Micrococcus Luteus Uspa-like Protein usp712 Controls Exponential Growth by up Regulation of Required Pathways Including Resuscitation Promoting Factor

Bacterial growth can be divided into four phases; the lag, exponential, stationary, and dormancy phase. The lag phase plays an important role in the shelf life of food products and the pathogenicity of bacterial infection. The lag phase is characterized by considerable metabolic activity with cells not actively dividing. The Micrococcus luteus (M. luteus) universal stress protein (UspA616) while not seen in exponential growth is among the most highly expressed proteins as the bacteria enters into dormancy. Another Universal stress protein from ML, UspA712, has been shown to be expressed in exponential growth but is not expressed when cells enter a dormant state by nutrient starvation. Knockout of the uspA712 gene by cassette replacement produces a phenotype that shows a prolonged lag phase compared to wild type ML when grown on rich (LB) media. Growth on minimal pyruvate shows no lag in growth while minimal acetate, lactate, and succinate show considerably smaller lag phases. The mechanism of universal stress proteins in regulating lag phase of bacteria have never been shown. The extended lag phase in MLusp712::kan cells was found to be shortened upon the addition of growth factors, first in the form of spent media and then purified fractions in picomole amounts of the resuscitation promoting factor (RPF). Higher concentrations of RPF was found to inhibit the growth of the bacteria. Transcriptomic analysis was used to study changes in gene transcription at different time points or stages of M. luteus growth, grown on either rich (LB) media or acetate minimal media (AMM) media and compared these results with the MLusp712::kan knockout strain transcriptomics data. The results show that several amino acid transporter genes are downregulated in the lag phase of the mutants suggesting M. luteus’s inability to incorporate amino acid metabolites into the cells which are present in LB media. Our results also showed that genes controlling the cell division machinery and key metabolic pathway genes are also downregulated. The Usp712 was purified as a dimer, crystals were obtained, and diffracted at a 1.5 Å resolution. The single domain Usp712 aligns to the N-terminal domain of the Usp616 protein with the possibility of an in vivo interaction. We conclude that this interaction and the different stages of both proteins’ expression might be assisting in switching between exponential growth and dormancy. I hypothesize that UspA712 binds to UspA616 preventing dormancy while the loss of UspA712 expression allows UspA616 to interact with another protein X in dormancy to upregulate gene transcription similar to Eukaryotic transcription factors and this protein interaction allows expression of genes needed for bacterial dormancy.Biology and Biochemistry, Department o