Cross-complementation study of the flagellar type III export apparatus membrane protein FlhB

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• The bacterial type III export apparatus is crucial for flagellar assembly and virulence in Gram-negative bacteria.
• Its precise mechanism, particularly the role of membrane proteins like FlhB and FlhA, remains incompletely understood.
• This system is essential for secreting proteins involved in flagellar construction and, in some pathogens, for delivering effector proteins into host cells.

Purpose of the Study:

• To investigate the functional interplay between the type III export apparatus and cellular metabolic pathways.
• To identify genetic factors that can suppress defects in flagellar biogenesis caused by alterations in the FlhB protein.
• To elucidate the role of ubiquinone biosynthesis in the function of the type III export system.

Main Methods:

• Cross-complementation assays were performed using Salmonella flagellar systems and FlhB orthologs/chimeras from Aquifex aeolicus.
• Suppressor mutants with restored motility were isolated from cells expressing a chimeric FlhB protein.
• Whole genome sequencing was employed to identify mutations in suppressor mutants.
• Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to quantify the quinone pool.

Main Results:

• Substitution of Salmonella FlhB with an Aquifex aeolicus ortholog or a chimeric protein (AquSalFlhB) significantly reduced flagella numbers and motility.
• Gain-of-function mutations in FlhA and genes involved in ubiquinone biosynthesis (4-hydroxybenzoate octaprenyltransferase, ubiquinone/menaquinone biosynthesis methyltransferase, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase) were identified in suppressor mutants.
• These mutations decreased the ubiquinone pool in the cytoplasmic membrane.
• Restoring ubiquinone biosynthesis by adding exogenous 4-hydroxybenzoate reversed the flagellar biogenesis defect caused by the AquSalFlhB chimera.

Conclusions:

• The respiratory chain quinone pool plays a critical role in flagellar biogenesis mediated by the type III export apparatus.
• Alterations in ubiquinone biosynthesis can compensate for defects in the type III export system, suggesting a metabolic link to flagellar assembly.
• FlhA function is sensitive to the ubiquinone levels within the cell membrane.