Bacterial test-system for a primary screening of the substances with potential anti-cancer activity

A principle possibility of antitumour activity test in bacterial system represented by Escherichia coil of the wild type and its MS2-induced mutant has been shown. The initial bacterial strain is an indicator of toxic properties of the tested substances and the mutant one is a specific test-culture modelling a tumour cell. The comparison of the data described for eucaryotes with the data obtained using the proposed bacterial test system confirms an adequate response of both strains to the substances with the proved antitumour properties. The data are considered as very promising for the further improvement of this test system towards its application for primary screening of antitumour substances.     

Antimicrobial activities of daunorubicin and adriamycin derivatives on bacterial and protoplast type L-form cells of Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI. Structure--activity relationship

The antibacterial activity of ten N-alkylated derivatives of daunorubicin and adriamycin as well as of 5-iminodaunorubicin has been tested by using Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI and their stable protoplast type L-forms in an agar diffusion test. Eight of the substances showed similar activities against B. subtilis and the L-forms of all test organisms, but no activity against the bacterial forms of E. coli and P. mirabilis. The cell wall of these gram-negative bacteria is responsible for this resistance by not allowing the antibiotics to enter the cells. The piperidino compound N-(CH2)5 daunorubicin shows 2-4 times higher activity against B. subtilis and all L-forms in comparison to daunorubicin and the other derivatives. Five of the substances were inactive against all test strains. Their inactivity seems to be associated with the larger substituents at the C-3' position. Relations between molecular structure and activity are discussed considering data about the interaction with DNA and the antitumor activity. Stable protoplast type L-forms and their bacterial forms represent a suitable and effective test system to screen for more effective substances and to get more information about their mode of action.     

Cp-S316菌株抗细菌活性物质对大肠杆菌的抑制作用及高产突变株选育

通过测定大肠杆菌K12 (Escherichia coli K12)菌悬液的OD260的变化, 研究了多粘类芽孢杆菌(Paenibacillus polymyxa)Cp-S316抗细菌活性物质对其细胞膜完整性的影响, 结果表明Cp-S316抗细菌活性物质可损伤大肠杆菌K12的细胞膜, 从而引起胞内RNA、DNA等大分子物质的泄漏。为获得抗细菌活性物质高产菌株, 以Cp-S316为出发菌株, 通过紫外诱变以及对自身产生的抗细菌活性物质的抗性筛选法进行预筛、摇瓶初筛和复筛, 获得突变株多粘类芽孢杆菌A17, 其发酵效价比出发菌株Cp-S316提高91%, 该突变株的高产遗传性状稳定。     

Mutagenic activity of dioxydine]

A previous evaluation of mutagenic activity of some drugs and perspective substances is carried out using indicator microorganisms. The mutagenicity of dioxydine, a drag with discovered antibacterial activity, is investigated. Dioxydine is shown to induce reversions in mutant of Salmonella typhimurium TA-1950, the indicator strain which demonstrates mutagenic activity of agents, producing mutations of base pair substitution type. Dioxydine proved to affect logariphmiically growing bacterial culture with great activity. Mutageni effect of dioxydine is not modified itself in microsomal oxidation system in vitro. Some data concerning participation of excision reparation enzyme (uvr-B+ gene product) in repair of lethal damages induced by dioxydine, have been obtained. The dioxydine ability to cause bacterial gene mutations in host mediated assay as well as dominant and recessive sex-linked lethal mutations in Drosophila is demonstrated. Dioxydine is capable of inducing chromosome aberrations in bone marrow cells and dominant lethal mutations in mouse germ cells.     

Histo-Blood Group Antigen-Like Substances of Human Enteric Bacteria as Specific Adsorbents for Human Noroviruses

Histo-blood group antigens (HBGAs) have been suggested to be receptors or coreceptors for human noroviruses (HuNoVs) expressed on the intestinal epithelium. We isolated an enteric bacterium strain (SENG-6), closely related to Enterobacter cloacae, bearing HBGA-like substances from a fecal sample of a healthy individual by using a biopanning technique with anti-HBGA antibodies. The binding capacities of four genotypes of norovirus-like particles (NoVLPs) to Enterobacter sp. SENG-6 cells were confirmed by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy demonstrated that NoVLPs bound mainly to extracellular polymeric substances (EPS) of Enterobacter sp. SENG-6, where the HBGA-like substances were localized. EPS that contained HBGA-like substances extracted from Enterobacter sp. SENG-6 was shown by enzyme-linked immunosorbent assay (ELISA) to be capable of binding to NoVLPs of a GI.1 wild-type strain (8fIIa) and a GII.6 strain that can recognize A antigen but not to an NoVLP GI.1 mutant strain (W375A) that loses the ability to bind to A antigen. Enzymatic cleavage of terminal N-acetyl-galactosamine residues in the bacterial EPS weakened bacterial EPS binding to the GI.1 wild-type strain (8fIIa). These results indicate that A-like substances in the bacterial EPS play a key role in binding to NoVLPs. Since the specific binding of HuNoVs to HBGA-positive enteric bacteria is likely to affect the transmission and infection processes of HuNoVs in their hosts and in the environment, further studies of human enteric bacteria and their binding capacity to HuNoVs will provide a new scientific platform for understanding interactions between two types of microbes that were previously regarded as biologically unrelated.     

百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究

【目的】筛选对百合枯萎病具有抑菌活性的拮抗细菌,对其抑菌活性物质进行初步分离纯化分析。【方法】以强致病力的百合尖孢镰刀菌(Fusarium oxysporum)为靶菌,采用系列稀释法和平板对峙法初筛拮抗细菌,并通过产铁载体能力、水解酶活性、土壤定殖力等多种生防特性指标进行复筛,结合形态学特征、生理生化指标和16S rRNA基因序列比对鉴定其分类地位;利用百合尖孢镰刀菌作为靶菌进行活性追踪,结合酸沉淀、快速柱色谱、HPLC等分离纯化手段,对菌发酵液中的抑菌活性物质进行纯化分析。【结果】在64株百合根际细菌和386株海洋细菌中进行初筛,得到9株对百合镰刀菌具有较强拮抗活性的菌株,最后筛选了1株拮抗活性较强且产铁载体能力和水解酶活性、土壤定殖能力较高的菌株11B91,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其抑菌活性物质初步推测可能为iturin和fengycin脂肽类化合物。【结论】菌株11B91在百合枯萎病的生物防治中具有潜在的应用价值,证实海洋来源的微生物也具有防治陆地植物病原菌的潜力,为植物病害的防治拓宽了思路。     

Possible involvement of red pigments in defense against mercury in Pseudomonas K-62

Abstract To study the physiological role of the red pigments in soil strain Pseudomonas K-62, we isolated a red pigment-deficient white mutant from the soil strain by treatment with mitomycin C and compared the phenotypic properties of the mutant and parent strain. The red pigments, which were classified as one of carotenoids based on their physicochemical properties, were separated into two groups, designated pigment A and B respectively on NH-Chromatorex HPLC.The crude pigments and pigment B which could react with Hg²⁺ in the wild-type Pseudomonas K-62 and its mercury-resistant plasmid-deficient strain were enhanced by the addition of Hg²⁺. The white mutant thus obtained showed a greater sensitivity to Hg²⁺ than the wild-type reddish strain despite containing the resistant plasmids. The major component in pigment B was identified by mass spectrometric analysis as 1-hydroxy-1-methoxy-1,2, 1',2',7',8'-hexahydro-ψ,ψ-caroten-4-one, a carotenoid monoketone. These results suggested that red pigments, especially pigment B, may account, at least partially, for defense against Hg²⁺ in the bacterial environments.     

Mutation of pfm affects the adherence of Pseudomonas aeruginosa to host cells and the quorum sensing system

The Pseudomonas aeruginosa quorum sensing (QS) system is controlled by the signal molecules acyl homoserine lactones (AHLs) that are synthesized from acyl enoyl-acyl carrier proteins (acyl-ACPs) provided by the fatty acid biosynthesis cycle. Pfm (PA2950), an enoyl-CoA reductase, has previously been shown to affect swimming mobility and fatty acid biosynthesis. In this report, we further show that pfm influences bacterial adherence to human cells. Microarray assay results suggest that pfm affects bacterial adherence through its influence on the QS system. Further experiments confirmed that the pfm mutant strain produces significantly less QS signal molecules than the corresponding wild-type strain. Using strains Escherichia coli DH5α(pECP64, lasB'-lacZ) and E.?coli DH5α(pECP61.5, rhlA'-lacZ), biosensors for N-(3-oxododecanoyl) homoserine lactone (3O-C(12) -HSL) and N-butyryl homoserine lactone (C(4) -HSL), respectively, we found that pfm mutant strain produces decreased amounts of both signal molecules. Elastase activity and pyocyanin measurements further confirmed the reduced levels of 3O-C(12) -HSL and C(4) -HSL in the pfm mutant. Finally, bacterial virulence, as assessed by the Caenorhabditis elegans worm killing assay, is decreased in the pfm mutant. Taken together, these data indicate that pfm can be an important target for the control of P.?aeruginosa infectivity.     

Isolation of a fosfomycin-hypersensitive mutant and production of α-d-glucose-l-phosphate from Bacillus sp. BA-3796 screened by its use

The development of a very sensitive and highly specific screening method for detection of new cell wall inhibitors of the fosfomycin type is described. A fosfomycin-hypersensitive mutant, f-360, was isolated from Staphylococcus aureus Newman by selection with fosfomycin, an antibiotic that inhibits synthesis of the bacterial cell wall. The mutant f-360 was 50-fold more sensitive than the parent strain to fosfomycin. The mutant was constitutive for the hexose phosphate transport system. Using the organism in screening, BA-3796, which had an antibacterial activity against mutant f-360 was found to be produced by a bacterium designated Bacillus sp. BA-3796. Starch and beef extract were the most suitable carbon and nitrogen sources for BA-3796 production and the amount of BA-3796 reached 3 g/l at a maximum level. The purified BA-3796 was identified as α-d-glucose-l-phosphate by its various physiochemical properties. α-d-Glucose-1-phosphate showes an antibacterial activity against Staphylococci in the presence of a slight amount pf α-d-glucose-6-phosphate.     

The Mycobacterium tuberculosis sigJ gene controls sensitivity of the bacterium to hydrogen peroxide

Sigma factors are important global regulators which control bacterial gene expression during growth and in response to stress. Previous work showed that mRNA of the sigJ gene was up-regulated in late stationary-phase and after rifampicin treatment. In order to verify the function of SigJ, we constructed a Mycobacterium tuberculosis mutant lacking the sigJ gene. In a microaerophilic stationary-phase model, the sigJ mutant showed the same growth pattern as the wild-type strain. In an immune stasis murine model in which the bacterial number plateaued between the second and the 15th week, the mutant showed a similar growth curve to the wild-type strain. However, the sigJ mutant was more susceptible to killing by H2O2 than its parental strain. The parental level of sensitivity to H2O2 was recovered in the sigJ complemented strain. These data suggest that the SigJ protein is not essential for survival in long-term stationary phase or in bacterial stasis in mice. However, the sigJ gene may control an alternative H2O2 resistance pathway.     

拮抗菌SB1的鉴定及其抗菌物质的分析

对番茄根系菌株SB1的抗菌活性进行测定,结果表明该菌株对多种植物病原真菌、细菌具有明显的抑制作用,表现出广谱抗菌活性。通过菌体形态、生理生化反应及16SrDNA序列分析,鉴定菌株SB1为枯草芽孢杆菌内生亚种。以青枯雷尔氏菌为指示菌,测定了菌株SB1抗菌物质的理化性质及组成。结果表明,其抗菌物质表现出良好的热稳定性、水溶性和醇溶性,且对紫外线照射和蛋白酶K处理不敏感。高效液相色谱分析结果进一步显示菌株SB1的抗菌物质中含有抗菌肽Surfactin。     

一株肠膜明串珠菌的分离鉴定及其抑菌特性

[背景] 水产病原细菌严重威胁水产动物健康且制约水产养殖业发展,细菌性鱼病的有效防治成为水产养殖领域亟待解决的问题。[目的] 筛选对水产病原细菌有抑制效果的菌株,并研究其抑菌特性及其在水产细菌病害防治中的实际效果。[方法] 通过16S rRNA基因测序、构建系统发育树和生理生化鉴定确定筛选菌株的进化地位,通过乙酸乙酯萃取获得抑菌物质粗提物,通过偶氮酪蛋白法检测菌株胞外蛋白酶活力,采用结晶紫染色法对菌株的生物膜形成能力进行测定,通过浸浴攻毒模型确定所筛菌株对维氏气单胞菌的防治作用。[结果] 从泡菜发酵物中筛选出一株乳酸菌DH,经16S rRNA基因测序、发育树分析和生理生化鉴定确定其为肠膜明串珠菌,该菌分泌的胞外抑菌物质对鼠伤寒沙门氏菌、大肠埃希氏菌、铜绿假单胞菌、杀鲑气单胞菌、希瓦氏菌和维氏气单胞菌表现出抑菌效果,其抑菌物质能被乙酸乙酯萃取并且具有热稳定性。菌株DH能够显著抑制待测菌株的蛋白酶产量和生物膜形成能力,并且对维氏气单胞菌浸浴攻毒有防治作用。[结论] 肠膜明串珠菌DH通过分泌抑菌物质抑制水产病原细菌的生长,能够为细菌性鱼病的防治提供一定的理论和应用潜力。     

Biochemical characterization of a thialysine-resistant clone of CHO cells

The intracellular transport and the activation of lysine, thialysine and selenalysine have been investigated in a thialysine-resistant CHO cell mutant strain in comparison with the parental strain. The cationic amino acid transport system responsible for the transport of these 3 amino acids shows no differences between the 2 strains as regards its affinity for each of these amino acids. On the other hand the Vmax of the transport system in the mutant is about double that in the parental strain. The lysyl-tRNA synthetase, assayed both as ATP = PPi exchange reaction and lysyl-tRNA synthesis, shows a lower affinity for thialysine and selenalysine than for lysine in both strains; in the mutant, however, the difference is even greater. Thus the thialysine resistance of the mutant is mainly due to the properties of its lysyl-tRNA synthetase, which shows a greater difference of the affinities for lysine and thialysine with respect to the parental strain.     

Selection of bacterial wilt-resistant tomato through tissue culture

Bacterial wilt-resistant plants were obtained using a tomato tissue culture system. A virulent strain ofPseudomonas solanacearum secreted some toxic substances into the culture medium. Leaf explant-derived callus tissues which were resistant to these toxic substances in the culture filtrate were selectedin vitro and regenerated into plants. These plants expressed bacterial wilt resistance at the early infection stage to suppress or delay the growth of the inoculated bacteria. On the other hand, complete resistance was obtained in self-pollinated progeny of regenerants derived from non-selected callus tissues. These plants showed a high resistance when inoculated with this strain, and were also resistant when planted in a field infested with a different strain of the pathogen.     

Evaluation of the properties of potassium ferrate used for water purification by luminescence bioassay

Characteristics of four natural water samples from urban and rural areas and the efficiency of a new purifying agent, potassium ferrate K₂FeO₄, were studied by bacterial luminescence bioassay for 30 minutes. It was revealed that two samples of water from the urban areas are toxic, while the other two samples (one from urban and one from rural environment) are nontoxic. Numerous data obtained on the increase in toxicity index with time allow reasonable conclusions to be made about the chemical nature of substances present in the test water samples. Toxic natural water samples were likely to contain heavy metals and were well purified using potassium ferrate, including via their adsorption. In nontoxic natural water samples, toxic complexes with organic compounds present in water could form at the addition of potassium ferrate. The obtained data call for further studying the properties of potassium ferrate complexes with organic compounds. Bacterial luminescence bioassay is a promising method for the rapid assessment of properties of various water sources (their integral toxicity and presumable chemical composition) and new reagents for their purification (effective concentrations, bactericidal properties, and mechanisms of interacting with heavy metals and organic substances in water).     

Accumulation of a Bioactive Benzoisochromanequinone Compound Kalafungin by a Wild Type Antitumor-Medermycin-Producing Streptomycete Strain

Medermycin and kalafungin, two antibacterial and antitumor antibiotics isolated from different streptomycetes, share an identical polyketide skeleton core. The present study reported the discovery of kalafungin in a medermycin-producing streptomycete strain for the first time. A mutant strain obtained through UV mutagenesis showed a 3-fold increase in the production of this antibiotic, compared to the wild type strain. Heterologous expression experiments suggested that its production was severely controlled by the gene cluster for medermycin biosynthesis. In all, these findings suggested that kalafungin and medermycin could be accumulated by the same streptomycete and share their biosynthetic pathway to some extent in this strain.     

Biological properties of antibiotic-resistant mutants of the mildly pathogenic Rickettsia prowazekii E strain

The use of R. prowazekii strain E with low pathogenicity as live vaccine against exanthematous typhus is limited by its high specific reactogenicity, which is probably due to the reversion of the virulence of the strain. One of the approaches to the stabilization of the avirulent properties of strain E is obtaining its mutants with stable decreased pathogenic properties. The article presents the data on the infectious properties of R. prowazekii antibiotic-resistant strain E mutants obtained in earlier experiments, in respect of chick embryos and laboratory animals, as well as the capacity of this strain for producing immunity to challenge with R. prowazekii virulent strain in guinea pigs. The study has revealed that the erythromycin-resistant mutant of R. prowazekii strain E, induced by nitrosoguanidine (NG), has lower infective capacity for chick embryos, guinea pigs, cotton rats and white mice. The infective capacity of the NG-induced rifampicin-resistant and spontaneous erythromycin-resistant mutants of R. prowazekii strain E is similar to the infective capacity of the initial strain. The rifampicin-resistant and spontaneous erythromycin-resistant mutants of R. prowazekii strain E possess immunogenicity similar to that of the initial strain E, and the NG-induced erythromycin-resistant mutant possesses lower, but sufficiently pronounced immunogenicity despite its decreased infective capacity for guinea pigs.     

Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen fixation and indole-3-acetic acid production

Abstract Inoculation of wheat seedlings with Azospirillum brasilense Sp6 produced an increase in the number and length of the lateral roots as a plant response. Inoculation with a Nif⁻ mutant, A. brasilense SpF103, which is producer of indole-3-acetic acid (IAA), yielded a very similar plant response. However, inoculation with a Nif⁻ mutant, A. brasilense SpF57, which is a low producer of IAA, did not elitic any response from the plant. The data suggest that the root system response of wheat seedlings to bacterial inoculation is due mainly to production of auxin-type substances by the microorganism.     

The role of the vacB gene in the pathogenesis of Brucella abortus

Brucella species are important zoonotic pathogens affecting a wide variety of mammals. Therefore, the identification of new Brucella virulence factors is of great interest in understanding bacterial pathogenesis and immune evasion. In this study, we have identified Brucella abortus vacB gene that presents 2343 nucleotides and 781 amino acids and it shows 39% identity with Shigella flexneri vacB gene that encodes an exoribonuclease RNase R involved in bacterial virulence. Further, we have inactivated Brucella vacB by gene replacement strategy generating a deletion mutant strain. In order to test the role of Brucella vacB in pathogenesis, BALB/c and interferon regulatory factor-1 (IRF-1) knockout (KO) mice received Brucella vacB mutant, the virulent parental strain 2308 or the vaccine strain RB51 and the bacterial CFU numbers in spleens and mous survival were monitored. Our results demonstrated that the B. abortus DeltavacB mutant and the wild type strain 2308 showed similar CFU numbers in BALB/c mice. Additionally, IRF-1 KO mice that received either the vacB mutant or S2308 strain died in 12-14 days postinfection; in contrast, all animals that received the RB51 vaccine strain survived for 30 days postinoculation. In summary, this study reports that the vacB gene in B. abortus has no impact on bacterial pathogenesis.     

Complementation studies with human ClpP in Bacillus subtilis

ATP-dependent intracellular proteolysis is essential for all living organisms. ClpP, the proteolytic subunit of the ATP-dependent Clp proteases, shares 56% protein identity between B. subtilis and man. The aim of this study was to verify, whether human ClpP (HClpP) is able to substitute the bacterial pendant, BClpP, irrespectively of the huge evolutionary distance. For this reason hclpP was expressed from the natural B. subtilis promoters at the original chromosomal site. Growth at 37 °C as well as sporulation in the presence of hclpP depict an intermediate phenotype between wild type and clpP mutant suggesting a partial functional substitution of BClpP by HClpP. Northern as well as Western blot analyses show a similar induction pattern of both, bclpP and hclpP during heat stress on the mRNA as well as on the protein levels. Co-immunoprecipitation experiments imply specific interaction of HClpP with bacterial ClpC, ClpX and ClpE during control as well as heat stress conditions. Radioactive pulse-chase labeling and immunoprecipitation revealed that a ClpXP substrate, the short-living regulatory protein MgsR, is degraded by HClpP, although with an extremely slower rate in comparison to BClpP. The occurrence of an exceptional thickened cell wall of a clpP mutant can be almost fully reversed by the complementation with HClpP. The utilization of the HClpP expressing strain as a test system for new biological or synthetic active substances targeting BClpP is discussed.