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

Older Adults and Information and Communication Technologies in the Global North

At all ages, people are incorporating information and communication technologies (ICTs) into their lives. It is not that they have stopped talking with each other in-person, it is that ICTs complement their interactions when they cannot be together face-to-face. Since the 1990s, email has provided a routine way to stay in touch and sustain meaningful contact over distance. But not all age groups have adopted ICTs with the same enthusiasm. Research in the Global North has consistently reported that age plays an important role in ICT adoption and use (Anderson and Perrin 2017). For example, older adults have been the least likely to use ICTs, and even when they do use ICTs, they are less active in their use (Blank and Groselji 2014; Haight, Quan-Haase, and Corbett 2014; Schreurs, Quan-Haase, and Martin 2017). Yet, this is changing. As more older adults use ICTs, analysts are wondering how such ICTs affect older adults’ social networks (Wang, Zhang and Wellman 2018; Wellman, Quan-Haase and Harper forthcoming): Are ICTs helping older adults build, maintain, or diminish personal networks? And how are they supporting or limiting the exchange of social support both for local and long-distance social networks? Moreover, are ICTs affecting different types of social ties differently—be they kin, friend, neighbor, workmate, or churchgoer; or strong or weak

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

Characterization of genes from Rhodococcus sp. involved in the degradation of environmental pollutants.

Submitted to the University of the Witwatersrand, Johannesburg for the degree of Masters of Science in BiotechnologyThe work described in this research report is an investigation into the microbial degradation of environmental pollutants, specifically : (a, the decolorization of azo dyes, (b) the desulfurization of dibenzothiophene and (c) the degradaticn of nylon monomers. (Abbreviation abstract)Andrew Chakane 201

Nonoptimal Microbial Response to Antibiotics Underlies Suppressive Drug Interactions

SummarySuppressive 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 of ribosomal genes under DNA stress as a side effect of their optimal regulation in different nutrient environments. These results reveal the genetic mechanism underlying an important class of suppressive drug interactions

Active Transcription of rRNA Operons Condenses the Nucleoid in Escherichia coli: Examining the Effect of Transcription on Nucleoid Structure in the Absence of Transertion▿

In Escherichia coli the genome must be compacted ∼1,000-fold to be contained in a cellular structure termed the nucleoid. It is proposed that the structure of the nucleoid is determined by a balance of multiple compaction forces and one major expansion force. The latter is mediated by transertion, a coupling of transcription, translation, and translocation of nascent membrane proteins and/or exported proteins. In supporting this notion, it has been shown consistently that inhibition of transertion by the translation inhibitor chloramphenicol results in nucleoid condensation due to the compaction forces that remain active in the cell. Our previous study showed that during optimal growth, RNA polymerase is concentrated into transcription foci or “factories,” analogous to the eukaryotic nucleolus, indicating that transcription and RNA polymerase distribution affect the nucleoid structure. However, the interpretation of the role of transcription in the structure of the nucleoid is complicated by the fact that transcription is implicated in both compacting forces and the expansion force. In this work, we used a new approach to further examine the effect of transcription, specifically from rRNA operons, on the structure of the nucleoid, when the major expansion force was eliminated. Our results showed that transcription is necessary for the chloramphenicol-induced nucleoid compaction. Further, an active transcription from multiple rRNA operons in chromosome is critical for the compaction of nucleoid induced by inhibition of translation. All together, our data demonstrated that transcription of rRNA operons is a key mechanism affecting genome compaction and nucleoid structure

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

Systematic Perturbation of Cytoskeletal Function Reveals a Linear Scaling Relationship between Cell Geometry and Fitness

SummaryDiversification of cell size is hypothesized to have occurred through a process of evolutionary optimization, but direct demonstrations of causal relationships between cell geometry and fitness are lacking. Here, we identify a mutation from a laboratory-evolved bacterium that dramatically increases cell size through cytoskeletal perturbation and confers a large fitness advantage. We engineer a library of cytoskeletal mutants of different sizes and show that fitness scales linearly with respect to cell size over a wide physiological range. Quantification of the growth rates of single cells during the exit from stationary phase reveals that transitions between “feast-or-famine” growth regimes are a key determinant of cell-size-dependent fitness effects. We also uncover environments that suppress the fitness advantage of larger cells, indicating that cell-size-dependent fitness effects are subject to both biophysical and metabolic constraints. Together, our results highlight laboratory-based evolution as a powerful framework for studying the quantitative relationships between morphology and fitness