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Identification of Members of the Metabolically Active Microbial Populations Associated with Beggiatoa Species Mat Communities from Gulf of Mexico Cold-Seep Sediments. Heath J. Mills, 2004.In this study, the composition of the metabolically active fraction of the microbial community occurring in Gulf of Mexico marine sediments (water depth, 550 to 575 m) with overlying filamentous bacterial mats was determined . The mats were mainly composed of either orange- or white-pigmented Beggiatoa spp . Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from three different sediment depths (0 to 2, 6 to 8, and 10 to 12 cm) that had been subjected to reverse transcription-PCR amplification . Domain-specific 16S PCR primers were used to construct 12 different 16S crDNA libraries containing 333 Archaea and 329 Bacteria clones . Analysis of the Archaea clones indicated that all sediment depths associated with overlying orange- and white-pigmented microbial mats were almost exclusively dominated by ANME-2 (95% of total Archaea clones), a lineage related to the methanogenic order Methanosarcinales . In contrast, bacterial diversity was considerably higher, with the dominant phylotype varying by sediment depth . An equivalent number of clones detected at 0 to 2 cm, representing a total of 93%, were related to the  and
 classes of Proteobacteria, whereas clones related to
-Proteobacteria dominated the metabolically active fraction of the bacterial community occurring at 6 to 8 cm (79%) and 10 to 12 cm (85%) . This is the first phylogenetics-based evaluation of the presumptive metabolically active fraction of the Bacteria and Archaea community structure investigated along a sediment depth profile in the northern Gulf of Mexico, a hydrocarbon-rich cold-seep region .
Conserved Low-Affinity Nickel-Binding Amino Acids Are Essential for the Function of the Nickel Permease NixA of Helicobacter pylori. Lutz Wolfram, 2002.Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori . The nickel permease NixA of H . pylori is a member of the single-component nickel-cobalt transporter family . To identify functionally relevant amino acids of NixA, single-site exchanges were introduced into NixA via PCR-based mutagenesis . This study investigated one of the recognition motifs for this family in transmembrane segment III and other conserved amino acids, mostly with possible nickel-binding capacities . The mutant alleles were expressed in Escherichia coli, and activity of the altered permeases was analyzed by measuring nickel accumulation and urease activity . Expression was checked by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a NixA-specific antibody . Replacement of Phe-75 and His-79—both part of the characteristic sequence motif—and of Asn-127, Thr-195, and Ser-197 with alanine abolished nickel uptake in the E . coli system . The results were unchanged if these amino acids were replaced with residues more similar to the original amino acid . The phenotype of the null mutants was independent of the culture medium . Mutation of Val-82, Tyr-242, Thr-260, His-181, and His-15 strongly affected uptake activity under nickel limitation on complex Luria-Bertani medium but had little effect in minimal medium . Eight other conserved amino acids (Ser-80, Ser-81, Phe-119, Trp-180, Tyr-183, Trp-244, Pro-249, and Asn-256) were found to be dispensable for the function of NixA . These results show that atypical nickel-binding amino acids play an important function in nickel uptake and that most of the essential amino acids are clustered in conserved motifs .
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