The VIMSS/ESPP2 project requires understanding of the microbial communities at contaminated field sites and, among other methods, will employ metagenomics in this endeavor. Metagenomics projects that seek to elucidate the population structure of microbial ecosystems are faced with the related computational challenges of classifying the sequences obtained and quantifying which organisms are present within a sample. Individually low-proportion species usually make up a large fraction of microbial communities, complicating their classification and quantification using traditional phylogenetic marker approaches. Such species usually don"t yield sufficient read depth to assemble into longer sequences, leaving fragments that rarely contain traditional markers such as the small subunit (SSU) rRNA gene. BLAST-based approaches for analysis of metagenomic sequences [1] compensate for this rarity of traditional markers, but may be confounded by genes that are subject to horizontal transfer or duplication. Another approach instead makes use only of reliable non-transferred single-copy genes [2] to classify and quantify the organisms present within a sample, but the application has so far been limited to the use of a fairly small set of universal genes found in all organisms. In this work, we have extended the latter approach, boosting the set of reliable marker genes from only about 30-40 universal genes to several hundred by identifying sets of single-copy genes that are not subject to inter-clade horizontal transfer through investigation of finished bacterial and archaeal genomes. These clade-oriented sequence markers allow for a method, which we have named "MicroCOSM", that greatly increases the probability that a marker will be found in any given sequence and therefore offers improved coverage for phylogenetic classification and quantification of microbial types in an environmental sample.

The metaMicrobesOnline database (freely available at http://meta.MicrobesOnline.org) offers phylogenetic analysis of genes from microbial genomes and metagenomes. Gene trees are constructed for canonical gene families such as COG and Pfam. Such gene trees allow for rapid homologue analysis and subfamily comparison of genes from multiple metagenomes and comparisons with genes from microbial isolates. Additionally, the genome browser permits genome context comparisons, which may be used to determine the closest sequenced genome or suggest functionally associated genes. Lastly, the domain browser permits rapid comparison of protein domain organization within genes of interest from metagenomes and complete microbial genomes.

Tropical forest soils decompose litter rapidly with frequent episodes of anoxia, making it likely that bacteria using alternate terminal electron acceptors (TEAs) such as iron play a large role in supporting decomposition under these conditions. The prevalence of many types of metabolism in litter deconstruction makes these soils useful templates for improving biofuel production. To investigate how iron availability affects decomposition, we cultivated feedstock-adapted consortia (FACs) derived from iron-rich tropical forest soils accustomed to experiencing frequent episodes of anaerobic conditions and frequently fluctuating redox. One consortium was propagated under fermenting conditions, with switchgrass as the sole carbon source in minimal media (SG only FACs), and the other consortium was treated the same way but received poorly crystalline iron as an additional terminal electron acceptor (SG + Fe FACs). We sequenced the metagenomes of both consortia to a depth of about 150 Mb each, resulting in a coverage of 26× for the more diverse SG + Fe FACs, and 81× for the relatively less diverse SG only FACs. Both consortia were able to quickly grow on switchgrass, and the iron-amended consortium exhibited significantly higher microbial diversity than the unamended consortium. We found evidence of higher stress in the unamended FACs and increased sugar transport and utilization in the iron-amended FACs. This work provides metagenomic evidence that supplementation of alternative TEAs may improve feedstock deconstruction in biofuel production.

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated "Enterobacter lignolyticus" SCF1 on minimal media with alkali lignin as the sole source of carbon. This organism was isolated anaerobically from tropical forest soils collected from the Short Cloud Forest site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are net methane producers. Because of its ability to grow on lignin anaerobically, we sequenced the genome. The genome of "E. lignolyticus" SCF1 is 4.81 Mbp with no detected plasmids, and includes a relatively small arsenal of lignocellulolytic carbohydrate active enzymes. Lignin degradation was observed in culture, and the genome revealed two putative laccases, a putative peroxidase, and a complete 4-hydroxyphenylacetate degradation pathway encoded in a single gene cluster.

Background

Microbial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO(2) make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways.

Results

We engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO(2).

Conclusions

We have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts.

AbstractBackgroundMicrobial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO2 make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways.ResultsWe engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO2.ConclusionsWe have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts.

In the version of this article initially published, the affiliation for Qizhi Zhang and Chris S. Henry was incorrectly shown as the University of Tennessee instead of Argonne National Laboratory. The affiliations have been corrected in the HTML and PDF versions of the article.