The role of microbial dormancy autoinducers in metabolism blockade

Alkyl-substituted hydroxybenzenes (AHBs), which are autoinducers of microbial dormancy (d ₁ factors), were found to stabilize the structure of protein macromolecules and modify the catalytic activity of enzymes. In vitro experiments showed that C₆-AHB at concentrations from 10⁻⁴ to 10⁻² M, at which it occurs in the medium as a true solution and a micellar colloid, respectively, nonspecifically inhibited the activity of chymotrypsin, RNase, invertase, and glucose oxidase. C⁶-AHB-induced conformational alterations in protein macromolecules were due to the formation of complexes, as evidenced by differences in the fluorescence spectra of individual RNase and C₆-AHB and their mixtures and in the surface tension isotherms of C₆-AHB and trypsin solutions. Data on the involvement of dormancy autoinducers in the posttranslational modification of enzymes and their inhibition will provide further insight into the mechanisms of development and maintenance of dormant microbial forms.     

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.     

Involvement of alkylhydroxybenzenes in the <Emphasis Type="Italic">Escherichia coli</Emphasis> response to the lethal effect of UV irradiation

This work is concerned with the role of alkylhydroxybenzenes (AHBs), chemical analogs of the autoregulatory microbial d ₁ factors, on the development of the stress response of bacterial cells to UV irradiation, including SOS system induction, preservation of cell viability, and S → R phase transitions of the Escherichia coli test strain with the bioluminescence genes cloned under the control of the recA gene promoter. UV irradiation, a natural stress factor, and an increase in AHB concentrations were found to elicit uniform responses in bacteria, indicating that AHBs function as alarmones, i.e., alarm signals. It was revealed that preincubating bacteria with alkylhydroxybenzenes considerably enhanced their viability upon irradiation with lethal UV doses; this was accompanied by a relative decrease in the SOS response activity and a concomitant increase in the frequency of phase transitions. The efficiency of the protective action of AHBs increased with an increase in their hydrophobicity degree. The probable mechanism of the protective effect of AHBs is discussed, based on their capacity for the interaction with biopolymers, which results in changing their structural organization and conferring resistance to a broad spectrum of stress factors. Such a “passive” protective mechanism reduces the susceptibility of DNA to UV irradiation, causing a decrease in the parameters related to the SOS system induction that is responsible for the “active” protective mechanism in bacterial cells. As a result, viability retention under the lethal influence of UV irradiation is possible at minimal values of repair activity and is accompanied by an increase in the phenotypic variability of the surviving part of a bacterial population.     

Involvement of Microbial Alkyl Hydroxybenzenes in the Regulation of Autolytic Degradation of Yeast Cells

A comparative study was performed of the processes of autolytic degradation of the cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe under conditions simulating the phase of cell death in microbial cultures: (1) during autolysis induced by oleic acid, which is the chemical analogue of factors d₂ (autolysis autoinducer), (2) under the effect of extracellular microbial proteinases (enzymatic lysis), and (3) under the concomitant effect of the enzymes of the endogenous autolytic complex and exogenous proteinases (heterolysis). Regulatory mechanisms controlling the rate and profundity of autolysis were elucidated, relying on the stabilization of hydrolytic enzymes and enhancement of their activity in their complexes with a chemical analogue of microbial autoregulatory factors d₁, which belong to alkylhydroxybenzenes and fulfill functions of chemical chaperones. The changes in the activity of proteinases and enzymes of the autolytic complex were shown to be dependent on the concentration of the analogue at the moment of complex formation.     

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 an anabiotic state, was studied using the Ames test. In the range of concentrations studied (0.1 to 100 g/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.     

Stabilization of enzymes by anabiosis autoinducers as a possible mechanism of resistance of resting microbial forms

Alkyl-substituted hydroxybenzenes (AHBs), auto-inducers of microbial dormancy (or d1 factors), were found to stabilize the structure of protein macromolecules, making them metabolically less active and more resistant to stresses. In vitro experiments with the Bacillus intermedius ribonuclease and chymotrypsin showed that the degree of the physical and chemical stability of these enzymes treated with AHBs depends on their concentration and incubation time. Experiments with RNase, which is capable of refolding, i.e., renaturation after heat denaturation, revealed that AHBs efficiently interact with both intact and denatured proteins. The data obtained allow the inference to be made that d1 factors may play the role of natural chemical chaperons, blocking metabolism in dormant cells through the formation of catalytically inactive thermostable complexes with enzymes.     

The Role of Bacterial Growth Autoregulators (Alkyl Hydroxybenzenes) in the Response of Staphylococci to Stresses

The investigation of the response of a batch culture of Staphylococcus aureus to exogenous alkyl-substituted hydroxybenzenes (AHBs), chemical analogues of anabiosis autoinducers, showed that C₁-AHB at concentrations from 5 M to 1.5 mM did not influence the culture growth, whereas the more hydrophobic C₆-AHB inhibited it at concentrations of 0.5 mM and higher. Either of the AHBs drastically enhanced the phenotypic dissociation of staphylococcal cultures, which manifested itself in an increase in the fraction of cells producing small nonhemolyzing colonies of G type when plated on solid media with erythrocytes. In a submerged staphylococcal culture, the relative number of cells producing G-type colonies varied from 10 to 90%, depending on the concentration of the AHB added. The growth of S. aureus in the presence of AHBs also enhanced cell tolerance to heat shock (heating at 45 or 60°C for 10 min). The role of AHBs, which are structural modifiers of membranes and possess chaperone activity, in the mechanisms responsible for cell tolerance and phenotypic dissociation of microbial populations is discussed.     

Changes in physicochemical properties of proteins, caused by modification with alkylhydroxybenzenes

Kinetic characteristics of model enzymes and physicochemical properties of globular proteins modified by chemical analogues of low-molecular-weight microbial autoregulators (alkylhydroxybenzenes, AHBs) have been studied. C₇ and C₁₂ AHB homologues were used, differing in the length of the alkyl radical and the capacity for weak physicochemical interactions. Both homologues affected the degree of protein swelling, viscosity, and the degree of hydrophobicity. The effects depended on the structure of AHBs, their concentration, and pH of the solution, which likely reflects changes in the charge of the protein globule and its solvate cover. Variations of hydrophobicity indices of AHB-modified enzymes (trypsin and lysozyme) were coupled to changes in the catalytic activity. The values of K _M, measured for the enzymes within both AHB complexes, did not change, whereas V _max increased (in the case of C₇ complexes) or decreased (C₁₂ complexes). Possible molecular mechanisms of changes in the physicochemical and catalytic parameters of enzymatically active proteins, induced by modification with structurally distinct AHBs, are described, with emphasis on targeted regulation of functional activity.     

Role of alkylhydroxybenzenes in bacterial adaptation to unfavorable growth conditions

The adaptogenic effect of the chemical analogues of alkylhydroxybenzenes (AHBs), bacterial extracellular autoregulators (the individual compound C7-AHB and its technical preparation Sidovit), was demonstrated for two pseudomonad species, Pseudomonas aeruginosa and P. fluorescens. The protective effect of AHBs resulted in increased growth rate and biomass accumulation in bacteria grown under suboptimal conditions within the species tolerance range. The adaptogenic effect of AHBs (10–50 μmg/l) was more pronounced under more unfavorable growth conditions. In the case of P. fluorescens, the individual compound C7-AHB increased the biomass yield by 30% under alkaline conditions (pH 9.5), when the growth rate decreased by 80–90% compared to the optimum (pH 5.5–7.5). The Sidovit preparation, containing a mixture of natural AHBs with C7-AHB as the main component, increased the growth rate of P. aeruginosa by 40–60% at nonoptimal temperatures (45 and 10°C) or under enhanced salinity (1% NaCl). The action of AHBs as regulators of the rpoS and SOS response stress regulons was demonstrated to be among the mechanisms of their adaptogenic effect, as was demonstrated with the relevant reporter genes in the model strains E. coli C600 thi, thr, leuΔ(pro-lac) with the osmE-lacZ and umuD-lacZ hybrid operons, respectively. AHBs are technologically and economically acceptable as adaptogenic supplements for bacterial preparations used in soil bioremediation and oil spillage removal under conditions unfavorable for microbial growth, including increased salinity, extreme pH, and fluctuating sub- or supraoptimal temperatures.     

Enzyme Modification by Natural Chemical Chaperons of Microorganisms

We demonstrated for the first time that alkyl hydroxybenzenes (the d₁ microbial autoregulatory factors involved in stress responses of cells) are capable of stabilizing enzymes in aqueous media and increasing their catalytic activity. The stabilizing effect of a chemical analogue of alkyl hydroxybenzenes, C₇-AHB, was established in in vitro studies with enzymes of microbial origin: a protease produced by Bacillus licheniformis, cellulase produced by Trichoderma viride, and -amylase produced by Bacillus subtilis. This effect manifested itself in considerable extension of the temperature and pH ranges of the enzymatic activity. The modulation of the catalytic activities of the stabilized enzymes depended on the C₇-AHB concentration and on the time of preincubation of the complexes obtained. We demonstrated that not only enzymes but also their polymeric substrates formed complexes with C₇-AHB and this significantly influenced the efficiency of hydrolytic reactions. We also conducted comparative studies on the efficiency of hydrolytic reactions in systems in which the structure of enzymes and/or substrates was modified with C₇-AHB.     

The boxing glove shape of subunit <Emphasis Type="Italic">d</Emphasis> of the yeast V-ATPase in solution and the importance of disulfide formation for folding of this protein

The low resolution structure of subunit d (Vma6p) of the Saccharomyces cerevisiae V-ATPase was determined from solution X-ray scattering data. The protein is a boxing glove-shaped molecule consisting of two distinct domains, with a width of about 6.5 nm and 3.5 nm, respectively. To understand the importance of the N- and C-termini inside the protein, four truncated forms of subunit d (d _11–345, d _38–345, d _1–328 and d _1–298) and mutant subunit d, with a substitution of Cys329 against Ser, were expressed, and only d _11–345, containing all six cysteine residues was soluble. The structural properties of d depends strongly on the presence of a disulfide bond. Changes in response to disulfide formation have been studied by fluorescence- and CD spectroscopy, and biochemical approaches. Cysteins, involved in disulfide bridges, were analyzed by MALDI-TOF mass spectrometry. Finally, the solution structure of subunit d will be discussed in terms of the topological arrangement of the V₁V_O ATPase.     

Characterization of Bacillus cereusDissociants

The autoregulation of the phenotypic (populational) variability of the Bacillus cereusstrain 504 was studied. The isolated colonial morphotypes of this bacterium were found to differ in their growth characteristics and the synthesis of extracellular proteases. The phenotypic variabilities of vegetative proliferating cells and those germinated from endospores and cystlike refractory cells were different. Bacterial variants also differed in the production of the d₁and d₂factors (the autoinducers of anabiosis and autolysis, respectively) and sensitivity to them. The possible role of these factors in the dissociation of microorganisms is discussed.     

Pollination services of Africanized honey bees and native Melipona beecheii to buzz‐pollinated annatto (Bixa orellana L.) in the neotropics

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Low-Molecular-Weight Autoregulatory Factors in the Haloalkaliphilic Bacteria Thioalkalivibrio versutus and Thioalkalimicrobium aerophilum

The haloalkaliphilic, lithoautotrophic, sulfur-oxidizing gram-negative bacteria Thioalkalivibrio versutus and Thioalkalimicrobium aerophilum were found to possess a special system for the autoregulation of their growth. The system includes the extracellular autoinducers of anabiosis (the d₁ factor) and autolysis (the d₂ factor). The principal components of the d₁ factor are alkylhydroxybenzenes, as evidenced by specific qualitative reactions. The principal components of the d₂ factor are free unsaturated fatty acids dominated by oleic acid isomers. Like the respective autoregulators of neutrophilic bacteria, the d₁ factor of haloalkaliphilic bacteria presumably controls their growth and transition to anabiotic state, while the d₂ factor controls autolytic processes. Alkylhydroxybenzenes of both microbial and chemical origin were found to influence bacterial respiration. The low-molecular-weight osmoprotectant glycine betaine enhanced the thermostability of trypsin. This suggests that both glycine betaine and the d₁ factor serve as chemical chaperones.     

Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

Purification and interaction analyses of two human lysosomal vitamin B12 transporters: LMBD1 and ABCD4

Mutations in human LMBRD1 and ABCD4 prevent lysosomal export of vitamin B₁₂ to the cytoplasm, impairing the vitamin B₁₂-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. The gene products of LMBRD1 and ABCD4 are implicated in vitamin B₁₂ transport at the lysosomal membrane and are proposed to act in complex. To address the mechanism for lysosomal vitamin B₁₂ transport, we report the novel recombinant production of LMBD1 and ABCD4 for detailed biophysical analyses. Using blue native PAGE, chemical crosslinking, and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we show that both detergent-solubilized LMBD1 and detergent-solubilized ABCD4 form homodimers. To examine the functional binding properties of these proteins, label-free surface plasmon resonance (SPR) provides direct in vitro evidence that: (i) LMBD1 and ABCD4 interact with low nanomolar affinity; and (ii) the cytoplasmic vitamin B₁₂-processing protein MMACHC also interacts with LMBD1 and ABCD4 with low nanomolar affinity. Accordingly, we propose a model whereby membrane-bound LMBD1 and ABCD4 facilitate the vectorial delivery of lysosomal vitamin B₁₂ to cytoplasmic MMACHC, thus preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions.     

Production of Resting Forms by the Gram-Negative Chemolithoautotrophic Bacteria Thioalkalivibrio versutusand Thioalkalimicrobium aerophilum

The haloalkaliphilic chemolithoautotrophic gram-negative bacteria Thioalkalivibrio versutus, strain AL2, and Thioalkalimicrobium aerophilum, strain AL3, were shown to possess the capacity to produce resting forms, namely cystlike refractile cells (CRC), whose production was controlled by the level of the d₁ extracellular factors exhibiting the function of anabiosis autoinducers. The conditions were elucidated that promote the formation of CRC in the developmental cycles of the cultures studied, in condensed cell suspensions undergoing autolysis, and under the action of exogenously introduced chemical analogues of anabiosis autoinducers (alkylhydroxybenzenes). The peculiarities of the fine structure of the resting cells obtained were studied. Distinctions were revealed (with respect to viability and thermotolerance) between the CRC formed under different conditions. The relationship between the growth strategy and survival strategy of extremophilic bacteria is discussed taking into account the effect of the d₁ autoregulatory factors. A new model of CRC formation is proposed: CRC production in the life cycle of bacteria developing under conditions of increased concentration of anabiosis autoinducers.     

7-Amino-actinomycin D complexes with deoxynucleotides as models for the binding of the drug to DNA

Fluorescence, CD, absorption, and ¹H-nmr studies are reported for complexes of 7-amino-actinomycin D with deoxydinucleotides, deoxytetranucleotides, and poly(dG-dC)· poly(dG-dC). The optical spectra for the 7-amino-actinomycin D complex with pdG-dC, pdG-dC-dG-dC and pdC-dG-dC-dG are similar in shape to the 7-amino-actinomycin D complex with either DNA or poly(dG-dC). The changes in the ¹H chemical shifts of the 7-amino-actinomycin D and the pdG-dC resonances that accompany complex formation show that 7-amino-actinomycin D forms a minature intercalated complex with two pdG-dC molecules. The magnitudes of the induced chemical shifts for the 7-amino-actinomycin D complex formation with pdG-dC are similar to, but slightly different from, the induced chemical shifts which are obtained when actinomycin D forms a minature intercalated complex with two pdG-dC molecules. The pdN-dG dinucleotides (N = C, A, or T) form stacked complexes with 7-amino-actinomycin D. The presence of the 7-amino-group results in a larger dimerization constant (in aqueous solution) for 7-amino-actinomycin D [K_D(6°C) = 4.4 × 10³M^?1], as compared to actinomycin D [K_D(6°C) = 1.7 × 10³M^?1]; the chemical shifts which accompany dimer formation indicate that the chromophores stack in an inverted manner. Intercalation of 7-amino-actinomycin D into minature double helices, as well as into calf thymus DNA, poly(dG-dC)·poly(dG-dC), and poly(dA-dC)·poly(dG-dT), results in an enhancement of the relative fluorescence intensity and a shift in both the absorbance and corrected emission spectra.     

Functional characteristics of chlorophyll d-predominating photosynthetic apparatus in intact cells of Acaryochloris marina

Functional organization of the photosynthetic apparatus in the unique chlorophyll d-predominating prokaryote, Acaryochloris marina, was studied using polarographic measurements of single-turnover flash yields, action spectra and optical cross sections for PS-specific reactions. O₂ evolution was indicative of PS II activity, while reversible photoinhibition of respiratory O₂ uptake under aerobic conditions in the presence of DCMU and H₂ photoevolution by anaerobically adapted cells were the indicatives of PS I activity. O₂ evolution in the cells upon single-turnover flashes followed the normal S-state cycle with a period-4 oscillation. Analysis of action spectra for the partial reactions of photosynthesis revealed that: (1) distinct spectral forms of Chl d are nonuniformly distributed between PS I and PS II, e.g. Chl d-695 and Chl d-735 are preferentially located in PS II and PS I, respectively; (2) a minor fraction of Chl a in the cells belongs mostly to PS II; (3) biliproteins transfer excitation energy both to PS II and, with a lower efficiency, PS I; (4) the efficiency of energy transfer from biliproteins to PS II depends on the light quality growth conditions and is larger in white light (WL)-grown cells compared to the red light (RL)-grown cells. Content of functional O₂ evolving PS II centers decreases 2 times in the RL-grown cells relative to the WL-grown cells, whereas content of competent PS I centers involved in photoinhibition of respiration remains almost the same in both the cultures. The effective antenna size of PS I was estimated to be 80–90 Chl d including 3–10 molecules absorbing at 735 nm. The effective optical cross-section of PS II corresponded to 90–100 Chl d and, presumably, 4 Chl a + 2 Pheo a [Mimuro et al. (1999) Biochim Biophys Acta 1412: 37–46]. Optical cross-section measurements indicated that the functional PS II units of A. marina attach one rod of four hexameric units of biliproteins. This revised version was published online in June 2006 with corrections to the Cover Date.