Genomic and population biology of dehalogenating Chloroflexi in deep sea sediments using single cell sorting and genome amplification
Final Report Abstract
Dehalogenating Chloroflexi, such as Dehalococcoidetes (Dhc), were originally discovered as the key microbes mediating reductive dehalogenation of the prevalent groundwater contaminants tetrachloroethene and trichloroethene to ethene via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. Molecular and genomic studies on reductive dehalogenases, encoded by rdh genes, have provided evidence for a rapid adaptive evolution of Dhc and rdh towards degradation of anthropogenic organohalogens. Since 2009, these microorganisms have also been recognized in a number of non-contaminated deep sea sediments. The frequent detection of Dhc-related 16S rRNA and rdh genes in the marine subsurface implied a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other and the metabolic life style of Dhc in the absence of said contaminants remained unknown. In order to provide fundamental insights into the (eco)physiology, genomic population structure and evolution of this unique and important microbial species as they naturally occur we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3 m below the seafloor by a single cell genomic (SCG) approach. The results present for the first time single cell genomic data on four deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Three of the single cells were considered to be part of a local Dsc population and assembled together into a 1.28-Mb genome, which appears to be at least 80% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dhc, no evidence for catabolic reductive dehalogenation was found. Only one putative rdh with weak similarities to known terestrial rdh genes was discovered. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle, with multiple genes found forming the Wood-Ljungdahl pathway of acetogens, including an Rnf/Etf enzyme complex. Surprisingly, the assembled genome revealed the presence of multiple predicted haloacid dehalogenase (HAD) genes. This class of enzymes catalyzes the hydrolytic dehalogenation of halogenated organic acids. The HAD and Rdh families are non-homologous, mechanistically different, and have evolved independently. The HAD may confer the ability to utilize iodated, chlorinated and/or brominated compounds, which occur naturally in sediments, by converting halogenated organic compounds into halogen-free organic compounds, which can then be used in canonical carbon degradation pathways. Intriguingly, also genes encoding for putative HAD superfamily enzymes are found, one being located next to the putative rdh. HAD superfamily enzymes are also present in known terrestrial Dhc strains. Finally, we were able to get a fourth single cell from the same environment that showed 100% nucleotide sequence identity with two of the other cells. When sequenced and assembled together, the genome revealed an Rnf/Etf complex, known in literature to be responsible for energy conservation in acetogens, but not to be found in terrestrial Dhc. These findings indicate, that despite not having a complete genome sequence for Dsc, that lifestyle and energy conserving mechanism of terrestrial and marine strains differ completely. This is a novel finding, since based on metagenomic and qPCR it was assumed that marine Dhc contain rdh genes and live as halorespirers in deep sea sediments. The ability to isolate individual microbes as single cells from a complex environmental sample and study their genomes provides a powerful tool to probe biological “dark matter” and holds the promise to greatly expand our understanding of microorganisms, complex biological systems and phenomena in situ. SCG is therefore an essential complement to cultivationbased, metagenomic, and microbial community-focused research approaches. I started my own research to answer questions about life within anaerobic and oligotrophic environments, e.g. the marine subsurface with this novel technique.
Publications
- „Single cell genomic study of Dehalococcoidites species in deep-sea sediments of the Peruvian Margin“, The ISME Journal
Kaster AK, Meyer-Blackwell K, Passarelli B, Spormann A
(See online at https://doi.org/10.1038/ismej.2014.24)
