Understanding Transcriptional Regulatory Units in Pseudomonas Putida Under Changing Environmental Conditions

Pseudomonas putida is a Gram-negative rhizobial bacterium capable of soil bioremediation and industrial biosynthesis. P. putida has a very diverse metabolism allowing it to perform these roles, but much is still unknown about its transcriptional regulation. This research aims to address the transcriptional programs that contribute to P. putida's resilience and to develop a synthetic genetic construct to increase the survival rate of genetically engineered P. putida in the complex soil environment. To identify genes with consistent stable transcription, we performed a meta-analysis on P. putida KT2440 transcriptomic datasets and determined genes with low log-fold changes. A rank-based analysis was then carried out to determine highly expressed genes. The resulting top gene was found downstream of the lapA promoter region, the largest gene in P. putida whose transcriptional regulation is unclear and promoter region highly complex. Another key question is whether P. putida change their TSS selection or operon usage in response to the environment or competition from the native microbiome. We are currently investigating P. putida's gene regulation by comparing operon-based expression in LB versus soil-extracted solubilized organic matter (SESOM), and will elucidate the differences between short Illumina and long Nanopore RNA-seq in the context of operons prediction. Development of these methods will enable us to dissect the role of the soil metabolic environment from the effects of competition from the microbial community in the context of genetic regulation. Our studies aim to uncover more about the relationship between environmental changes and the regulatory gene network of P. putida.