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Summary Production of chemicals in microbes often employs potent biosynthetic enzymes, which can interact with the microbial native metabolism to affect cell fitness and product yield. However, production optimization largely relies on data collected from wild-type strains in the absence of metabolic perturbations, thus limiting their relevance to specific conditions. Here, we address this issue by coupling cell fitness to the production of thiamine diphosphate in Escherichia coli using a synthetic RNA biosensor. We use this strategy to interrogate a library of transposon mutants and elucidate the native gene network influencing both cell fitness and thiamine production. Ultimately, we identify effectors of the OxyR-Fur stress response that limit thiamine biosynthesis via alternative regulation of iron storage and Fe-S cluster inclusion in enzymes. This study presents a new approach for the reliable high-throughput identification of genetic targets of both known and unknown function that are directly relevant to a specific biosynthetic process. [ABSTRACT FROM AUTHOR]

Summary: Predictable operation of engineered biological circuitry requires the knowledge of host factors that compete or interfere with designed function. Here, we perform a detailed analysis of the interaction between constitutive expression from a test circuit and cell-growth properties in a subset of genetic variants of the bacterium Escherichia coli. Differences in generic cellular parameters such as ribosome availability and growth rate are the main determinants (89%) of strain-specific differences of circuit performance in laboratory-adapted strains but are responsible for only 35% of expression variation across 88 mutants of E. coli BW25113. In the latter strains, we identify specific cell functions, such as nitrogen metabolism, that directly modulate circuit behavior. Finally, we expose aspects of carbon metabolism that act in a strain- and sequence-specific manner. This method of dissecting interactions between host factors and heterologous circuits enables the discovery of mechanisms of interference necessary for the development of design principles for predictable cellular engineering. [Copyright &y& Elsevier]

Summary: We have used a human artificial chromosome (HAC) to manipulate the epigenetic state of chromatin within an active kinetochore. The HAC has a dimeric α-satellite repeat containing one natural monomer with a CENP-B binding site, and one completely artificial synthetic monomer with the CENP-B box replaced by a tetracycline operator (tetO). This HAC exhibits normal kinetochore protein composition and mitotic stability. Targeting of several tet-repressor (tetR) fusions into the centromere had no effect on kinetochore function. However, altering the chromatin state to a more open configuration with the tTA transcriptional activator or to a more closed state with the tTS transcription silencer caused missegregation and loss of the HAC. tTS binding caused the loss of CENP-A, CENP-B, CENP-C, and H3K4me2 from the centromere accompanied by an accumulation of histone H3K9me3. Our results reveal that a dynamic balance between centromeric chromatin and heterochromatin is essential for vertebrate kinetochore activity. [Copyright &y& Elsevier]

Cleavage factor Im (CF Im) is required for the first step in pre-mRNA 3′-end processing and can be reconstituted in vitro from its heterologously expressed 25- and 68- kDa subunits. The binding of CF Im to the pre-mRNA is one of the earliest steps in the assembly of the cleavage and polyadenylation machinery and facilitates the recruitment of other processing factors. We identified regions in the subunits of CF Im involved in RNA binding, protein-protein interactions, and subcellular localization. CF Im68 has a modular domain organization consisting of an N-terminal RNA recognition motif and a C-terminal alternating charge domain. However, the RNA recognition motif of CF Im68 on its own is not sufficient to bind RNA but is necessary for association with the 25-kDa subunit. RNA binding appears to require a CF Im68/25 heterodimer. Whereas multiple protein interactions with other 3′-end-processing factors are detected with CF Im25, CF Im68 interacts with SRp20, 9G8, and hTra2β, members of the SR family of splicing factors, via its C-terminal alternating charge domain. This domain is also required for targeting CF Im68 to the nucleus. However, CF Im68 does not concentrate in splicing speckles but in foci that partially colocalize with paraspeckles, a subnuclear component in which other proteins involved in transcriptional control and RNA processing have been found. [ABSTRACT FROM AUTHOR]