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Characterization of the Chlorate Reductase from Pseudomonas chloritidismutans. Arthur F. W. M. Wolterink, 2003.A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans . Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P . chloritidismutans reduced only chlorate and bromate . Differences were also found in N-terminal sequences, molecular weight, and subunit composition . Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are also found in other dissimilatory oxyanion reductases . Metabolically Active Eukaryotic Communities in Extremely Acidic Mine Drainage. Brett J. Baker, 2004.Acid mine drainage (AMD) microbial communities contain microbial eukaryotes (both fungi and protists) that confer a biofilm structure and impact the abundance of bacteria and archaea and the community composition via grazing and other mechanisms . Since prokaryotes impact iron oxidation rates and thus regulate AMD generation rates, it is important to analyze the fungal and protistan populations . We utilized 18S rRNA and beta-tubulin gene phylogenies and fluorescent rRNA-specific probes to characterize the eukaryotic diversity and distribution in extremely acidic (pHs 0.8 to 1.38), warm (30 to 50°C), metal-rich (up to 269 mM Fe2+, 16.8 mM Zn, 8.5 mM As, and 4.1 mM Cu) AMD solutions from the Richmond Mine at Iron Mountain, Calif . A Rhodophyta (red algae) lineage and organisms from the Vahlkampfiidae family were identified . The fungal 18S rRNA and tubulin gene sequences formed two distinct phylogenetic groups associated with the classes Dothideomycetes and Eurotiomycetes . Three fungal isolates that were closely related to the Dothideomycetes clones were obtained . We suggest the name "Acidomyces richmondensis" for these isolates . Since these ascomycete fungi were morphologically indistinguishable, rRNA-specific oligonucleotide probes were designed to target the Dothideomycetes and Eurotiomycetes via fluorescent in situ hybridization (FISH) . FISH analyses indicated that Eurotiomycetes are generally more abundant than Dothideomycetes in all of the seven locations studied within the Richmond Mine system . This is the first study to combine the culture-independent detection of fungi with in situ detection and a demonstration of activity in an acidic environment . The results expand our understanding of the subsurface AMD microbial community structure . In Situ Monitoring of Streptothricin Production by Streptomyces rochei F20 in Soil and Rhizosphere. Usanee Anukool, 2004.The onset of streptothricin (ST) biosynthesis in Streptomyces rochei F20 was studied by using reverse transcription-PCR (RT-PCR) to detect transcripts of ST genes during growth in liquid medium, soil, and the rhizosphere . In situ results correlated with those obtained in vitro, illustrating the growth phase-dependent manner of ST production by F20 . Maximal transcription of ST resistance (sttR) and biosynthesis (sttA) genes occurred during the transition between the exponential and stationary phases of growth, when the specific growth rate (µ) started to decline . A higher level of gene expression of sttR versus sttA was observed in all experiments . In liquid culture, maximal transcript accumulation of the sttA gene was only ca . 40% that of the sttR gene . sttA and sttR mRNAs were detected in soil containing approximately 106 CFU of growing cells g of soil–1 . sttR mRNA was detected in sterile and nonsterile rhizosphere colonized with growing mycelium of F20 at 1.2 x 106 and 4.0 x 105 CFU g of soil–1, respectively . However, neither sttR nor sttA transcripts were detected by RT-PCR in the rhizoplane, which supported a lower population density of F20 than the rhizosphere . Mutational Loss of a K+ and NH4+ Transporter Affects the Growth and Endospore Formation of Alkaliphilic Bacillus pseudofirmus OF4. Yi Wei, 2003.A putative transport protein (Orf9) of alkaliphilic Bacillus pseudofirmus OF4 belongs to a transporter family (CPA-2) of diverse K+ efflux proteins and cation antiporters . Orf9 greatly increased the concentration of K+ required for growth of a K+ uptake mutant of Escherichia coli . The cytoplasmic K+ content of the cells was reduced, consistent with an efflux mechanism . Orf9-dependent translocation of K+ in E . coli is apparently bidirectional, since ammonium-sensitive uptake of K+ could be shown in K+-depleted cells . The upstream gene product Orf8 has sequence similarity to a subdomain of KTN proteins that are associated with potassium-translocating channels and transporters; Orf8 modulated the transport capacities of Orf9 . No Orf9-dependent K+(Na+)/H+ antiport activity was found in membrane vesicles . Nonpolar deletion mutants in the orf9 locus of the alkaliphile chromosome exhibited no K+-related phenotype but showed profound phenotypes in medium containing high levels of amine-nitrogen . Their patterns of growth and ammonium content suggested a physiological role for the orf9 locus in bidirectional ammonium transport . Orf9-dependent ammonium uptake was observed in right-side-out membrane vesicles of the alkaliphile wild type and the mutant with an orf8 deletion . Uptake was proton motive force dependent and was inhibited by K+ . Orf9 is proposed to be designated AmhT (ammonium homeostasis) . Ammonium homeostasis is important in high-amine-nitrogen settings and is particularly crucial at high pH since cytosolic ammonium accumulation interferes with cytoplasmic pH regulation . Endospore formation in amino-acid-rich medium was significantly defective and germination was modestly defective in the orf9 and orf7-orf10 deletion mutants .
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