While unique responses in microorganisms are well documented for short duration culture, the impact of long duration, multigenerational growth of microorganisms in the chronic stress of microgravity…

This is a $2,508,591.16 grant awarded by the National Aeronautics and Space Administration (NASA) to Arizona State University (ASU) to study the long-term, multigenerational effects of microgravity on microbial populations. The research aims to characterize genomic, epigenetic, and transcriptomic changes in the model pathogen Salmonella Typhimurium in response to chronic stress and selective pressures in the spaceflight environment. This grant addresses two key research emphases from NASA's ISS National Laboratory Opportunities announcement: ISS Microbial Observatory Studies and Spaceflight Omics Studies. The research will provide insight into the potential impacts of microbial evolution on crew health and vehicle system performance during long-duration space missions. No set-aside designation was used for this award. As a comprehensive public research university, ASU has a long history of supporting NASA's space biology research through both contract and grant funding.

While unique responses in microorganisms are well documented for short duration culture, the impact of long duration, multigenerational growth of microorganisms in the chronic stress of microgravity has not been investigated. The implications of this GAP in knowledge are tremendous, as the response to short duration growth in microgravity alters gene expression, pathogenesisrelated stress responses (EX, biofilm formation and resistance to antibiotics) and virulence. Moreover, IT is known that stress responses (including chronic stress) can increase mutagenesis in bacteria leading to genomic plasticity, which may provide novel mechanistic insight into genomic instability underlying resistance to antibiotics, pathogenicity and virulence. We propose that long-term growth in spaceflight culture will induce heritable genomic and epigenetic changes in microorganisms with adverse phenotypic outcomes that could have a profound impact on the risk to crew health and vehicle system performance. Our previous studies aboard the space shuttle investigated the response of the model enteric pathogen, salmonella enterica serovar typhimurium (s. Typhimurium) to short duration spaceflight culture and demonstrated a significant increase in virulence in a murine model of infection, in addition to biofilm formation, as compared to identically grown cultures on the ground. In addition, global microarray and proteomic analyses revealed that 167 transcripts (including small noncoding rnas/ncrnas) and 73 proteins changed expression, with the evolutionarily conserved protein HFQ identified as a likely global regulator involved in the response to this environment. HFQ binds to small ncrnas thereby facilitating their association with mrnas, the outcome of which plays a diverse role in regulating gene expression, virulence, stress responses, antibiotic resistance, DNA methylation and genome stability. We hypothesize that long-term multigenerational culture of s. Typhimurium in the spaceflight environment will result in stress-induced mutations and genomic instability as reflected in changes in the genomic, epigenetic, transcriptomic and select phenotypic characteristics (including virulence) that will reflect a central role for HFQ as a global regulator of these heritable responses. To address this hypothesis, we propose to characterize the impact of long term spaceflight culture (~300 generations) of an s. Typhimurium wild type and HFQ mutant on the genomic, epigenetic, transcriptomic, virulence and pathogenesis-related characteristics of these strains. This proposal addresses research emphasis 2 - ISS microbial observatory studies from section b.3 of this announcement, as IT is designed to take advantage of the technological maturity, low cost, and speed of genomic analyses and the rapid generation time of microbes to monitor the evolution of microbial genomic changes in response to the selective pressures present in the spaceflight environment. In addition, by employing genomic, transcriptomic, and epigenomic analysis, this proposal also addresses the research emphasis 1 spaceflight omics studies in section b.2. In its design, this proposal will address the NASA space biology plan, overarching questions CMM-1A, CMM-1D and CMM5. This proposal also directly addresses the highest priority recommendations from the decadal survey for the establishment of an ISS microbial observatory conducting long-term, multigenerational studies of microbial population dynamics (priority P1). In addition, this study analyzes microbial growth and physiological responses to the multiple stimuli encountered in spaceflight environments, as described by the panel in priority P2.