Abstract
asdA mutants of Gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd+ plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd+ plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric Gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd+ expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd+ vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd+ plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.
Highlights
Aspartate b-semialdehyde deshydrogenase (Asd; EC 1.2.1.11), a highly conserved homodimeric enzyme encoded by the asd gene, is involved in the conversion of b-aspartyl phosphate to aspartate bsemialdehyde
Asd is an enzyme common to the biosynthesis of the essential amino acids lysine, threonine, methionine, and isoleucine. It performs a key step in the production of diaminopimelic acid (DAP), a required component for the peptidoglycan synthesis of Gram-negative and some Gram-positive bacterial cell walls [1,2,3,4] and an immediate precursor to lysine. asd mutants have an obligate requirement for DAP, and in the absence of DAP they undergo lysis
Deletion of the asdA gene precluded the growth of E. ictaluri in absence of DAP (Fig. 4), indicating that asdB does not encode for a functional protein related to DAP synthesis
Summary
Aspartate b-semialdehyde deshydrogenase (Asd; EC 1.2.1.11), a highly conserved homodimeric enzyme encoded by the asd gene, is involved in the conversion of b-aspartyl phosphate to aspartate bsemialdehyde. Asd is an enzyme common to the biosynthesis of the essential amino acids lysine, threonine, methionine, and isoleucine It performs a key step in the production of diaminopimelic acid (DAP), a required component for the peptidoglycan synthesis of Gram-negative and some Gram-positive bacterial cell walls [1,2,3,4] and an immediate precursor to lysine. Threonine, methionine, and isoleucine are essential amino acids in the diet of teleostei fish [12,13,14,15,16,17], suggesting the absence of both the DAP/lysine synthesis pathway and Asd enzyme in fish cells
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