Forests accumulate and store large amounts of carbon (C), and a substantial fraction of this stock is contained in deadwood. This transient pool is subject to decomposition by deadwood-associated organisms, and in this process it contributes to CO2 emissions. Although fungi and bacteria are known to colonize deadwood, little is known about the microbial processes that mediate carbon and nitrogen (N) cycling in deadwood. In this study, using a combination of metagenomics, metatranscriptomics, and nutrient flux measurements, we demonstrate that the decomposition of deadwood reflects the complementary roles played by fungi and bacteria. Fungi were found to dominate the decomposition of deadwood and particularly its recalcitrant fractions, while several bacterial taxa participate in N accumulation in deadwood through N fixation, being dependent on fungal activity with respect to deadwood colonization and C supply. Conversely, bacterial N fixation helps to decrease the constraints of deadwood decomposition for fungi. Both the CO2 efflux and N accumulation that are a result of a joint action of deadwood bacteria and fungi may be significant for nutrient cycling at ecosystem levels. Especially in boreal forests with low N stocks, deadwood retention may help to improve the nutritional status and fertility of soils. IMPORTANCE Wood represents a globally important stock of C, and its mineralization importantly contributes to the global C cycle. Microorganisms play a key role in deadwood decomposition, since they possess enzymatic tools for the degradation of recalcitrant plant polymers. The present paradigm is that fungi accomplish degradation while commensalist bacteria exploit the products of fungal extracellular enzymatic cleavage, but this assumption was never backed by the analysis of microbial roles in deadwood. This study clearly identifies the roles of fungi and bacteria in the microbiome and demonstrates the importance of bacteria and their N fixation for the nutrient balance in deadwood as well as fluxes at the ecosystem level. Deadwood decomposition is shown as a process where fungi and bacteria play defined, complementary roles.

SummaryWhen recombinant DNA technology was developed more than 40 years ago, no one could have imagined the impact it would have on both society and the scientific community. In the field of genetic engineering, the most important tool developed was the plasmid vector. This technology has been continuously expanding and undergoing adaptations. Here, we provide a detailed view following the evolution of vectors built throughout the years destined to study microorganisms and their peculiarities, including those whose genomes can only be revealed through metagenomics. We remark how synthetic biology became a turning point in designing these genetic tools to create meaningful innovations. We have placed special focus on the tools for engineering bacteria and fungi (both yeast and filamentous fungi) and those available to construct metagenomic libraries. Based on this overview, future goals would include the development of modular vectors bearing standardized parts and orthogonally designed circuits, a task not fully addressed thus far. Finally, we present some challenges that should be overcome to enable the next generation of vector design and ways to address it.

Regulation of gene expression in bacteria results from the interplay between hundreds of transcriptional factors (TFs) at target promoters. However, how the arrangement of binding sites for TFs generates the regulatory logic of promoters is not well-known. Here, we generated and fully characterized a library of synthetic complex promoters for the global regulators, CRP and IHF, in Escherichia coli, which are formed by a weak -35/-10 consensus sequence preceded by four combinatorial binding sites for these two TFs. Using this approach, we found that while cis-elements for CRP preferentially activate promoters when located immediately upstream of the promoter consensus, binding sites for IHF mainly function as "UP" elements and stimulate transcription in several different architectures in the absence of this protein. However, the combination of CRP- and IHF-binding sites resulted in emergent properties in these complex promoters, where the activity of combinatorial promoters cannot be predicted from the individual behavior of its components. Taken together, the results presented here add to the information on architecture-logic of complex promoters in bacteria.

No abstract

Bacterial promoters are usually formed by multiple cis-regulatory elements recognized by a plethora of transcriptional factors (TFs). From those, global regulators are key elements since these TFs are responsible for the regulation of hundreds of genes in the bacterial genome. For instance, Fis and IHF are global regulators that play a major role in gene expression control in Escherichia coli, and usually, multiple cis-regulatory elements for these proteins are present at target promoters. Here, we investigated the relationship between the architecture of the cis-regulatory elements for Fis and IHF in E. coli. For this, we analyze 42 synthetic promoter variants harboring consensus cis-elements for Fis and IHF at different distances from the core −35/−10 region and in various numbers and combinations. We first demonstrated that although Fis preferentially recognizes its consensus cis-element, it can also recognize, to some extent, the consensus-binding site for IHF, and the same was true for IHF, which was also able to recognize Fis binding sites. However, changing the arrangement of the cis-elements (i.e., the position or number of sites) can completely abolish the non-specific binding of both TFs. More remarkably, we demonstrated that combining cis-elements for both TFs could result in Fis and IHF repressed or activated promoters depending on the final architecture of the promoters in an unpredictable way. Taken together, the data presented here demonstrate how small changes in the architecture of bacterial promoters could result in drastic changes in the final regulatory logic of the system, with important implications for the understanding of natural complex promoters in bacteria and their engineering for novel applications.