Selwyn Quan

Nonoptimal Microbial Response to Antibiotics Underlies Suppressive Drug Interactions

Suppressive drug interactions, in which one antibiotic can actually help bacterial cells to grow faster in the presence of another, occur between protein and DNA synthesis inhibitors. Here, we show that this suppression results from nonoptimal regulation of ribosomal genes in the presence of DNA stress. Using GFP-tagged transcription reporters in Escherichia coli, we find that ribosomal genes are not directly regulated by DNA stress, leading to an imbalance between cellular DNA and protein content. To test whether ribosomal gene expression under DNA stress is nonoptimal for growth rate, we sequentially deleted up to six of the seven ribosomal RNA operons. These synthetic manipulations of ribosomal gene expression correct the protein-DNA imbalance, lead to improved survival and growth, and completely remove the suppressive drug interaction. A simple mathematical model explains the nonoptimal regulation in different nutrient environments. These results reveal the genetic mechanism underlying an important class of suppressive drug interactions.

Adaptive Evolution of the Lactose Utilization Network in Experimentally Evolved Populations of Escherichia coli

Adaptation to novel environments is often associated with changes in gene regulation. Nevertheless, few studies have been able both to identify the genetic basis of changes in regulation and to demonstrate why these changes are beneficial. To this end, we have focused on understanding both how and why the lactose utilization network has evolved in replicate populations of Escherichia coli. We found that lac operon regulation became strikingly variable, including changes in the mode of environmental response (bimodal, graded, and constitutive), sensitivity to inducer concentration, and maximum expression level. In addition, some classes of regulatory change were enriched in specific selective environments. Sequencing of evolved clones, combined with reconstruction of individual mutations in the ancestral background, identified mutations within the lac operon that recapitulate many of the evolved regulatory changes. These mutations conferred fitness benefits in environments containing lactose, indicating that the regulatory changes are adaptive. The same mutations conferred different fitness effects when present in an evolved clone, indicating that interactions between the lac operon and other evolved mutations also contribute to fitness. Similarly, changes in lac regulation not explained by lac operon mutations also point to important interactions with other evolved mutations. Together these results underline how dynamic regulatory interactions can be, in this case evolving through mutations both within and external to the canonical lactose utilization network.

Transcriptional Polarity in rRNA Operons of Escherichia coli nusA and nusB Mutant Strains

Synthesis of ribosomes in Escherichia coli requires an antitermination system that modifies RNA polymerase to achieve efficient transcription of the genes specifying 16S, 23S, and 5S rRNA. This modification requires nucleotide signals in the RNA and specific transcription factors, such as NusA and NusB. Transcription of rrn operons in strains lacking the ability to produce either NusA or NusB was examined by electron microscopy. The distribution and numbers of RNA polymerase molecules on rrn operons were determined for each mutant. Compared to the wild type, the 16S gene in the nusB mutant strain had an equivalent number of RNA polymerase molecules, but the number of RNA polymerase molecules was reduced 1.4-fold for the nusA mutant. For both mutant strains, there were twofold-fewer RNA polymerase molecules on the 23S RNA gene than for the wild type. Overall, the mutant strains each had 1.6-fold-fewer RNA polymerase molecules on their rrn operons than did the wild type. To determine if decreased transcription of the 23S gene observed by electron microscopy also affected the 30S/50S ribosomal subunit ratio, ribosome profiles were examined by sucrose gradient analysis. The 30S/50S ratio increased 2.5- to 3-fold for the nus mutant strains over that for wild-type cells. Thus, strains carrying either a nusA mutation or a nusB mutation have defects in transcription of 23S rRNA.

Tools for Characterizing Bacterial Protein Synthesis Inhibitors

ABSTRACT Many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. However, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. Furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. In order to avoid these pitfalls and improve the process of dereplication of chemically complex extracts, we designed a two-pronged approach for the characterization of inhibitors of protein synthesis (ChIPS) that is suitable for the rapid identification of the site and mode of action on the bacterial ribosome. First, we engineered antibiotic-hypersensitive Escherichia coli strains that contain only one rRNA operon. These strains are used for the rapid isolation of resistance mutants in which rRNA mutations identify the site of the antibiotic action. Second, we show that patterns of drug-induced ribosome stalling on mRNA, monitored by primer extension, can be used to elucidate the mode of antibiotic action. These analyses can be performed within a few days and provide a rapid and efficient approach for identifying the site and mode of action of translation inhibitors targeting the bacterial ribosome. Both techniques were validated using a bacterial strain whose culture extract, composed of unknown metabolites, exhibited protein synthesis inhibitory activity; we were able to rapidly detect the presence of the antibiotic chloramphenicol.

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.

Evolutionary Comparison of Ribosomal Operon Antitermination Function

Transcription antitermination in the ribosomal operons of Escherichia coli results in the modification of RNA polymerase by specific proteins, altering its basic properties. For such alterations to occur, signal sequences in rrn operons are required as well as individual interacting proteins. In this study we tested putative rrn transcription antitermination-inducing sequences from five different bacteria for their abilities to function in E. coli. We further examined their response to the lack of one known rrn transcription antitermination protein from E. coli, NusB. We monitored antitermination activity by assessing the ability of RNA polymerase to read through a factor-dependent terminator. We found that, in general, the closer the regulatory sequence matched that of E. coli, the more likely there was to be a successful antitermination-proficient modification of the transcription complex. The rrn leader sequences from Pseudomonas aeruginosa, Bacillus subtilis, and Caulobacter crescentus all provided various levels of, but functionally significant antitermination properties to, RNA polymerase, while those of Mycobacterium tuberculosis and Thermotoga maritima did not. Possible RNA folding structures of presumed antitermination sequences and specific critical bases are discussed in light of our results. An unexpected finding was that when using the Caulobacter crescentus rrn leader sequence, there was little effect on terminator readthrough in the absence of NusB. All other hybrid antitermination system activities required this factor. Possible reasons for this finding are discussed.