Abstract
Riemerella anatipestifer is a bacterial pathogen responsible for major economic losses within the duck industry. Recent studies have revealed that biotin biosynthesis is critical for the bacterium’s survival and virulence. We previously found that R. anatipestifer AS87_RS09170, a putative bioF gene, is important for bacterial virulence. In the present study, we characterized the AS87_RS09170 gene in R. anatipestifer strain Yb2. Sequence analysis indicated that the AS87_RS09170 gene is highly conserved among R. anatipestifer strains; the deduced protein harbored the conserved pyridoxal 5′-phosphate binding pocket of 8-amino-7-oxononanoate synthase. Western blot analysis demonstrated that the biotin-dependent enzyme was present in smaller quantities in the mutant strain Yb2ΔbioF compared to that of the wide-type strain Yb2, suggesting that the biotin biosynthesis was defective. The mutant strain Yb2ΔbioF displayed a decreased growth rate at the exponential phase in tryptic soy broth culture and in BeaverBeads Streptavidin treated tryptic soy broth culture, but recovered when biotin was supplemented. In addition, the mutant strain Yb2ΔbioF showed an enhanced biofilm formation, as well as increased adhesion and invasion capacities to duck embryo fibroblasts. Moreover, the mutant strain Yb2ΔbioF exhibited irregular shapes with budding vegetations and relatively thickened cell walls under scanning and transmission electron microscope observation, as well as a reduced capacity to establish systemic infection in a duck infection model. These results provide the first evidence that the R. anatipestifer AS87_RS09170 gene is responsible for biotin synthesis, bacterial morphology and virulence.
Highlights
Riemerella anatipestifer is a Gram-negative, non-spore-forming, rod-shaped bacterium that mainly affects commercially-important birds such as ducks and turkeys[1,2]
The sequence alignment demonstrated that the putative R. anatipestifer amino-7-oxononanoate synthase (AONS) protein harbored the conserved pyridoxal 5′-phosphate binding pocket composed of amino acids G97, Y98, N101, E168, D197, H200, T228 and K231 (Fig. 1)
An indispensable nutrient found in all living cells, is synthesized de novo in many microorganisms, plants, and a few fungi
Summary
Riemerella anatipestifer is a Gram-negative, non-spore-forming, rod-shaped bacterium that mainly affects commercially-important birds such as ducks and turkeys[1,2]. Several virulence factors of R. anatipestifer have been identified, including VapD5, CAMP cohemolysin[6], outer membrane protein A7, putative genes associated with lipopolysaccharide (LPS) synthesis[8,9,10] and nicotinamidase[11]. Transponson Tn4351 disrupted the AS87_ RS09170 gene, which encodes a predicted 8-amino-7-oxononanoate synthase (AONS). AONS catalyzes the decarboxylative condensation of L-alanine and pimeloyl-CoA to form 8 (S)-amino-7-oxononanoate, the first committed step in conserved biotin biosynthesis. Biotin is intimately associated with lipid synthesis where the products form key components of the mycobacterial cell membrane that are critical for bacterial survival and pathogenesis[15]. We described a mutant strain of R. anatipestifer Yb2ΔbioF in which the homologous bioF gene AS87_RS09170 was disrupted. The bacterial growth, protein biotinylation, biofilm formation, adherence and invasion capabilities, morphology, gene expression as well as colonization and development during infection of the mutant strain Yb2ΔbioF were characterized
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