STREPTOCOCCUS MUTANS IS A MEMBER OF THE HUMAN ORAL MICROBIOTA AND A MAJOR ETIOLOGICAL AGENT OF DENTAL CARIES (CAVITIES) DUE TO ITS ABILITY TO FORM BIOFILM ADAPT TO ORAL CAVITY STRESSES AND RAPIDLY FERMENT CARBOHYDRATES. THESE CHARACTERISTICS PROMOTE ITS PREDOMINATION OF DENTAL PLAQUE BIOFILM AND COMBINED WITH REPEATED CYCLES OF FERMENTATIVE ACID PRODUCTION LEADS TO TOOTH DEMINERALIZATION AND CAVITY FORMATION. USING HIGH ASPECT ROTATING VESSELS (HARVS) IN THE LOW-SHEAR MODELLED MICROGRAVITY (LSMMG) MODEL WE PREVIOUSLY DEMONSTRATED THAT S. MUTANS DISPLAYS SEVERAL PHYSIOLOGICAL CHANGES IN RESPONSE TO SIMULATED MICROGRAVITY INCLUDING ALTERED CELL AGGREGATION ENHANCED SENSITIVITY TO OXIDATIVE STRESS AND GLOBAL ALTERATIONS IN GENE EXPRESSION. FURTHERMORE SIMULATED MICROGRAVITY EXPOSURE HAS BEEN SHOWN BY OTHERS TO CAUSE INCREASED MANDIBULAR AND ALVEOLAR BONE LOSS AND DECREASED SALIVA FLOW FACTORS THAT COULD PREDISPOSE ASTRONAUTS TO CARIES. THEREFORE THE GOAL OF THIS APPLICATION IS TO CHARACTERIZE THE PHYSIOLOGICAL AND GENETIC RESPONSE OF S. MUTANS TO SIMULATED MICROGRAVITY FOCUSING SPECIFICALLY ON BIOFILM DEVELOPMENT STRESS RESISTANCE AND IDENTIFYING KEY GENE(S) AND/OR THE CORE TRANSCRIPTOMIC RESPONSE THAT FAVORS S. MUTANS SURVIVAL DURING MICROGRAVITY GROWTH. THIS WILL BE ACCOMPLISHED BY THREE OBJECTIVES: (1) S. MUTANS WILL BE CULTURED IN BIOFILM-PROMOTING MEDIA UNDER SIMULATED MICROGRAVITY CONDITIONS USING THE LSMMG AND RANDOM-POSITIONING MACHINE (RPM) GROUND-BASED MODELS OF GROWTH. CHANGES IN CELL AGGREGATION STRESS RESISTANCE AND GENE EXPRESSION PROFILING (VIA RNASEQ) WILL BE ASSESSED WITH RESPECT TO NORMAL GRAVITY CONTROL CULTURES AND THESE RESULTS WILL BE COMPARED USING BIOINFORMATICS AND STATISTICAL TECHNIQUES. THIS ANALYSIS WILL ALLOW US TO MAKE MORE REFINED PREDICTIONS AS TO WHICH PHYSIOLOGICAL AND GENE EXPRESSION CHANGES ARE LIKELY TO OCCUR IN S. MUTANS DURING SPACE FLIGHT AND HOW THIS MAY IMPACT ITS PATHOGENIC POTENTIAL; (2) CHANGES IN S. MUTANS BIOFILM DEVELOPMENT IN RESPONSE TO SIMULATED MICROGRAVITY WILL BE STUDIED USING 2 GROUND-BASED MODELS (LSMMG RPM). S. MUTANS WILL BE ASSESSED IN THESE MODELS FOR BIOFILM VIABILITY AND ULTRASTRUCTURE EXOPOLYSACCHARIDE PRODUCTION AND FOR ITS ABILITY TO COMPETE WITH NON-CARIOGENIC ORAL STREPTOCOCCI; (3) A S. MUTANS TN-SEQ LIBRARY WILL BE SCREENED FOR MUTANTS THAT LOSE COMPETITIVE FITNESS DURING LSMMG AND RPM-BASED SIMULATED MICROGRAVITY GROWTH. THIS WILL BE ACCOMPLISHED BY USING HIGH-THROUGHPUT DNA SEQUENCING OF POOLED BATCHES OF TN MUTANTS PRE- AND POST-HARV GROWTH UNDER BOTH SIMULATED MICROGRAVITY AND NORMAL GRAVITY CONDITIONS. THIS RESEARCH IS DIRECTED AT UNDERSTANDING THE PHYSIOLOGICAL AND GENETIC RESPONSE OF S. MUTANS TO MICROGRAVITY ESPECIALLY WITH RESPECT TO MULTICELLULAR BEHAVIOR (BIOFILM FORMATION) STRESS RESISTANCE AND THE GENES THAT FAVOR S. MUTANS SURVIVAL DURING MICROGRAVITY GROWTH. THEREFORE THIS APPLICATION DIRECTLY ADDRESSES GUIDING QUESTIONS MB-1 MB-3 AND MB-4 OF THE 2016-2025 SB SCIENCE PLAN EACH OF WHICH ALIGNS WITH PRIORITY P1 OF THE NRCS 2011 DECADAL SURVEY REPORT.