Bacteria such as Lactococcus lactis have d-aspartate (d-Asp) or its amidated derivative d-asparagine (d-Asn), in their peptidoglycan (PG) interpeptide crossbridge. We performed a subtractive genome analysis to identify L. lactis gene yxbA, orthologues of which being present only in bacteria containing d-amino acids in their PG crossbridge, but absent from those that instead insert l-amino acids or glycine. Inactivation of yxbA required a complementing Streptococcus pneumoniae murMN genes, which express enzymes that incorporate l-Ser-l-Ala or l-Ala-l-Ala in the PG crossbridge. Our results show that (i) yxbA encodes d-Asp ligase responsible for incorporation of d-Asp in the PG crossbridge, and we therefore renamed it as aslA, (ii) it is an essential gene, which makes its product a potential target for specific antimicrobials, (iii) the absence of d-Asp may be complemented by l-Ser-l-Ala or l-Ala-l-Ala in the L. lactis PG, indicating that the PG synthesis machinery is not selective for the side-chain residues, and (iv) lactococcal strains having l-amino acids in their PG crossbridge display defects in cell wall integrity, but are able to efficiently anchor cell wall proteins, indicating relative flexibility of lactococcal transpeptidation reactions with respect to changes in PG side-chain composition.