Comparative Leaching of Chalcopyrite by Selected Acidophilic Bacteria and Archaea

A systematic study of the bioleaching of chalcopyrite (CuFeS 2 ) was conducted using axenic cultures of 11 species of acidophilic Bacteria and Archaea to obtain a direct comparison of the microbial chalcopyrite leaching capabilities of the different cultures and to determine the factors that affect Cu release. The characteristics of chalcopyrite leaching by the moderate thermophile Sulfobacillus thermosulfidooxidans , the mesophile Acidithiobacillus ferrooxidans , and the thermophile Acidianus brierleyi were used to elucidate the leaching process. Moderately thermophilic cultures of Sulfobacillus acidophilus, Acidimicrobium ferrooxidans , and Acidithiobacillus caldus were used to study the effects of different metabolic capabilities and relate those to leaching efficiency. The greatest rate of Cu solubilization from chalcopyrite was achieved at high temperatures (up to 70°C) at redox potentials below +550 mV (Ag/AgCl). The enhanced Cu solubilization observed at high temperatures resulted from accelerated chemical reaction rates, rather than from the rates at which individual acidophiles generated the mineral leaching reactants such as Fe 3+ .     

Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS₂) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing Thiobacillus spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph Thiobacillus ferrooxidans. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph Thiobacillus thiooxidans promoted pyrite dissolution; since neither strains T-21 and T-23 nor T. thiooxidans could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph Thiobacillus acidophilus also oxidized pyrite but to a lesser extent than did mixed cultures containing T. thiooxidans. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.     

Acidophilic, Heterotrophic Bacteria of Acidic Mine Waters

Obligately acidophilic, heterotrophic bacteria were isolated both from enrichment cultures developed with acidic mine water and from natural mine drainage. The bacteria were grouped by the ability to utilize a number of organic acids as sole carbon sources. None of the strains were capable of chemolithotrophic growth on inorganic reduced iron and sulfur compounds. All bacteria were rod shaped, gram negative, nonencapsulated, motile, capable of growth at pH 2.6 but not at pH 6.0, catalase and oxidase positive, strictly aerobic, and capable of growth on citric acid. The bacteria were cultivatable on solid nutrient media only if agarose was employed as the hardening agent. Bacterial densities in natural mine waters ranged from approximately 20 to 250 cells per ml, depending upon source and culture medium. Ferric hydrates and stream vegetation contained from 1,500 to over 7 × 10⁶ cells per g.     

Glucose Catabolism in Strains of Acidophilic, Heterotrophic Bacteria

Pathways of glucose catabolism, potentially operational in six strains of obligately aerobic, acidophilic bacteria, including Acidiphilium cryptum strain Lhet2, were investigated by short-term radiorespirometry and enzyme assays. Short-term radiorespirometry was conducted at pH 3.0 with specifically labeled [¹⁴C]glucose. The high rate and yield of C-1 oxidized to CO₂ indicated that the Entner-Doudoroff, pentose phosphate, or both pathways were operational in all strains. Apparent nonequivalent yields of CO₂ from C-1 and estimated CO₂ from C-4 (C-1 > C-4) were suggestive of simultaneous glucose catabolism by both pathways in all strains tested. Variation in the relative contribution of the two pathways of glucose catabolism appears to account for observed strain differences. Calculation of the actual percent pathway participation was not feasible. Enzyme assays were completed with crude extracts of glucose-grown cells to substantiate the results obtained by radiorespirometry. The key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase) and the Entner-Doudoroff pathway (2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrase) were present in all strains examined (PW2, Lhet2, KLB, OP, and QBP). However, none of the strains exhibited detectable levels of the key enzyme of the Embden-Meyerhof-Parnas pathway, 6-phosphofructokinase. All strains contained glucose-6-phosphate dehydrogenase and fructose bisphosphate aldolase. The results of the enzyme study supported the contention that the pentose phosphate and Entner-Doudoroff pathways are operational for glucose catabolism in the acidophilic heterotrophs, and that the Embden-Meyerhof-Parnas pathway is apparently absent.     

Temperature Sensitivity Conferred by ligA Alleles from Psychrophilic Bacteria upon Substitution in Mesophilic Bacteria and a Yeast Species

We have assembled a collection of 13 psychrophilic ligA alleles that can serve as genetic elements for engineering mesophiles to a temperature-sensitive (TS) phenotype. When these ligA alleles were substituted into Francisella novicida, they conferred a TS phenotype with restrictive temperatures between 33 and 39°C. When the F. novicidaligA hybrid strains were plated above their restrictive temperatures, eight of them generated temperature-resistant variants. For two alleles, the mutations that led to temperature resistance clustered near the 5′ end of the gene, and the mutations increased the predicted strength of the ribosome binding site at least 3-fold. Four F. novicida ligA hybrid strains generated no temperature-resistant variants at a detectable level. These results suggest that multiple mutations are needed to create temperature-resistant variants of these ligA gene products. One ligA allele was isolated from a Colwellia species that has a maximal growth temperature of 12°C, and this allele supported growth of F. novicida only as a hybrid between the psychrophilic and the F. novicidaligA genes. However, the full psychrophilic gene alone supported the growth of Salmonella enterica, imparting a restrictive temperature of 27°C. We also tested two ligA alleles from two Pseudoalteromonas strains for their ability to support the viability of a Saccharomyces cerevisiae strain that lacked its essential gene, CDC9, encoding an ATP-dependent DNA ligase. In both cases, the psychrophilic bacterial alleles supported yeast viability and their expression generated TS phenotypes. This collection of ligA alleles should be useful in engineering bacteria, and possibly eukaryotic microbes, to predictable TS phenotypes.     

Evaluation of a Fluorescent Lectin-Based Staining Technique for Some Acidophilic Mining Bacteria

A fluorescence-labeled wheat germ agglutinin staining technique (R. K. Sizemore et al., Appl. Environ. Microbiol. 56:2245–2247, 1990) was modified and found to be effective for staining gram-positive, acidophilic mining bacteria. Bacteria identified by others as being gram positive through 16S rRNA sequence analyses, yet clustering near the divergence of that group, stained weakly. Gram-negative bacteria did not stain. Background staining of environmental samples was negligible, and pyrite and soil particles in the samples did not interfere with the staining procedure.     

Floating Filters, a Novel Technique for Isolation and Enumeration of Fastidious, Acidophilic, Iron-Oxidizing, Autotrophic Bacteria

Nuclepore polycarbonate filters floating on a liquid, FeSO₄-containing medium (pH 1.6) were used to isolate a moderately thermophilic bacterium from a pyrite-oxidizing enrichment culture. The isolate failed to grow on any of the conventional solid media tried. To test the general applicability of the method, the enumeration of a fastidious acidophilic organism, Thiobacillus ferrooxidans, was carried out and the results compared with those obtained with other filters, solid media, and the most probable number technique. T. ferrooxidans showed better viability on the floating polycarbonate filters and grew in a much shorter time (4 to 5 days) than with the other techniques.     

DNA Transport and Natural Transformation in Mesophilic and Thermophilic Bacteria

Comparative genome analyses revealed a massive DNA exchange between microbes of distant evolutionary lineages. This phenomenon known as horizontal, or lateral, gene transfer has a tremendous impact in the evolution of prokaryotes. Here, the process of DNA transport via genetic transformation is discussed. This review will focus on the process of DNA uptake mediated by type IV pilin-like proteins in Gram-positive and Gram-negative bacteria. Three tentative models of transformation machineries comprising components similar to proteins of type IV pili and type II secretion are presented. A comparative discussion of the structure of DNA translocators and the underlying mechanism of transfer of free DNA in mesophilic and extremely thermophilic bacteria highlights conserved and distinctive features of the DNA translocators in mesophilic and thermophilic bacteria.     

A Combined Immunofluorescence-DNA-Fluorescence Staining Technique for Enumeration of Thiobacillus ferrooxidans in a Population of Acidophilic Bacteria

An antiserum raised against whole cells of Thiobacillus ferrooxidans was allowed to react with a variety of acidophilic and nonacidophilic bacteria in an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay. Both experiments demonstrated that the antiserum was specific at the species level. This preparation was used to evaluate the role of T. ferrooxidans in the microbial desulfurization process. Leaching experiments were performed, and the numbers of T. ferrooxidans cells and other bacteria were estimated by using a combined immunofluorescence-DNA-fluorescence staining technique that was adapted for this purpose. Nonsterile coal samples inoculated with T. ferrooxidans yielded high concentrations of soluble iron after 16 days. After this period, however, T. ferrooxidans cells could no longer be detected by the immunofluorescence assay, whereas the DNA-fluorescence staining procedure demonstrated a large number of microorganisms on the coal particles. These results indicate that T. ferrooxidans is removed by competition with different acidophilic microorganisms that were originally present on the coal.     

Thermophilic Iron-Oxidizing Bacteria Found in Copper Leaching Dumps

Rod-shaped bacteria capable of oxidizing ferrous iron at 55°C were cultured from samples of a copper mine leach dump. Yeast extract or cysteine was required by these Thiobacillus-like bacteria for growth, using ferrous iron as an energy source.     

Studies on the Inactivation of Protein Synthesis in Mesophilic Bacteria Induced by Chilling

The mechanism of inactivation of in vivo protein synthesis (incorporationof phenylalanine into protein at 20C), by chilling at 0C,in Escherichia coli Q13 and Pseudomonas aeruginosa was studied.In vitro protein synthesis with poly(U) or R17 phage RNA asmRNA showed that the protein-synthesizing system itself wasnot damaged by the chilling. In contrast, the ability of E.coli Q13 and P. aeruginosa to take up phenylalanine decreasedby 100% and 90%, respectively, after the 8-day chilling period.A significant part of the phenylalanine pool leaked out of thecells during the chilling period. Intracellular ATP levels andthe energy balance did not alter greatly in E. coli Q13, butchanged considerably in P. aeruginosa due to chilling. These results strongly suggest that the inactivation of thein vivo protein synthesis is due to damage of membrane functions,probably those related to the amino acid transport system, andnot to the inactivation of the protein-synthesizing system itself. (Received January 28, 1985; Accepted August 6, 1985)     

Accuracy,Precision, and Method Detection Limits of Quantitative PCR for Airborne Bacteria and Fungi

Real-time quantitative PCR (qPCR) for rapid and specific enumeration of microbial agents is finding increased use in aerosol science. The goal of this study was to determine qPCR accuracy, precision, and method detection limits (MDLs) within the context of indoor and ambient aerosol samples. Escherichia coli and Bacillus atrophaeus vegetative bacterial cells and Aspergillus fumigatus fungal spores loaded onto aerosol filters were considered. Efficiencies associated with recovery of DNA from aerosol filters were low, and excluding these efficiencies in quantitative analysis led to underestimating the true aerosol concentration by 10 to 24 times. Precision near detection limits ranged from a 28% to 79% coefficient of variation (COV) for the three test organisms, and the majority of this variation was due to instrument repeatability. Depending on the organism and sampling filter material, precision results suggest that qPCR is useful for determining dissimilarity between two samples only if the true differences are greater than 1.3 to 3.2 times (95% confidence level at n = 7 replicates). For MDLs, qPCR was able to produce a positive response with 99% confidence from the DNA of five B. atrophaeus cells and less than one A. fumigatus spore. Overall MDL values that included sample processing efficiencies ranged from 2,000 to 3,000 B. atrophaeus cells per filter and 10 to 25 A. fumigatus spores per filter. Applying the concepts of accuracy, precision, and MDL to qPCR aerosol measurements demonstrates that sample processing efficiencies must be accounted for in order to accurately estimate bioaerosol exposure, provides guidance on the necessary statistical rigor required to understand significant differences among separate aerosol samples, and prevents undetected (i.e., nonquantifiable) values for true aerosol concentrations that may be significant.Real-time quantitative PCR (qPCR) is an analytical method for the rapid and potentially sensitive enumeration of broad and specific microbial populations in environmental samples (17). For bioaerosol analysis, this method allows for detection and enumeration independent of culturing, thereby circumventing the significant concerns surrounding the unculturability of environmental microorganisms and loss of culturability due to aerosol sampling (1, 2, 18, 34, 46, 55). Over the last decade, the application of qPCR has advanced research in the human health, environmental, and the national security arenas by enabling the specific measurement of airborne allergenic mold, pathogenic bacteria, and human viruses (6, 7, 9, 13, 37, 45).The quantitative nature of this technique as well as the documented advantages over culturing provides the potential for integrating microbial measurements with physical and chemical aerosol processes to understand exposure and to describe the fate and sources of biological aerosols in indoor environments and the atmosphere. However, the logarithmic amplification that is the basis of qPCR results in significant standard deviations among repeated qPCRs (25, 50). This variability rarely constrains the use of qPCR in aquatic and terrestrial systems, where biological growth typically dictates concentrations above detection limits and multiple order-of-magnitude differences in microorganism concentrations between treatments. However, the volume concentrations of biological agents in air (10³ to 10⁶ per m³ of air) are dramatically more dilute than those measured in environmental waters (10¹² to 10¹⁴ per m³ of water) (4, 5, 10, 12, 20, 52), and processes that result in indoor and atmospheric bioaerosol concentrations are growth independent. These processes include aerosol infiltration and exfiltration, resuspension, and deposition and typically result in less than an order of magnitude of variability in aerosol or biological particulate matter (PM) concentrations (22, 29, 41). As qPCR becomes more commonly used in indoor and outdoor air quality research, it is necessary to know the analytical variability and method detection limits (MDLs) to determine whether the method is suitable for estimating exposure and delineating the experimental differences observed in aerosol processes.The goal of this study was to estimate the accuracy, precision, and MDLs associated with qPCR of aerosol samples. These concepts were applied to air sampling filters loaded with three test organisms, including spores of Aspergillus fumigatus and vegetative bacterial cells from the Gram-negative Escherichia coli and Gram-positive Bacillus atrophaeus. The efficiencies associated with DNA extraction and with extraction of whole cells from aerosol filters were measured to describe the statistical accuracy of common qPCR bioaerosol protocols. Overall precision (reproducibility) as well as instrument repeatability were determined, and a binary method for describing MDLs was developed and applied to fungal spores and bacterial cells. Such experimental and statistical treatment of qPCR-based aerosol measurements is expected to guide improved estimates of human exposure, incorporate limits of qPCR precision into experimental design, and provide a context for undetected (i.e., nonquantifiable) values.     

食品级乳酸菌表达系统研究进展

乳酸菌表达系统是近几年发展起来的食品级高效表达系统。乳酸菌具有益生菌特征,因此该表达系统与其他细菌表达系统相比有很多优点。介绍了糖诱导表达系统、噬菌体Φ31爆发式诱导的表达系统、乳链球菌素调控表达系统、温控表达系统等的研究进展,以及这些系统的应用前景。     

Luminescence-Based Systems for Detection of Bacteria in the Environment

Abstract

The development of techniques for detection and tracking of microorganisms in natural environments has been accelerated by the requirement for assessment of the risks associated with environmental release of genetically engineered microbial inocula. Molecular marker systems are particularly appropriate for such studies and luminescence-based markers have the broadest range of applications, involving the introduction of prokaryotic (lux) or eukaryotic (luc) genes for the enzyme luciferase.

Lux or luc genes can be detected on the basis of unique DNA sequences by gene probing and PCR amplification, but the major advantage of luminescence-based systems is the ability to detect light emitted by marked organisms or by luciferase activity in cell-free extracts. Luminescent colonies can be detected by eye, providing distinction from colonies of indigenous organisms, and the sensitivity of plate counting can be increased greatly by CCD imaging. Single cells or microcolonies of luminescent organisms can also be detected in environmental samples by CCD image-enhanced microscopy, facilitating study of their spatial distribution. The metabolic activity of luminescence-marked populations can be quantified by luminometry and does not require extraction of cells or laboratory growth. Metabolic activity, and potential activity, of marked organisms therefore can be measured during colonization of soil particles and plant material in real time without disturbing the colonization process.

In comparison with traditional activity techniques, luminometry provides significant increases in sensitivity, accuracy, and, most importantly, selectivity, as activity can be measured in the presence of indigenous microbial communities. The sensitivity, speed, and convenience of luminescence measurements make this a powerful technique that is being applied to the study of an increasingly wide range of ecological problems. These include microbial survival and recovery, microbial predation, plant pathogenicity, phylloplane and rhizosphere colonization and reporting of gene expression in environmental samples.     

Low Temperature Decreases the Phylogenetic Diversity of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofiltration Systems

The phylogenetic diversity and species richness of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were examined with aquarium biofiltration systems. Species richness, deduced from rarefaction analysis, and diversity indices indicated that the phylogenetic diversity and species richness of AOA are greater than those of AOB; the diversity of AOA and of AOB is minimized in cold-water aquaria. This finding implies that temperature is a key factor influencing the population structure and diversity of AOA and AOB in aquarium biofiltration systems.     

生物源性基因疫苗的运送系统

基因疫苗具有很多独特的优点,已经成为疫苗研究领域的热点。但由于其免疫原性相对较弱,限制了基因疫苗的广泛应用。人们一直在寻求一种理想的基因疫苗运送系统,它不仅能将基因疫苗导入体内,还能提高基因疫苗的免疫原性,诱导机体产生持续高水平的免疫应答反应。