Alanine racemase from ''E. coli''
Usage and Biology
The alanine racemase Alr (EC 22.214.171.124) from the Gram-negative enteric bacteria Escherichia coli is a racemase, which catalyses the reversible conversion of L-alanine into the enantiomer D-alanine (see Figure 1). For this reaction, the cofactor pyridoxal-5'-phosphate (PLP) is necessary. The constitutively expressed alanine racemase (alr) is naturally responsible for the accumulation of D-alanine. This compound is an essential component of the bacterial cell wall, because it is used for the cross-linkage of peptidoglycan (Walsh, 1989).
So in fact the alanine racemase (alr) can be used as:
- an antibiotic free selection marker in D-alanine auxotrophic strains, like E. coli ∆alr ∆dadX to obtain a higher plasmid stability.
- part of a Biosafety-Plasmid like BBa_K1172909 to create powerful Biosafety-Systems.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 331
- 21Illegal BglII site found at 255
Illegal BamHI site found at 957
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 373
Illegal AgeI site found at 673
- 1000Illegal BsaI.rc site found at 130
Characterizations of the alanine racemase
The konstitutive Alanin-Racemase (alr) and the catabolic Alanine-Racemase (dadX) were deleted in E. coli K-12 leading to the Strain K-12 ∆alr ∆dadX.
To avoid a second recombination of the alanine racemase (alr) from the plasmid with the genome, the whole coding sequence was deleted in the genome and the characterization of the alanine racemase was performed with the antibiotic chloramphenicol. For the complementation the alanine racemase (alr) was brought under the control of the Ptac promoter, resulting in the BioBrick BBa_K1172902. The Ptac promoter is a fusion promoter of the -35 region of the trp promoter and the -10 region the lac promoter, so that there only slight repression and the expression of the alanine racemase is highly activated (De Boer et al., 1983). Therefore an induction with IPTG was not necessary on M9 minimal medium, but surprisingly it was essential on LB-agar.
The deletion of the constitutive alanine racemase (alr) and the catabolic alanine racemase (dadX) in E. coli leads to a strict dependence on the amino acid D-alanine, as expected. As shown in Figure 2 below the bacteria with this deletions are not any more able to grow on normal M9 medium without D-alanine supplementation (purple curve), whereas the wild type does (red curve). The auxotrophic Safety-Strain grows only on media with D-alanine (5 mM) supplemented (blue curve) or by a complementation of the alanine racemase via plasmid. Furthermore, it could be demonstrated, that the auxotrophic mutant K-12 ∆alr ∆dadX grows slightly slower, than the wild type K-12. In contrast the bacteria containing the alanine racemase (alr) on the plasmid BBa_K1172902 does hardly show a disadvantage in the cell division compared to the wild type.
- Cava F, Lam H, de Pedro MA, Waldor MK (2011) Emerging knowledge of regulatory roles of d-amino acids in bacteria Cell and Molecular Life Sciences 68: 817 - 831.
- De Boer Hermann, Comstock J. Lisa and Vasser Mark (1983) The tac promoter: a functional hybrid derived from the trp and lac promoters. Proceedings of the National Academy of Science of the United States of America 80: 21 - 25.
- Link, A.J., Phillips, D. and Church, G.M. (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: Application to open reading frame characterization. Journal of Bacteriology 179: 6228-6237.
- Walsh, Christopher (1989) Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. Journal of biological chemistry 264: 2393 - 2396.