Characterization of carABpyrB operon and role of pyrE in Francisella novicida biofilm.

Abstract

Pyrimidine biosynthesis is essential for bacterial growth, but its role in regulating biofilm formation in Francisella (F.) novicida remains poorly defined. In this study, we experimentally defined the carABpyrB operon in F. novicida and investigated how disruption of the de novo pyrimidine biosynthesis pathway affects growth and biofilm formation under nutrient-restricted conditions. Reverse transcriptase PCR confirmed co-transcription of carA, carB, and pyrB, and promoter prediction identified two putative σ70-dependent promoter regions upstream of carA. Transposon mutants disrupted in carA, carB, and pyrB exhibited pronounced growth defects in Chamberlain's Defined Medium that were restored by uracil supplementation, confirming pyrimidine auxotrophy and functional disruption of de novo pyrimidine biosynthesis. We then extended this analysis to additional genes in the pyrimidine biosynthetic pathway and assessed biofilm formation in modified Mueller-Hinton broth, a nutrient-restricted condition. In this medium, carA, carB, pyrB, and pyrE mutants exhibited growth deficiencies; however, the pyrE mutant uniquely produced significantly more biofilm than the wild type. This phenotype remained evident even without growth normalization, with the pyrE mutant producing 3.3-fold more biofilm than wild type, despite impaired growth, and increased to 11.8-fold when normalized to growth. Quantitative PCR demonstrated that uracil supplementation represses carA, carB, and pyrB transcription, consistent with feedback regulation of the pathway. Together, these findings indicate that pyrimidine limitation is not simply a growth-limiting condition but can alter biofilm regulation, with pyrE disruption revealing a strong association between de novo pyrimidine biosynthesis and biofilm formation.