Microbial community structure and sulfur biogeochemistry in mildly-acidic sulfidic geothermal springs in Yellowstone National Park.

Geothermal and hydrothermal waters often contain high concentrations of dissolved sulfide, which reacts with oxygen (abiotically or biotically) to yield elemental sulfur and other sulfur species that may support microbial metabolism. The primary goal of this study was to elucidate predominant biogeochemical processes important in sulfur biogeochemistry by identifying predominant sulfur species and describing microbial community structure within high-temperature, hypoxic, sulfur sediments ranging in pH from 4.2 to 6.1. Detailed analysis of aqueous species and solid phases present in hypoxic sulfur sediments revealed unique habitats containing high concentrations of dissolved sulfide, thiosulfate, and arsenite, as well as rhombohedral and spherical elemental sulfur and/or sulfide phases such as orpiment, stibnite, and pyrite, as well as alunite and quartz. Results from 16S rRNA gene sequencing show that these sediments are dominated by Crenarchaeota of the orders Desulfurococcales and Thermoproteales. Numerous cultivated representatives of these lineages, as well as the Thermoproteales strain (WP30) isolated in this study, require complex sources of carbon and respire elemental sulfur. We describe a new archaeal isolate (strain WP30) belonging to the order Thermoproteales (phylum Crenarchaeota, 98% identity to Pyrobaculum/Thermoproteus spp. 16S rRNA genes), which was obtained from sulfur sediments using in situ geochemical composition to design cultivation medium. This isolate produces sulfide during growth, which further promotes the formation of sulfide phases including orpiment, stibnite, or pyrite, depending on solution conditions. Geochemical, molecular, and physiological data were integrated to suggest primary factors controlling microbial community structure and function in high-temperature sulfur sediments.

Linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments.

The identification and characterization of genes involved in the microbial oxidation of arsenite will contribute to our understanding of factors controlling As cycling in natural systems. Towards this goal, we recently characterized the widespread occurrence of aerobic arsenite oxidase genes (aroA-like) from pure-culture bacterial isolates, soils, sediments and geothermal mats, but were unable to detect these genes in all geothermal systems where we have observed microbial arsenite oxidation. Consequently, the objectives of the current study were to measure arsenite-oxidation rates in geochemically diverse thermal habitats in Yellowstone National Park (YNP) ranging in pH from 2.6 to 8, and to identify corresponding 16S rRNA and aroA genotypes associated with these arsenite-oxidizing environments. Geochemical analyses, including measurement of arsenite-oxidation rates within geothermal outflow channels, were combined with 16S rRNA gene and aroA functional gene analysis using newly designed primers to capture previously undescribed aroA-like arsenite oxidase gene diversity. The majority of bacterial 16S rRNA gene sequences found in acidic (pH 2.6-3.6) Fe-oxyhydroxide microbial mats were closely related to Hydrogenobaculum spp. (members of the bacterial order Aquificales), while the predominant sequences from near-neutral (pH 6.2-8) springs were affiliated with other Aquificales including Sulfurihydrogenibium spp., Thermocrinis spp. and Hydrogenobacter spp., as well as members of the Deinococci, Thermodesulfobacteria and beta-Proteobacteria. Modified primers designed around previously characterized and newly identified aroA-like genes successfully amplified new lineages of aroA-like genes associated with members of the Aquificales across all geothermal systems examined. The expression of Aquificales aroA-like genes was also confirmed in situ, and the resultant cDNA sequences were consistent with aroA genotypes identified in the same environments. The aroA sequences identified in the current study expand the phylogenetic distribution of known Mo-pterin arsenite oxidase genes, and suggest the importance of three prominent genera of the order Aquificales in arsenite oxidation across geochemically distinct geothermal habitats ranging in pH from 2.6 to 8.

Isolation and distribution of a novel iron-oxidizing crenarchaeon from acidic geothermal springs in Yellowstone National Park.

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75 degrees C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65 degrees C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80 degrees C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-like 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP.

Mass vaccination and herd immunity: cattle and buffalo.

The design of effective programmes for emergency response to incursion of epizootic diseases of cattle, for exclusion of such diseases and for implementation of progressive control in enzootic situations leading to eventual virus elimination, is currently largely empirical. This needs to be remedied to provide more cost-effective use of vaccines and more effective control. At population level, protective effects of immunisation can extend well beyond the individual, influencing the dynamics of viral propagation within the whole population, non-vaccinated as well as vaccinated. This concept of herd immunity and application of the resulting epidemiological principles, combined with experience gained from disease control programmes such as the Global Rinderpest Eradication Programme has much to offer in designing effective science-based control programmes. This paper explores practical exploitation of the herd immunity principle by considering some of the factors which militate against mass vaccination achieving effective levels of herd immunity and, with these in mind, suggesting ways to optimise the efficiency of mass vaccination programmes.

Experience with eradicating rinderpest by vaccination.

Rinderpest was such a devastating disease throughout Africa, Asia and Europe, capable of shaping the destinies of governments as well as the livelihoods of producers and consumers alike, that all sectors of society demanded that scientists should strive to develop a means of protecting cattle against the constant risk. The history of vaccination as a tool for the control of rinderpest is a long one but finally spawned a vaccine which certainly ranks highly among the safest and most efficacious of vaccines. Having this Tissue Culture Rinderpest Vaccine (TCRV) available generated aspirations of global rinderpest control and even eradication, which could now be considered feasible.

Peste des petits ruminants has been widely present in southern India since, if not before, the late 1980s.

Because previous authorities had suggested that small ruminants were playing a part in the dissemination of rinderpest, and a rinderpest-eradication campaign was about to begin, it was necessary to make precise virus identifications from a number of small-ruminant "rinderpest" outbreaks. When this was done using a database created from passive disease reports, we found that epidemics-reportedly due to rinderpest-were in fact due to peste des petits ruminants (PPRs). Although such cases had been common in India for a number of years, earlier clinical and laboratory reports no longer should be regarded as definitive. PPR outbreaks have been frequent in recent years. Further, we suggest that PPR is not a recent invader of India.

The control of rinderpest in Tanzania between 1997 and 1998.

In January 1997, Tanzania requested international assistance against rinderpest on the grounds that the virus had probably entered the country from southern Kenya. Over the next few months, a variety of attempts were made to determine the extent of the incursion by searching for serological and clinical evidence of the whereabouts of the virus. At the clinical level, these attempts were hampered by the low virulence of the strain, and at the serological level by the lack of a baseline against which contemporary interpretations could be made. Once it became apparent that neither surveillance tool was likely to produce a rapid result, an infected area was declared on common-sense grounds and emergency vaccination was initiated. The vaccination programme had two objectives, firstly to prevent any further entry across the international border, and secondly to contain and if possible eliminate rinderpest from those districts into which it had already entered. On the few occasions that clinical rinderpest was subsequently found, it was always within this provisional infected area. Emergency vaccination campaigns within the infected area ran from January to the end of March 1997 but were halted by the onset of the long rains. At this time, seromonitoring in two districts showed that viral persistence was still theoretically possible and therefore a second round of emergency vaccination was immediately organized. Further seromonitoring then indicated a large number of villages with population antibody prevalences of over 85%. These populations were considered to have been 'immunosterilized'. Although no clinical disease had been observed in them, it was decided to undertake additional vaccination in a group of districts to the south of the infected area. Serosurveillance indicated that rinderpest could have been present in a number of these districts prior to vaccination. Serosurveillance in 1998 suggested that numerous vaccinated animals had probably moved into districts outside the infected and additional vaccination areas, but did not rule out the continued presence of field infection.

Substrate ambiguity of 3-deoxy-D-manno-octulosonate 8-phosphate synthase from Neisseria gonorrhoeae revisited.

Homogeneous, recombinant 3-deoxy-D-manno-octulosonate 8-phosphate synthase from Neisseria gonorrhoeae is shown to catalyze the formation of 3-deoxy-D-manno-octulosonate 8-phosphate from phosphoenolpyruvate and D-arabinose 5-phosphate as determined from (1)H-nuclear magnetic resonance analysis of the product. This enzyme does not catalyze the condensation of D-erythrose 4-phosphate and phosphoenolpyruvate to form 3-deoxy-D-ribo-heptulosonate 7-phosphate, as was previously reported (P. S. Subramaniam, G. Xie, T. Xia, and R. A. Jensen, J. Bacteriol. 180:119-127, 1998).

Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.