of wild-type and mutant Lac operator fragments

Assignment of the 31P resonances of a series of six sequenced-related tetradecamer DNA duplexes, d(TGTGAGCGCTCACA)2, d(TATGAGCGCTCATA)2, d(TCTGAGCGCTCAGA)2, d(TGTGTGCGCACACA)2, d(TGTGACGCGTCACA)2 and d(CACAGTATACTGTG)2, related to the lac operator DNA sequence was determined either by site-specific 17O labeling of the phosphoryl groups or by two-dimensional 1H-31P pure absorption phase constant time (PAC) heteronuclear correlation spectroscopy. J(H3'-P) coupling constants for each of the phosphates of the tetradecamers were obtained from 1H-31P J-resolved selective proton flip 2D spectra. By use of a modified Karplus relationship the C4'-C3'-O3'-P torsional angles (epsilon) were obtained. Comparison of the 31P chemical shifts and J(H3'-P) coupling constants of these sequences has allowed greater insight into those various factors responsible for 31P chemical shift variations in oligonucleotides and provided an important probe of the sequence-dependent structural variation of the deoxyribose phosphate backbone of DNA in solution. These sequence-specific variations in the conformation of the DNA sugar phosphate backbone of various lac operator DNA sequences can possibly explain the sequence-specific recognition of DNA by DNA binding proteins, as mediated through direct contacts between the phosphates and the protein.     

Effect of alkylhydroxybenzenes,microbial anabiosis inducers,on the structural organization of <Emphasis Type="Italic">Pseudomonas aurantiaca</Emphasis> DNA and on the induction of phenotypic dissociation

We revealed a relationship between alkylhydroxybenzene (AHB)-induced changes in the structural organization of supramolecular complexes (SC) of the DNA of Pseudomonas aurantiaca and the phenotypic dissociation of this bacterium. The addition of 0.1–0.3 mM hexylresorcinol (C₆-AHB), a chemical analogue of microbial anabiosis autoinducers, caused the formation of cystlike refractile cells (CRC) in these gram-negative, nonsporulating bacteria. Inoculating pseudomonad CRC on solid nutrient media resulted in phenotypic dissociation of the microbial population that yielded several variants with different colony structure and morphology. This manifested itself in the conversion of the original S-colony-forming phenotype into the R form and in the formation of less pigmented colonies. These transitions were possibly linked to AHB-induced structural changes in the DNA. In vitro studies revealed that AHB could interact with DNA SC, resulting in their structural modification that manifested itself in changes in their viscoelasticity. DNA supramolecular complexes isolated from proliferating, stationary-phase, and anabiotic P. aurantiaca cells differed in their viscoelasticity and capacity to interact with AHB homologues with different hydrophobicity, such as hexylresorcinol and methylresorcinol (C₁-AHB). The DNA SC from actively proliferating cells were characterized by smaller viscoelasticity compared with those from stationary-phase and anabiotic cells, due to the difference in the DNA superspiralization degree and the physiological age of the bacteria involved. C₆-AHB produced a pronounced relaxing effect on the DNA SC from exponential-phase P. aurantiaca cells. The less hydrophobic C₁-AHB produced a similar relaxing effect on the DNA SC from stationary-phase cells. The curve of the dose-effect dependence of C₆-AHB had a breaking point within the submillimolar (10^–4 M) concentration range. These concentrations induce the formation of cystlike anabiotic pseudomonad cells that are characterized by an unstable phenotype and dissociate into distinct variants upon inoculation on solid media.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 157–165.Original Russian Text Copyright © 2005 by Mulyukin, Vakhrushev, Strazhevskaya, Shmyrina, Zhdanov, Suzina, Duda, Kozlova, El-Registan.