Unraveling the stratification of an iron-oxidizing microbial mat by metatranscriptomics

Abstract

A metatranscriptomic approach was used to study community gene expression in a naturally occurring iron-rich microbial mat. Total microbial community RNA was reversely transcribed and sequenced by pyrosequencing. Characterization of expressed gene sequences provided accurate and detailed information of the composition of the transcriptionally active community and revealed phylogenetic and functional stratifications within the mat. Comparison of 16S rRNA reads and delineation of OTUs showed significantly lower values of metatranscriptomic-based richness and diversity in the upper parts of the mat than in the deeper regions. Taxonomic affiliation of rRNA sequences and mRNA genome recruitments indicated that iron-oxidizing bacteria affiliated to the genus Leptothrix, dominated the community in the upper layers of the mat. Surprisingly, type I methanotrophs contributed to the majority of the sequences in the deep layers of the mat. Analysis of mRNA expression patterns showed that genes encoding the three subunits of the particulate methane monooxygenase (pmoCAB) were the most highly expressed in our dataset. These results provide strong hints that iron-oxidation and methane-oxidation occur simultaneously in microbial mats and that both groups of microorganisms are major players in the functioning of this ecosystem.

Figure 1. Relative abundance and distribution of microbial taxa in the SSU and LSU rRNA transcript libraries.

The rRNA reads from each triplicate are combined and average values shown. (a) Comparison of the five samples at the phylum level. (b) Comparison of the combined two surfaces (S1 and S2) and the subjacent depth (D1 and D2) samples at the phylum, class and order level. Error bars indicate the standard deviations among the replicates.

Figure 2. Estimation of the bacterial community diversity.

Ribosomal RNA reads covering the variable 16S rRNA gene regions V3, V4 and V5 (393–925 E. coli positions) were analyzed on 462 positions (56.600 sequences). (a) Rarefaction curves for all five samples with combined replicates. For illustration purposes the curves were shorted to 7.000 sequences sampled. (b) Rank abundance distribution of OTUs from Methylococcales, Burkholderiales and Nitrosomonadales. All five samples and replicates were combined. (c) OTU abundance ratio of Methylococcales, Burkholderiales and Nitrosomonadales. Parentheses: Number of sequences included. Error bars: standard deviation. P-value from Wilcoxon signed rank test for each OTU group comparing surface versus depth: Nitrosomonadales: p<10⁻⁵, Burkholderiales p<10⁻⁸, Methylococcales p<10⁻⁷.

Figure 3. Linkage of community diversity to metabolic function.

(a) Relative abundance of mRNA reads matching selected genomes from bacteria with relevant characteristic metabolisms: iron oxidation (red), methanotrophy (blue) and anaerobic iron respiration (green). (b) Relative abundance of pmoCAB matching transcripts coding for the three subunits of the methane monooxygenase. Error bars correspond to standard deviation from the replicates.

Figure 4. Schematic profile of the iron rich microbial mat with sampling sites.