Genome-Wide Association Analyses in the Model Rhizobium Ensifer meliloti.

Genome-wide association studies (GWAS) can identify genetic variants responsible for naturally occurring and quantitative phenotypic variation. Association studies therefore provide a powerful complement to approaches that rely on de novo mutations for characterizing gene function. Although bacteria should be amenable to GWAS, few GWAS have been conducted on bacteria, and the extent to which nonindependence among genomic variants (e.g., linkage disequilibrium [LD]) and the genetic architecture of phenotypic traits will affect GWAS performance is unclear. We apply association analyses to identify candidate genes underlying variation in 20 biochemical, growth, and symbiotic phenotypes among 153 strains of Ensifer meliloti For 11 traits, we find genotype-phenotype associations that are stronger than expected by chance, with the candidates in relatively small linkage groups, indicating that LD does not preclude resolving association candidates to relatively small genomic regions. The significant candidates show an enrichment for nucleotide polymorphisms (SNPs) over gene presence-absence variation (PAV), and for five traits, candidates are enriched in large linkage groups, a possible signature of epistasis. Many of the variants most strongly associated with symbiosis phenotypes were in genes previously identified as being involved in nitrogen fixation or nodulation. For other traits, apparently strong associations were not stronger than the range of associations detected in permuted data. In sum, our data show that GWAS in bacteria may be a powerful tool for characterizing genetic architecture and identifying genes responsible for phenotypic variation. However, careful evaluation of candidates is necessary to avoid false signals of association.IMPORTANCE Genome-wide association analyses are a powerful approach for identifying gene function. These analyses are becoming commonplace in studies of humans, domesticated animals, and crop plants but have rarely been conducted in bacteria. We applied association analyses to 20 traits measured in Ensifer meliloti, an agriculturally and ecologically important bacterium because it fixes nitrogen when in symbiosis with leguminous plants. We identified candidate alleles and gene presence-absence variants underlying variation in symbiosis traits, antibiotic resistance, and use of various carbon sources; some of these candidates are in genes previously known to affect these traits whereas others were in genes that have not been well characterized. Our results point to the potential power of association analyses in bacteria, but also to the need to carefully evaluate the potential for false associations.

Select and resequence reveals relative fitness of bacteria in symbiotic and free-living environments.

Assays to accurately estimate relative fitness of bacteria growing in multistrain communities can advance our understanding of how selection shapes diversity within a lineage. Here, we present a variant of the "evolve and resequence" approach both to estimate relative fitness and to identify genetic variants responsible for fitness variation of symbiotic bacteria in free-living and host environments. We demonstrate the utility of this approach by characterizing selection by two plant hosts and in two free-living environments (sterilized soil and liquid media) acting on synthetic communities of the facultatively symbiotic bacterium Ensifer meliloti We find (i) selection that hosts exert on rhizobial communities depends on competition among strains, (ii) selection is stronger inside hosts than in either free-living environment, and (iii) a positive host-dependent relationship between relative strain fitness in multistrain communities and host benefits provided by strains in single-strain experiments. The greatest changes in allele frequencies in response to plant hosts are in genes associated with motility, regulation of nitrogen fixation, and host/rhizobia signaling. The approach we present provides a powerful complement to experimental evolution and forward genetic screens for characterizing selection in bacterial populations, identifying gene function, and surveying the functional importance of naturally occurring genomic variation.

Transport Function of Rice Amino Acid Permeases (AAPs).

The transport function of four rice (Oryza sativa) amino acid permeases (AAPs), OsAAP1 (Os07g04180), OsAAP3 (Os06g36180), OsAAP7 (Os05g34980) and OsAAP16 (Os12g08090), was analyzed by expression in Xenopus laevis oocytes and electrophysiology. OsAAP1, OsAAP7 and OsAAP16 functioned, similarly to Arabidopsis AAPs, as general amino acid permeases. OsAAP3 had a distinct substrate specificity compared with other rice or Arabidopsis AAPs. OsAAP3 transported the basic amino acids lysine and arginine well but selected against aromatic amino acids. The transport of basic amino acids was further analyzed for OsAAP1 and OsAAP3, and the results support the transport of both neutral and positively charged forms of basic amino acids by the rice AAPs. Cellular localization using the tandem enhanced green fluorescent protein (EGFP)-red fluorescent protein (RFP) reporter pHusion showed that OsAAP1 and OsAAP3 localized to the plasma membrane after transient expression in onion epidermal cells or stable expression in Arabidopsis.

Innovation under cap-and-trade programs.

Policies incentivizing the private sector to reach its innovative potential in "clean" technologies are likely to play a key role in achieving climate stabilization. This article explores the relationship between innovation and cap-and-trade programs (CTPs)--the world's most prominent climate policy instrument--through empirical evidence drawn from successful CTPs for sulfur dioxide and nitrogen oxide control. The article shows that before trading began for these CTPs, analysts overestimated the value of allowances in a pattern suggestive of the frequent a priori overestimation of the compliance costs of regulation. When lower-than-expected allowance prices were observed, in part because of the unexpected range of abatement approaches used in the lead-up to trading, emissions sources chose to bank allowances in significant numbers and reassess abatement approaches going forward. In addition, commercially oriented inventive activity declined for emissions-reducing technologies with a wide range of costs and technical characteristics, dropping from peaks before the establishment of CTPs to nadirs a few years into trading. This finding is consistent with innovators deciding during trading that their research and development investments should be reduced, based on assessments of future market conditions under the relevant CTPs. The article concludes with a discussion of the results and their implications for innovation and climate policy.

Environmental risks of nanotechnology: National Nanotechnology Initiative funding, 2000-2004.

By considering risk in the early stages of a technology, costs of identifying important health and environmental impacts after a technology has widely diffused can be avoided. Nanotechnology, involving materials and objects less than 100 nm in size, is an important case in point. In this paper we analyze the research priorities discussed by various interest groups concerned with the environmental risks of nanotechnology, evaluate the distribution of federal environmental nanotechnology R&D funding, and discuss research in this field. Overall federal environmental R&D funding to date is limited and focuses more on the positive environmental applications of nanotechnology than on basic knowledge/research, tools for nanoenvironmental research, or the potential risks of nanotechnology. The situation began to change in 2004 when a significant increase occurred in federal R&D funding for the environmental implications of engineered nanomaterials. Though literature exits on the exposure, transport, and toxicity of incidental nanoparticles, little work has been published on the environmental risks of engineered nanoparticles.

Technology innovations and experience curves for nitrogen oxides control technologies.

This paper reviews the regulatory history for nitrogen oxides (NOx) pollutant emissions from stationary sources, primarily in coal-fired power plants. Nitrogen dioxide (NO2) is one of the six criteria pollutants regulated by the 1970 Clean Air Act where National Ambient Air Quality Standards were established to protect public health and welfare. We use patent data to show that in the cases of Japan, Germany, and the United States, innovations in NOx control technologies did not occur until stringent government regulations were in place, thus "forcing" innovation. We also demonstrate that reductions in the capital and operation and maintenance (O&M) costs of new generations of high-efficiency NOx control technologies, selective catalytic reduction (SCR), are consistently associated with the increasing adoption of the control technology: the so-called learning-by-doing phenomena. The results show that as cumulative world coal-fired SCR capacity doubles, capital costs decline to approximately 86% and O&M costs to 58% of their original values. The observed changes in SCR technology reflect the impact of technological advance as well as other factors, such as market competition and economies of scale.

Effect of government actions on technological innovation for SO2 control.

The relationship between government actions and innovation in environmental control technology is important for the design of cost-effective policies to achieve environmental goals. This paper examines such relationships for the case of sulfur dioxide control technology for U.S. coal-fired power plants. The study employs several complementary research methods, including analyses of key government actions, technology patenting activity, technology performance and cost trends, knowledge transfer activities, and expert elicitations. Our results indicate that government regulation appears to be a greater stimulus to inventive activity than government-sponsored research support alone, and that the anticipation of regulation also spurs inventive activity. Regulatory stringency focuses this activity along particular technical pathways and is a key factor in creating markets for environmental technologies. We also find that with greater technology adoption, both new and existing systems experience notable efficiency improvements and capital cost reductions. The important role of government in fostering knowledge transfer via technical conferences and other measures is also seen as an important factor in promoting environmental technology innovation.