Phylogenetic clustering of small low nucleic acid-content bacteria across diverse freshwater ecosystems

Abstract

Here we used flow cytometry (FCM) and filtration paired with amplicon sequencing to determine the abundance and composition of small low nucleic acid (LNA)-content bacteria in a variety of freshwater ecosystems. We found that FCM clusters associated with LNA-content bacteria were ubiquitous across several ecosystems, varying from 50 to 90% of aquatic bacteria. Using filter-size separation, we separated small LNA-content bacteria (passing 0.4 µm filter) from large bacteria (captured on 0.4 µm filter) and characterized communities with 16S amplicon sequencing. Small and large bacteria each represented different sub-communities within the ecosystems' community. Moreover, we were able to identify individual operational taxonomical units (OTUs) that appeared exclusively with small bacteria (434 OTUs) or exclusively with large bacteria (441 OTUs). Surprisingly, these exclusive OTUs clustered at the phylum level, with many OTUs appearing exclusively with small bacteria identified as candidate phyla (i.e. lacking cultured representatives) and symbionts. We propose that LNA-content bacteria observed with FCM encompass several previously characterized categories of bacteria (ultramicrobacteria, ultra-small bacteria, candidate phyla radiation) that share many traits including small size and metabolic dependencies on other microorganisms.

Fig. 1

Sample collection, treatment, and statistical analysis. a A total of 47 samples were collected from 22 sampling sites classified in 5 ecosystems. b Each sample was processed in duplicate, and for each duplicate, 3 different groups were collected: “All bacteria”, which was filtered directly onto a 0.2 µm filter, “Large bacteria” (red), which was filtered directly onto a 0.4 µm filter, and “Small bacteria” (blue), which was the filtrate from the 0.4 µm filter captured on a 0.2 µm filter. c Each OTU from the community sequencing data were classified into 5 categories based on its appearance in the large and small bacteria group of a filter pair or sample. For all categories, it was permissible that an OTU appeared in both the large and small bacteria groups of a filter pair. Unclassifiable was a catch-all for OTUs not meeting the criteria of the other categories, and eliminated OTUs did not meet abundance cutoffs

Fig. 2

Typical flow cytometric density and histogram plots from the five investigated natural and engineered freshwater ecosystems (groundwater a, c, river water b. d, lake water e, h, tap water f, i, wastewater g, j) stained with SYBR Green I. Dotted black lines indicate electronic gates separating bacteria from background. Blue and red gates/dotted lines indicate electronic gates separating LNA and HNA content bacteria. FL1-A indicates green fluorescence intensity, FL3-A indicates red fluorescence intensity

Fig. 3

Scanning electron microscopy (SEM) image of bacteria from a stagnant pond sample rich in LNA content bacteria (>90%), filtered onto a 0.2 µm pore-size filter. Filter pores are visible as black holes, bacteria are highlighted in blue/purple shades and extracellular filaments are highlighted in green. Colors were added articifially, and the original image can be found in Supplementary Figure S13

Fig. 4

Non-metric multidimensional scaling (NMDS) of bacterial communities (characterized with 16 S amplicon sequencing) calculated with Bray–Curtis dissimilarity between samples from five different ecosystems (marked by color: Groundwater, Wastewater, River water, Lake water, and Tap water), with three different size groups by shape. ‘All bacteria’ is the total community, directly filtered onto a 0.2 µm filter. Large bacteria is the HNA-dominated community collected on a 0.4 µm filter, and Small bacteria is the LNA-dominated community in the 0.4 µm filtrate, collected on a 0.2 µm filter. In NMDS plots, points that are closer together represent bacterial communities more similar to each other than those further away. A low stress value indicates a robust diagram

Fig. 5

Non-metric multidimensional scaling (NMDS) of bacterial communities (characterized with 16 S amplicon sequencing) calculated with Bray–Curtis dissimilarity between samples from four different rivers (Site A-D), with three different groups by filter pore size. Color is by sampling site, and shape is by size group. ‘All bacteria’ is the total community, directly filtered onto a 0.2 µm filter. Large bacteria is the HNA-dominated community collected on a 0.4 µm filter, and Small bacteria is the LNA-dominated community in the 0.4 µm filtrate, collected on a 0.2 µm filter. In NMDS plots, points that are closer together represent bacterial communities more similar to each other than those further away. A low stress value indicates a robust diagram

Fig. 6

Phylogenetic tree colored by OTU occurrences in each size constructed with 1'224 of >40'000 OTUs found in water samples. Circle area represents the number of samples (both technical duplicates) in which an OTU was consistently exclusively appearing with either large bacteria (0.4 µm filter, red) or small bacteria (0.2 µm filtered after 0.4 µm filter, blue), with this number ranging from 1 to 16 samples. OTUs, which were at times exclusive to both sizes in a filter pair (non-exclusive) are marked in green. Extraneous OTU branches that never met these criteria (unclassifiable, eliminated) were removed from the figure. Several phyla and a class of interest are labeled. For more detailed phylogenetic identification, see Supplementary Figure S10a

Fig. 7

Relative abundances of OTUs classified with the described exclusivity criteria (exclusively small [blue], exclusively large [red], non-exclusive [green], and unclassifiable. eliminated and rare OTUs[white/gray]) in each size group of each ecosystem. For each ecosystem (Groundwater, Wastewater, River water, Lake water, and Tap water), the total relative abundance for all filters in a particular size (small, large bacteria) is shown. Large bacteria is the HNA-dominated community collected on a 0.4 µm filter, and Small bacteria is the LNA-dominated community in the 0.4 µm filtrate, collected on a 0.2 µm filter. Non-exclusive OTUs are further divided into whether they occur in duplicate (I) or not (II). OTUs not meeting initial cutoffs are marked as “rare”, and not meeting secondary cutoffs are marked as “eliminated”. Overlap (e.g., exclusively large OTUs in the small bacteria community) is due to leniency that OTUs may occur on both filters (0.2 and 0.4 µm filters) of a filter-pair, so long as it does not appear exlcusviely on the opposite fitler anywhere (e.g., non-exclusive OTU)