Stress-Induced {beta}-Lactam Antibiotic Resistance Mutation and Sequences of Stationary-Phase Mutations in the Escherichia coli Chromosome

doi:10.1128/JB.00732-09

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	Articles by Petrosino, J. F.
	Articles by Rosenberg, S. M.

Stress-Induced β-Lactam Antibiotic Resistance Mutation and Sequences of Stationary-Phase Mutations in the Escherichia coli Chromosome

Joseph F. Petrosino,¹^,2^, Rodrigo S. Galhardo,¹^, Liza D. Morales,¹^, and Susan M. Rosenberg¹^,2^,3^*

Departments of Molecular and Human Genetics,¹ Molecular Virology and Microbiology,² Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030-3411³

Received 4 June 2009/ Accepted 21 July 2009

In some enterobacterial pathogens, but not in Escherichia coli,loss-of-function mutations are a common route to clinicallyrelevant β-lactam antibiotic resistance. We previouslyconstructed an assay system for studying enterobacterial β-lactamresistance mutations using the well-developed genetics of E.coli by integrating enterobacterial ampRC genes into the E.coli chromosome. Like the cells of other enterobacteria, E.coli cells acquire β-lactam resistance by ampD mutation.Here we show that starvation and stress responses provoke ampDβ-lactam resistance mutagenesis. When starved on lactosemedium, Lac^– strains used in mutagenesis studies accumulateampD β-lactam resistance mutations independent of Lac reversion.DNA double-strand break repair (DSBR) proteins and the SOS andRpoS stress responses are required for this mutagenesis, inagreement with the results obtained for lac reversion in thesecells. Surprisingly, the stress-induced ampD mutations requireDinB (DNA polymerase IV) and partially require error-prone DNApolymerase V, unlike lac mutagenesis, which requires only DinB.This assay demonstrates that real-world stressors, such as starvation,can induce clinically relevant resistance mutations. Finally,we used the ampD system to observe the true forward-mutationsequence spectrum of DSBR-associated stress-induced mutagenesis,for which previously only frameshift reversions were studied.We found that base substitutions outnumber frameshift mutations,as seen in other experimental systems showing stress-inducedmutagenesis. The important evolutionary implication is thatnot only loss-of-function mutations but also change-of-functionmutations can be generated by this mechanism.

* Corresponding author. Mailing address: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Room S809A, Mail Stop BCM225, Houston, TX 77030-3411. Phone: (713) 798-6924. Fax: (713) 798-8967. E-mail: smr{at}bcm.edu

Published ahead of print on 31 July 2009.

J.F.P. and R.S.G. contributed equally to this work.

Present address: M.D. Anderson University of Texas Health ScienceCenter, Houston, TX 77030.