is an important zoonotic pathogen, however, an efficient markerless genetic manipulation system is still lacking for further physiological and pathological studies on this bacterium. Several techniques have been developed for markerless genetic manipulation of utilizing either a temperature-sensitive vector or a counterselectable markers (CSMs), however, at present, the efficiency of these techniques is not very satisfactory. In this study, we developed a strategy for markerless genetic manipulation of employing a CSM based on a conditionally lethal mutant allele of , which encodes the α-subunit of phenylalanyl-tRNA synthetase (PheS). This mutant , , was constructed by introducing site-directed mutations for a T261S/A315G double-substitution and a number of silent mutations to decrease its similarity with the endogenous wild type gene (). Additionally, five potentially strong promoters from were screened for their ability to drive high-level expression of mPheS, thus endowing the carrier strain with sufficient sensitivity to the phenylalanine analog -chloro-phenylalanine (-Cl-phe). Insertion of these P- cassettes into a vector or into the chromosomal locus a linked erythromycin resistance gene revealed that allele driven by promoters P and P renders sensitive to as low as 0.01% (or 0.5 mM) of -Cl-phe. This offers two potential CSMs for with -Cl-phe as a counterselective agent. P- was revealed to be 100% efficient for counter-selection in by application in a precise gene deletion. Using P- as a CSM, a two-step insertion and excision strategy for markerless genetic manipulation of were developed, supplying a powerful tool for markerless genetic manipulation of .