The Target of Rapamycin (TOR) protein is a Ser/Thr kinase that functions in two distinct multiprotein complexes: TORC1 and TORC2. These conserved complexes regulate many different aspects of cell growth in response to intracellular and extracellular cues. Here we report that the AGC kinase Sch9 is a substrate of yeast TORC1. Six amino acids in the C terminus of Sch9 are directly phosphorylated by TORC1. Phosphorylation of these residues is lost upon rapamycin treatment as well as carbon or nitrogen starvation and transiently reduced following application of osmotic, oxidative, or thermal stress. TORC1-dependent phosphorylation is required for Sch9 activity, and replacement of residues phosphorylated by TORC1 with Asp/Glu renders Sch9 activity TORC1 independent. Sch9 is required for TORC1 to properly regulate ribosome biogenesis, translation initiation, and entry into G0 phase, but not expression of Gln3-dependent genes. Our results suggest that Sch9 functions analogously to the mammalian TORC1 substrate S6K1 rather than the mTORC2 substrate PKB/Akt.

Yeast cells sense the amount and quality of external nutrients through multiple interconnected signaling networks, which allow them to adjust their metabolism, transcriptional profile and developmental program to adapt readily and appropriately to changing nutritional states. We present our current understanding of the nutritional sensing networks yeast cells rely on for perceiving the nutritional landscape, with particular emphasis on those sensitive to carbon and nitrogen sources. We describe the means by which these networks inform the cell's decision among the different developmental programs available to them-growth, quiescence, filamentous development, or meiosis/sporulation. We conclude that the highly interconnected signaling networks provide the cell with a highly nuanced view of the environment and that the cell can interpret that information through a sophisticated calculus to achieve optimum responses to any nutritional condition.

Significance: The incidence, cost, morbidity, and mortality associated with nonhealing of chronic skin wounds are dramatic. With the increasing numbers of people with obesity, chronic medical conditions, and an increasing life expectancy, the healthcare cost of non-healing ulcers has recently been estimated at $25 billion annually in the United States. The role played by bacterial biofilm in chronic wounds has been emphasized in recent years, particularly in the context of the prolongation of the inflammatory phase of repair. Recent Advances: Rapid high-throughput genomic approaches have revolutionized the ability to identify and quantify microbial organisms from wounds. Defining bacterial genomes and using genetic approaches to knock out specific bacterial functions, then studying bacterial survival on cutaneous wounds is a promising strategy for understanding which genes are essential for pathogenicity. Critical Issues: When an animal sustains a cutaneous wound, understanding mechanisms involved in adaptations by bacteria and adaptations by the host in the struggle for survival is central to development of interventions that favor the host. Future Directions: Characterization of microbiomes of clinically well characterized chronic human wounds is now under way. The use of in vivo models of biofilm-infected cutaneous wounds will permit the study of the mechanisms needed for biofilm formation, persistence, and potential synergistic interactions among bacteria. A more complete understanding of bacterial survival mechanisms and how microbes influence host repair mechanisms are likely to provide targets for chronic wound therapy. SCOPE AND SIGNIFICANCEThis review provides a current overview of how microbial biofilm may participate in the pathogenesis of chronic wounds. It will consider how prolonged host inflammatory responses to microbes may also participate in the pathogenesis of chronic wounds.Better understanding of microbial biofilm and host responses to biofilm may provide therapeutic targets in the management of chronic wounds. TRANSLATIONAL RELEVANCEUnderstanding mechanisms by which microbial biofilms may prevent cutaneous wounds from healing and finding strategies that control excessive host inflammation are likely to provide therapeutic approaches to improve chronic wound healing. For example, gallium-based agents have anti-Pseudomonas biofilm effects in vitro and may be an adjunct to antibiotic treatment of biofilms in chronic wounds, where antibiotic resistance is very high.

Addition of glucose to yeast cells increases their growth rate and results in a massive restructuring of their transcriptional output. We have used microarray analysis in conjunction with conditional mutations to obtain a systems view of the signaling network responsible for glucose-induced transcriptional changes. We found that several well-studied signaling pathways-such as Snf1 and Rgt-are responsible for specialized but limited responses to glucose. However, 90% of the glucoseinduced changes can be recapitulated by the activation of protein kinase A (PKA) or by the induction of PKB (Sch9). Blocking signaling through Sch9 does not interfere with the glucose response, whereas blocking signaling through PKA does. We conclude that both Sch9 and PKA regulate a massive, nutrient-responsive transcriptional program promoting growth, but that they do so in response to different nutritional inputs. Moreover, activating PKA completely recapitulates the transcriptional growth program in the absence of any increase in growth or metabolism, demonstrating that activation of the growth program results solely from the cell's perception of its nutritional status.

The essential functions of a bacterial pathogen reflect the most basic processes required for its viability and growth, and represent potential therapeutic targets. Most screens for essential genes have assayed a single condition-growth in a rich undefined medium-and thus have not distinguished genes that are generally essential from those that are specific to this particular condition. To help define these classes for Pseudomonas aeruginosa, we identified genes required for growth on six different media, including a medium made from cystic fibrosis patient sputum. The analysis used the Tn-seq circle method to achieve high genome coverage and analyzed more than 1,000,000 unique insertion positions (an average of one insertion every 6.0 bp). We identified 352 general and 199 condition-specific essential genes. A subset of assignments was verified in individual strains with regulated expression alleles. The profile of essential genes revealed that, compared with Escherichia coli, P. aeruginosa is highly vulnerable to mutations disrupting central carbon-energy metabolism and reactive oxygen defenses. These vulnerabilities may arise from the stripped-down architecture of the organism's carbohydrate utilization pathways and its reliance on respiration for energy generation. The essential function profile thus provides fundamental insights into P. aeruginosa physiology as well as identifying candidate targets for new antibacterial agents.Tn-seq | ESKAPE | cystic fibrosis | antibiotic target | sputum