The Aspartate-Semialdehyde Dehydrogenase of Edwardsiella ictaluri and Its Use as Balanced-Lethal System in Fish Vaccinology

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

Aspartokinase genes lysC alpha and lysC beta overlap and are adjacent to the aspartate beta-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum

Kalinowski J, Bachmann B, Thierbach G, Pühler A. Aspartokinase genes lysC alpha and lysC beta overlap and are adjacent to the aspartate beta-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum. MOLECULAR & GENERAL GENETICS. 1990;224(3):317-324.A 2.1 kb DNA fragment of the recombinant plasmid pCS2, isolated from an aminoethyl cysteine (AEC)-resistant and lysine-producing Corynebacterium glutamicum mutant strain, and which confers AEC resistance and lysine production on the wild-type G. glutamicum ATCC 13032 was analysed. DNA sequence analysis of this fragment revealed three large open reading frames (ORFs). The incomplete ORF1 does not contain the 5' end of the coding region. ORF2, which uses the same reading frame as ORF1, is identical to the 3' end of ORF1 and encodes a putative protein of 172 amino acids (aa) and of M(r) 18584. ORF3 encodes a putative protein of 344 aa and of M(r) 36275. The amino acid sequences deduced from ORF1 and ORF2 display strong homologies to those of the alpha- and beta-subunits of the Bacillus subtilis aspartokinase II. It is therefore proposed that the incomplete ORF1, termed lysC-alpha, encodes part of the alpha-subunit of the C. glutamicum aspartokinase whereas the complete ORF2, termed lysC-beta, encodes the beta-subunit of the same enzyme. ORF2 is responsible for AEC resistance and lysine production due to a feedback-resistant aspartokinase. The amino acid sequence deduced from ORF3, termed asd, is highly homologous to that of the Streptococcus mutans aspartate beta-semialdehyde dehydrogenase (ASD). Plasmids carrying the C. glutamicum asd gene complemented Escherichia coli asd mutants. Increase in ASD activity by a factor of 30-60 was measured for C. glutamicum cells harbouring high copy-number plasmids with the C. glutamicum asd gene