Rapid screening for freshwater bacterial groups by using reverse line blot hybridization.
The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.
Zwart
Gabriel
G
Centre for Limnology, NIOO-KNAW, 3631 AC Nieuwersluis, The Netherlands. g.zwart@nioo.knaw.nl
van Hannen
Erik J
EJ
Kamst-van Agterveld
Miranda P
MP
Van der Gucht
Katleen
K
Lindström
Eva S
ES
Van Wichelen
Jeroen
J
Lauridsen
Torben
T
Crump
Byron C
BC
Han
Suk-Kyun
SK
Declerck
Steven
S
D000818:Animals; D001419:Bacteria / Q000145:classification* / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D005060:Europe; D005618:Fresh Water / Q000382:microbiology*; D020459:Genes, rRNA; D005810:Multigene Family; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics*; D010933:Plankton / Q000145:classification / Q000235:genetics; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S