Abstract
The bacterial type III export apparatus is found in the flagellum and in the needle complex of some pathogenic Gram-negative bacteria. In the needle complex its function is to secrete effector proteins for infection into Eukaryotic cells. In the bacterial flagellum it exports specific proteins for the building of the flagellum during its assembly. The export apparatus is composed of about five membrane proteins and three soluble proteins. The mechanism of the export apparatus is not fully understood. The five membrane proteins are well conserved and essential. Here a cross-complementation assay was performed: substituting in the flagellar system of Salmonella one of these membrane proteins, FlhB, by the FlhB ortholog from Aquifex aeolicus (an evolutionary distant hyperthermophilic bacteria) or a chimeric protein (AquSalFlhB) made by the combination of the trans-membrane domain of A. aeolicus FlhB with the cytoplasmic domain of Salmonella FlhB dramatically reduced numbers of flagella and motility. From cells expressing the chimeric AquSalFlhB protein, suppressor mutants with enhanced motility were isolated and the mutations were identified using whole genome sequencing. Gain-of-function mutations were found in the gene encoding FlhA, another membrane protein of the type III export apparatus. Also, mutations were identified in genes encoding 4-hydroxybenzoate octaprenyltransferase, ubiquinone/menaquinone biosynthesis methyltransferase, and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, which are required for ubiquinone biosynthesis. The mutations were shown by reversed-phase high performance liquid chromatography to reduce the quinone pool of the cytoplasmic membrane. Ubiquinone biosynthesis could be restored for the strain bearing a mutated gene for 4-hydroxybenzoate octaprenyltransferase by the addition of excess exogenous 4-hydroxybenzoate. Restoring the level of ubiquinone reduced flagella biogenesis with the AquSalFlhB chimera demonstrating that the respiratory chain quinone pool is responsible for this phenomenon.
Highlights
The bacterial flagellum is an intricate nanomachine, which is made of about 30 different proteins [1]
The FlhB protein of S. typhimurium (SalFlhB) was replaced with the FlhB protein of A. aeolicus (AquFlhB) or a chimera of the trans-membrane domain of A. aeolicus FlhB fused to the cytoplasmic domain of Salmonella FlhB (AquSalFlhB) (Figure 1A)
A cross-complementation analysis was performed with the type III export apparatus protein FlhB to investigate the level of conservation between different type III export apparatuses, and the type of properties which could allow a host flagellar system to function better with a foreign homolog
Summary
The bacterial flagellum is an intricate nanomachine, which is made of about 30 different proteins [1]. It rotates like a propeller to enable the cells to swim [2], [3]. The type III secretion system, sometimes referred to as the needle complex, which is found in some pathogenic Gram-negative bacteria uses a type III export apparatus. This highly homologous export apparatus is used to inject proteins for infection into Eukaryotic host cells, which is a major source of pathogenicity [4], [5]. The proton motive force is the energy source for the export apparatus of both flagellar assembly and the needle complex, and in bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) it is the energy source for the rotation of the fully-formed flagellum [1], [6], [7]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
