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Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization. Raju Sekar, 2004.We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH) . In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338) . Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the ß-proteobacteria (probe BET42a) . A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry . The purity of sorted cells was >95% . 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library) . Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%) . No enrichment of ß-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library . These bacteria, together with members of marine clade OM43, represented >90% of the ß-proteobacteria in the water sample, as determined by CARD-FISH with specific probes . This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats . Stochastic Assembly of Two-Component Staphylococcal  -Hemolysin into Heteroheptameric Transmembrane Pores with Alternate Subunit Arrangements in Ratios of 3:4 and 4:3.Noriko Sugawara-Tomita, 2002.Self-assembling, pore-forming toxins from Staphylococcus aureus are illustrative molecules for the study of the assembly and membrane insertion of oligomeric transmembrane proteins . On the basis of previous studies, we have shown that the two-component -hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of ca . 200 kDa . Here we show that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3 . High-resolution electron microscopic images of negatively stained pore complexes clearly revealed a heptameric structure . When adjacent monomers in the pore complexes were randomly cross-linked by using glutaraldehyde, LukF-LukF, LukF-Hlg2, and Hlg2-Hlg2 dimers were detected in an approximate ratio of 1:12:1, suggesting that LukF and Hlg2 were alternately arranged in the pore complex in molar ratios of 3:4 and 4:3 . The alternate arrangements of LukF and Hlg2 in molar ratios of 3:4 and 4:3 were also visualized under electron microscope with the pore complexes consisting of glutathione S-transferase fusion protein of LukF or Hlg2 and wild-type protein of Hlg2 or LukF, respectively .
Mob Psychology. Stephen C. Winans, 2002. ldpA Encodes an Iron-Sulfur Protein Involved in Light-Dependent Modulation of the Circadian Period in the Cyanobacterium Synechococcus elongatus PCC 7942. Mitsunori Katayama, 2003.We generated random transposon insertion mutants to identify genes involved in light input pathways to the circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942 . Two mutants, AMC408-M1 and AMC408-M2, were isolated that responded to a 5-h dark pulse differently from the wild-type strain . The two mutants carried independent transposon insertions in an open reading frame here named ldpA (for light-dependent period) . Although the mutants were isolated by a phase shift screening protocol, the actual defect is a conditional alteration in the circadian period . The mutants retain the wild-type ability to phase shift the circadian gene expression (bioluminescent reporter) rhythm if the timing of administration of the dark pulse is corrected for a 1-h shortening of the circadian period in the mutant . Further analysis indicated that the conditional short-period mutant phenotype results from insensitivity to light gradients that normally modulate the circadian period in S . elongatus, lengthening the period at low light intensities . The ldpA gene encodes a polypeptide that predicts a 7Fe-8S cluster-binding motif expected to be involved in redox reactions . We suggest that the LdpA protein modulates the circadian clock as an indirect function of light intensity by sensing changes in cellular physiology .
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