THE OBJECTIVE OF THIS PROPOSAL IS TO UNDERSTAND THE MECHANISMS USED BY MYCOBACTERIA TO ADAPT TO LIFE UNDER LSMMG AND TO DETERMINE WHETHER THEIR ABILITY TO FORM BIOFILM IS ALTERED UNDER LSMMG. M. MARINUM WILL BE THE MODEL ORGANISM. IT IS A GOOD MODEL FOR THE MORE POTENT HUMAN PATHOGENIC MYCOBACTERIA AS IT IS A BSL2 ORGANISM. M. MARINUM WILL BE GROWN IN HIGH ASPECT RATIO VESSELS USING THE ROTATING CELL CULTURE SYSTEM TO SIMULATE MICROGRAVITY. PREVIOUSLY WE WERE FUNDED BY NASA FOR 1 YEAR TO EXAMINE HOW M. MARINUM ALTERED GROWTH AND GENE EXPRESSION UNDER LSMMG. OUR PREVIOUS TRANSCRIPTOME ANALYSIS SUGGESTS THAT THE REL AND SIGMA FACTOR H (SIGH) STRESS RESPONSE PATHWAYS ARE ACTIVATED WHEN M. MARINUM ARE SUBJECTED TO LSMMG. WE PLAN TO EXTEND THESE STUDIES WITH THIS PROPOSAL. AIM 1 WILL DECREASE TRANSCRIPTION FROM THE REL GENE AND DETERMINE HOW THE LOSS OF THIS PATHWAY ALTERS M. MARINUM SURVIVAL AND ANTIBIOTIC RESISTANCE UNDER LSMMG. WE WILL USE CRISPR INTERFERENCE (CRISPRI) TO DISRUPT REL TRANSCRIPTION AND RNASEQ TO EXAMINE THE TRANSCRIPTOME. THIS WILL CONFIRM OUR HYPOTHESIS THAT THE REL STRINGENT RESPONSE IS NECESSARY FOR THE SURVIVAL OF LSMMG-GROWN M. MARINUM. WE ARE CURRENTLY STUDYING LSMMG AND THE SIGH TRANSCRIPTOME USING A SMALL GRANT FROM THE LASPACE CONSORTIUM. AIM 2 WILL EXAMINE BIOFILM FORMATION UNDER LSMMG USING MICROCARRIERS FOR BACTERIA ATTACHMENT IN THE HARVS. WE WILL ALSO EXAMINE THE TRANSCRIPTOME RESPONSE OF THE BACTERIA IN BIOFILMS. WE WILL USE WILD-TYPE AND CRISPRI-MODIFIED M. MARINUM WITH DECREASED SIGH OR REL TRANSCRIPTS IN AIM 2. WE HYPOTHESIZE THAT BIOFILM FORMATION WILL BE ENHANCED UNDER LSMMG AND THAT THE REL AS WELL AS SIGH STRESS RESPONSE PATHWAYS WILL BE REQUIRED FOR BIOFILM FORMATION. THIS WILL EXTEND OUR UNDERSTANDING OF MYCOBACTERIA SUBJECTED TO SPACE TRAVEL AND POTENTIALLY REVEAL MECHANISMS TO TARGET MYCOBACTERIUM INFECTIONS IN SPACE AND ON EARTH. THE PROPOSED WORK IS THEREFORE CONSISTENT WITH THE OBJECTIVES OF THE NASA SPACE BIOLOGY PROGRAM.