Investigating the Role of OrbF in Biofilm Biosynthesis and Regulation of Biofilm-Associated Genes in Bacillus cereus BC1

Abstract

Bacillus cereus (B. cereus), a prevalent foodborne pathogen, constitutes a substantial risk to food safety due to its pronounced resilience under adverse environmental conditions such as elevated temperatures and ultraviolet radiation. This resilience can be attributed to its capacity for biofilm synthesis and sustained high viability. Our research aimed to elucidate the mechanisms governing biofilm biosynthesis in B. cereus. To this end, we constructed a 5088-mutant library of the B. cereus strain BC1 utilizing the transposon TnYLB-1. Systematic screening of this library yielded mutants exhibiting diminished biofilm formation capabilities. Twenty-four genes associated with the biofilm synthesis were identified by reverse PCR in these mutants, notably revealing a significant reduction in biofilm synthesis upon disruption of the orbF gene in B. cereus BC1. Comparative analysis between the wild type and orbF-deficient BC1 strains (BC1ΔorbF) indicated a marked downregulation (decreased by 11.7% to 96.7%) in the expression of genes implicated in biofilm formation, flagellar assembly, and bacterial chemotaxis in the BC1ΔorbF. Electrophoretic mobility shift assay (EMSA) further corroborated the role of OrbF, demonstrating its binding to the promoter region of the biofilm gene cluster, subsequently leading to the suppression of transcriptional activity of biofilm-associated genes in B. cereus BC1. Our findings underscore the pivotal role of orbF in biofilm biosynthesis in B. cereus, highlighting its potential as a target for strategies aimed at mitigating biofilm formation in this pathogen.

Keywords: Bacillus cereus; biofilm formation; regulator OrbF; regulatory mechanism; transposon TnYLB-1.

Figure 1

Schematic representation of the regulatory network controlling biofilm formation in B. cereus. Yellow proteins represent transcription factors that play pivotal roles in orchestrating the genetic regulation of biofilm formation, purple circles represent metabolites involved in the biofilm synthesis pathway, contributing to the intricate molecular processes, and light blue rectangles denote open reading frames (ORFs) associated with genes participating in biofilm formation. The question mark indicates that clpY affects biofilm formation through an unknown mechanism. (This figure has been modified based on the reference [19]).

Figure 2

Workflow for screening biofilm formation defect mutants using the TnYLB-1 mutant library.

Figure 3

Discovery of the biofilm synthesis regulatory factor OrbF in BC1. (A) Relative positions of J8Y18_01575, J8Y18_01580, J8Y18_01585 and J8Y18_01590 in the B. cereus BC1 strain. The locus inserted by the transposon is marked with a triangle arrow. (B) The biofilm formation by strain BC1, strain BC1ΔorbF and strain BC1ΔorbF::orbF. (C) Growth pattern of strain BC1, strain BC1ΔorbF and strain BC1ΔorbF::orbF in Luria–Bertani (LB) medium.

Figure 4

RT-qPCR analysis of (A) biofilm, (B) flagellar assembly, (C) bacterial chemotaxis, (D) quorum sensing-related gene transcription levels. The statistically significant differences were observed at a 5% confidence level of significance.

Figure 5

Swarming motility of wild type, BC1ΔorbF, and BC1ΔorbF::orbF.

Figure 6

SDS-PAGE validation of transcription factor OrbF. M: Protein marker; Lane 1: Purified protein; Lane 2: E. coli BL21/pET-28a.

Figure 7

Binding of OrbF to the fla/che operon promoter.