Lateral Gene Transfer in a Heavy Metal-Contaminated-Groundwater Microbial Community

Abstract

Unraveling the drivers controlling the response and adaptation of biological communities to environmental change, especially anthropogenic activities, is a central but poorly understood issue in ecology and evolution. Comparative genomics studies suggest that lateral gene transfer (LGT) is a major force driving microbial genome evolution, but its role in the evolution of microbial communities remains elusive. To delineate the importance of LGT in mediating the response of a groundwater microbial community to heavy metal contamination, representative Rhodanobacter reference genomes were sequenced and compared to shotgun metagenome sequences. 16S rRNA gene-based amplicon sequence analysis indicated that Rhodanobacter populations were highly abundant in contaminated wells with low pHs and high levels of nitrate and heavy metals but remained rare in the uncontaminated wells. Sequence comparisons revealed that multiple geochemically important genes, including genes encoding Fe(2+)/Pb(2+) permeases, most denitrification enzymes, and cytochrome c553, were native to Rhodanobacter and not subjected to LGT. In contrast, the Rhodanobacter pangenome contained a recombinational hot spot in which numerous metal resistance genes were subjected to LGT and/or duplication. In particular, Co(2+)/Zn(2+)/Cd(2+) efflux and mercuric resistance operon genes appeared to be highly mobile within Rhodanobacter populations. Evidence of multiple duplications of a mercuric resistance operon common to most Rhodanobacter strains was also observed. Collectively, our analyses indicated the importance of LGT during the evolution of groundwater microbial communities in response to heavy metal contamination, and a conceptual model was developed to display such adaptive evolutionary processes for explaining the extreme dominance of Rhodanobacter populations in the contaminated groundwater microbiome.

Importance: Lateral gene transfer (LGT), along with positive selection and gene duplication, are the three main mechanisms that drive adaptive evolution of microbial genomes and communities, but their relative importance is unclear. Some recent studies suggested that LGT is a major adaptive mechanism for microbial populations in response to changing environments, and hence, it could also be critical in shaping microbial community structure. However, direct evidence of LGT and its rates in extant natural microbial communities in response to changing environments is still lacking. Our results presented in this study provide explicit evidence that LGT played a crucial role in driving the evolution of a groundwater microbial community in response to extreme heavy metal contamination. It appears that acquisition of genes critical for survival, growth, and reproduction via LGT is the most rapid and effective way to enable microorganisms and associated microbial communities to quickly adapt to abrupt harsh environmental stresses.

FIG 1

Heat maps of gene abundances of key functional groups in Rhodanobacter genomes and OR-IFRC metagenomes. Abundance is measured as relative abundance against all sequenced bacterial genomes and as the odds ratio of each COG number against all COG categories for the selected organism/community. Asterisks indicate significantly overabundant COGs (chi-square test and 1-tailed Fisher’s exact test). The x axis shows the biological samples, with “All Rhodanobacter” representing all Rhodanobacter genomes combined and red font indicating OR-IFRC isolates or metagenomes.

FIG 2

Genome map of R. denitrificans 2APBS1 showing putative LGT events. Regions of anomalous nucleotide composition were computed by using Alien_Hunter on the genome of Rhodanobacter denitrificans 2APBS1. The denitrification genes were not found to overlap the putative laterally transferred regions. The HMR gene cluster near the 2-Mb mark was found to overlap the putative laterally transferred regions and was confirmed independently, using DarkHorse, to be putatively laterally transferred (see Fig. S2 in the supplemental material).

FIG 3

Nitrate/nitrite transporter genes of Rhodanobacter species. Sequences corresponding to COG2223 (nitrate/nitrite transporter) were extracted from the Rhodanobacter strains, and homologous reference sequences were identified by BLASTp analysis and extracted from GenBank. The sequences separated into 4 clades and 8 subclades, and sequences from Rhodanobacter strains are labeled in color. The tree was inferred in MEGA 5.2 using the maximum-likelihood method based on the JTT matrix-based model. The tree with the highest log likelihood (−13,400.3677) is shown. A discrete gamma distribution was used to model the evolutionary rate differences among sites (5 categories [+G parameter = 2.7633]). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 0.1999% sites). The analysis involved 55 amino acids with a total of 354 positions in the final data set.

FIG 4

Recombinational hot spot in Rhodanobacter genomes that confers heavy metal resistance. The y axis shows the organism analyzed, and the x axis shows the gene analyzed, with genes organized roughly in the direction of the leading strand of R. denitrificans. Cells colored red indicate genes that are present based on annotation, yellow indicates truncated genes or unannotated conserved regions, and white indicates the absence of the gene.

FIG 5

A conceptual model of Rhodanobacter population dynamics in contaminated-groundwater systems. Populations are distinguished by different colors, with Rhodanobacter shown in blue. Fuzzy red dots indicate the proposed recombinational hot spot in the Rhodanobacter pangenome. (A) Background state with high biodiversity and low abundance of Rhodanobacter. (B) Contaminated state with chronic exposure to uranium, nitrate, and low pH in which the environment selects for Rhodanobacter. (C) Lateral transfer and duplication of geochemical resistance genes (indicated by arrows) within and between Rhodanobacter populations, which initiates subsequent rounds of selection.