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Applied and Environmental Microbiology, August 2007, p. 5138-5145, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.00751-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Use of a Dominant rpsL Allele Conferring Streptomycin Dependence for Positive and Negative Selection in Thermus thermophilus{triangledown} ,{dagger}

Emilio Blas-Galindo, Felipe Cava, Eduardo López-Viñas, Jesús Mendieta, and José Berenguer*

Centro de Biologia Molecular Severo Ochoa, CSIC-UAM, Campus de Cantoblanco, 28049 Madrid, Spain

Received 30 March 2007/ Accepted 19 June 2007

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.

* Corresponding author. Mailing address: Centro de Biologia Molecular Severo Ochoa, CSIC-UAM, Campus de Cantoblanco, 28049 Madrid, Spain. Phone: 34 91 4978099. Fax: 34 91 4978087. E-mail: jberenguer{at}cbm.uam.es

{triangledown} Published ahead of print on 29 June 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, August 2007, p. 5138-5145, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.00751-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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