Markerless Escherichia coli rrn Deletion Strains for Genetic Determination of Ribosomal Binding Sites.

Single-copy rrn strains facilitate genetic ribosomal studies in Escherichia coli. Consecutive markerless deletion of rrn operons resulted in slower growth upon inactivation of the fourth copy, which was reversed by supplying transfer RNA genes encoded in rrn operons in trans. Removal of the sixth, penultimate rrn copy led to a reduced growth rate due to limited rrn gene dosage. Whole-genome sequencing of variants of single-copy rrn strains revealed duplications of large stretches of genomic DNA. The combination of selective pressure, resulting from the decreased growth rate, and the six identical remaining scar sequences, facilitating homologous recombination events, presumably leads to elevated genomic instability.

The majority of antibiotic classes currently in clinical use act by inhibiting ribosome function. Their utility is threatened by the emergence of microbial resistance, mainly due to compound efflux (Thaker ), covalent modification of the ribosome (Goossens 2009), or alteration of the antibiotic (Zhanel ). The binding interactions of the antibiotics with the ribosome are established predominantly with its RNA components (Poehlsgaard and Douthwaite 2005), which in Escherichia coli are encoded by seven virtually identical rrn operons: rrnA through rrnH (rrnF was renamed as rrnG) (supporting information, Figure S1A). This sevenfold redundancy limits the introduction of resistance caused by binding site mutations, mainly due to the recessive nature of resistance mutations that are suppressed by the presence of six wild-type alleles that lead to rapid cell death (Orelle ). In addition, the multiplicity of wild-type alleles will tend to revert the mutant allele due to rapid gene conversion. This extremely low-resistance frequency is from a clinical perspective a very attractive feature for an antibiotic, and as a consequence the ribosome has remained an attractive antibacterial target despite decades of macrolide and aminoglycoside usage. From a drug-discovery perspective, however, this redundancy makes definition of a structural foundation guiding iterative chemistry programs challenging.

One recent example is negamycin, a natural compound that mediates its antibacterial activity by inhibition of translation (Mizuno ) and for which as many as 10 different ribosomal-binding sites have been described (Schroeder ; Olivier ; Polikanov ). The location of a single binding site through which the compound mediated its activity, required for structure-based design of improved analogs, remained elusive for 40 years. Negamycin-resistant mutants of wild-type E. coli have been isolated, but the mutations affected transport of the compound into the cell rather than binding to the ribosome (McKinney ). To obtain genetic evidence for negamycin’s functional binding site, helix 34 of the small subunit, the redundancy of wild-type E. coli ultimately needed to be circumvented by the use of markerless single rrn strains (Olivier ; Polikanov ). Although this tool has found use in various seminal scientific advances of ribosomal biology (Bollenbach ; Orelle ; Polikanov ; Orelle ), its construction was mentioned in brief (Bollenbach ). Here we describe the construction in detail, showing that sequential deletion of four rrn genes leads to a reduced growth rate due to limiting expression of transfer RNAs (tRNAs), and that after deletion of the sixth copy the cellular ribosomal content becomes growth-limiting. Furthermore, although colony morphology suggested stability of the strains (Bollenbach ), whole-genome analyses revealed elevated genomic instability caused by homologous recombination between the scars remaining upon rrn deletion.

Materials and Methods