Article Figures & Data
Figures
- FIG. 1.
Phylogenetic trees for α-, β-, and γ-Proteobacteria (A) and Nitrospira, Verrucomicrobia, GNSB, and the CFB group (B), showing the positions of the clones obtained from the autotrophic nitrifying biofilm. The tree was generated by using 1,309 bp (A) and 1,086 bp (B) of the 16S rRNA genes and the neighbor-joining method. Scale bar = 5% sequence divergence. The values at the nodes are bootstrap values (100× resampling analysis). The Methanobacterium congolense sequence served as the outgroup for rooting the tree. The numbers in parentheses indicate the frequencies of appearance of identical clones in the clones analyzed.
- FIG. 2.
Confocal laser scanning microscope images showing the in situ spatial organization of nitrifying bacteria (AOB plus NOB) and heterotrophic bacteria in the autotrophic nitrifying biofilm. (A) FISH with TRITC-labeled probes Nso190 and Ntspa1026 (specific for AOB and Nitrospira, respectively) and FITC-labeled probe ALF1b (specific for α-Proteobacteria). (B) FISH with TRITC-labeled probes BET42a and Ntspa1026 (specific for β-Proteobacteria and Nitrospira, respectively) and FITC-labeled probe GAM42a (specific for γ-Proteobacteria). (C) FISH with TRITC-labeled probes Nso190 and Ntspa1026 (specific for AOB and Nitrospira, respectively) and FITC-labeled probes CF319a/b, S-*-CFB-0665-a-A-18, and S-*-CFB-0730-a-A-18 (specific for the CFB group). (D) FISH with TRITC-labeled probes Nso190 and Ntspa1026 (specific for AOB and Nitrospira, respectively) and FITC-labeled probes S-*-GNS-0667-a-A-18, GNSB-941, and CFX1223 (specific for GNSB). Bars = 10 μm. The surface of the biofilm cannot be seen in the photographs.
- FIG. 3.
Microbial community composition for the autotrophic nitrifying biofilm as determined quantitatively by FISH. The number of cells that hybridized with a probe in each microscopic field was expressed as a percentage of the total surface area of the bacteria that hybridized with the EUB338 probe. Unidentified refers to the surface area of cells detectable with probe EUB338 but not with any other specific probe.
- FIG. 4.
Combined MAR-FISH images of homogenized autotrophic nitrifying biofilm samples. (A) Uptake of [14C]bicarbonate by nitrifying bacteria when NH4+ was used as the sole electron donor. In situ hybridization was performed with FITC-labeled probe Ntspa1026 (green), which was used simultaneously with TRITC-labeled probe Nso190 (red). (B) Uptake of [14C]acetic acid by members of the γ-Proteobacteria group. In situ hybridization was performed with FITC-labeled probe EUB338 (green) and TRITC-labeled probe GAM42a (red). (C) Uptake of 14C-amino acids by the GNSB. In situ hybridization was performed with FITC-labeled probe EUB338 (green) and TRITC-labeled probes S-*-GNS-0667-a-A-18, GNSB-941, and CFX1223 (red). (D) Uptake of [14C]NAG by the GNSB. In situ hybridization was performed with FITC-labeled probe EUB338 (green) and TRITC-labeled probes S-*-GNS-0667-a-A-18, GNSB-941, and CFX1223 (red). Yellow signals resulted from binding of both FITC- and TRITC-labeled probes to one cell. Bars = 10 μm.
- FIG. 5.
Patterns of substrate uptake by the major phylogenetic bacterial groups identified by using group-specific probes. (A) Fractions of group-specific-probe-hybridized cells that simultaneously took up radioactive substrates (MAR-positive cells), expressed as percentages of the total bacteria that were detected with probe EUB338 and simultaneously took up radioactive substrates. (B) Fractions of probe-hybridized and MAR-positive cells, expressed as percentages of each group-specific-probe-hybridized cell count. AOB, AOB that hybridized with probe Nso190; NOB, NOB that hybridized with probe Ntspa1026; α-Proteobacteria hybridized with probe ALF1b; γ-Proteobacteria hybridized with probe GAM42a; GNSB, GNSB that hybridized with probes S-*-GNS-0667-a-A-18, GNSB-941, and CFX1223; CFB, members of the CFB group that hybridized with probes CF319a/b, S-*-CFB-0665-a-A-18, and S-*-CFB-0730-a-A-18. The error bars indicate the standard errors (n = 10).
Tables
- TABLE 2.
16S and 23S rRNA-targeted oligonucleotide probes used
Probea Sequence (5′ to 3′) FA (%)b Specificity Reference EUB338 GCTGCCTCCCGTAGGAGT —c Most bacteria 3 BET42ad GCCTTCCCACTTCGTTT 35 β-Proteobacteria 23 Nso190 CGATCCCCTGCTTTTCTCC 35e Ammonia oxidizers 26 GAM42af GCCTTCCCACATCGTTT 35 γ-Proteobacteria 23 ALF1b CGTTCGYTCTGAGCCAG 20 Many α-Proteobacteria, including Nitrospira 23 CF319 a/b TGGTCCGTRTCTCAGTAC 35 CFB, including OTU 7 24 S-*-CFB-0655-a-A-18 CGCTCACCTCCACAACAT 20 OTU 8 of CFB This study S-*-CFB-0730-a-A-18 TACAGKCTAGYAAGCTGC 20 OTU 9 of CFB This study Ntspa1026 AGCACGCTGGTATTGCTA 20 Nitrospira moscoviensis 18 GNSB-941 AAACCACACGCTCCGCT 35 Phylum Chloroflexi (GNSB) 11 CFX1223 CCATTGTAGCGTGTGTGTMG 35 Phylum Chloroflexi (GNSB) 6 S-*-GNS-0667-a-A-18 CACCCSGAATTCCACRTT 35 OTU 11 and 12 of GNSB This study S-*-Opitu-0744-a-A-18 GCCTGAGCGTCAGCAATT 5 OTU 13 of Verrucomicrobia This study ↵ a Probes designed in this study were named according to the recommendations of Alm et al. (1).
↵ b FA, formamide concentration in the hybridization buffer.
↵ c The probe can be used at any formamide concentration.
↵ d Unlabeled probe GAM42a was used as a competitor to enhance specificity.
↵ e Although the original description of the use of Nso190 indicated that 55% formamide should be used, we experimentally confirmed that 35% formamide was sufficient to discriminate ammonia-oxidizing β-proteobacteria in our autotrophic nitrifying biofilm, which is consistent with other studies in which Nso190 was used at much lower stringencies than that described originally (39).
↵ f Unlabeled probe BET42a was used as a competitor to enhance specificity.
- TABLE 3.
Detection frequency and phylogenetic relatives of the clones analyzed
OTUa No. of clonesb Group Closest taxon Accession no. 10 39 Nitrospirales, Nitrospira Nitrospira sp. Y14639 1 14 β-Proteobacteria, Nitrosomonas Nitrosomonas sp. strain R7c140 AF386753 2 11 β-Proteobacteria, Nitrosomonas Nitrosomonas europaea AF353160 11 11 GNSB Uncultured antarctic bacterium LB3-17 AF173820 4 7 γ-Proteobacteria, Xanthomonas Frateuria aurantia AJ010481 9 4 CFB group Uncultured bacterium PHOS-HE21 AF314419 8 4 CFB group Uncultured bacterium clone HP1B19 AF502210 12 3 GNSB Uncultured sludge bacterium S9 AF234718 3 2 γ-Proteobacteria Lucina nassula gill symbiont X95229 5 2 α-Proteobacteria Uncultured sludge bacterium S43 AF234739 6 1 α-Proteobacteria, Hyphomonas Hyphomonas polymorpha AJ227813 7 1 CFB group Uncultured Cytophagales clone AF289153 13 1 Verrucomicrobia, subdivision IV Opitutus sp. X99391
