title: Coupling 16S-ITS rDNA clone libraries and automated ribosomal intergenic spacer analysis to show marine microbial diversity: development and application to a time series.
abstract: We outline an approach to simultaneously assess multilevel microbial diversity patterns utilizing 16S-ITS rDNA clone libraries coupled with automated ribosomal intergenic spacer analysis (ARISA). Sequence data from 512 clones allowed estimation of ARISA fragment lengths associated with bacteria in a coastal marine environment. We matched 92% of ARISA peaks (each comprising >1% total amplified product) with corresponding lengths from clone libraries. These peaks with putative identification accounted for an average of 83% of total amplified community DNA. At 16S rDNA similarities <98%, most taxa displayed differences in ARISA fragment lengths >10 bp, readily detectable and suggesting ARISA resolution is near the 'species' level. Prochlorococcus abundance profiles from ARISA were strongly correlated (r2=0.86) to Prochlorococcus cell counts, indicating ARISA data are roughly proportional to actual cell abundance within a defined taxon. Analysis of ARISA profiles for 42 months elucidated patterns of microbial presence and abundance providing insights into community shifts and ecological niches for specific organisms, including a coupling of ecological patterns for taxa within the Prochlorococcus, the Gamma Proteobacteria and Actinobacteria. Clade-specific ARISA protocols were developed for the SAR11 and marine cyanobacteria to resolve ambiguous identifications and to perform focused studies. 16S-ITS data allowed high-resolution identification of organisms by ITS sequence analysis, and examination of microdiversity.
authors.affiliation: Department of Biological Science and Wrigley Institute for Marine Studies, University of Southern California, 3616 Trousdale Parkway, AHF B4, Los Angeles, CA 90089, USA. mbrown@ifa.hawaii.edu
authors.forename: Mark V
authors.initials: MV
authors.lastname: Brown
authors.forename: Michael S
authors.initials: MS
authors.lastname: Schwalbach
authors.forename: Ian
authors.initials: I
authors.lastname: Hewson
authors.forename: Jed A
authors.initials: JA
authors.lastname: Fuhrman
mesh_terms: D001331:Automation; D002140:California; D003001:Cloning, Molecular; D021903:DNA, Ribosomal Spacer / Q000032:analysis*; D015723:Gene Library; D008969:Molecular Sequence Data; D010802:Phylogeny; D020560:Proteobacteria / Q000145:classification* / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D012621:Seasons; D012623:Seawater / Q000382:microbiology*; D017422:Sequence Analysis, DNA
chemical_list: D021903:DNA, Ribosomal Spacer; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S