Abstract
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 σ
70
‐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.