Microbial ecology of an extreme acidic environment,the Tinto River

The Tinto River (Huelva, southwestern Spain) is an extreme environment with a rather constant acidic pH along the entire river and a high concentration of heavy metals. The extreme conditions of the Tinto ecosystem are generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt. Molecular ecology techniques were used to analyze the diversity of this microbial community. The community's composition was studied by denaturing gradient gel electrophoresis (DGGE) using 16S rRNA and by 16S rRNA gene amplification. A good correlation between the two approaches was found. Comparative sequence analysis of DGGE bands showed the presence of organisms related to Leptospirillum spp., Acidithiobacillus ferrooxidans, Acidiphilium spp., "Ferrimicrobium acidiphilum," Ferroplasma acidiphilum, and Thermoplasma acidophilum. The different phylogenetic groups were quantified by fluorescent in situ hybridization with a set of rRNA-targeted oligonucleotide probes. More than 80% of the cells were affiliated with the domain Bacteria, with only a minor fraction corresponding to ARCHAEA: Members of Leptospirillum ferrooxidans, Acidithiobacillus ferrooxidans, and Acidiphilium spp., all related to the iron cycle, accounted for most of the prokaryotic microorganisms detected. Different isolates of these microorganisms were obtained from the Tinto ecosystem, and their physiological properties were determined. Given the physicochemical characteristics of the habitat and the physiological properties and relative concentrations of the different prokaryotes found in the river, a model for the Tinto ecosystem based on the iron cycle is suggested.     

Novel nickel resistance genes from the rhizosphere metagenome of plants adapted to acid mine drainage

Metal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment.     

Screening of anaerobic activities in sediments of an acidic environment: Tinto River

The Tinto River (Huelva, Spain) is a natural acidic rock drainage environment produced by the bio-oxidation of metallic sulfides from the Iberian Pyritic Belt. A geomicrobiological model of the different microbial cycles operating in the sediments was recently developed through molecular biological methods, suggesting the presence of iron reducers, methanogens, nitrate reducers and hydrogen producers. In this study, we used a combination of molecular biological methods and targeted enrichment incubations to validate this model and prove the existence of those potential anaerobic activities in the acidic sediments of Tinto River. Methanogenic, sulfate-reducing, denitrifying and hydrogen-producing enrichments were all positive at pH between 5 and 7. Methanogenic enrichments revealed the presence of methanogenic archaea belonging to the genera Methanosarcina and Methanobrevibacter. Enrichments for sulfate-reducing microorganisms were dominated by Desulfotomaculum spp. Denitrifying enrichments showed a broad diversity of bacteria belonging to the genera Paenibacillus, Bacillus, Sedimentibacter, Lysinibacillus, Delftia, Alcaligenes, Clostridium and Desulfitobacterium. Hydrogen-producing enrichments were dominated by Clostridium spp. These enrichments confirm the presence of anaerobic activities in the acidic sediments of the Tinto River that are normally assumed to take place exclusively at neutral pH.     

Macrofilamentous microbial communities in the metal-rich and acidic River Tinto, Spain

A novel type of macroscopic microbial community consisting of large dendritic filaments (up to 1.5 m) in a pH 2.0 dam of the River Tinto (South-western Spain) is described. The combined use of 16S rRNA-gene surveys and fluorescent in situ hybridisation (FISH) suggested that gamma-proteobacteria and a relative large diversity of alpha-proteobacteria dominated these structures. beta-Proteobacteria, Actinobacteria and Firmicutes were also detected. Whereas acidophilic bacteria of the genera Acidithiobacillus, Leptospirillum and Acidiphilium, and archaea belonging to the Thermoplasmatales dominate mine acid drainage waters and streamers (riverbed filamentous biofilms), none of the lineages identified in this study affiliate to typical acid mine drainage acidophilic bacteria. Bacteria of the Tinto macrofilaments might be heterotrophic, and could be feeding on the organic matter entrapped in the filamentous structure.     

Methanogenesis in the sediments of Rio Tinto, an extreme acidic river

Río Tinto (Iberian Pyritic Belt, SW Spain) is well known for its low pH (mean pH 2.3), high redox potential (> +400 mV) and high concentration of heavy metals. In this work we describe and analyse the presence of methanogenic archaea in the extreme acidic and oxidizing environment of the Tinto basin. Methane formation was measured in microcosms inoculated with sediments from the Rio Tinto basin. Methanol, formate, volatile fatty acids and lactate stimulated the production of methane. Methane formation was associated with a decrease of redox potential and an increase in pH. Cores showed characteristic well-defined black bands in which a high acetate concentration was measured among the otherwise reddish-brown sediments with low acetate concentration. Methanosaeta concilii was detected in the black bands. In enrichment cultures, M. concilii (enriched with a complex substrate mixture), Methanobacterium bryantii (enriched with H(2)) and Methanosarcina barkeri (enriched with methanol) were identified. Our results suggest that methanogens thrive in micro-niches with mildly acidic and reducing conditions within Rio Tinto sediments, which are, in contrast, immersed in an otherwise extremely acidic and oxidizing environment.     

Biodiversity and geochemistry of an extremely acidic, low-temperature subterranean environment sustained by chemolithotrophy

The geochemical dynamics and composition of microbial communities within a low-temperature (≈ 8.5°C), long-abandoned (> 90 years) underground pyrite mine (Cae Coch, located in north Wales) were investigated. Surface water percolating through fractures in the residual pyrite ore body that forms the roof of the mine becomes extremely acidic and iron-enriched due to microbially accelerated oxidative dissolution of the sulfide mineral. Water droplets on the mine roof were found to host a very limited diversity of exclusively autotrophic microorganisms, dominated by the recently described psychrotolerant iron/sulfur-oxidizing acidophile Acidithiobacillus ferrivorans, and smaller numbers of iron-oxidizing Leptospirillum ferrooxidans. In contrast, flowing water within the mine chamber was colonized with vast macroscopic microbial growths, in the form of acid streamers and microbial stalactites, where the dominant microorganisms were Betaproteobacteria (autotrophic iron oxidizers such as 'Ferrovum myxofaciens' and a bacterium related to Gallionella ferruginea). An isolated pool within the mine showed some similarity (although greater biodiversity) to the roof droplets, and was the only site where archaea were relatively abundant. Bacteria not previously associated with extremely acidic, metal-rich environments (a Sphingomonas sp. and Ralstonia pickettii) were found within the abandoned mine. Data supported the hypothesis that the Cae Coch ecosystem is underpinned by acidophilic, mostly autotrophic, bacteria that use ferrous iron present in the pyrite ore body as their source of energy, with a limited role for sulfur-based autotrophy. Results of this study highlight the importance of novel bacterial species (At. ferrivorans and acidophilic iron-oxidizing Betaproteobacteria) in mediating mineral oxidation and redox transformations of iron in acidic, low-temperature environments.     

Microbial Ecology of an Extreme Acidic Environment, the Tinto River

The Tinto River (Huelva, southwestern Spain) is an extreme environment with a rather constant acidic pH along the entire river and a high concentration of heavy metals. The extreme conditions of the Tinto ecosystem are generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt. Molecular ecology techniques were used to analyze the diversity of this microbial community. The community's composition was studied by denaturing gradient gel electrophoresis (DGGE) using 16S rRNA and by 16S rRNA gene amplification. A good correlation between the two approaches was found. Comparative sequence analysis of DGGE bands showed the presence of organisms related to Leptospirillum spp., Acidithiobacillus ferrooxidans, Acidiphilium spp., “Ferrimicrobium acidiphilum,” Ferroplasma acidiphilum, and Thermoplasma acidophilum. The different phylogenetic groups were quantified by fluorescent in situ hybridization with a set of rRNA-targeted oligonucleotide probes. More than 80% of the cells were affiliated with the domain Bacteria, with only a minor fraction corresponding to Archaea. Members of Leptospirillum ferrooxidans, Acidithiobacillus ferrooxidans, and Acidiphilium spp., all related to the iron cycle, accounted for most of the prokaryotic microorganisms detected. Different isolates of these microorganisms were obtained from the Tinto ecosystem, and their physiological properties were determined. Given the physicochemical characteristics of the habitat and the physiological properties and relative concentrations of the different prokaryotes found in the river, a model for the Tinto ecosystem based on the iron cycle is suggested.     

Novel extremely acidic lipases produced from Bacillus species using oil substrates

The extremely acidophilic microorganisms Bacillus pumilus and Bacillus subtilis were isolated from soil collected from the commercial edible oil and fish oil extraction industry. Optimization of conditions for acidic lipase production from B. pumilus and B. subtilis using palm oil and fish oil, respectively, was carried out using response surface methodology. The extremely acidic lipases, thermo-tolerant acidic lipase (TAL) and acidic lipase (AL), were produced by B. pumilus and B. subtilis, respectively. The optimum conditions for B. pumilus obtaining the maximum activity (1,100 U/mL) of TAL were fermentation time, 96 h; pH, 1; temperature, 50 °C; concentration of palm oil, 50 g/L. After purification, a 7.1-fold purity of lipase with specific activity of 5,173 U/mg protein was obtained. The molecular weight of the TAL was 55 kDa. The AL from B. subtilis activity was 214 U/mL at a fermentation time of 72 h; pH, 1; temperature, 35 °C; concentration of fish oil, 30 g/L; maltose concentration, 10 g/L. After purification, an 11.4-fold purity of lipase with specific activity of 2,189 U/mg protein was obtained. The molecular weight of the extremely acidic lipase was 22 kDa. The functional groups of lipases were determined by Fourier transform-infrared (FT-IR) spectroscopy.     

Ecological study of the fungal populations of the acidic Tinto River in southwestern Spain

The characterization of the microbial ecology of the Tinto River, an extreme habitat with an extremely low pH and a high concentration of heavy metals, revealed an unexpected level of microbial richness. A variety of microbial eukaryotes was isolated, among them several fungal strains that were identified and their physiological characteristics studied. Ninety strains of yeast were isolated from the Tinto River. Fifty-two percent of them were capable of growth in vitro using medium amended with river water. They belong to 6 genera of basidiomycetes (Rhodotorula, Cryptococcus, Tremella, Holtermannia, Leucosporidium, and Mrakia) and 2 of ascomycetes (Candida and Williopsis). In addition, 349 strains of hyphomycetes belonging to 17 genera (most of them ascomycetes) were isolated and studied. Forty-four percent of the isolated filamentous fungi (154 strains) were capable of growing in vitro using medium amended with Tinto River water. Of this percentage, 19% (29 strains) belonged to the genus Penicillium (16 species) and 66% (102 strains) were included in the genera Scytalidium, Bahusakala, Phoma, and Heteroconium or showed dark sterile mycelia, which probably are of dematiaceous hyphomycetes. In addition, we characterized strains of the ascomycete genera Lecythophora and Acremonium and of the zygomycete genus Mortierella, all of them capable of growing in medium amended with river water. Statistical correlation of biological and physicochemical variables suggested a positive relationship between the dematiaceous hyphomycetes and the most extreme physicochemical conditions found in the Tinto River. Principal components analysis confirmed this relationship and also showed that the Acremonium and Lecythophora groups had environmental preferences similar to those of dematiaceous fungi. The spatial positions of the sampling sites were grouped in 2 main clusters: (i) sampling sites in the mine zone in which most of the dematiaceous, Acremonium, and Lecythophora strains were isolated and (ii) sites that were not in the mine zone and sampling station 5 from which were isolated mainly strains of fungi that were not capable of growing in the medium amended with river water and species of the Penicillium genus.     

On the ecology of the rotifer Cephalodella hoodi from an extremely acidic lake

1. The biovolume‐specific carbon content, relative egg volume (a measure of per‐offspring reproductive investment), growth and grazing rates, and the gross growth efficiency (GGE) of the rotifer Cephalodella hoodi, isolated from an extremely acidic habitat (pH 2.65), were determined and compared with literature values for rotifers living in circum‐neutral habitats in order to reveal potential special features or adaptations related to the extreme habitat of C. hoodi. 2. Of the two dominant phytoflagellates (Ochromonas sp. and Chlamydomonas acidophila) that occur in the natural habitat of C. hoodi, only C. acidophila promoted positive growth and reproduction and, thus, the following results were obtained with C. acidophila as a food alga. 3. The body volume‐specific carbon content of C. hoodi is in the range of that found in rotifers from circum‐neutral lakes, suggesting that no costly carbon investment, brought about by the thickening of the lorica, for example, was required to withstand low pH. 4. The egg volume of C. hoodi exhibited no phenotypic plasticity dependent on the food concentration and, thus, C. hoodi allocated a constant, absolute amount of energy to each individual offspring. No adaptation to low food densities was found. 5. A dome‐shaped type II functional response curve was found to describe the ingestion of Chlamydomonas as a source of food. 6. Compared with other rotifers, C. hoodi had a high threshold and half‐saturating food concentration (=low affinity) but also a high maximum growth rate and a relatively high GGE, suggesting no severe adverse effect of low pH.     

Eukaryotic community distribution and its relationship to water physicochemical parameters in an extreme acidic environment, Rio Tinto (southwestern Spain)

The correlation between water physicochemical parameters and eukaryotic benthic composition was examined in Río Tinto. Principal component analysis showed a high inverse relationship between pH and most of the heavy metals analyzed as well as Dunaliella sp., while Chlamydomonas sp. abundance was positively related. Zn, Cu, and Ni clustered together and showed a strong inverse correlation with the diversity coefficient and most of the species analyzed. These eukaryotic communities seem to be more influenced by the presence of heavy metals than by the pH.     

环境抗生素抗性基因研究进展

抗生素耐药性及其在全球范围内的传播已成为国际关注的热点。本文结合最新文献,综述了抗生素抗性基因在环境中的来源、传播、分布以及新型抗性基因的发现等方面的研究进展。环境中抗生素抗性基因的来源主要是环境中细菌的内在抗性基因及随人或动物粪便排到体外的抗性细菌。功能宏基因组学技术的应用极大地丰富了人们对抗生素抗性组学的认知,并已从环境中筛选到多种新型抗性基因。近年来,由于抗生素在医疗以及养殖业中的大量使用,增加了抗性基因在环境中的丰度和多样性,加速了抗性基因在环境中的传播,在多种环境介质(如养殖水域、污水处理厂、河流、沉积物和土壤等)均检测到多种高丰度的抗生素抗性基因。我们建议今后在以下方面开展深入研究:(1)抗性基因传播和扩散的机制;(2)新型抗性基因筛选和抗性机制;(3)抗生素和抗性基因环境风险评估体系等。     

Naphthalene-utilizing and mercury-resistant bacteria isolated from an acidic environment

Soil samples were taken from areas of low pH (2.5–3.5) surrounding an outdoor coal storage pile. These samples were added to medium with naphthalene as the sole carbon source to enrich for organisms capable of degrading polycyclic aromatic hydrocarbons (PAH) at low pH. Five such bacterial strains were isolated. Sequencing of the 16S rDNA showed them to be members of the genera Clavibacter, Arthrobacter and Acidocella. These organisms were all capable of growth with naphthalene as a sole carbon source at low pH. The genes nahAc, nahAd, phnAc, nahH, xylE or GST, which are known to be associated with PAH degradation were not detected. Isolate 10, the Acidocella strain, tolerated high levels of mercury. PCR amplification and sequencing of genes from the mer operon from isolate 10 DNA suggested that mercury is transported into the bacterial cell and subsequently detoxified since the enzymes encoded by genes in this operon are involved in these processes.     

Microbial diversity in anaerobic sediments at Rio Tinto, a naturally acidic environment with a high heavy metal content

The Tinto River is an extreme environment located at the core of the Iberian Pyritic Belt (IPB). It is an unusual ecosystem due to its size (100 km long), constant acidic pH (mean pH, 2.3), and high concentration of heavy metals, iron, and sulfate in its waters, characteristics that make the Tinto River Basin comparable to acidic mine drainage (AMD) systems. In this paper we present an extensive survey of the Tinto River sediment microbiota using two culture-independent approaches: denaturing gradient gel electrophoresis and cloning of 16S rRNA genes. The taxonomic affiliation of the Bacteria showed a high degree of biodiversity, falling into 5 different phyla: Proteobacteria, Firmicutes, Bacteroidetes, Acidobacteria, and Actinobacteria; meanwhile, all the Archaea were affiliated with the order Thermoplasmatales. Microorganisms involved in the iron (Acidithiobacillus ferrooxidans, Sulfobacillus spp., Ferroplasma spp., etc.), sulfur (Desulfurella spp., Desulfosporosinus spp., Thermodesulfobium spp., etc.), and carbon (Acidiphilium spp., Bacillus spp., Clostridium spp., Acidobacterium spp., etc.) cycles were identified, and their distribution was correlated with physicochemical parameters of the sediments. Ferric iron was the main electron acceptor for the oxidation of organic matter in the most acid and oxidizing layers, so acidophilic facultative Fe(III)-reducing bacteria appeared widely in the clone libraries. With increasing pH, the solubility of iron decreases and sulfate-reducing bacteria become dominant, with the ecological role of methanogens being insignificant. Considering the identified microorganisms-which, according to the rarefaction curves and Good's coverage values, cover almost all of the diversity-and their corresponding metabolism, we suggest a model of the iron, sulfur, and organic matter cycles in AMD-related sediments.     

蔗糖富集环境中蔗糖磷酸化酶的酶学性质研究与改造

【背景】蔗糖富集土壤是蔗糖磷酸化酶的重要来源之一。此酶能以较廉价的蔗糖为底物进行转葡萄糖基反应,改善受体底物的理化性质,因而具有重要的应用价值。【目的】研究蔗糖富集土壤中蔗糖磷酸化酶的酶学性质和转糖苷活性,为其更优质的改造提供材料和理论基础。【方法】将宏基因组中获得的蔗糖磷酸化酶基因克隆到表达载体上构建重组大肠杆菌,诱导表达并进行镍亲和层析纯化蛋白。以蔗糖为底物测量重组酶的基本酶学性质,研究其对糖类底物的转糖苷活性。通过底物通道分析,利用反向PCR技术对其第155位点进行饱和突变,并测定突变体基本酶学性质和转糖苷活性。【结果】纯化的酶蛋白分子量大小约为56 kD,活性状态时以三聚体形式存在。在以蔗糖为底物时,最适温度和最适pH值分别为55°C和6.5;Km和Vmax值分别为23.1±2.4 mmol/L和407.9±8.5μmol/(mg·min),对第155位点进行了定点饱和突变,获得了部分酶学性质或转糖苷活性改善的突变体。【结论】宏基因组中蔗糖磷酸化酶的研究丰富了酶学数据,并通过分子改造获得了部分性质更优的突变体,为转糖苷活性关键氨基酸的研究和该酶的工业应用奠定了基础。     

Expression of leucine genes from an extremely thermophilic bacterium in Escherichia coli

Summary The organisation of the leucine genes in Thermus thermophilus HB8 was analysed by examining the ability of recombinant DNAs to complement Escherichia coli mutations. The arrangement of the genes is different from that in the mesophilic bacteria E. coli and Salmonella typhimurium. The promoter responsible for the expression of the leuB, leuC and leuD genes of Thermus HB8 in E. coli was identified. The sequence of Thermus DNA containing this promoter revealed structural similarities to the promoter and attenuator regions of the E. coli leucine operon.     

Novel motifs in amino acid permease genes from Leishmania

Eight amino acid permease genes from the protozoan parasite Leishmania donovani (AAPLDs) were cloned, sequenced, and shown to be expressed in promastigotes. Seven of these belong to the amino acid transporter-1 and one to the amino acid polyamino-choline superfamilies. Using these sequences as well as known and characterized amino acid permease genes from all kingdoms, a training set was established and used to search for motifs, using the MEME motif discovery tool. This study revealed two motifs that are specific to the genus Leishmania, four to the family trypanosomatidae, and a single motif that is common between trypanosomatidae and mammalian systems A1 and N. Interestingly, most of these motifs are clustered in two regions of 50-60 amino acids. Blast search analyses indicated a close relationship between the L. donovani and Trypanosoma brucei amino acid permeases. The results of this work describe the cloning of the first amino acid permease genes in parasitic protozoa and contribute to the understanding of amino acid permease evolution in these organisms. Furthermore, the identification of genus-specific motifs in these proteins might be useful to better understand parasite physiology within its hosts.     

Microbial Community Composition and Ecology of an Acidic Aquatic Environment: The Tinto River, Spain

We studied the correlation between physicochemical and biological characteristics of an acidic river, the Tinto River, in Southwestern Spain. The Tinto River is an extreme environment characterized by its low pH (mean of 2.2) and high concentrations of heavy metals (Fe 2.3 g/L, Zn 0.22 g/L, Cu 0.11 g/L). These extreme conditions are the product of the metabolic activity of chemolithotrophic microorganisms, including iron- and sulfur-oxidizing bacteria, that can be found in high concentrations in its waters. The food chain in the river is very constrained and exclusively microbial. Primary productivity in the Tinto River is the sum of photosynthetic and chemolithotrophic activity. Heterotrophic bacteria and fungi are the major decomposers and protists are the major predators. A correlation analysis including the physicochemical and biological variables suggested a close relationship between the acidic pH values and abundance of both chemolithotrophic bacteria and filamentous fungi. Chemolithotrophic bacteria correlated with the heavy metals found in the river. A principal component analysis of the biotic and abiotic variables suggested that the Tinto River ecosystem can be described as a function of three main groups of variables: pH values, metal concentrations, and biological productivity.     

一种来自于土壤宏基因组文库的AprN基因的鉴定

宏基因组文库技术的发展拓宽了酶学的研究范围,从而导致了一系列新型的生物催化剂被发现和鉴定。采用蛋白酶的活性筛选策略,从已构建的碱性污染土壤宏基因组文库分离得到了一个表达蛋白酶的阳性克隆。生物信息学分析表明该克隆所包含的外源DNA片断编码一个由381个氨基酸编码组成的多肽,该多肽大约为39kDa,等电点为8.91。BLAST软件分析该外源DNA片断包含一个完整的碱性蛋白酶基因（ap01）,GC含量为46.3%。相关酶学数据表明该阳性克隆所分泌的碱性蛋白酶最适作用pH值为9.5,最佳作用温度为40℃。     

Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora

A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.