石油污染土壤生物修复过程中微生物生态研究

The analysis on microbiological ecology for four types of oil contaminates soils showed that the bacteria utilizing the oil as carbon sources increase,wheras the fugi become less .Zooloea and Bacillu are the dominant bacteria ; Mocor and Cunninghamella ,and Fursarium are the dominant fungi streptomyces take the superiority among the actinomyces.The anaiysis on esterase activity showed that the microbes above mentioned have abilies of degrading esters. The biodeg radationrates are 55.45%,56.74%,38.37% and 45.19%respectively,after 53 days,the biodegradation rate can be increased by 12.6% when the dominant microbes are added.

Chandrananimycins A-C: Production of novel anticancer antibiotics from a marine Actinomadura sp isolate M048 by variation of medium composition and growth conditions

In our screening of marine actinomycetes for bioactive principles, three novel antibiotics designated as chandrananimycin A (3c), B (3d) and C (4) were isolated from the culture broth of a marine Actinomadura sp. isolate M045. The structures of the new antibiotics were determined by detailed interpretation of mass, 1 D and 2 D NMR spectra.

胶州湾海洋链霉菌抑菌活性筛选、2株抑菌活性菌株的鉴定及次级代谢产物的研究

本文对256株胶州湾海洋链霉菌进行了抑菌活性的筛选，并选取10株典型菌株进行化学筛选，获得2株有研究价值的菌株。通过大规模发酵，获得纯化的次级代谢产物，进行了结构解析。通过与其他5株活性菌株的16S rRNA基因序列比较，并结合生理生化、形态特征和培养特征分析，探讨了这两株菌的分类地位。 　　采用液体扩散法，选用金黄色葡萄球菌、大肠杆菌、绿脓杆菌、八叠球菌、隐球菌、白色念珠菌、Mucor miehei (TÜ 284)和Streptomyces viridochromogenes (TÜ57) 8株受试菌进行抑菌活性的筛选，结果22%的菌株显示出对至少一种受试菌具有抑制作用(抑菌圈Æ ³ 8 mm)。根据菌株的形态特征和抑菌活性特点，选择M024、M028、M042、M083、M086、M095、M097、M124、M134和M226 10株链霉菌进行化学筛选。考察了8种培养基和4种培养条件，结果发现菌株M095在Meat extract培养基、pH 6.5、28℃和95 r/min条件下，菌株M097在Meat extract培养基、pH 7.8、 28℃、95 r/min(条件Ⅰ)和M2+培养基、pH 7.8、 35℃、110 r/min(条件Ⅱ)条件下，可供进一步研究。 对菌株M095(24 L规模)和M097(Ⅰ为30 L规模，Ⅱ为14 L规模)进行发酵，采用乙酸乙酯提取和柱层析分离纯化次级代谢产物，通过ESI-MS、EI-MS、1H-NMR和13C-NMR等波谱解析，鉴定出次级代谢产物的结构。发现菌株M095产生一抑菌活性很强的化合物全霉素，首次证实该全霉素具有抑制丝状真菌的作用；菌株M097主要产生10个化合物，其中8个具有不同程度的生物活性，另外两个化合物中，Aloesaponaria Ⅱ为首次从微生物野生菌株(wild strain)中获得，化合物Cui D为一新结构的蒽醌类化合物。 经分子鉴定，初步认为本实验分离的7株活性海洋链霉菌分属于4个链霉菌类群，结合生理生化、形态特征和培养特征分析，认为菌株M095可能为灰色链霉菌的变种，M097可能为球孢类群中的一个新种。

四株海洋放线菌遗传转化体系的研究

作者所在的课题组，自1998年以来从胶州湾海泥中陆续分离了800株海洋放线菌，并从4株放线菌中分离出了12个新结构活性化合物。选择产生新颖抗肿瘤抗生素的海洋放线菌M045和M048，产全霉素的海洋放线菌M095和产蒽醌类化合物的海洋放线菌M097为研究材料，建立了海洋放线菌的遗传转化体系，为海洋放线菌的遗传工程操作及天然化合物组合生物合成奠定了基础。 （1）通过接合转移建立了菌株M045的遗传转化体系。用来源于蓝藻Anacystis nidulans UTEX625的别藻蓝蛋白基因验证了转化体系的有效性。通过PCR及基因组步移方法获得长度为1709bp的部分聚酮合成酶（PKS）基因，分析其同放射菌素基因具有同源性，利用基因中断插入失活该基因，但未获得突变株。因此尝试通过反向遗传学方法，克隆该菌株中新骨架抗肿瘤抗生素——中国霉素的生物合成基因簇，本研究已经构建了该菌株Fosmid基因组文库，对基因组文库的筛选工作正在进行中。 （2）利用PEG-介导的质粒pIJ702转化原生质体和接合转移两种方法均成功获得菌株M048的转化子，其中接合转移率高达10-4。菌株M048来源于高盐的海洋环境，维持原生质体所需渗透压与模式菌株—变铅青链霉菌（Streptomyces lividans）有很大差异，本研究对菌株M048原生质体形成和再生的各种因素进行了优化，获得了渗透压稳定剂蔗糖最佳浓度为0.4M。 质粒pIJ8600整合于菌株M048染色体上，对该转化株的抑菌活性、薄层层析（TLC）以及HPLC-MS进行了分析。结果表明，同野生菌株相比，该转化株对7种受试菌的抑菌活性显著增强，TLC显示差异的化合物条带，HPLC-MS显示化合物组分有差异。因此质粒pIJ8600的整合，引起菌株次级代谢产物生物合成途径的改变，使有抑菌活性的化合物大量累积。 从菌株M048染色体上克隆获得了1196bp的部分PKS基因，通过基因中断插入失活该基因，结果显示M048突变株次级代谢产物抑菌活性增强，HPLC分析发现显著差异。初步分析该PKS基因的中断使菌株体内某些生物合成途径受阻，而大量合成抗菌活性强的chandrananimycin C，或者产生了抑菌活性强的其它化合物。 （3）本研究成功建立了菌株M095的接合转移体系。M095/pIJ8600转化株的生物学活性分析并未发现差异，表明该菌株染色体上的整合位点（attB）是中性（neutral）的。通过PCR以及基因组步移的方法克隆获得了该菌株的部分糖基转移酶基因，该基因中断突变株对4株受试菌的抑菌活性增强，HPLC显示有差异，表明该糖基转移酶基因参与了菌株M095活性次级代谢产物的生物合成过程。 （4）对于菌株M097，用接合转移法成功获得了转化子。实现了别藻蓝蛋白基因的重组表达，并纯化了表达产物，体外试验表明其具有清除羟基自由基能力。结果表明来源于蓝藻的外源基因可以在海洋放线菌体内有效表达和正确折叠，初步验证了本研究所建立的海洋放线菌遗传转化体系的稳定性及有效性。对M097/pIJ8600转化株的生物学活性分析，未发现差异，表明该菌株染色体上的整合位点是中性的。 本论文首次将基因工程技术引入四株海洋放线菌，建立了海洋放线菌自身的基因转移系统，为利用基因工程技术改造海洋放线菌的天然化合物生物合成途径提供了方法。对部分PKS基因中断突变株的生物学活性及化学分析，初步揭示了通过遗传转化方法进行化合物组合生物合成的可行性。

青海高寒草甸土壤放线菌区系研究

2001～2002年从海北高寒草甸生态系统采集土样,用不同方法从中分离放线菌300余株,根据其形态和分类特征,分别归入小单孢菌属(Micromonospora)、诺卡氏菌属(Nocardia)、糖多孢菌属(Saccharopolyspora)、原小单孢菌属(Promicromonospora)和链霉菌属(Streptomyces),并将链霉菌归入7个类群.同时对230株中温菌和110株低温菌的部分酶活性及其对真菌和细菌的拮抗性进行了测定,发现链霉菌不仅具有许多酶活性,而且对真菌和细菌有拮抗性.

Biodiscovery of natural products from microbes associated with Irish coastal sponges

Marine sponges have been an abundant source of new metabolites in recent years. The symbiotic association between the bacteria and the sponge has enabled scientists to access the bacterial diversity present within the bacterial/sponge ecosystem. This study has focussed on accessing the bacterial diversity in two Irish coastal marine sponges, namely Amphilectus fucorum and Eurypon major. A novel species from the genus Aquimarina has been isolated from the sponge Amphilectus fucorum. The study has also resulted in the identification of an α–Proteobacteria, Pseudovibrio sp. as a potential producer of antibiotics. Thus a targeted based approach to specifically cultivate Pseudovibrio sp. may prove useful for the development of new metabolites from this particular genus. Bacterial isolates from the marine sponge Haliclona simulans were screened for anti–fungal activity and one isolate namely Streptomyces sp. SM8 displayed activity against all five fungal strains tested. The strain was also tested for anti–bacterial activity and it showed activity against both against B. subtilis and P. aeruginosa. Hence a combinatorial approach involving both biochemical and genomic approaches were employed in an attempt to identify the bioactive compounds with these activities which were being produced by this strain. Culture broths from Streptomyces sp. SM8 were extracted and purified by various techniques such as reverse–phase HPLC, MPLC and ash chromatography. Anti–bacterial activity was observed in a fraction which contained a hydroxylated saturated fatty acid and also another compound with a m/z 227 but further structural elucidation of these compounds proved unsuccessful. The anti–fungal fractions from SM8 were shown to contain antimycin–like compounds, with some of these compounds having different retention times from that of an antimycin standard. A high–throughput assay was developed to screen for novel calcineurin inhibitors using yeast as a model system and three putative bacterial extracts were found to be positive using this screen. One of these extracts from SM8 was subsequently analysed using NMR and the calcineurin inhibition activity was con rmed to belong to a butenolide type compound. A H. simulans metagenomic library was also screened using the novel calcineurin inhibitor high–throughput assay system and eight clones displaying putative calcineurin inhibitory activity were detected. The clone which displayed the best inhibitory activity was subsequently sequenced and following the use of other genetic based approaches it became clear that the inhibition was being caused by a hypothetical protein with similarity to a hypothetical Na+/Ca2+ exchanger protein. The Streptomyces sp. SM8 genome was sequenced from a fragment library using Roche 454 pyrosequencing technology to identify potential secondary metabolism clusters. The draft genome was annotated by IMG/ER using the Prodigal pipeline. The Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMPN00000000. The genome contains genes which appear to encode for several polyketide synthases (PKS), non–ribosomal peptide synthetases (NRPS), terpene and siderophore biosynthesis and ribosomal peptides. Transcriptional analyses led to the identification of three hybrid clusters of which one is predicted to be involved in the synthesis of antimycin, while the functions of the others are as yet unknown. Two NRPS clusters were also identified, of which one may be involved in gramicidin biosynthesis and the function of the other is unknown. A Streptomyces sp. SM8 NRPS antC gene knockout was constructed and extracts from the strain were shown to possess a mild anti–fungal activity when compared to the SM8 wild–type. Subsequent LCMS analysis of antC mutant extracts confirmed the absence of the antimycin in the extract proving that the observed anti–fungal activity may involve metabolite(s) other than antimycin. Anti–bacterial activity in the antC gene knockout strain against P. aeruginosa was reduced when compared to the SM8 wild–type indicating that antimycin may be contributing to the observed anti–bacterial activity in addition to the metabolite(s) already identified during the chemical analyses. This is the first report of antimycins exhibiting anti–bacterial activity against P. aeruginosa. One of the hybrid clusters potentially involved in secondary metabolism in SM8 that displayed high and consistent levels of gene–expression in RNA studies was analysed in an attempt to identify the metabolite being produced by the pathway. A number of unusual features were observed following bioinformatics analysis of the gene sequence of the cluster, including a formylation domain within the NRPS cluster which may add a formyl group to the growing chain. Another unusual feature is the lack of AT domains on two of the PKS modules. Other unusual features observed in this cluster is the lack of a KR domain in module 3 of the cluster and an aminotransferase domain in module 4 for which no clear role has been hypothesised.

A coiled-coil-repeat protein 'Ccrp' in Bdellovibrio bacteriovorus prevents cellular indentation, but is not essential for vibroid cell morphology

Bdellovibrio bacteriovorus are small, vibroid, predatory bacteria that grow within the periplasmic space of a host Gram-negative bacterium. The intermediate-filament (IF)-like protein crescentin is a member of a broad class of IF-like, coiled-coil-repeat-proteins (CCRPs), discovered in Caulobacter crescentus, where it contributes to the vibroid cell shape. The B. bacteriovorus genome has a single ccrp gene encoding a protein with an unusually long, stutter-free, coiled-coil prediction; the inactivation of this did not alter the vibriod cell shape, but caused cell deformations, visualized as chiselled insets or dents, near the cell poles and a general 'creased' appearance, under the negative staining preparation used for electron microscopy, but not in unstained, frozen, hydrated cells. Bdellovibrio bacteriovorus expressing 'teal' fluorescent protein (mTFP), as a C-terminal tag on the wild-type Ccrp protein, did not deform under negative staining, suggesting that the function was not impaired. Localization of fluorescent Ccrp-mTFP showed some bias to the cell poles, independent of the cytoskeleton, as demonstrated by the addition of the MreB-specific inhibitor A22. We suggest that the Ccrp protein in B. bacteriovorus contributes as an underlying scaffold, similar to that described for the CCRP protein FilP in Streptomyces coelicolor, preventing cellular indentation, but not contributing to the vibroid shape of the B. bacteriovorus cells.

A tale of two bioprocesses

Bioprocesses use microorganisms or cells in order to produce and/or obtain some desired products. Nowadays these strategies appear as a fundamental alternative to the traditional chemical processes. Amongst the many advantages associated to their use in the chemical, oil or pharmaceutical industries, their low cost, easily scale-up and low environmental impact should be highlighted. This work reports two examples of bioprocesses as alternatives to traditional chemical processes used by the oil and pharmaceutical industries. In the first part of this work it was studied an example of a bioprocess based on the use of microorganisms in enhanced oil recovery. Currently, due to high costs of oil and its scarcity, the enhanced oil recovery techniques become very attractive. Between the available techniques the use of microbial enhanced oil recovery (MEOR) has been highlighted. This process is based on the stimulation of indigenous microorganisms or by the injection of microorganism consortia to produce specific metabolites and hence increase the amount of oil recovered. In the first chapters of this work the isolation of several microorganisms from samples of paraffinic Brazilian oils is described, and their tensioactive and biodegradability properties are presented. Furthermore, the chemical structures of the biosurfactants produced by those isolates were also characterized. In the final chapter of the first part, the capabilities of some isolated bacteria to enhance the oil recovery of paraffinic Brazilian oils entrapped in sand-pack columns were evaluated. In the second part of this work it was investigated aqueous two-phase systems or aqueous biphasic systems (ABS) as extractive strategies for antibiotics directly from the fermented broth in which they are produced. To this goal, several aqueous two-phase systems composed of ionic liquids (ILs) and polymers were studied for the first time and their phase diagrams were determined. The novel ATPS appear as effective and economic methods to extract different biomolecules or/and biological products. Thus, aiming the initial antibiotics extraction purpose it was studied the influence of a wide range of ILs and polymers in the aqueous two-phase formation ability, as well as their influence in the partitioning of several type-molecules, such as amino acids, alkaloids and dyes. As a final chapter it is presented the capacity of these novel systems to extract the antibiotic tetracycline directly from the fermented broth of Streptomyces aureofaciens.

Distribution and bioactive potential of soil actinomycetes from different ecological habitats

Emergence of drug resistance among pathogenic bacteria to currently available antibiotics has intensified the search for novel bioactive compounds from unexplored habitats. In the present study actinomycetes were isolated from two relatively unexplored and widely differing habitats such as mountain and wetlands and their ability to produce antibacterial substances were analyzed. Pure cultures of actinomycetes were identified by morphological and biochemical tests. Various genera of actinomycetes encountered included Nocardia, Pseudonocardia, Streptomyces, Nocardiopsis, Streptosporangium, Micromonospora, Rhodococcus, Actinosynnema, Nocardiodes, Kitasatosporia, Gordona, Intrasporangium and Streptoalloteichus. The frequency of occurrence of each genus was found to vary with sample. About 47% of wetland isolates and 33% of mountain isolates were identified as various species of Nocardia. The isolated strains differed among themselves in their ability to decompose proteins and amino acids and also in enzyme production potential. Antibiotic activities of these actinomycetes were evaluated against 12 test pathogenic bacteria by well diffusion method using agar wells in glycerol-yeast extract agar. About 95% of actinomycete isolates from wetland ecosystem and 75% of highland isolates suppressed in different degrees the growth of test pathogens. Relatively high antibacterial activity among these isolates underlined their potential as a source of novel antibiotics.

Actinomycete Isolates from Arabian Sea and Bay of Bengal: Biochemical, Molecular and Functional Characterization

The thesis is comprised of seven chapters. Chapter 1 gives a general introduction to marine actinomycetes; Chapter 2 gives an account on the morphological, biochemical and physiological characterization of marine actinomycetes. Comprehensive description of molecular identification and phylogenetic analysis of actinomycetes is dealt with in Chapter 3. The antimicrobial property with special reference to antivibrio activity is described in Chapter 4. Chapter 5 explores the melanin production ability of marine actinomycetes, characterization of melanin and evaluation of its bioactivity. Chapter 6 illustrates the study on chitinolytic Streptomyces as antifungal and insecticidal agents. Summary and Conclusion of the study is presented in Chapter 7, followed by References and Appendices.The present study provides an insight into the various actinomycetes occurring in the sediments of Arabian Sea and Bay of Bengal. Streptomyces was found to be the dominant group followed by Nocardiopsis. Eventhough generic level identification is possible by traditional phenotypic methods, species level identification necessitate a polyphasic approach including both phenotypic and genotypic characterization. Antibiotic production coupled with biogranulation property helped in the effective utilization of the actinomycetes for the control of vibrios. Melanin from Streptomyces bikiniensis was proved to be a promising antioxidant and photoprotectant. Marine actinomycetes were found to be a good source of hydrolytic enzymes and the chitinolytic isolates could be explored as biocontrol agents in terms of antifungal and insecticidal property. The present study explored the potential of marine actinomycetes especially Streptomycetes as a promising source of bioactive molecules for application in aquaculture and pharmaceutical industry.

Brucella melitensis 16M: characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant

The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined. An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified. DNA sequences flanking the B. melitensis galE gene shared no identity with other gal genes and, as for B. abortus, were located adjacent to a mazG homologue. A plasmid which encoded the B. melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity. The galE gene on the B. melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed. One B. melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain. CD-1 mice immunised with B. melitensis 16M galE were protected against B. melitensis 16M challenge. Crown Copyright (C) 1999 Published by Elsevier Science B.V.

Water, irrigation and plant diseases

Irrigation is a major husbandry tool, vital for world food production and security. The purpose of this review is twofold:- firstly drawing attention to the beneficial and deleterious aspects of irrigation resulting from interactions with the microbial world; secondly, forming a basis for encouraging further research and development. Irrigation is for example, a valuable component in the control of some soil borne pathogens such as Streptomyces scabies, the cause of potato common scab and Fusarium cubense, a cause of banana wilt. By contrast, applying irrigation encourages some foliar pathogens and factors such as splash dispersal of propagules and the retention of leaf wetness are important elements in the successful establishment of disease foci. Irrigation applied at low levels in the canopy directly towards the stem bases and root zones of plants also provides means encouraging disease development. Irrigation also offers means for the direct spread of microbes such as water borne moulds, Oomycetes, and plasmodial pathogens coming from populations present in the water supply. The presence of plant disease causing microbes in sources of irrigation has been associated with outbreaks of diseases such as clubroot (Plasmodiophora brassicae). Irrigation can be utilised as a means for applying agrochemicals, fungigation. The developing technologies of water restriction and root zone drying also have an impact on the success of disease causing organisms. This is an emerging technology and its interactions with benign and pathogenic microbes require consideration.

Enzyme assisted extraction of chitin from shrimp shells (Litopenaeus vannamei)

BACKGROUND: Chemical chitin extraction generates large amounts of wastes and increases partial deacetylation of the product. Therefore, the use of biological methods for chitin extraction is an interesting alternative. The effects of process conditions on enzyme assisted extraction of chitin from the shrimp shells in a systematic way were the focal points of this study. RESULTS: Demineralisation conditions of 25C, 20 min, shells-lactic acid ratio of 1:1.1 w/w; and shells-acetic acid ratio of 1:1.2 w/w, the maximum demineralisation values were 98.64 and 97.57% for lactic and acetic acids, respectively. A total protein removal efficiency of 91.10% by protease from Streptomyces griseus with enzyme-substrate ratio 55 U/g, pH 7.0 and incubation time 3 h is obtained when the particle size range is 50-25 μm, which was identified as the most critical factor. The X-ray diffraction and 13C NMR spectroscopy analysis showed that the lower percent crystallinity and higher degree of acetylation of chitin from enzyme assisted extraction may exhibit better solubility properties and less depolymerisation in comparison with chitin from the chemical extraction. CONCLUSION: The present work investigates the effects of individual factors on process yields, and it has shown that, if the particle size is properly controlled a reaction time of 3 h is more than enough for deproteination by protease. Physicochemical analysis indicated that the enzyme assisted production of chitin seems appropriate to extract chitin, possibly retaining its native structure.

Polyhydroxyalkanoates production by actinobacteria isolated from soil

Polyhydroxyalkanoates (PHAs) are biodegradable and renewable polymers produced by a wide range of bacterial groups. New microbial bioprospection approaches have become an important way to find new PHA producers and new synthesized polymers. Over the past years, bacteria belonging to actinomycetes group have become known as PHA producers, such as Nocardia and Rhodococcus species, Kineosphaera limosa Liu et a]. 2002, and, more recently, Streptomyces species. In this paper, we disclose that there are more actinobacteria PHA producers in addition to the genera cited. Some unusual genera, such as Streptoalloteichus, and some genera frequently present in soil, such as Streptacidiphilus, have been found. Thirty-four isolates were able to accumulate poly(3-hydroxybutyrate) and a number of these have traces of poly(3-hydroxyvalerate) when cultivated on glucose or glucose and casein as carbon source. Furthermore, some strains showed traces of medium chain length PHA. Transmission electron microscopy demonstrated that the PHA accumulation occurs in hyphae and spores.

DNA damage induced by the anthracycline cosmomycin D in DNA repair-deficient cells

Anthracyclines have been widely used as antitumor agents, playing a crucial role in the successful treatment of many types of cancer, despite some side effects related to cardiotoxicity. New anthracyclines have been designed and tested, but the first ones discovered, doxorubicin and daunorubicin, continue to be the drugs of choice. Despite their extensive use in chemotherapy, little is known about the DNA repair mechanisms involved in the removal of lesions caused by anthracyclines. The anthracycline cosmomycin D is the main product isolated from Streptomyces olindensis, characterized by a peculiar pattern of glycosylation with two trisaccharide rings attached to the A ring of the tetrahydrotetracene. We assessed the induction of apoptosis (Sub-G(1)) by cosmomycin D in nucleotide excision repair-deficient fibroblasts (XP-A and XP-C) as well as the levels of DNA damage (alkaline comet assay). Treatment of XP-A and XP-C cells with cosmomycin D resulted in apoptosis in a time-dependent manner, with highest apoptosis levels observed 96 h after treatment. The effects of cosmomycin D were equivalent to those obtained with doxorubicin. The broad caspase inhibitor Z-VAD-FMK strongly inhibited apoptosis in these cells, and DNA damage induced by cosmomycin D was confirmed by alkaline comet assay. Cosmomycin D induced time-dependent apoptosis in nucleotide excision repair-deficient fibroblasts. Despite similar apoptosis levels, cosmomycin D caused considerably lower levels of DNA damage compared to doxorubicin. This may be related to differences in structure between cosmomycin D and doxorubicin.