ABSTRACTExogenous spermine was reported to enhance the killing of methicillin-resistantStaphylococcus aureus(MRSA) by β-lactams through a strong synergistic effect of unknown nature. Spermine alone also exerts an antimicrobial activity againstS. aureusin a pH-dependent manner. MIC measurements revealed stronger effects of spermine under alkaline conditions, suggesting the nucleophilic property of spermine instead of its positive charge as the cause of adverse effects. A spontaneous suppressor mutant (MuM) of MRSA Mu50 was selected for spermine resistance and conferred complete abolishment of spermine–β-lactam synergy. In comparison to that in Mu50, the spermine MIC in MuM remained constant (64 mM) at pH 6 to 8; however, MuM, a heat-sensitive mutant, also grew in a very narrow pH range. Furthermore, MuM acquired a unique phenotype of vancomycin-spermine synergy. Genome resequencing revealed a 7-bp deletion inpbpB, which results in a truncated penicillin-binding protein 2 (PBP 2) without the transpeptidase domain at the C terminus while the N-terminal transglycosidase domain remains intact. The results of fluorescent Bocillin labeling experiments confirmed the presence of this defective PBP 2 in MuM. All the aforementioned phenotypes of MuM were reverted to those of Mu50 after complementation by the wild-typepbpBcarried on a recombinant plasmid. The anticipated changes in cell wall metabolism and composition in MuM were evidenced by observations that the cell wall of MuM was more susceptible to enzyme hydrolysis and that MuM exhibited a lower level of autolytic activities. Pleiotropic alterations in gene expression were revealed by microarray analysis, suggesting a remarkable flexibility of MuM to circumvent cell wall damage by triggering adaptations that are complex but completely different from that of the cell wall stress stimulon. In summary, these results reveal phenotypic changes and transcriptome adaptations in a uniquepbpBmutant and provide evidence to support the idea that exogenous spermine may perturb normal cell wall formation through its interactions with PBP 2.