Terahertz-infrared spectroscopy of Shewanella oneidensis MR-1 extracellular matrix.

Employing optical spectroscopy we have performed a comparative study of the dielectric response of extracellular matrix and filaments of electrogenic bacteria Shewanella oneidensis MR-1, cytochrome c, and bovine serum albumin. Combining infrared transmission measurements on thin layers with data of the terahertz spectra, we obtain the dielectric permittivity and AC conductivity spectra of the materials in a broad frequency band from a few cm-1 up to 7000 cm-1 in the temperature range from 5 to 300 K. Strong absorption bands are observed in the three materials that cover the range from 10 to 300 cm-1 and mainly determine the terahertz absorption. When cooled down to liquid helium temperatures, the bands in Shewanella oneidensis MR-1 and cytochrome c reveal a distinct fine structure. In all three materials, we identify the presence of liquid bound water in the form of librational and translational absorption bands at ≈ 200 and ≈ 600 cm-1, respectively. The sharp excitations seen above 1000 cm-1 are assigned to intramolecular vibrations.

Electroanalysis of Shewanella oneidensis MR-1.

Electrochemical parameters of bacterial cells Shewanella oneidensis MR-1 were investigated. For registration of the direct electron transfer between S. oneidensis MR-1 and electrode, bacterial cells were pretreated with didodecyldimethylammonium bromide (DDAB), a synthetic membrane-like substance of polycationic nature that exhibits membrane-loosening properties. Such pretreatment of S. oneidensis MR-1 allowed increasing the efficiency of extracellular electron transfer by the proteobacterium due to better availability of electroactive proteins for registration of electron transfer processes. The electroanalysis of bacterial cells S. oneidensis MR-1 under anaerobic conditions allows registering redox-active proteins and biomolecules in the range of potentials of-0.40,-0.16, and-0 V, which corresponds to flavohemoproteins, quinone derivatives, and c-type cytochromes of the external membrane of S. oneidensis MR-1 cells.

[Study of some aspects of the mechanism of bacterial synthesis of silver sulfide nanoparticles by mMetal-reducing bacteria Shewanella oneidensis MR-1].

In the present work it was shown that biosynthesis of silver sulfide nanoparticles from silver nitrate and sodium thiosulfate solutions of millimolar concentration occurs efficiently by living Shewanella oneidensis MR-1 cells, as well as by ultrasonically-disrupted cells and by the membrane fraction of the cells. The size of nanoparticles synthesized in the presence of living cells was 7.8 ± 1.5 nm, while in the presence of ultrasonically-disrupted cells--it was 6.52 nm. The shape of nanoparticles in both cases was close to spherical. It was also shown, that synthesis of nanoparticles occurs in a cell-free solution of sodium thiosulfate that has been incubated with cells previously and to which then a silver nitrate solution was added. In this case the nanoparticles were of elongated shape and their size was (11 ± 4) x (24 ± 6) nm. In the control experiment, when only silver nitrate and sodium thiosulfate solutions not incubated with cells were used, the nanoparticles were not detected. It was shown that biosynthesis of nanoparticles occurs both in aerobic and anaerobic conditions. Nanoparticles are not formed by using thermally inactivated cells as it was shown by us previously. The results show the important role of the native structures of cells for the nanoparticles formation.

[Bion-M1. Biological activities of microorganisms under the conditions of a 30-day space flight].

It was stated that spaceflight factors (SFF) affect the chromosomal DNA interchange during Streptomyces crossing. Cross polarity and primary input of a parent chromosome fragment in recombinant generation imply a more lasting cells contact in microgravity and a broader horizontal transport of genetic material. SFF had no effect on recombination frequency and mutation in a model of parental auxotrophic markers reversion to prototrophism. It was demonstrated that SFF boosted the fC31 phage exit from S. lividans 66 (fC31) and did not influence phage induction in S. coelicolor A3(2) (fC31). SFF inhibited synthesis of antiobiotic actinorhodin in lisogenic S. coelicolor A3(2), and tylosin and desmicosin in S. fradiae. Survivability of electrogenic bacteria Shewanella oneidensis MR-1 in space flight was higher compared with the synchronous control experiment. The reduction activity of S. oneidensis MR-1 as an indicator of electron generation effectiveness was identical in flight and laboratory samples.

[Microbial fuel cells as an alternative power supply].

Purpose of the work was designing and prototyping of microbial fuel cells (MFC) and comparative evaluation of the electrogenic activity of wastewater autochthonous microorganisms as well as bacterial monocultures. Objects were model electrogenic strain Shewanella oneidensis MR-1, and an Ochrobactrum sp. strain isolated from the active anode biofilm of MFC composed as an electricity generating system. The study employed the methods typically used for aerobic and anaerobic strains, current measurement, identification of new electrogenic strains in microbial association of wastewater sludge and species definition by rRNA 16-S. As a result, two MFCs prototypes were tried out. Besides, it was shown that electrogenic activity of S. oneidensis MR-1 and Ochrobactrum sp. monocultures is similar but differs from that of the microbial association of the anode biofilm.

Observation of dielectric universalities in albumin, cytochrome C and Shewanella oneidensis MR-1 extracellular matrix.

The electrodynamics of metals is well understood within the Drude conductivity model; properties of insulators and semiconductors are governed by a gap in the electronic states. But there is a great variety of disordered materials that do not fall in these categories and still respond to external field in an amazingly uniform manner. At radiofrequencies delocalized charges yield a frequency-independent conductivity σ 1(ν) whose magnitude exponentially decreases while cooling. With increasing frequency, dispersionless conductivity starts to reveal a power-law dependence σ 1(ν)∝ν s with s < 1 caused by hopping charge carriers. At low temperatures, such Universal Dielectric Response can cross over to another universal regime with nearly constant loss ε″∝σ1/ν = const. The powerful research potential based on such universalities is widely used in condensed matter physics. Here we study the broad-band (1-1012 Hz) dielectric response of Shewanella oneidensis MR-1 extracellular matrix, cytochrome C and serum albumin. Applying concepts of condensed matter physics, we identify transport mechanisms and a number of energy, time, frequency, spatial and temperature scales in these biological objects, which can provide us with deeper insight into the protein dynamics.

[Autoregulation of phenotypic dissiciation in Bacillus licheniformis].

Regulation of phenotypic variability of Bacillus licheniformis mediated by autoinducers of anabiosis, d1-factors was investigated. These factors are represented by alkylhydroxybenzenes. Colonial morphological variants of B. licheniformis were obtained and described (of R,S,M-types) in the first passage of both vegetative proliferative and resting cells. Resting cells were of different type, spores and cyst-like refractile cells induced by autoinducers of anabiosis. The possibility to manage the spectrum of dissociants by the mean of autoinducers of anabiosis was demonstrated.

[Comparative analysis of in vitro antifungal activity and in vivo acute toxicity of the nystatin analogue S44HP produced via genetic engineering].

New polyene macrolide S44HP was purified from the culture of recombinant Streptomyces noursei strain with engineered nystatin polyketide synthase. S44HP, nystatin (NYS), and amphotericin B (Amph-B) were tested against 19 clinical fungal isolates in agar diffusion assay, which demonstrated clear differences in antifungal activities of these antibiotics. Sodium deoxycholate suspensions of all three antibiotics were subjected to acute toxicity studies in vivo upon intravenous administration in mice. NYS exhibited the lowest acute toxicity in mice in these experiments, while both Amph-B and S44HP were shown to be 4 times more toxic as judged from the LD50 values. While the acute toxicity of S44HP was higher than that of Amph-B, the data analysis revealed a significantly increased LD10 to LD50 dose interval for S44HP compared to Amph-B. The data revealed structural features of polyene macrolides, which might have an impact on both the activity and toxicity profiles of these antibiotics. These results represent the first example of preclinical evaluation of an "engineered" polyene macrolide, and can be valuable for rational design of novel antifungal drugs with improved pharmacological properties.

[Conjugative transfer of a plasmid from Escherichia coli to various strains of the order Actinomycetales]. / Kon''iugativnyi perenos plazmid iz Escherichia coli v razlichnye shtammy poriadka Actinomycetales.

The conjugal transfer of autonomous and integrative plasmids from the donor strain Escherichia coli S17-1 to strains of genera Actinomadura, Arthrobacter, Kitasatoa, Micromonospora, Nocardia, Rhodococcus, Saccharopolyspora, and to 16 strains of the genus Streptomyces was demonstrated. The status of plasmids in recipient strains and the stability of their inheritance were analyzed. Plasmids constructed for strains of the genus Streptomyces were shown to function in a large number of strains belonging to the order Actinomycetales. The well-developed system of Streptomyces vector molecules and cloned genes of antibiotic biosynthesis allows their transfer to those microorganisms for which conventional techniques of plasmid transfer by regenerated protoplast transformation or electroporation have not been developed or are inefficient.

[Elements of regulatory systems of antibiotic biosynthesis in Streptomyces]. / Elementy sistem reguliatsii biosinteza antibiotikov u Streptomyces.

Studies of regulation of antibiotic synthesis involved both basic and applied aspects of biology of antibiotic producers. This paper is a survey of published experimental data and key hypotheses for the regulation of antibiotic synthesis in bacteria of the genus Streptomyces. Summarized data on transcriptional regulatory systems of gene clusters responsible for biosynthesis of some antibiotics in these microorganisms are presented. Results of our previous research of the regulation of antibiotic bialaphos production in the S. hygroscopicus strain are considered as well. On the basis of these results, a hypothetical scheme of the organization of lower levels of the regulatory system of the bialaphos biosynthetic cluster was constructed. It is assumed that an integrase-like product of the brpB gene cloned by the authors mediates transcriptional initiation of this gene cluster.

[Actinophage phi C31 restriction and modification]. / Restriktsiia i modifikatsiia aktinofaga Fi C31.

Actinophage phi C31 was shown to be insensitive to a number of restriction and modification systems (RM). Phage sensitivity to RM systems of those strains to which phage cannot absorb, may be tested using protoplast transfection. For instance, the absence of phi C13 transfection in Streptomyces griseus Kr15 protoplasts, as compared to efficient transfection in protoplasts of R- M+ mutant of this strain seems to imply the sensitivity of phi C31 to the RM system of S. griseus KR15. Restriction of mutant phi C31 phage modified by S. albus G Rm system has been detected in S. coelicolor A3(2). This effect being dependent on a previous host may indicate that the mutant phage was rendered sensitive to an Rm system of S. coelicolor A3(2).

[Development of a system of plasmid transfer in the strains Streptomyces avermitilis ATCC 31272 and Saccharopolyspora erythraea ATCC 11635 by the method of intergeneric conjugation]. / Razrabotka sistemy peredachi plazmid metodom mezhrodovoi kon''iugatsii v shtammy Streptomyces avermitilis ATCC 31272 i Saccharopolyspora erythraea ATCC 11635.

A new method of plasmid DNA transfer from the donor strain Escherichia coli S17-1 to the erythomycin-producing strain Saccharopolyspora erythraea and avermectin-producing strain Streptomyces avermitilis via intergeneric conjugation was proposed. The optimal parameters of the method were chosen for increasing the efficiency of crosses and ensuring easily reproducible results. The behavior of the multicopy plasmid pPM803 and the integration vector pTO1 along with a number of new plasmids specially created by us, was examined in these strains. A new plasmid vector (pSI60) capable of integrating into the chromosome of actinomycetes at the integration site of the temperate actinophage phi C31 was constructed. This vector possesses unique sites convenient for cloning and may be stably maintained in exconjugants of S. avermitilis and in the model strain Streptomyces lividans. The gene encoding resistance to spectinomycin and streptomycin was cloned into the vector pSI60 in this strain. For cloning in strain Sac. erythraea, vectors pSI261-280, which integrate into the chromosome via homology with the cloned DNA and can be stably maintained in exconjugants, were constructed.

[Molecular-genetic study of the functional role of the regulatory gene brpB of the bialaphos producer Streptomyces hygroscopicus ATCC21705]. / Molekuliarno-geneticheskoe issledovanie funktsional'noi roli reguliatornogo gena brpB u produtsenta bialafosa Streptomyces hygroscopicus ATCC21705.

A chromosomal determinant of Streptomyces hygroscopicus, functioning as a positive regulator of the bialaphos-resistance gene bar in the model strain S. lividans, was conjugatively transferred into a bialaphos (BAP) producer through pVGTB24 plasmid. The plasmid is unable to replicate autonomously in S. hygroscopicus, but integrates into its chromosome via homology with the fragment containing this determinant. Integration of pVGTB24 was accompanied by a decrease in resistance of S. hygroscopicus exconjugant clones to the self-produced antibiotic and also by a decrease in their antibiotic productivity.

[Molecular genetic study of the strain Streptomyces virginiae-- the producer of virginiamycin]. / Molekuliarno-geneticheskoe izuchenie shtamma Streptomyces virginiae--produtsenta virdzhiniamitsina.

The level and the character of the Streptomyces virginiae virginiamycin-producing strain's resistance to self-produced antibiotic and a number of antibiotics from different groups were determined. S. virginiae was shown to display constitutive and inducible resistance to self-produced antibiotic. The phenomenon of cross-inducible resistance of the strain to virginiamycin and the antibiotics erythromycin, oleandomycin, and thiostrepton was demonstrated. The pTO1 and pVGTB24 plasmids were introduced into the strain by the method of intergeneric conjugation with Escherichia coli. Site-specific integration of the pTO1 vector into the S. virginiae chromosomal attB site without disturbance of growth, differentiation, and productivity of the strain was shown. The multicopy autonomously replicating plasmids pIJ699, pIJ702, pWOR109, and the integrative pZAT22 plasmid were introduced into the strain via electrotransformation of germinating spores. The efficiency of transformation was 1-5 x 10(3) transformants per 1 microgram DNA. The stable inheritance of the plasmids in S. virginiae without structural rearrangements was shown. These results allow the use of these plasmids to clone genes into S. virginiae.

[Identification of the restriction and modification systems in Streptomyces strains]. / Identifikatsiia sistem restriktsii i modifikatsii u shtammov Streptomyces.

Host range of actinophage phi C31, VP5 and Pg81 in respect to 109 strains of Streptomyces genus and hybrid strain Rcg2 from the cross S. coelicolor A3(2)XS. griseus Kr was studied. The existence of RM-systems in strains S. griseus Kr15, S. griseus Kr20, Rcg2, S. griseofovillus 43 was shown using phage Pg81. Mutants of Pg81 were observed which to some extent lost snesitivity to RM-system in the strain Rcg2. The presence of RM-system in S. lividans 67 was demonstrated by the phage VP5.

[Hybridization in Actinomycetes]. / Gibridizatsiia u aktinomitsetov

Ways of transferring genetic information in Actinomycetes and their use for the transmission of genetic material in intervarietal crosses are discussed. The producing of hybrids is shown to be an efficient method to obtain Actinomycetes strains with new properties. Properties of recombinants obtained in intervarietal crosses Streptomyces coelicolor A3(2)XS. lividans 66 are shown to possess new antibiotic properties differing from those of parental strains: they suppress a number of Actinomycetes and bacterial strains which are not affected by parental strains. It is found that at least two groups of genes located on chromosomes of S. coelicolor A3(2) and S. lividans 66 participate in the synthesis of the antibiotic. The obtaining of recombinants between S. coelicolor A3(2) and S. griseus 15 makes possible to select variants which are capable to produce the antibiotic grisine and are resistant to actinophages, specifically attacking S. griseus. Properties of recombinants between S. coelicolor A3(2) and S. griseus 15 make possible to localize on parental chromosomes regions containing genes which control the synthesis of the antibiotic, the formation of a receptor for the adsorption of actinophages, and genes controlling the restriction and t he modification of actinophage development in S. coelicolor and S. griseus. A sex plasmid (SGP1) is found in S. griseus 15.

[Use of a plasmid with integrative function of phage phiC31 for transfer of cloned genes into Streptomyces strains]. / Ispol'zovanie plazmid s integrativnoi funktsiei faga phiC31 dlia perenosa klonirovannykh genov v shtammy Streptomyces.

Opportunities for application of integrative vectors carrying the attP site and the int gene of the temperate actinophage phi C31 for cloning genes in Streptomyces strains were demonstrated. The behavior of the integrative vectors pZAT22 and pTO1 in the model strain S. lividans TK64 and in the bialaphos-producing strain S. hygroscopicus, respectively, was characterized. Restriction maps of the S. lividans and S. hygroscopicus chromosomal regions containing attB sites were constructed. The bar gene of resistance to bialaphos was incorporated into the chromosome of the model strain S. lividans via the integrative pZAT22 vector, the level of expression of this gene within the chromosome of a heterologous host was determined. The possibility of amplification of the bar gene in the chromosome of the strain S. lividans was shown. The conjugative integrative vector pTO1, which carried the cloned bar gene, was introduced into the bialaphos-producing strain by intergeneric matings of Escherichia coli and S. hygroscopicus. The effect of an additional copy of the gene in the chromosome of S. hygroscopicus on strain resistance and productivity was studied.

[Effect of the gene for bacterial hemoglobin vhb on the effectiveness of the process of Escherichia coli-Streptomyces interspecies conjugation and production of antibiotics in streptomycetes]. / Vliianie gena bakterial'nogo gemoglobina vhb na éffektivnost' protsessa mezhrodovoi kon''iugatsii Escherichia coli-Streptomyces i produktsiiu antibiotikov u streptomitsetov.

The bacterial hemoglobin vhb gene was cloned from sliding bacterium Vitreoscilla sp. as an element of the system ensuring survival of this microorganism in an environment that contains insufficient amount of oxygen. The vhb gene was transferred from Escherichia coli to some Streptomyces strains, producers of antibiotics, by the method of intergeneric conjugation using conjugative-integrative plasmid vectors pIH1 and pCH2. The stability of plasmid DNA inheritance was analyzed in the genomes of exconjugants. A positive effect of the vhb gene on processes of conjugation and antibiotic production in a number of examined strains was shown.

[Intergeneric Escherichia coli-Streptomyces conjugation as a means for the transfer of conjugative plasmids into producers of antibiotics chlortetracycline and bialaphos]. / Mezhrodovaia kon"iugatsiia Escherichia coli-Streptomyces kak sposob peredachi kon"iugativnykh plazmid v produtsenty antibiotikov khlortetratsiklina i bialafosa.

A system for introduction of plasmids into industrial producers of antibiotics chlortetracycline and bialaphos using intergeneric conjugation of Escherichia coli and Streptomyces was developed. Low level stability of inheritance of autonomously replicating DNA in recipient strains was shown. Site-specific integration of the conjugative-integrative vector pTO1 provided stable plasmid maintenance within the chromosomes of Streptomyces aureofaciens and S. hygroscopicus. Phenomenon of disturbance in differentiation and antibiotic production, resulting from pTO1 integration into S. hygroscopicus chromosome, was discovered.

[Determinants of resistance to chlortetracycline and other antibiotics in chlortetracycline-producing strain of Streptomyces aureofaciens]. / Determinanty ustoichivosti k khlortetratsiklinu i drugim antibiotikam u shtamma Streptomyces aureofaciens--produtsenta khlortetratsiklina.

Data are presented on resistance of Streptomyces aureofaciens strain TB-633 FU--the producer of chlortetracycline (CTC) to autogenous antibiotics and a number of other antibiotics. It is demonstrated that resistance to CTC is specified by ctr genes of constitutive expression as well as by inducible genes. CTC and ethidium bromide may serve as efficient inductors of inducible ctr genes. The induction process is accompanied by increase in antibiotic biosynthesis level. Genes responsible for strain resistance to a number of macrolide antibiotics and thiostrepton are inducible and only function in the presence of appropriate antibiotics in the medium. The action of inducible mtr gene(s) is described in detail. The gene(s) simultaneously ensure increase in resistance to CTC and a number of macrolide antibiotics in the presence of exogenous inductors in media, such as both CTC and macrolide antibiotics. Mutants have been isolated which provide constitutive level of resistance to these antibiotics. A series of ctr and mtr mutants have increased CTC biosynthesis as compared to the initial level. Data on comparative analysis of the results obtained from hybridization of fragments of S. aureofaciens and S. rimosus DNAs to actI and actIII genes, responsible for polyketide synthases' synthesis, demonstrate that genes for CTC and OTC biosynthesis are situated on DNA fragments of similar size. This determines the strategy for cloning ctr and mtr genes as well as genes for CTC biosynthesis from S. aureofaciens.