Synthetic biology aims to design and construct bacterial genomes harboring the minimum number of genes required for self-replicable life. However, the genome-reduced bacteria often show impaired growth under laboratory conditions that cannot be understood based on the removed genes. The unexpected phenotypes highlight our limited understanding of bacterial genomes. Here, we deploy adaptive laboratory evolution (ALE) to re-optimize growth performance of a genome-reduced strain. The basis for suboptimal growth is the imbalanced metabolism that is rewired during ALE. The metabolic rewiring is globally orchestrated by mutations in rpoD altering promoter binding of RNA polymerase. Lastly, the evolved strain has no translational buffering capacity, enabling effective translation of abundant mRNAs. Multi-omic analysis of the evolved strain reveals transcriptome- and translatome-wide remodeling that orchestrate metabolism and growth. These results reveal that failure of prediction may not be associated with understanding individual genes, but rather from insufficient understanding of the strain’s systems biology.

Pyruvate is an important intermediate of central carbon metabolism and connects a variety of metabolic pathways in Escherichia coli. Although the intracellular pyruvate concentration is dynamically altered and tightly balanced during cell growth, the pyruvate transport system remains unclear. Here, we identified a pyruvate transporter in E. coli using high-throughput transposon sequencing. The transposon mutant library (a total of 5 × 105 mutants) was serially grown with a toxic pyruvate analog (3-fluoropyruvate [3FP]) to enrich for transposon mutants lacking pyruvate transport function. A total of 52 candidates were selected on the basis of a stringent enrichment level of transposon insertion frequency in response to 3FP treatment. Subsequently, their pyruvate transporter function was examined by conventional functional assays, such as those measuring growth inhibition by the toxic pyruvate analog and pyruvate uptake activity. The pyruvate transporter system comprises CstA and YbdD, which are known as a peptide transporter and a conserved protein, respectively, whose functions are associated with carbon starvation conditions. In addition to the presence of more than one endogenous pyruvate importer, it has been suggested that the E. coli genome encodes constitutive and inducible pyruvate transporters. Our results demonstrated that CstA and YbdD comprise the constitutive pyruvate transporter system in E. coli, which is consistent with the tentative genomic locus previously suggested and the functional relationship with the extracellular pyruvate sensing system. The identification of this pyruvate transporter system provides valuable genetic information for understanding the complex process of pyruvate metabolism in E. coli.IMPORTANCE Pyruvate is an important metabolite as a central node in bacterial metabolism, and its intracellular levels are tightly regulated to maintain its functional roles in highly interconnected metabolic pathways. However, an understanding of the mechanism of how bacterial cells excrete and transport pyruvate remains elusive. Using high-throughput transposon sequencing followed by pyruvate uptake activity testing of the selected candidate genes, we found that a pyruvate transporter system comprising CstA and YbdD, currently annotated as a peptide transporter and a conserved protein, respectively, constitutively transports pyruvate. The identification of the physiological role of the pyruvate transporter system provides valuable genetic information for understanding the complex pyruvate metabolism in Escherichia coli.

Pollination in angiosperms does not involve direct contact between the pollen grain and the ovule. Pollen matures within the anthers and is transported, often by insects, birds, or other animals, to the stigma of another flower. When pollen reaches the stigma, it germinates, and a pollen tube grows down, carrying the sperm nuclei to the embryo sac. After double fertilization takes place, development of the embryo and endosperm begins. The seed matures within the ripening fruit; the germination of the seed initiates another life cycle. Successful pollination in many angiosperms depends on the regular attraction of pollinators such as insects, birds, and other animals, so that pollen is transferred between plants of the same species. When animals disperse pollen, they perform the same functions for flowering plants that they do for themselves when they actively search out mates. The relationship between plant and pollinator can be quite intricate. Mutations in either partner can block reproduction. If a plant flowers at the "wrong" time, the pollinator may not be available. If the morphology of the flower or pollinator is altered, there may be physical barriers to pollination. Clearly floral morphology has coevolved with pollinators and the result is much more complex and diverse than the initiation of four distinct whorls of organs described in chapter 41.