The diversity of the Limnohabitans genus, an important group of freshwater bacterioplankton, by characterization of 35 isolated strains

Abstract

Bacteria of the genus Limnohabitans, more precisely the R-BT lineage, have a prominent role in freshwater bacterioplankton communities due to their high rates of substrate uptake and growth, growth on algal-derived substrates and high mortality rates from bacterivory. Moreover, due to their generally larger mean cell volume, compared to typical bacterioplankton cells, they contribute over-proportionally to total bacterioplankton biomass. Here we present genetic, morphological and ecophysiological properties of 35 bacterial strains affiliated with the Limnohabitans genus newly isolated from 11 non-acidic European freshwater habitats. The low genetic diversity indicated by the previous studies using the ribosomal SSU gene highly contrasted with the surprisingly rich morphologies and different patterns in substrate utilization of isolated strains. Therefore, the intergenic spacer between 16S and 23S rRNA genes was successfully tested as a fine-scale marker to delineate individual lineages and even genotypes. For further studies, we propose the division of the Limnohabitans genus into five lineages (provisionally named as LimA, LimB, LimC, LimD and LimE) and also additional sublineages within the most diversified lineage LimC. Such a delineation is supported by the morphology of isolated strains which predetermine large differences in their ecology.

Figure 1. Basic morphotypes of isolated Limnohabitans spp. strains.

(A, B) Lineage LimA, (C) lineage LimE, (D) lineage LimB, (E–L) lineage LimC – strains 2KL-17 (unaffiliated), CEP5 (LimC3), 2KL-27 (LimC5), LI2-55 (LimC2), Hin4 (LimC4), 2KL-16 (LimC1), 2KL-1 (unaffiliated) and WS1 (LimC6). The strain-specific codes refer to the codes assigned to isolates in the overview Table 2. Microphotographs, 1000×magnification, scale bar represents 2 µm.

Figure 2. Metabolic characteristics of newly isolated Limnohabitans spp. and described species.

The characteristics of L. australis strain MWH-BRAZ-DAM-2D, L. curvus MWH-C5, L. parvus II-B4 and L. planktonicus II-D5 were taken from , and , respectively. Biomass increase of strains was scored as++(highly positive, >180% of control treatments),+(positive, 50–180%), w (weak, 10–50%), − (no growth, -20–10%), – (light inhibition, −40–−20%) and inhib (severe inhibition,<-40%). Strains and substrates are sorted according to their relative number of hits when growth growth was observed (<20% biomass increase), i.e. more opportunist on the left and more specialist on the right, and more preferred substrate on the top and less on the bottom.

Figure 3. Phylogenetic tree of isolated Limnohabitans spp. strains, environmental clones and described species based on 16S rRNA gene.

GKS16 cluster is composed of the homonymous clone and other 19 environmental sequences. The consensus tree was constructed by Bayesian algorithm with 8 million generations, when 2000 trees were removed as burnin. The scale bar correspond to 50 base substitutions per 100 nucleotide positions. Bootstrap values for Bayesian probability at the branching points are given. The tree was rooted by Polynucleobacter necessarius subsp. asymbioticus, Ralstonia eutropha and Herbaspirillum putei. Detailed description of used dataset is available in Table S3 in File S1.

Figure 4. Microdiversity of Limnohabitans genus based on 40 isolated strains.

The simplified phylogeny schema was build on analyses of 16S rRNA gene and IGS1 sequences. The phylogeny was constructed on the base of Bayesian algorithm with 5 million generations, when 1000 trees were removed as burnin. Bootstrap values for Bayesian probability/Maximum Parsimony/Maximum Likelihood at the branching points are given. The tree was rooted by Polynucleobacter necessarius subsp. asymbioticus. Symbol “ω” with a number stands as reference for isolated strains obtained by K. Watanabe. Question marks stands for polyphyletic groups of strains with similar morphologies. Listed habitats originate from GenBank/EMBL sequence databases.

Figure 5. Biovolume of the R-BT065-positive-cells compared to other bacteria and their relative contribution in natural bacterial community.

(A) Boxes represent 25% and 75% quartils, whiskers 5% and 95% quintiles, full circles outliers. Dashed lines represent means, whereas full lines are medians. (B) Relative proportions (%) of the R-BT065 bacteria targeted to total cells (black bar) and to total carbon biomass (white bar). Note that due to very low proportion of the R-BT bacteria in humic pond Dolní Kočvarů there is also incorporated a fine-scale resolution insert.