UNDERSTANDING THE ORIGIN EVOLUTION AND ORGANIZATION OF MULTICELLULARITY HAS BEEN IDENTIFIED AS A KEY OBJECTIVE BY THE ASTROBIOLOGY COMMUNITY AND NASA. PAST STUDIES ON THE TRANSITION FROM SINGLE CELLS TO MULTICELLULAR ENTITIES MOSTLY FOCUSED ON VOLVOCINE ALGAE AND EARLY RADIATING ANIMAL TAXA AS EXPERIMENTAL SYSTEMS. WHILE MULTICELLULAR FORMS OF BACTERIA EXIST THEIR ORGANIZATION APPEARSCOMPARATIVELY SIMPLE AND THE PHENOMENON SEEMS TO OCCUR ONLY AS AN ADAPTATION TO CHANGING ENVIRONMENTAL CONDITIONS OR AS A SINGLE STEP IN A COMPLEX LIFE CYCLE.A GROUP OF DELTA PROTEOBACTERIA TERMED MULTICELLULAR MAGNETOTACTIC BACTERIA MMB CONSTITUTES THE ONLY KNOWN EXCEPTION TO THIS VIEW. MMB ARE MONO SPECIES CONSORTIA THAT ARE COMPRISED OF 10 60 CELLS ARRANGED IN SYMMETRY AROUND A CENTRAL A CELLULAR COMPARTMENT. THE LIFE CYCLE OF MMB HAS NO KNOWN UNICELLULAR STAGE. DIVISION OCCURS BY SEPARATION OF A MMB INTO TWO APPARENTLY IDENTICAL DAUGHTER CONSORTIA WHILE DISAGGREGATED CELLS RAPIDLY LOSE VIABILITY. THESE CHARACTERISTICS RENDER MMB THE ONLY IDENTIFIED BACTERIA WITH AN OBLIGATE MULTICELLULAR LIFESTYLE AND MAKE THEM A PRIME SUBJECT FOR THE STUDY OF THE EARLY EVOLUTION OF ADVANCED LIFE MOST IMPORTANTLY THE ORIGINS OF AND ENVIRONMENTAL FACTORS DRIVING MULTICELLULARITY AS WELL AS THE CELLULAR ORGANIZATION OF COMPLEX LIFE. WHILE SEVERAL GENERA EXHIBITING THIS LIFE STRATEGY EXIST IN THE ENVIRONMENT WE ARE LARGELY IGNORANT OF THE BIOLOGY OF THESE UNCULTURED BACTERIA.THE MOST IMPORTANT GOALS OF THIS PROJECT ARE(I) TO UNDERSTAND THE ECOPHYSIOLOGY OF MMB DETERMINE SUBSTRATES THAT SUSTAIN THEIR METABOLISM AND IDENTIFY THE GEOCHEMICAL PARAMETERS THAT DRIVE THEIR ECOLOGY(II) TO DETERMINE WHETHER MMB CONSORTIA EXHIBIT CELLULAR COOPERATION OR METABOLIC DIVISION OF LABOR TWO KEY CHARACTERISTICS OF MULTICELLULAR LIFE; AND(III) TO DEVELOP HYPOTHESES ON WHICH GENETIC TRAITS AND EVOLUTIONARY EVENTS MIGHT HAVE BEEN INVOLVED IN THE DEVELOPMENT OF MULTICELLULARITY IN MMB.THIS PROJECT ADDRESSES NASA GOALS 4 AND 5 IN PARTICULAR OBJECTIVES 4.2. FOUNDATIONS OF COMPLEX LIFE 5.1. ENVIRONMENT-DEPENDENT MOLECULAR EVOLUTION IN MICROORGANISMS AND 5.2. CO EVOLUTION OF MICROBIAL COMMUNITIES.WE PROPOSE TO STUDY THE BIOLOGY OF MMB VIA A COMBINATION OF CUTTING EDGE MOLECULAR BIOLOGICAL APPROACHES THAT DO NOT DEPEND ON THE AVAILABILITY OF A PURE CULTURE. RELYING ON THE MAGNETOTACTIC PROPERTIES OF MMB WE WILL USE METAGENOMICS TARGETED AT MAGNETICALLY ENRICHED CONSORTIA TO OBTAIN DRAFT GENOMES FROM THREE DIFFERENT LOCATIONS WITH THE STATES OF CALIFORNIA AND MASSACHUSETTS. BY COMPARING THE GENOMES OF UNICELLULAR RELATIVES AS WELL AS OTHER MICROBES CAPABLE OF MULTICELLULAR GROWTH TO THOSE OF MMB WE WILL IDENTIFY POTENTIAL FACTORS UNDERLYING THEIR MULTICELLULAR LIFESTYLE. THESE INCLUDE FOR EXAMPLE CANDIDATE MOLECULES INVOLVED IN INTER- CELLULAR COMMUNICATION AND CELL ADHESION THAT MIGHT BE INVOLVED IN SHAPING CONSORTIA MORPHOLOGY AS WELL AS PROTEINS POTENTIALLY INVOLVED IN TRANSCRIPTIONAL CONTROL AND REGULATION PROCESSES THAT HAVE BEEN SHOWN TO BE OF IMPORTANCE IN OTHER MULTICELLULAR ORGANISMS.THE GENOMES WILL BE USED TO FORMULATE HYPOTHESES OF POTENTIAL ENERGY CONSERVING METABOLISMS. THESE WILL BE TESTED IN SITU USING A COMBINATION OF BIOORTHOGONAL NON-CANONICAL AMINO ACID TAGGING (BONCAT) IN COMBINATION WITH FLUORESCENCE MICROSCOPY AS WELL AS STABLE ISOTOPE LABELING IN COMBINATION WITH NANO SCALE SECONDARY ION MASS SPECTROMETRY. THESE EXPERIMENTS WILL REVEAL WHETHER ALL CELLS WITHIN A CONSORTIUM ARE EQUALLY METABOLICALLY ACTIVE OR INDIVIDUAL CELLS ARE DIFFERENTIATED AND EXHIBIT METABOLIC DIVISION OF LABOR.BONCAT WILL FURTHER BE USED TO TEST HOW SPECIFIC GEOCHEMICAL PARAMETERS (E.G. TEMPERATURE NITROGEN SOURCE TRACE METALS AVAILABILITY) INFLUENCE MMB ACTIVITY AND GROWTH. THE RESULTS OF THESE EXPERIMENTS WILL GUIDE US IN THE DEVELOPMENT OF NOVEL ENRICHMENT MEDIA TOULTIMATELY BRING M