Machine learning analysis of RB-TnSeq fitness data predicts functional gene modules in Pseudomonas putida KT2440.

There is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules ("functional modules"), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida. Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets. IMPORTANCE: This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

Richness and density jointly determine context dependence in bacterial interactions.

Pairwise interactions are often used to predict features of complex microbial communities due to the challenge of measuring multi-species interactions in high dimensional contexts. This assumes that interactions are unaffected by community context. Here, we used synthetic bacterial communities to investigate that assumption by observing how interactions varied across contexts. Interactions were most often weakly negative and showed a phylogenetic signal among genera. Community richness and total density emerged as strong predictors of interaction strength and contributed to an attenuation of interactions as richness increased. Population level and per-capita measures of interactions both displayed such attenuation, suggesting factors beyond systematic changes in population size were involved; namely, changes to the interactions themselves. Nevertheless, pairwise interactions retained some explanatory power across contexts, provided those contexts were not substantially divergent in richness. These results suggest that understanding the emergent properties of microbial interactions can improve our ability to predict the features of microbial communities.

Novel integrative elements and genomic plasticity in ocean ecosystems.

Horizontal gene transfer accelerates microbial evolution. The marine picocyanobacterium Prochlorococcus exhibits high genomic plasticity, yet the underlying mechanisms are elusive. Here, we report a novel family of DNA transposons-"tycheposons"-some of which are viral satellites while others carry cargo, such as nutrient-acquisition genes, which shape the genetic variability in this globally abundant genus. Tycheposons share distinctive mobile-lifecycle-linked hallmark genes, including a deep-branching site-specific tyrosine recombinase. Their excision and integration at tRNA genes appear to drive the remodeling of genomic islands-key reservoirs for flexible genes in bacteria. In a selection experiment, tycheposons harboring a nitrate assimilation cassette were dynamically gained and lost, thereby promoting chromosomal rearrangements and host adaptation. Vesicles and phage particles harvested from seawater are enriched in tycheposons, providing a means for their dispersal in the wild. Similar elements are found in microbes co-occurring with Prochlorococcus, suggesting a common mechanism for microbial diversification in the vast oligotrophic oceans.