Whole Genome-scale Assessment of Gene Fitness of Novosphingobium Aromaticavorans During Spaceflight

In microgravity, bacteria undergo intriguing physiological adaptations. There have been few attempts to assess global bacterial physiological responses to microgravity, with most studies only focusing on a handful of individual systems. This study assessed the fitness of each gene in the genome of the aromatic compound-degrading Alphaproteobacterium Novosphingobium aromaticavorans during growth in spaceflight. This was accomplished using Comparative TnSeq, which involves culturing the same saturating transposon mutagenized library under two different conditions. To assess gene fitness, a novel comparative TnSeq analytical tool was developed, named TnDivA, that is particularly useful in leveraging biological replicates. In this approach, transposon diversity is represented numerically using a modified Shannon diversity index, which was then converted into effective transposon density. This transformation accounts for variability in read distribution between samples, such as cases where reads were dominated by only a few transposon inserts. Effective density values were analyzed using multiple statistical methods, including log-fold change, least-squares regression analysis, and Welch's t-test. The results obtained across applied statistical methods show a difference in the number of significant genes identified. However, the functional categories of genes important to growth in microgravity showed similar patterns. Lipid metabolism and transport, energy production, transcription, translation, and secondary metabolite biosynthesis and transport were shown to have high fitness during spaceflight. This suggests that core metabolic processes, including lipid and secondary metabolism, play an important role adapting to stress and promoting growth in microgravity.