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Contingency locus in ctsR of Listeria monocytogenes Scott A: a strategy for occurrence of abundant piezotolerant isolates within clonal populations
In a recent study we demonstrated that a high-hydrostatic-pressure-tolerant isolate of Listeria monocytogenes
lacks a codon in the class 3 heat shock regulator gene ctsR. This mutation in the region that encodes four
consecutive glycines was directly responsible for the observed piezotolerance, increased stress resistance, and
reduced virulence. The aim of the present study was to determine whether mutations in ctsR are frequently
associated with piezotolerance in L. monocytogenes. Wild-type cultures of L. monocytogenes were therefore
exposed to 350 MPa for 20 min, and the piezotolerance of individual surviving isolates was assessed. This
rendered 33 isolates with a stable piezotolerant phenotype from a total of 84 survivors. Stable piezotolerant
mutants were estimated to be present in the initial wild-type population at frequencies of >10�5. Subsequent
sequencing of the ctsR gene of all stable piezotolerant isolates revealed that two-thirds of the strains (i.e., n �
21) had mutations in this gene. The majority of the mutations (16 of 21 strains) consisted of a triplet deletion
in the glycine-encoding region of ctsR, identical to what was found in our previous study. Interestingly, 2 of 21
mutants contained a codon insertion in this repeat region. The remaining three stable piezotolerant strains
showed a 19-bp insertion in the glycine repeat region, a 16-bp insertion downstream of the glycine repeat area
(both leading to frameshifts and a truncated ctsR), and an in-frame 114-bp deletion encoding a drastically
shortened carboxy terminus of CtsR. In four instances it was not possible to generate a PCR product. A
piezotolerant phenotype could not be linked to mutations in ctsR in 8 of 33 isolates, indicating that other
thus-far-unknown mechanisms also lead to stable piezotolerance. The present study highlights the importance
of ctsR in piezotolerance and stress tolerance of L. monocytogenes, and it demonstrates that short-sequence
repeat regions contribute significantly to the occurrence of a piezotolerant and stress-tolerant subpopulation
within L. monocytogenes cultures, thus playing an important role in survival