Comparative genomics hints at dispensability of multiple essential genes in two Escherichia coli L-form strains.

Despite the critical role of bacterial cell walls in maintaining cell shapes, certain environmental stressors can induce the transition of many bacterial species into a wall-deficient state called L-form. Long-term induced Escherichia coli L-forms lose their rod shape and usually hold significant mutations that affect cell division and growth. Besides this, the genetic background of L-form bacteria is still poorly understood. In the present study, the genomes of two stable L-form strains of E. coli (NC-7 and LWF+) were sequenced and their gene mutation status was determined and compared with their parental strains. Comparative genomic analysis between two L-forms reveals both unique adaptions and common mutated genes, many of which belong to essential gene categories not involved in cell wall biosynthesis, indicating that L-form genetic adaptation impacts crucial metabolic pathways. Missense variants from L-forms and Lenski's long-term evolution experiment (LTEE) were analyzed in parallel using an optimized DeepSequence pipeline to investigate predicted mutation effects (α) on protein functions. We report that the two L-form strains analyzed display a frequency of 6-10% (0% for LTEE) in mutated essential genes where the missense variants have substantial impact on protein functions (α<0.5). This indicates the emergence of different survival strategies in L-forms through changes in essential genes during adaptions to cell wall deficiency. Collectively, our results shed light on the detailed genetic background of two E. coli L-forms and pave the way for further investigations of the gene functions in L-form bacterial models.