Microbial community gene expression in ocean surface waters

Abstract

Metagenomics is expanding our knowledge of the gene content, functional significance, and genetic variability in natural microbial communities. Still, there exists limited information concerning the regulation and dynamics of genes in the environment. We report here global analysis of expressed genes in a naturally occurring microbial community. We first adapted RNA amplification technologies to produce large amounts of cDNA from small quantities of total microbial community RNA. The fidelity of the RNA amplification procedure was validated with Prochlorococcus cultures and then applied to a microbial assemblage collected in the oligotrophic Pacific Ocean. Microbial community cDNAs were analyzed by pyrosequencing and compared with microbial community genomic DNA sequences determined from the same sample. Pyrosequencing-based estimates of microbial community gene expression compared favorably to independent assessments of individual gene expression using quantitative PCR. Genes associated with key metabolic pathways in open ocean microbial species-including genes involved in photosynthesis, carbon fixation, and nitrogen acquisition-and a number of genes encoding hypothetical proteins were highly represented in the cDNA pool. Genes present in the variable regions of Prochlorococcus genomes were among the most highly expressed, suggesting these encode proteins central to cellular processes in specific genotypes. Although many transcripts detected were highly similar to genes previously detected in ocean metagenomic surveys, a significant fraction ( approximately 50%) were unique. Thus, microbial community transcriptomic analyses revealed not only indigenous gene- and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.

Fig. 1.

Community-level gene expression profile based on GOS peptide database. (A) GOS protein clusters with DNA or cDNA matches at bit scores ≥40 are shown in the Venn diagram. Numbers of reads assigned to GOS protein clusters, when >70, are plotted for both cDNA-unique protein clusters and DNA-unique protein clusters. GOS protein clusters shared by DNA and cDNA libraries (shaded in gray) were further illustrated in B. (B) GOS protein clusters shared by cDNA and DNA libraries were ranked by their cluster-based expression ratio (representation of each cluster in the cDNA library normalized by its representation in the DNA library). Furthermore, each protein cluster was categorized (and color-coded) according to its abundance in the DNA library. Representative protein clusters were highlighted from each category and discussed in the text.

Fig. 2.

Distribution of different phylogenetic groups in DNA and cDNA libraries. Percentages of the different phylogenetic groups were calculated from the MEGAN analysis results at the phylum level cutoff (SI Table 5 shows a detailed list of the distribution of number of hits and percentages for all phyla). Not assigned reads are sequences with an NR hit but a bit score <40.

Fig. 3.

Prochlorococcus gene and transcript abundance using strain MIT9301 as a reference genome. (A) Rank abundance of the 20 genes with highest frequency in the raw cDNA, reflecting transcription of the entire Prochlorococcus population. (B) Frequency of DNA hits from the natural sample along the genome of MIT9301 normalized to gene length. (C) Frequency of cDNA hits from the natural sample normalized to the DNA values in B. Gray bars indicate the location of genomic islands identified through whole-genome analysis of cultured isolates (6). Core genes, genes present in all genomes of Prochlorococcus sequenced, are shown in blue. Flexible genes, genes not present in all genomes of Prochlorococcus sequenced, are shown in pink.