污灌和农药对农田土壤假单胞菌种群的影响

随着环境污染问题的日益严重，微生物修复起到越来越重要的作用。假单胞菌是土壤微生物中最重要、研究最多的细菌之一，能降解简单和复杂的有机物，它们因此而广泛的存在于土壤和水体。但有关于石油、重金属及农药污染物对农田土壤假单胞菌多样性及种群结构的影响却缺乏全面和系统的认识。 本论文首次采用传统微生物培养方法与PCR-DGGE等现代微生物分子生态学研究方法相结合的手段，系统评价了长期含石油和重金属污水灌溉对中国最大的石油、重金属污灌区——沈抚、张士灌区农田土壤中的假单胞菌多样性及其种群结构的影响。同时，本论文也研究了乙草胺、甲胺磷对黑土假单胞菌多样性及种群结构的影响。得出以下结果： 石油污灌区土壤中总的假单胞菌多样性显著高于重金属污灌区；石油污灌区旱田土壤假单胞菌多样性接近于对照清洁土壤,同时低于相似污染程度的石油污灌区水田土壤。进一步测序发现，Pseudomonas mendocina、Pseudomonas stutzeri、Pseudomonas aeruginosa是所分析石油和重金属污灌区土壤中的优势类群，说明在长期污染胁迫下这3种假单胞菌分别得到了不同程度的富集。DGGE 结果显示石油和重金属污染土壤样品的可培养假单胞菌多样性没有显著差异，但均低于对照清洁土壤样品。对各个土壤样品可培养假单胞菌菌株进行REP-PCR基因分型，结果表明这些假单胞菌之间有显著的遗传差异。进一步测序表明，土壤样品中可培养假单胞菌优势类群中含有Pseudomonas. fluorescens 和Pseudomonas. Putida两种。 黑土农田土壤中使用乙草胺会严重降低总的及可培养假单胞菌群落的多样性，而且在5周内不能恢复。而甲胺磷处理土壤与对照相比则差异不显著，并且经过一段时间的适应，土壤中的总的及可培养假单胞菌种群不仅得到恢复而且超过对照。对各处理土壤总的及可培养假单胞菌DGGE谱带类型聚类分析，发现乙草胺、甲胺磷处理土壤样品均各自聚为一簇，说明农药污染类型是影响土壤中假单胞菌种群结构的重要因素。

一株嗜杂醇油细菌的分离、鉴定和特性研究

为结合一般白酒存在的杂醇油含量偏高或超标问题，作者试图寻找能嗜高醇类(含杂醇油)的有效菌株加以开发利用。本文研究了一株从富集分离、定向诱导得到的嗜异戊醇的W-2菌，该菌能以异戊醇或其它某些多碳醇作为唯一的碳源和能源进行生长。该菌在PH8.5的环境条件下，异戊醇的利用率可高达84.4%。此外，该菌还能广泛利用糖类、醇、有机酸、酯、盐等有机物作为唯一的碳源和能源。一般不利用—碳化合物。该菌具单极生鞭毛，呈杆状，适宜生长温度范围4℃—43℃。最适生长温度36℃，在PH4.0—11.0均能生长，最适生长PH为8.5—9.0，在培养基产生蓝绿色色素。W-2菌经形态，生理生化鉴定为假单胞菌属中的铜绿假单胞菌的一个变种。初步定名为Pseudomonas aeruginosa W-2。本文最后讨论了该菌的开发应用前景。

Biosorption of uranium by chemically modified Rhodotorula glutinis

The present paper reports the biosorption of uranium onto chemically modified yeast cells, Rhodotorula glutinis, in order to study the role played by various functional groups in the cell wall. Esterification of the carboxyl groups and methylation of the amino groups present in the cells were carried out by methanol and formaldehyde treatment, respectively. The uranium sorption capacity increased 31% for the methanol-treated biomass and 11% for the formaldehyde-treated biomass at an initial uranium concentration of 140 mg/L The enhancement of uranium sorption capacity was investigated by Fourier transform infrared (FTIR) spectroscopy analysis, with amino and carboxyl groups were determined to be the important functional groups involved in uranium binding. The biosorption isotherms of uranium onto the raw and chemically modified biomass were also investigated with varying uranium concentrations. Langmuir and Freundlich models were well able to explain the sorption equilibrium data with satisfactory correlation coefficients higher than 0.9. (C) 2010 Elsevier Ltd. All rights reserved.

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction, Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Intra-habitat heterogeneity of microbial food web structure under the regime of eutrophication and sediment resuspension in the large subtropical shallow Lake Taihu, China

Planktonic microbial community structure and classical food web were investigated in the large shallow eutrophic Lake Taihu (2338 km(2), mean depth 1.9 m) located in subtropical Southeast China. The water column of the lake was sampled biweekly at two sites located 22 km apart over a period of twelve month. Site 1 is under the regime of heavy eutrophication while Site 2 is governed by wind-driven sediment resuspension. Within-lake comparison indicates that phosphorus enrichment resulted in increased abundance of microbial components. However, the coupling between total phosphorus and abundance of microbial components was different between the two sites. Much stronger coupling was observed at Site 1 than at Site 2. The weak coupling at Site 2 was mainly caused by strong sediment resuspension, which limited growth of phytoplankton and, consequently, growth of bacterioplankton and other microbial components. High percentages of attached bacteria, which were strongly correlated with the biomass of phytoplankton, especially Microcystis spp., were found at Site 1 during summer and early autumn, but no such correlation was observed at Site 2. This potentially leads to differences in carbon flow through microbial food web at different locations. Overall, significant heterogeneity of microbial food web structure between the two sites was observed. Site-specific differences in nutrient enrichment (i.e. nitrogen and phosphorus) and sediment resuspension were identified as driving forces of the observed intra-habitat differences in food web structure.

Synthesis of acyl thiourea derivatives of chitosan and their antimicrobial activities in vitro

Three different acyl thiourea derivatives of chitosan (CS) were synthesized and their structures were characterized by FT-IR spectroscopy and elemental analysis. The antimicrobial behaviors of CS and its derivatives against four species of bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Sarcina) and four crop-threatening pathogenic fungi (Alternaria solani, Fusarium oxysporum f. sp. vasinfectum, Colletotrichum gloeosporioides (Penz.) Saec, and Phyllisticta zingiberi) were investigated. The results indicated that the antimicrobial activities of the acyl thiourea derivatives are much better than that of the parent CS. The minimum value of MIC and MBC of the derivatives against E coli was 15.62 and 62.49 mu g/mL, respectively. All of the acyl thiourea derivatives had a significant inhibitory effect on the fungi in concentrations of 50 - 500 mu g/mL; the maximum inhibitory index was 66.67%. The antifungal activities of the chloracetyl thiourea derivatives of CS are noticeably higher than the acetyl and benzoyl thiourea derivatives. The degree of grafting of the acyl thiourea group in the derivatives was related to antifungal activity; higher substitution resulted in stronger antifungal activity. (c) 2007 Elsevier Ltd. All rights reserved.

Towards the identification of the common features of bacterial biofilm development

UPNa. Instituto de Agrobiotecnología. Laboratorio de Biofilms Microbianos

Bactérias e as suas redes sociais

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Infeções em Cateteres de Hemodialisados

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Biobased additives as biodegradability enhancers with application in TPU-based footwear components

Among the wide variety of materials employed in the manufacture of shoes, thermoplastic polyurethanes (TPUs) are one of the most widely used. Given its widespread use, and associated waste management problems, the development of more biodegradable and evironmentally compatible solutions is needed. In this work, a polyester-based TPU used in the footwear industry for outsoles production was modified by compounding with lignin, starch and cellulose at content of 4% (w/w). The biodegradability was evaluated by using agar plate tests with the fungi Aspergillus niger ATCC16404, the Gram-negative bacteria Pseudomonas aeruginosa ATCC9027 and an association of both (consortium), and soil tests at 37 °C and 58 °C. The obtained results evidenced a positive effect of the tested biobased additives, the most favourable results being registered with lignin. These results were corroborated by the structural modifications observed by FTIR analysis. Additionally, mechanical tests prove the suitability of using the lignin modified TPUs for footwear outsoles production.

Capillary electrophoresis with boron doped diamond electrode for amperometric detection of low molecular weight biological substrate

The research work in this thesis included the sensitive and selective separation of biological substance by capillary electrophoresis with a boron doped diamond electrode for amperometric detection. Chapter 1 introduced the capillary electrophoresis and electrochemical detection. It included the different modes of capillary electrophoresis, polyelectrolyte multilayers coating for open tubular capillary electrochromatography, different modes of electrochemical detection and carbon based electrodes. Chapter 2 showed the synthesized and electropolymerized N-acetyltyramine with a negatively charged sulfobutylether-β-cyclodextrin on a boron doped diamond (BDD) electrode followed by the electropolymerzation of pyrrole to form a stable and permselective film for selective dopamine detection. For comparison, a glassy carbon (GC) electrode with a combined electropolymerized permselective film of polytyramine and polypyrrole-1-propionic acid was used for selective detection of dopamine. The detection limit of dopamine was improved from 100 nM at a GC electrode to 5 nM at a BDD electrode. Chapter 3 showed field-amplified sample stacking using a fused silica capillary coated with gold nanoparticles embedded in poly(diallyldimethylammonium) chloride, which has been investigated for the electrophoretic separation of indoxyl sulphate, homovanillic acid and vanillylmandelic acid. The detection limit of the three analytes obtained by using a boron doped diamond electrode was around 75 nM, which was significantly below their normal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfereing chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration. Chapter 4 showed the selective detection of Pseudomonas Quinolone Signal, PQS for quorum sensing from its precursor HHQ, using a simply boron doped diamond electrode. Furthermore, by combining poly(diallyldimethylammonium) chloride modified fused silica capillary with a BDD electrode for amperometric detection, PQS was separated from HHQ and other analogues. The detection limit of PQS was as low as 65 nM. Different P. aeruginosa mutant strains were studied. Chapter 5 showed the separation of aminothiols by layer-by-layer coating of silica capillary with a boron doped diamond electrode. The capillary was layer-by-layer coated with the polycation poly(diallyldimethylammonium) chloride and negatively charged silica nanoparticles. All the aminothiols was separated and detected using a BDD electrode in an acidic electrolyte. It was a novel scheme for the separation and detection of glutathione reduced and oxidized forms, which is important for estimated overstressed level in the human system.

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.

The role of bacterial interspecies communication in polymicrobial infection of the cystic fibrosis lung

There is an increasing appreciation of the polymicrobial nature of bacterial infections associated with Cystic Fibrosis (CF) and of the important role for interactions in influencing bacterial virulence and response to therapy. Patients with CF are co-infected with Pseudomonas aeruginosa, Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. Previous studies showed that DSF from S. maltophilia leads to altered biofilm formation and increased tolerance to antibiotics in P. aeruginosa and that these responses require the P. aeruginosa sensor kinase PA1396. The work in this thesis aims of further elucidate the influence and mechanism of DSF signalling on P. aeruginosa and examine the role that such interspecies signalling play in infection of the CF airway. Next generation sequencing technologies targeting the 16S ribosomal RNA gene were applied to DNA and RNA isolated from sputum taken from cohorts of CF and non-CF subjects to characterise the bacterial community. In parallel, metabolomics analysis of sputum provided insight into the environment of the CF airway. This analysis revealed a number of observations including; that differences in metabolites occur in sputum taken from clinically stable CF patients and those with exacerbation and DNA- and RNA-based methods suggested that a strong relationship existed between the abundance of specific strict anaerobes and fluctuations in the level of metabolites during exacerbation. DSF family signals were also detected in the sputum and a correlation with the presence of DSFproducing organisms was observed. To examine the signal transduction mechanisms used by P. aeruginosa, bioinformatics with site directed mutagenesis were employed to identify signalling partners for PA1396. A pathway suggesting a role for a number of proteins in the regulation of several factors following DSF recognition by PA1396 were observed.