Bacterial influence on storage and mobilisation of metals in iron-rich mine tailings from the Salobo mine, Brazil.

In this study we investigated the potential effects of promoting bacterial activity on tailings from the Salobo iron-oxide copper­gold (IOCG) mine, Brazil. In particular we focussed on (1) the potential for mobilising additional Cu and (2) the effects of long-term storage on other metals. Unlike typical sulphide-ore tailings, the pH of the Salobo tailings is circumneutral and these tailings are dominated by Fe-bearing silicates and magnetite, with minor micrometre-scale encapsulated Cu-bearing sulphides. While these tailings do not produce acid mine drainage, an endemic strain of Acidithiobacillus ferrooxidans was isolated from the mine site. These bacteria were used in laboratory column leaching experiments of tailings material, which ran for up to 395 days, without the addition of ferrous iron. Bacteria-tailings interactions were typically maintained at a pH > 5, due to silicate-mediated pH buffering. This was eventually overcome after ~200 days by regular addition of acidic (pH 2.2) nutrient solution, as well as growth and acid generation by bacteria. Copper dissolution was not significantly enhanced by bacterial activity compared to abiotic control experiments while pH was >5. However, as the experiments were progressively acidified, additional Cu was mobilised in the biotic systems. The mineral alteration reactions produced abundant ferrihydrite precipitates within the tailings, which was enhanced by bacterial activity as the pH decreased. Adsorption of metal cations to these precipitates ensured that effluent solutions had only low levels (<0.5 mg/l) of dissolved trace metals such as As, Co, Pb, Zn, Se, Ni and Cr. These adsorption processes will strongly inhibit metal leaching from the tailings during long-term storage, as long as the iron oxidising bacteria are producing the requisite excess of ferrihydrite and the pH is >5. This case study shows that bacterially-mediated silicate weathering, in Fe(II)-bearing silicate rich tailings with only minor sulphides and Acidithiobacillus ferrooxidans can enhance the environmental stability of the tailings.

The influence of hydrogeological disturbance and mining on coal seam microbial communities.

The microbial communities present in two underground coal mines in the Bowen Basin, Queensland, Australia, were investigated to deduce the effect of pumping and mining on subsurface methanogens and methanotrophs. The micro-organisms in pumped water from the actively mined areas, as well as, pre- and post-mining formation waters were analyzed using 16S rRNA gene amplicon sequencing. The methane stable isotope composition of Bowen Basin coal seam indicates that methanogenesis has occurred in the geological past. More recently at the mine site, changing groundwater flow dynamics and the introduction of oxygen in the subsurface has increased microbial biomass and diversity. Consistent with microbial communities found in other coal seam environments, pumped coal mine waters from the subsurface were dominated by bacteria belonging to the genera Pseudomonas and the family Rhodocyclaceae. These environments and bacterial communities supported a methanogen population, including Methanobacteriaceae, Methanococcaceae and Methanosaeta. However, one of the most ubiquitous micro-organisms in anoxic coal mine waters belonged to the family 'Candidatus Methanoperedenaceae'. As the Archaeal family 'Candidatus Methanoperedenaceae' has not been extensively defined, the one studied species in the family is capable of anaerobic methane oxidation coupled to nitrate reduction. This introduces the possibility that a methane cycle between archaeal methanogenesis and methanotrophy may exist in the anoxic waters of the coal seam after hydrogeological disturbance.

Biomineralization of gold in biofilms of Cupriavidus metallidurans.

Cupriavidus metallidurans, a bacterium capable of reductively precipitating toxic, aqueous gold(I/III)-complexes, dominates biofilm communities on gold (Au) grains from Australia. To examine the importance of C. metallidurans biofilms in secondary Au formation, we assessed the biomineralization potential of biofilms growing in quartz-sand-packed columns to periodic amendment with Au(I)-thiosulfate. In these experiments, >99 wt % of Au, was retained compared to <30 wt % in sterilized and abiotic controls. Biomineralization of Au occurred in the presence of viable biofilms via the formation of intra- and extra-cellular spherical nanoparticles, which aggregated into spheroidal and framboidal microparticles of up to 2 µm in diameter. Aggregates of Au formed around cells, eventually encapsulating and ultimately replacing them. These particles were morphologically analogous to Au-particles commonly observed on natural Au grains. Bacterial cells were connected via exopolymer or nanowires to µm-sized, extracellular Au-aggregates, which would intuitively improve the flow of electrons through the biofilm. This study demonstrates the importance of C. metallidurans biofilms for the detoxification of Au-complexes and demonstrates a central role for bacterial biomineralization in the formation of highly pure Au in surface environments.

Mobile hydrocarbon microspheres from >2-billion-year-old carbon-bearing seams in the South African deep subsurface.

By ~2.9 Ga, the time of the deposition of the Witwatersrand Supergroup, life is believed to have been well established on Earth. Carbon remnants of the microbial biosphere from this time period are evident in sediments from around the world. In the Witwatersrand Supergroup, the carbonaceous material is often concentrated in seams, closely associated with the gold deposits and may have been a mobile phase 2 billion years ago. Whereas today the carbon in the Witwatersrand Supergroup is presumed to be immobile, hollow hydrocarbon spheres ranging in size from <1 µm to >50 µm were discovered emanating from a borehole drilled through the carbon-bearing seams suggesting that a portion of the carbon may still be mobile in the deep subsurface. ToF-SIMS and STXM analyses revealed that these spheres contain a suite of alkane, alkenes, and aromatic compounds consistent with the described organic-rich carbon seams within the Witwatersrand Supergroup's auriferous reef horizons. Analysis by electron microscopy and ToF-SIMS, however, revealed that these spheres, although most likely composed of biogenic carbon and resembling biological organisms, do not retain any true structural, that is, fossil, information and were formed by an abiogenic process.

The preservation and degradation of filamentous bacteria and biomolecules within iron oxide deposits at Rio Tinto, Spain.

One of the keys to understanding and identifying life on other planets is to study the preservation of organic compounds and their precursor micro-organisms on Earth. Rio Tinto in southwestern Spain is a well documented site of microbial preservation within iron sulphates and iron oxides over a period of 2.1 Ma. This study has investigated the preservation of filamentous iron oxidising bacteria and organics through optical microscopy, scanning electron microscopy (SEM) and Fourier transform infra-red (FTIR) spectroscopy, from laboratory cultures of natural samples to contemporary natural materials to million-year old river terraces. Up to 40% elemental carbon and >7% nitrogen has been identified within microbial filaments and cell clusters in all samples through SEM EDS analyses. FTIR spectroscopy identified C-H(x) absorption bands between 2960 and 2800 cm(-1), Amide I and II absorption bands at 1656 and 1535 cm(-1), respectively and functional group vibrations from within nucleic acids at 917, 1016 and 1124 cm(-1). Absorption bands tracing the diagenetic transformation of jarosite to goethite to hematite through the samples are also identified. This combination of mineralogy, microbial morphology and biomolecular evidence allows us to further understand how organic fossils are created and preserved in iron-rich environments, and ultimately will aid in the search for the earliest life on Earth and potential organics on Mars.

Modern carbonate microbialites from an asbestos open pit pond, Yukon, Canada.

Microbialites were discovered in an open pit pond at an abandoned asbestos mine near Clinton Creek, Yukon, Canada. These microbialites are extremely young and presumably began forming soon after the mine closed in 1978. Detailed characterization of the periphyton and microbialites using light and scanning electron microscopy was coupled with mineralogical and isotopic analyses to investigate the mechanisms by which these microbialites formed. The microbialites are columnar in form (cm scale), have an internal spherulitic fabric (mm scale), and are mostly made of aragonite, which is supersaturated in the subsaline pond water. Initial precipitation is seen as acicular aragonite crystals nucleating onto microbial biomass and detrital particles. Continued precipitation entombs benthic diatoms (e.g. Brachysira vitrea), filamentous algae (e.g. Oedogonium sp.), dinoflagellates, and cyanobacteria. The presence of phototrophs at spherulite centers strongly suggests that these microbes play an important initial role in aragonite precipitation. Substantial growth of individual spherulites occurs abiotically through periodic precipitation of aragonite that forms concentric laminations around spherulite centers while pauses in spherulite growth allow for colonization by microbes. Aragonite associated with biomass (δ(13)C = -4.6‰ VPDB) showed a (13)C-enrichment of 0.8‰ relative to aragonite exhibiting no biomass (δ(13)C = -5.4‰ VPDB), which suggests a modest removal of isotopically light dissolved inorganic carbon by phototrophs. The combination of a low sedimentation rate, high calcification rate, and low microbial growth rate appears to result in the formation of these microbialites. The formation of microbialites at an historic mine site demonstrates that an anthropogenically constructed environment can foster microbial carbonate formation.

A high-resolution chemical and structural study of framboidal pyrite formed within a low-temperature bacterial biofilm.

A novel, anaerobically grown microbial biofilm, scraped from the inner surface of a borehole, 1474 m below land surface within a South African, Witwatersrand gold mine, contains framboidal pyrite. Water flowing from the borehole had a temperature of 30.9 degrees C, a pH of 7.4, and an Eh of -50 mV. Examination of the biofilm using X-ray diffraction, field emission gun scanning electron microscope equipped for energy dispersive X-ray microanalysis demonstrated that the framboids formed within a matrix of bacteria and biopolymers. Focused ion beam sectioning of framboids followed by NEXAFS measurements using both scanning transmission X-ray microscopy and X-ray photoelectron emission microscopy revealed that the pyrite crystals grew within an organic carbon matrix consisting of exopolysaccharides and possibly extracellular DNA, which is intuitively important in sulfide mineral diagenesis. Growth of individual pyrite crystals within the framboid occurred inside organic templates confirms the association between framboidal pyrite and organic materials in low-temperature diagenetic environments and the important role of microenvironments in biofilms in regulating geochemical cycles.

Stars of the terrestrial deep subsurface: a novel 'star-shaped' bacterial morphotype from a South African platinum mine.

We study structure and function. Credit of course, goes to TJB, for it is from him that I inherited the habit of personifying bacteria and attempting to think like a bacterium, to better understand what they do. This work has taken us to wonderful places such as Yellowstone National Park, The Canadian Arctic, Australia, and the deep subsurface in the Republic of South Africa, the subject of this manuscript. From their perspective, why they do what they do is simple, to live. How they do it, is more challenging for us to understand, so it is something that we continue to work on. The marvel of bacteria is something that I, in turn, try to pass on to my students where I hope it will find fertile ground and provide as much enjoyment as it has given me - G. Southam. A biofilm (mine-slime) collected from the Northam Platinum mine in the Republic of South Africa contained a new bacterial morphotype. Mine-slimes are generally considered to be microbiologically compromised, subsurface samples due to the likelihood of contamination from the mining environment. However, careful examination of this biofilm demonstrated that it possessed a diverse bacterial population that included organisms that are consistent with the deep subsurface, suggesting that mine-slimes represent an underutilized, 'natural' bacterial enrichment. Using scanning and transmission electron microscopy, a novel, branching, filamentous, star-shape bacterium (in cross section) has been found, adding a new bacterial morphotype and strategy that bacteria have demonstrated to increase their surface area to volume ratio.

Scanning force microscopy studies of the colonization and growth of A. ferrooxidans on the surface of pyrite minerals.

We have used the technique of scanning force microscopy (SFM) for studying the interaction of the bacteria A. ferrooxidans with the surface of the mineral pyrite. These bacteria are important to study with regard to acidification of streams and the environmental impact of such acidification. A. ferrooxidans cells readily colonize the pyrite surface, forming a tight mineral seal between the cell and the pyrite substrate. These bacteria subsequently may grow under pH neutral conditions, biooxidizing the underlying pyrite; this process creates etch pits in the pyrite. On average, these etch pits are 1.2 microns in lateral dimension and approximately 220 nm deep.

Characterization of the cell wall of the sheathed methanogen Methanospirillum hungatei GP1 as an S layer.

The cell wall of Methanospirillum hungatei GP1 is a labile structure that has been difficult to isolate and characterize because the cells which it encases are contained within a sheath. Cell-sized fragments, 560 nm wide by several micrometers long, of cell wall were extracted by a novel method involving the gradual drying of the filaments in 2% (wt/vol) sodium dodecyl sulfate and 10% (wt/vol) sucrose in 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer containing 10 mM EDTA. The surface was a hexagonal array (a = b = 15.1 nm) possessing a helical superstructure with a ca. 2.5 degrees pitch angle. In shadowed relief, the smooth outer face was punctuated with deep pits, whereas the inner face was relatively featureless. Computer-based two-dimensional reconstructed views of the negatively stained layer demonstrated 4.0- and 2.0-nm-wide electron-dense regions on opposite sides of the layer likely corresponding to the openings of funnel-shaped channels. The face featuring the larger openings best corresponds to the outer face of the layer. The smaller opening was encircled by a stalk-like mass from which 2.2-nm-wide protrusions were resolved. The cell wall in situ was degraded at pH 9.6 at 56 degrees C but was unaffected at pH 7.4 at the same temperature. The cell wall was composed of two nonglycosylated polypeptides (114 and 110 kDa). The cell wall resembled an archaeal S layer and may function in regulating the passage of small (< 10-kDa) sheath precursor proteins.

Examination of Lipopolysaccharide (O-Antigen) Populations of Thiobacillus ferrooxidans from Two Mine Tailings.

Net acid-generating capacities of 39.74 kg of H(2)SO(4) per ton (ca. 0.05 kg/kg) (pH 2.68) for the Lemoine copper mine tailings (closed ca. 8 years ago; located 40 km west of Chibougamau, Quebec, Canada) and 16.07 kg of H(2)SO(4) per ton (ca. 0.02 kg/kg) (pH 3.01) for the Copper Rand tailings (in current use and 50 km distant [east] from those of Lemoine) demonstrate that these sulfide tailings can support populations of acidophilic thiobacilli. Oxidized regions in both tailings environments were readily visible, were extremely acidic (Lemoine, pH 2.36; Copper Rand, pH 3.07), and provided natural isolates for our study. A 10% (wt/vol) oxalic acid treatment, which solubilizes both ferric sulfate and ferric hydroxide precipitates (B. Ramsay, J. Ramsay, M. deTremblay, and C. Chavarie, Geomicrobiol. J. 6:171-177, 1988), enabled the recovery of intact bacterial cells from the tailings material and from liquid synthetic medium for lipopolysaccharide analysis. No viable cells could be cultured after this oxalic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electro-phoretic profiles of lipopolysaccharides extracted from the Lemoine tailings were complex, indicating a heterogeneous population of Thiobacillus ferrooxidans. Six T. ferrooxidans subspecies as identified by lipopolysaccharide analysis (i.e., lipopolysaccharide chemotypes) were eventually isolated from a total of 112 cultures from the Lemoine tailings. Using the same isolate and lipopolysaccharide typing techniques, we identified only a single lipopolysaccharide chemotype from 20 cultures of T. ferrooxidans isolated from the Copper Rand tailings. This homogeneity of lipopolysaccharide chemotype was much different from what was found for the older Lemoine tailings and may reflect a progressive lipopolysaccharide heterogeneity of Thiobacillus isolates as tailings leach and age.

Transmission electron microscopy, scanning tunneling microscopy, and atomic force microscopy of the cell envelope layers of the archaeobacterium Methanospirillum hungatei GP1.

Methanospirillum hungatei GP1 possesses paracrystalline cell envelope components including end plugs and a sheath formed from stacked hoops. Both negative-stain transmission electron microscopy (TEM) and scanning tunneling microscopy (STM) distinguished the 2.8-nm repeat on the outer surface of the sheath, while negative-stain TEM alone demonstrated this repeat around the outer circumference of individual hoops. Thin sections revealed a wave-like outer sheath surface, while STM showed the presence of deep grooves that precisely defined the hoop-to-hoop boundaries at the waveform nodes. Atomic force microscopy of sheath tubes containing entrapped end plugs emphasized the end plug structure, suggesting that the sheath was malleable enough to collapse over the end plugs and deform to mimic the shape of the underlying structure. High-resolution atomic force microscopy has revised the former idea of end plug structure so that we believe each plug consists of at least four discs, each of which is approximately 3.5 nm thick. PT shadow TEM and STM both demonstrated the 14-nm hexagonal, particulate surface of an end plug, and STM showed the constituent particles to be lobed structures with numerous smaller projections, presumably corresponding to the molecular folding of the particle.

Detection of growth sites in and protomer pools for the sheath of Methanospirillum hungatei GP1 by use of constituent organosulfur and immunogold labeling.

Methanospirillum hungatei GP1 integrated approximately 9% of cellular [35S]cysteine into its sheath. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels revealed that [35S]cysteine was confined to the proteins released by the sodium dodecyl sulfate-beta-mercaptoethanol-EDTA solubilization method (G. Southam and T. J. Beveridge, J. Bacteriol. 173:6213-6222, 1991) and was not present in the proteins released by treatment with phenol (G. Southam and T. J. Beveridge, J. Bacteriol. 174:935-946, 1992). Limited labeling of exposed sulfhydryl groups on hoops produced from sheath material suggested that most organosulfur groups occur within hoops and therefore help contribute to resilience. Electron microscopic autoradiography demonstrated that sheath growth, which is most active at the sites of cell division (spacer region), occurs through the de novo development of hoops. For growth to occur in the spacer region, sheath precursors must transverse several periodic envelope layers, including the cell wall (a single layer) and the various lamellae of the spacer plug (T. J. Beveridge, G. D. Sprott, and P. Whippey, J. Bacteriol. 173:130-140, 1991).

Enumeration of Thiobacilli within pH-Neutral and Acidic Mine Tailings and Their Role in the Development of Secondary Mineral Soil.

The Lemoine tailings of Chibougamau, Quebec, Canada, were deposited as a pH-neutral mineral conglomerate consisting of aluminum-silicates, iron-aluminum-silicates, pyrite, chalcopyrite, and sphalerite. These tailings are colonized by an active population of Thiobacillus ferrooxidans which is localized to an acid zone occupying 40% of the tailings' surface. This population peaked at 7 x 10 most probable number per gram of tailings during July and August 1990 and extended to a depth of 40 cm from the surface. Examination of samples over this depth profile by transmission electron microscopy and electron dispersive spectroscopy revealed a microbially mediated mineral transition from sulfides (below 40 cm) to chlorides and phosphates (at the surface). Silicate minerals were unaltered by microbial action. Transmission electron microscopy showed a tight association between Thiobacillus species and the sulfide minerals, which helps account for their prominence in tailings environments. Accurate enumeration of T. ferrooxidans from tailings required the disruption of their bonding to the mineral interface. Vortexing of a 10% aqueous suspension of the tailings material prior to most-probable-number analysis best facilitated this release. Even though heavy metals were highly mobile under acidic conditions at the Lemoine tailings, it was evident by transmission electron microscopy and electron dispersive spectroscopy that they were being immobilized as bona fide fine-grain minerals containing iron, copper, chlorine, phosphorus, and oxygen on bacterial surfaces and exopolymers. This biomineralization increased with increasing bacterial numbers and was most evident in the upper 3 cm of the acidic zone.

Characterization of novel, phenol-soluble polypeptides which confer rigidity to the sheath of Methanospirillum hungatei GP1.

Treatment of the Methanospirillum hungatei GP1 sheath with 90% (wt/vol) phenol resulted in the solubilization of a novel phenol-soluble group of polypeptides. These polypeptides were purified by the removal of insoluble material by ultracentrifugation and represented approximately 19% of the mass of the sheath. The phenol-insoluble material resembled untreated sheath but had lost its rigidity and cylindrical form. Recombination of phenol-soluble and phenol-insoluble fractions by dialysis to remove phenol resulted in cylindrical reassembly products. Although bona fide sheath (complete with the 2.8-nm lattice) was not produced, a role for the phenol-soluble polypeptides in the maintenance of sheath rigidity is implied. The phenol-soluble polypeptides have limited surface exposure as detected by antibodies on intact sheath; therefore, they are not responsible for the 2.8-nm repeat occurring on the outer face of the sheath. However, longitudinal and transverse linear labeling by protein A-colloidal gold on the outer and inner faces, respectively, occurred with monoclonal antibodies specific to the phenol-soluble polypeptides. Restricted surface exposure of phenol-soluble polypeptides on the sheath highlighted molecular defects in sheath architecture. These lattice faults may indicate sites of sheath growth to accommodate cell growth or division (longitudinal immunogold label) and filament division (transverse immunogold label). The identification of a second group of polypeptides within the infrastructure of the sheath suggests that the sheath is a trilaminar structure in which phenol-soluble polypeptides are sandwiched between sodium dodecyl sulfate-beta-mercaptoethanol-EDTA-soluble polypeptides (G. Southam and T. J. Beveridge, J. Bacteriol. 173:6213-6222, 1991) (phenol-insoluble material).

Dissolution and immunochemical analysis of the sheath of the archaeobacterium Methanospirillum hungatei GP1.

The sheath of Methanospirillum hungatei GP1 was degraded by three dissolution techniques, which produced a range of soluble products. By using 0.05 M L-arginine buffer (pH 12.6) at 90 degrees C for 10 min, 74% (dry weight) of the sheath was dissolved; however, the solubilized polypeptides were extensively degraded. Treatment with 2% beta-mercaptoethanol and 2% sodium dodecyl sulfate at 90 degrees C in 0.05 M 2(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 9.0) solubilized 42% (dry weight) of the sheath as a group of polypeptides of 30 to 40 kDa. At 100 degrees C for 2 h, 5% beta-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and 20 mM EDTA released 74% of the sheath's mass as a group of polypeptides of 10 to 40 kDa. All solubilized products were examined by SDS-polyacrylamide gel electrophoresis, and a range of high- and low-molecular-weight polypeptides was identified. None were glycoproteins. Hoops, which comprise the sheath's structure, were seen by electron microscopy after all of the attempted dissolutions. Monoclonal antibodies were produced against the 10- to 40-kDa range of solubilized products and against the approximately 40-kDa polypeptides, and polyclonal antiserum was produced against an 18-kDa polypeptide. These immunological markers were used in Western immunoblotting and protein A-colloidal gold-antibody probing by electron microscopy to identify the structural location of the various polypeptides. Native sheath, which possesses 2.8-nm particles on its outer surface (M. Stewart, T.J. Beveridge, and G.D. Sprott, J. Mol. Biol. 183:509-515, 1985; P.J. Shaw, G.J. Hills, J.A. Henwood, J.E. Harris, and D.B. Archer, J. Bacteriol. 161:750-757, 1985), presented a gentle wave-form surface in platinum-shadowed specimens. In contrast, the inner face of the sheath was highlighted by ridges lying perpendicular to the longitudinal axis of the sheath and likely corresponded to hoop boundaries. Both the polyclonal and monoclonal antibodies were specific for different faces; polyclonal antibodies labeled the inner face, whereas monoclonal antibodies labeled the outer face. Accordingly, the apparent asymmetry of structure between the two faces of the sheath can be correlated by our immunochemical probing with a distinct asymmetry in the distribution of exposed polypeptides between the faces. The possible implications of this asymmetry for growth and maturation of the sheath are explained.

High-resolution topography of the S-layer sheath of the archaebacterium Methanospirillum hungatei provided by scanning tunneling microscopy.

The inner and outer surfaces of the sheath of Methanospirillum hungatei GP1 have been imaged for the first time by using a bimorph scanning tunneling microscope (STM) on platinum-coated or uncoated specimens to a nominal resolution in height of ca. 0.4. nm. Unlike more usual types of microscopy (e.g., transmission electron microscopy), STM provided high-resolution topography of the surfaces, giving good depth detail which confirmed the sheath to be a paracrystalline structure possessing minute pores and therefore impervious to solutes possessing a hydrated radius of greater than 0.3 nm. STM also confirmed that the sheath consisted of a series of stacked hoops approximately 2.5 nm wide which were the remnants of the sheath after treatment with 2% (wt/vol) sodium dodecyl sulfate-2% (vol/vol) beta-mercaptoethanol (pH 9.0). No topographical infrastructure could be seen on the sides of the hoops. This research required the development of a new long-range STM capable of detecting small particles such as bacteria on graphite surfaces as well as a new "hopping" STM mode which did not deform the poorly conducting bacterial surface during high-resolution topographical analysis.

Isolation, characterization, and cellular insertion of the flagella from two strains of the archaebacterium Methanospirillum hungatei.

In high (45 mM)-phosphate medium, Methanospirillum hungatei strains GP1 and JF1 grew as very long, nonmotile chains of cells that did not possess flagella. However, growth in lower (3 or 30 mM)-phosphate medium resulted in the production of mostly single cells and short chains that were motile by means of two polar tufts of flagella, which transected the multilayered terminal plug of the cell. Electron microscopy of negatively stained whole mounts revealed a flagellar filament diameter of approximately 10 nm. Flagellar filaments were isolated from either culture fluid or concentrated cell suspensions that were subjected to shearing. Flagellar filaments were sensitive to treatment with both Triton X-100 and Triton X-114 at concentrations as low as 0.1% (vol/vol). The filaments of both strains were composed of two flagellins of Mr 24,000 and 25,000. However, variations in trace element composition of the medium resulted in the production of a third flagellin in strain JF1. This additional flagellin appeared as a ladderlike smear on sodium dodecyl sulfate-polyacylamide gels with a center of intensity of Mr 35,000 and cross-reacted with antisera produced from filaments containing only the Mr-24,000 and -25,000 flagellins. On sodium dodecyl sulfate-polyacrylamide gels, all flagellins stained by the thymol-sulfuric acid and Alcian blue methods, suggesting that they were glycosylated. This was further supported by chemical deglycosylation of the strain JF1 flagellins, which resulted in a reduction in their apparent molecular weight on sodium dodecyl sulfate-polyacylamide gels. Heterologous reactions to sera raised against the flagella from each strain were limited to the Mr-24,000 flagellins.

Survival and transport of bacteria in egg washwater.

An evaluation of methods for monitoring the quality of water used to wash eggs at grading stations was undertaken to improve maintenance of bacterial viability during overnight sample transport. Bacterial content of samples at analysis would then better reflect conditions at the time eggs were washed. The interactive effects of temperature and the highly alkaline water conditions upon viability were the subjects of this study. Nine transport methods were examined for their efficacy in recovering total and coliform bacteria from recycled water used to wash eggs, and these were compared with samples analyzed at two commercial egg grading stations. Samples were shipped under test to the laboratory for analysis the following day. The survival of Staphylococcus aureus and Escherichia coli was also examined, but in a synthetic washwater matrix under various combinations of temperature (6 to 32 degrees C) and pH (9.5 to 10.5) to determine whether there was likely to be a different response to variations in transport treatment among gram-positive and -negative bacteria. S. aureus was much more resistant to the lethal effects of high pH and moderate temperature than E. coli. These results indicated that samples of high pH should be held (transported) at less than or equal to 13 degrees C to optimize bacterial survival. Considering cost, ease of manipulation, and the ability to protect both coliforms and the bacterial population as a whole, the method of choice for transport of industrial samples was the direct addition of washwater to containers in which powdered KH2PO4 and Na2S2O3 had been placed to yield final concentrations, when dissolved, of 0.2 and 0.05% (wt/vol), respectively.

Production and characterization of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa.

Monoclonal antibodies against 12 of the 17 IATS serotype strains of Pseudomonas aeruginosa were produced. Eighty-seven hybridoma clones were isolated, and the antibodies secreted were found to be reactive with both Formalin-fixed whole cells and purified lipopolysaccharide of homologous strains in enzyme-linked immunosorbent assays. Among these monoclonal antibodies, the predominant antibody class was immunoglobulin M (IgM) (76%), although antibodies of the IgG2a and IgG3 isotypes were also produced. The monoclonal antibodies could further be divided into two groups based on their ability to agglutinate whole cells of homologous strains. The agglutinating monoclonal antibodies were found to immunoblot with the O side chains of homologous lipopolysaccharide, while the nonagglutinating monoclonal antibodies were found to be reactive with outer membrane protein-associated lipopolysaccharide. The applicability of monoclonal antibodies for serotyping was examined, and several antibodies were found to agglutinate whole cells and immunoblot with the O antigen of corresponding serotypes of clinical isolates from cystic fibrosis patients. In conclusion, a set of monoclonal antibodies against the IATS serotype strains of P. aeruginosa have been produced. These antibodies represent a bank of invaluable immunological reagents which may have application in serotyping, epitope mapping, lipopolysaccharide structural determination, and studies of protection against P. aeruginosa.