Temperature and Nutrient Levels Correspond with Lineage-Specific Microdiversification in the Ubiquitous and Abundant Freshwater Genus Limnohabitans

Abstract

Most freshwater bacterial communities are characterized by a few dominant taxa that are often ubiquitous across freshwater biomes worldwide. Our understanding of the genomic diversity within these taxonomic groups is limited to a subset of taxa. Here, we investigated the genomic diversity that enables Limnohabitans, a freshwater genus key in funneling carbon from primary producers to higher trophic levels, to achieve abundance and ubiquity. We reconstructed eight putative Limnohabitans metagenome-assembled genomes (MAGs) from stations located along broad environmental gradients existing in Lake Michigan, part of Earth's largest surface freshwater system. De novo strain inference analysis resolved a total of 23 strains from these MAGs, which strongly partitioned into two habitat-specific clusters with cooccurring strains from different lineages. The largest number of strains belonged to the abundant LimB lineage, for which robust in situ strain delineation had not previously been achieved. Our data show that temperature and nutrient levels may be important environmental parameters associated with microdiversification within the Limnohabitans genus. In addition, strains predominant in low- and high-phosphorus conditions had larger genomic divergence than strains abundant under different temperatures. Comparative genomics and gene expression analysis yielded evidence for the ability of LimB populations to exhibit cellular motility and chemotaxis, a phenotype not yet associated with available Limnohabitans isolates. Our findings broaden historical marker gene-based surveys of Limnohabitans microdiversification and provide in situ evidence of genome diversity and its functional implications across freshwater gradients.IMPORTANCELimnohabitans is an important bacterial taxonomic group for cycling carbon in freshwater ecosystems worldwide. Here, we examined the genomic diversity of different Limnohabitans lineages. We focused on the LimB lineage of this genus, which is globally distributed and often abundant, and its abundance has shown to be largely invariant to environmental change. Our data show that the LimB lineage is actually comprised of multiple cooccurring populations for which the composition and genomic characteristics are associated with variations in temperature and nutrient levels. The gene expression profiles of this lineage suggest the importance of chemotaxis and motility, traits that had not yet been associated with the Limnohabitans genus, in adapting to environmental conditions.

Keywords: Great Lakes; Limnohabitans; chemotaxis; environmental adaptation; freshwater; metagenomics; metatranscriptomics; microbial ecology; microdiversity; strain delineation; strain resolved.

FIG 1

Phylogenetic tree of EMIRGE reconstructed 16S rRNA gene sequences and reference betaproteobacterium 16S rRNA gene sequences based on previous findings (39). Color-coded bars at the top indicate Limnohabitans lineages. Sequences within these lineages were also classified according to the Newton et al. (28) taxonomic framework into “Lhab” tribes. The second row of color bars corresponds to the clustering of the EMIRGE reconstructed sequences at 97% average similarity. Pairwise percent identity distributions are provided for the LimB and Lhab-A4 metagenome reconstructed 16S rRNA gene sequences in the inset figure. The tree was rooted using sequences of Enterobacter cancerogenus LMG 2693 (Z96078.1) and Escherichia vulneris (AF530476.1) as outgroups.

FIG 2

Phylogenomic tree of putative Limnohabitans sp. MAGs and available reference Limnohabitans genomes. Only bootstrap values of <80 are shown. (A) Boxplots show the normalized relative abundance of each MAG across the 24 samples (square root scale). (B) Inferred (normalized) relative abundances of closely related populations to the reconstructed MAGs in 117 freshwater metagenomic data sets publicly available from NCBI (94.5% identity cutoff). (C) For each genome or MAG, the growth rate was predicted from growth-imprinted genomic traits. The tree was rooted in Chitinophaga niabensis (IMG TaxID 2636416022). MAG labels (MAGx.XX-YYY-AB) are comprised of a MAG identifier (MAGx) and environmental information on the sample from which it was reconstructed (XX, season; YYY, site; A, depth; B, day/night). DCM, deep chlorophyll maximum.

FIG 3

Absolute values of log₂-fold changes of genes differentially expressed (adjusted P value of <0.01) between the spring and fall seasons for each MAG (controlled for sampling station). The number and percentage of differentially expressed genes relative to the total DESeq selected genes of each MAG are indicated between parentheses. ***, pairwise Wilcoxon rank sum test (adjusted P value of ≤0.001) of MAG8 compared to all other MAGs. MAGs were ordered according to their position in the phylogenomic tree. MAG labels (MAGx.XX-YYY-AB) are comprised of a MAG identifier (MAGx) and environmental information on the sample from which it was reconstructed (XX, season; YYY, site; A, depth; B, day/night). DCM, deep chlorophyll maximum.

FIG 4

Competitive metagenomic read recruitment to each MAG visualized using violin plots with the bw.nrd0 rule-of-thumb bandwidth. For each MAG, the identity profiles of all sample reads were pooled into a single violin plot. The area of each violin plot was fixed in order to allow visual comparison between MAGs. MAGs were ordered according to their position in the phylogenomic tree. MAG labels (MAGx.XX-YYY-AB) are comprised of a MAG identifier (MAGx) and environmental information of the sample from which it was reconstructed (XX, season; YYY, site; A, depth; B, day/night). DCM, deep chlorophyll maximum.

FIG 5

(A) Pearson’s correlation (r_P) between Limnohabitans MAG frequencies and primary environmental parameters differentiating the environmental gradients of the studied transect. The size of labels is proportional to the correlation strength. PAR, photosynthetically active radiation. (B) Cooccurrence network of all Limnohabitans strains using a Fruchterman-Reingold layout. Colored regions highlight identified network modules (n = 24).

FIG 6

Spatiotemporal strain frequency dynamics of MAG8.SU-M110-DCMD as inferred from DESMAN. Only strains for which inference was robust are shown, and mean frequencies per site and season are indicated by horizontal bars.