Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1.

Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.

An uncertain role for Cu(II) in stimulating Mn(II) oxidation by Leptothrix discophora SS-1.

In an effort to improve understanding of the role of Cu(II) in bacterial Mn(II) oxidation, a model Mn(II)-oxidizing bacterium, Leptothrix discophora SS-1, was grown in presence of toxic and non-toxic concentrations of Cu(II), Cd(II) and Mn(II). Mn(II)-oxidizing activity increased by 40% when cells were grown in the presence of 0.05 microM of Cu(II) and increased twofold at 0.18 microM Cu(II). Toxic levels of Cd(II) did not stimulate Mn(II) oxidizing activity, indicating that Mn(II) oxidation is not a response to metal toxicity. Stimulation by Cu(II) confirms the specific role of Cu(II) in Mn(II) oxidation. Comparison of transcript levels of the multicopper oxidase mofA gene in the presence and absence of added Cu(II) do not indicate a statistically significant change in mofA transcript levels in cultures supplemented with Cu(II). Thus, the exact role of Cu(II) in Mn(II) oxidation and its affect on mofA gene expression remain uncertain.

Significance of wall structure, macromolecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum oocysts.

The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium- and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.

Iron requirement for Mn(II) oxidation by Leptothrix discophora SS-1.

A common form of biocatalysis of Mn(II) oxidation results in the formation of biogenic Mn(III, IV) oxides and is a key reaction in the geochemical cycling of Mn. In this study, we grew the model Mn(II)-oxidizing bacterium Leptothrix discophora SS-1 in media with limited iron (0.1 microM iron/5.8 mM pyruvate) and sufficient iron (0.2 microM iron/5.8 mM pyruvate). The influence of iron on the rate of extracellular Mn(II) oxidation was evaluated. Cultures in which cell growth was limited by iron exhibited reduced abilities to oxidize Mn(II) compared to cultures in medium with sufficient iron. While the extracellular Mn(II)-oxidizing factor (MOF) is thought to be a putative multicopper oxidase, Mn(II) oxidation in the presence of zero added Cu(II) was detected and the decrease in the observed Mn(II) oxidation rate in iron-limited cultures was not relieved when the medium was supplemented with Cu(II). The decline of Mn(II) oxidation under iron-limited conditions was not accompanied by siderophore production and is unlikely to be an artifact of siderophore complex formation with Mn(III). The temporal variations in mofA gene transcript levels under conditions of limited and abundant iron were similar, indicating that iron limitation did not interfere with the transcription of the mofA gene. Our quantitative PCR results provide a step forward in understanding the regulation of Mn(II) oxidation. The mechanistic role of iron in Mn(II) oxidation is uncertain; the data are consistent with a direct requirement for iron as a component of the MOF or an indirect effect of iron resulting from the limitation of one of many cellular functions requiring iron.

A quantitative analysis of DNA extraction and purification from compost.

We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost. The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification. Direct microscopic observation of pre- and post-lysis samples revealed that 95.3+/-2.3% of native cells was lysed. Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2+/-3.8 microg DNA/g of wet compost. The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27+/-4.7 ng humic acid/microl. The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA.

Preferential transport of Cryptosporidium parvum oocysts in variably saturated subsurface environments.

When oocysts of the protozoan Cryptosporidium parvum contaminate drinking water supplies, they can cause outbreaks of Cryptosporidiosis, a common waterborne disease. Of the different pathways by which oocysts can wind up in drinking water, one has received little attention to date; that is, because soils are often considered to be perfect filters, the transport of oocysts through the subsoil to groundwater is generally ignored. To evaluate the significance of this pathway, a series of laboratory experiments investigated subsurface transport of oocysts. Experiment 1 was carried out in a vertical 18-cm-long column filled either with glass beads or silica sand, under conditions known to foster fingered flow. Experiment 2 involved undisturbed, macroporous soil columns subjected to macropore flow. Experiment 3 aimed to study the lateral flow on an undisturbed soil block. The columns and soil samples were subjected to artificial rainfall and were allowed to reach steady state. At that point, feces of contaminated calves were applied at the surface along with a known amount of potassium chloride to serve as a tracer, and rainfall was continued at the same rate. The breakthrough of oocysts and chloride, monitored in the effluent, demonstrate the importance of preferential flow on the transport of oocysts. Compared with chloride, peak oocyst concentrations were not appreciably delayed and, in some cases, occurred even before the chloride peak. Recovery rates for oocysts were low, ranging from 0.1 to 10.4% of the oocysts originally applied on the columns. However, the numbers of oocysts present in the effluents were still orders of magnitude higher than 10 oocysts, the infectious dose considered by the U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, to be sufficient to cause Cryptosporidiosis in healthy adults. These results suggest that the transport of oocysts in the subsurface via preferential flow may create a significant risk of groundwater contamination in some situations.

Spatial distributions of copper in microbial biofilms by scanning electrochemical microscopy.

The spatial distribution of Cu was determined in Escherichia coli PHL628 biofilms using a scanning electrochemical microscope (SECM) consisting of a microelectrode in conjunction with a piezoelectric micropositioning system. Aqueous labile copper species were determined using voltametric stripping after reductive deposition of Cu for 4 min on the microelectrode at -0.7 V (vs Ag/AgCl). The position of the bulk solution-biofilm interface was determined from the change in current produced by 0.4 mM hydroxymethyl ferrocene that was added as a redox indicator. After a 2 h exposure to 0.2 mM copper, Cu was located in the upper region of the biofilm with a penetration depth less than 150 microm. A one-dimensional diffusive transport model adequately described the spatial distribution of copper in the biofilm, but the Cu retardation factor in the biofilm was more than 6-fold larger than that calculated from the isotherm for Cu binding to suspensions of E. coli PHL628 cells. There are several possible reasons for this difference, including an increase in the amount of extracellular polymer per cell within the biofilm and/or tortuosity that might hinder Cu transport into biofilms. The SECM technique in combination with model calculations provides direct evidence in support of the concept that formation of a biofilm may confer resistance to transient spikes in the bulk solution concentration of toxic metal species by retarding metal diffusion and reducing the metal exposure of cells within the biofilm.

An evaluation of web-based case studies in microscopy.

It is often difficult to provide students in introductory science courses with opportunities that mimic the investigative learning experience of doing research. This is particularly true in microbiology courses where advanced microscopy techniques are expensive and difficult to do. To that end, we developed three computer-based case studies around real-life scenarios. Our goals were to: (i) improve students' understanding of advanced microscopic techniques, (ii) give students practice analyzing and interpreting data, and (iii) model a scientific approach to how these techniques are applied to current issues in microbiology. Each case requires students to use references and interpret actual microscopic images, thus giving them a more realistic experience than we could previously provide. We analyzed student learning and perceptions to these case studies. After doing the case studies, students were more able to apply microscopic methods to a realistic problem, thus demonstrating an understanding of how the methods are used. Students appreciated the intellectual challenges presented by having to interpret and analyze actual microscopic images. This approach has allowed us to introduce new areas of content to our course and to stimulate critical thinking skills, a difficult task in a large introductory microbiology course.

Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy.

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 microm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 microm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 microm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).

Polaromonas naphthalenivorans sp. nov., a naphthalene-degrading bacterium from naphthalene-contaminated sediment.

Strain CJ2T, capable of growth on naphthalene as a sole carbon and energy source, was isolated from coal-tar-contaminated freshwater sediment. The Gram reaction of strain CJ2T was negative. The cells were non-spore-forming, non-motile cocci (without flagella). The isolate was found to be an aerobic heterotroph capable of utilizing glucose and other simple sugars. Growth was observed between 4 and 25 degrees C (optimum, 20 degrees C) and between pH 6.0 and 9.0 (optimum, pH 7.0-7.5). The G+C content of the genomic DNA was 61.5 mol% and the major quinone was ubiquinone-8. The peptidoglycan of strain CJ2T was determined as belonging to type A1-gamma, meso-diaminopimelic acid. The major fatty acids of strain CJ2T were 16:1omega7c (67.0%), 16:0 (19.6%), 18:1omega7c (approximately 7.9%) and 10:0 3-OH (approximately 2.5%). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Mycolic acid and glycolipids could not be detected. Comparative 16S rDNA analysis indicated that strain CJ2T is related to the family Comamonadaceae and that the nearest phylogenetic relative was Polaromonas vacuolata 34-PT (97.1% similarity). On the basis of the physiological and molecular properties, the naphthalene-degrading isolate was designated Polaromonas naphthalenivorans sp. nov. The type strain is CJ2T (=ATCC BAA-779T=DSM 15660T).

Effect of oxide formation mechanisms on lead adsorption by biogenic manganese (hydr)oxides, iron (hydr)oxides, and their mixtures.

The effects of iron and manganese (hydr)oxide formation processes on the trace metal adsorption properties of these metal (hydr)oxides and their mixtures was investigated by measuring lead adsorption by iron and manganese (hydr)oxides prepared by a variety of methods. Amorphous iron (hydr)oxide formed by fast precipitation at pH 7.5 exhibited greater Pb adsorption (gamma(max) = 50 mmol of Pb/mol of Fe at pH 6.0) than iron (hydr)oxide formed by slow, diffusion-controlled oxidation of Fe(II) at pH 4.5-7.0 or goethite. Biogenic manganese(III/IV) (hydr)oxide prepared by enzymatic oxidation of Mn(II) by the bacterium Leptothrix discophora SS-1 adsorbed five times more Pb (per mole of Mn) than an abiotic manganese (hydr)oxide prepared by oxidation of Mn(II) with permanganate, and 500-5000 times more Pb than pyrolusite oxides (betaMnO2). X-ray crystallography indicated that biogenic manganese (hydr)oxide and iron (hydr)oxide were predominantly amorphous or poorly crystalline and their X-ray diffraction patterns were not significantly affected by the presence of the other (hydr)oxide during formation. When iron and manganese (hydr)oxides were mixed after formation, or for Mn biologically oxidized with iron(III) (hydr)oxide present, observed Pb adsorption was similar to that expected for the mixture based on Langmuir parameters for the individual (hydr)oxides. These results indicate that interactions in iron/manganese (hydr)oxide mixtures related to the formation process and sequence of formation such as site masking, alterations in specific surface area, or changes in crystalline structure either did not occur or had a negligible effect on Pb adsorption by the mixtures.

Caldimonas manganoxidans gen. nov., sp. nov., a poly(3-hydroxybutyrate)-degrading, manganese-oxidizing thermophile.

A poly(3-hydroxybutyrate) (PHB)-degrading, gram-negative, aerobic bacterium, strain HS(T), was isolated from a hot spring and chemotaxonomically and phylogenetically characterized. The oxidase-positive, weakly catalase-positive, non-pigmented cells (0.6 x 2.6 microm) exhibited a single polar flagellum and accumulated PHB granules. Strain HS(T) was capable of manganese oxidation. Highest growth rate was attained at 50 degrees C. The optimum pH for growth was 7-8. The major respiratory quinone was ubiquinone-8 and major cellular fatty acids were C16:0, C16:1 and C18:1. The G+C content of the DNA was 66.2 mol%. Comparative 16S rDNA analysis indicated that strain HS(T) is related to the Rubrivivax subgroup and the family Comamonadaceae. The nearest phylogenetic relatives were Ideonella dechloratans (92.1% similarity), Leptothrix discophora (93.6%), Roseateles depolymerans (92.4%) and Rubrivivax gelatinosus (92.2%). On the basis of its phylogenetic and phenotypic properties, it is proposed that this isolate be designated Caldimonas manganoxidans gen. nov., sp. nov.; the type strain is HS(T) (= JCM 10698T = IFO 16448T = ATCC BAA-369T).