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While critical cellular components-such as the RNA moiety of bacterial ribonuclease P-can sometimes be replaced with a highly divergent homolog, the cellular response to such perturbations is often unexpectedly complex. RNase P is a ubiquitous and essential ribonucleoprotein involved in the processing of multiple RNA substrates, including tRNAs, small non-coding RNAs and intergenic operons. In Bacteria, RNase P RNAs have been subdivided-based on their secondary and tertiary structures-into two major groups (A and B), each with a distinct phylogenetic distribution. Despite the vast phylogenetic and structural gap that separates the two RNase P RNA classes, previous work suggested their interchangeability. Here, we explore in detail the functional and fitness consequences of replacing the endogenous Type-A Escherichia coli RNase P RNA with a Type-B homolog derived from Bacillus subtilis, and show that E. coli cells forced to survive with a chimeric RNase P as their sole source of RNase P activity exhibit extremely variable responses. The chimeric RNase P alters growth rates-used here as an indirect measure of fitness-in unpredictable ways, ranging from 3- to 20-fold reductions in maximal growth rate. The transcriptional behavior of cells harboring the chimeric RNAse P is also perturbed, affecting the levels of at least 79 different transcripts. Such transcriptional plasticity represents an important mechanism of transient adaptation which, when coupled with the emergence and eventual fixation of compensatory mutations, enables the cells to overcome the disruption of this tightly coevolving ribonucleoprotein.

Pathogenic bacteria resistant to many or all antibiotics already exist. With the decline in microbiological research at pharmaceutical companies, the high rate at which resistance has evolved and spread has demanded a novel approach to addressing this critical human health issue. In the present paper, we propose a new paradigm in antibiotic discovery and development, one that applies ecological and evolutionary theory to design antimicrobial drugs that are more difficult and/or more costly to resist. In essence, we propose to simply adopt the strategies invented and applied by bacteria for hundreds of millions of years. Our research focuses on bacteriocins, powerful biological weapons, and their use as alternative therapeutics in human health.

In an effort to develop derivatives of the Escherichia coli antimicrobial protein colicin E9 that exhibit novel interactions with a target cell, we mutagenized a 10-amino acid region located at the C terminus of the colicin receptor-binding domain. We subsequently selected for those colicin molecules that retain the antimicrobial phenotype and found that, despite a mutagenic strategy that alters every amino acid in the targeted domain, more than 70% of the engineered colicins retained antimicrobial activity. This result is all the more surprising given the extensive phylogenetic conservation of this receptor-binding domain, which originally suggested the operation of strong selective constraints on the amino acid sequence of this region. This apparent contradiction between our experimental results and the comparative data is resolved by exploring the fitness consequences of the experimentally induced amino acid substitutions. In 17 of 52 cases we examined, the fitness of cells harboring the functional engineered colicins was lower than that of our control line (containing wild-type colicin E9), and in 33 of 52 cases, equal to it. Paradoxically, two of the engineered colicins appear to confer a higher fitness to the producer cell lines. While the mechanism linking changes in the amino acid sequence of the colicin receptor-binding domain and the growth rate of the cells remains unclear, these results illustrate the surprising versatility of the colicin/receptor interaction and underscore the importance of distinguishing molecular function from organismal fitness.

Escherichia coli RNase P derivatives were evolved in vitro for DNA cleavage activity. Ribonucleoproteins sampled after ten generations of selection show a >400-fold increase in the first-order rate constant ( k cat ) on a DNA substrate, reflecting a significant improvement in the chemical cleavage step. This increase is offset by a reduction in substrate binding, as measured by K M . We trace the catalytic enhancement to two ubiquitous A → U sequence changes at positions 136 and 333 in the M1 RNA component, positions that are phylogenetically conserved in the Eubacteria. Furthermore, although the mutations are located in different folding domains of the catalytic RNA, the first in the substrate binding domain, the second near the catalytic core, their effect on catalytic activity is significantly influenced by the presence of the C5 protein. The activity of the evolved ribonucleoproteins on both pre-4.5 S RNA and on an RNA oligo substrate remain at wild-type levels. In contrast, improved DNA cleavage activity is accompanied by a 500-fold decrease in pre-tRNA cleavage efficiency ( k cat / K M ). The presence of the C5 component does not buffer this tradeoff in catalytic activities, despite the in vivo role played by the C5 protein in enhancing the substrate versatility of RNase P. The change at position 136, located in the J11/12 single-stranded region, likely alters the geometry of the pre-tRNA-binding cleft and may provide a functional explanation for the observed tradeoff. These results thus shed light both on structure/function relations in E. coli RNase P and on the crucial role of proteins in enhancing the catalytic repertoire of RNA.

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4. 5S RNA. In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA. This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates. This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding. The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available.

Bacterial RNase P is an essential ribonucleoprotein composed of a catalytic RNA component (encoded by the rnpB gene) and an associated protein moiety (encoded by rnpA). We construct a system that allows for the deletion of the essential endogenous rnpA copy and for its simultaneous replacement by a heterologous version of the gene. Using growth rate as a proxy, we explore the effects on fitness of heterologous replacement by increasingly divergent versions of the RNase P protein. All of the heterologs tested complement the loss of the endogenous rnpA gene, suggesting that all existing bacterial versions of the rnpA sequence retain the elements required for functional interaction with the RNase P RNA. All replacements, however, exact a cost on organismal fitness, and particularly on the rate of growth acceleration, defined as the time required to reach maximal growth rate. Our data suggest that the similarity of the heterolog to the endogenous version — whether defined at the sequence, structure or codon usage level — does not predict the fitness costs of the replacement. The common assumption that sequence similarity predicts functional similarity requires experimental confirmation and may prove to be an oversimplification.