Quorum sensing determines the choice of antiphage defense strategy in <i>Vibrio anguillarum</i>

Selection for phage resistance is a key driver of bacterial diversity and evolution, and phage-host interactions may therefore have strong influence on the genetic and functional dynamics of bacterial communities. In this study, we found that an important, but so far largely overlooked, determinant of the outcome of phage-bacterial encounters in the fish pathogen Vibrio anguillarum is bacterial cell-cell communication, known as quorum sensing. Specifically, V. anguillarum PF430-3 cells locked in the low-cell-density state (ΔvanT mutant) express high levels of the phage receptor OmpK, resulting in a high susceptibility to phage KVP40, but achieve protection from infection by enhanced biofilm formation. By contrast, cells locked in the high-cell-density state (ΔvanΟ mutant) are almost completely unsusceptible due to quorum-sensing-mediated downregulation of OmpK expression. The phenotypes of the two quorum-sensing mutant strains are accurately reflected in the behavior of wild-type V. anguillarum, which (i) displays increased OmpK expression in aggregated cells compared to free-living variants in the same culture, (ii) displays a clear inverse correlation between ompK mRNA levels and the concentration of N-acylhomoserine lactone quorum-sensing signals in the culture medium, and (iii) survives mainly by one of these two defense mechanisms, rather than by genetic mutation to phage resistance. Taken together, our results demonstrate that V. anguillarum employs quorum-sensing information to choose between two complementary antiphage defense strategies. Further, the prevalence of nonmutational defense mechanisms in strain PF430-3 suggests highly flexible adaptations to KVP40 phage infection pressure, possibly allowing the long-term coexistence of phage and host

Effects of Four Different Regulatory Mechanisms on the Dynamics of Gene Regulatory Cascades

Gene regulatory cascades (GRCs) are common motifs in cellular molecular networks. A given logical function in these cascades, such as the repression of the activity of a transcription factor, can be implemented by a number of different regulatory mechanisms. The potential consequences for the dynamic performance of the GRC of choosing one mechanism over another have not been analysed systematically. Here, we report the construction of a synthetic GRC in Escherichia coli, which allows us for the first time to directly compare and contrast the dynamics of four different regulatory mechanisms, affecting the transcription, translation, stability, or activity of a transcriptional repressor. We developed a biologically motivated mathematical model which is sufficient to reproduce the response dynamics determined by experimental measurements. Using the model, we explored the potential response dynamics that the constructed GRC can perform. We conclude that dynamic differences between regulatory mechanisms at an individual step in a GRC are often concealed in the overall performance of the GRC, and suggest that the presence of a given regulatory mechanism in a certain network environment does not necessarily mean that it represents a single optimal evolutionary solution

Transfer RNA is highly unstable during early amino acid starvation in <i>Escherichia coli</i>

Due to its long half-life compared to messenger RNA, bacterial transfer RNA is known as stable RNA. Here, we show that tRNAs become highly unstable as part of Escherichia coli's response to amino acid starvation. Degradation of the majority of cellular tRNA occurs within twenty minutes of the onset of starvation for each of several amino acids. Both the non-cognate and cognate tRNA for the amino acid that the cell is starving for are degraded, and both charged and uncharged tRNA species are affected. The alarmone ppGpp orchestrates the stringent response to amino acid starvation. However, tRNA degradation occurs in a ppGpp-independent manner, as it occurs with similar kinetics in a relaxed mutant. Further, we also observe rapid tRNA degradation in response to rifampicin treatment, which does not induce the stringent response. We propose a unifying model for these observations, in which the surplus tRNA is degraded whenever the demand for protein synthesis is reduced. Thus, the tRNA pool is a highly regulated, dynamic entity. We propose that degradation of surplus tRNA could function to reduce mistranslation in the stressed cell, because it would reduce competition between cognate and near-cognate charged tRNAs at the ribosomal A-site

Direct and indirect effects in the regulation of overlapping promoters

Optimal response to environmental stimuli often requires activation of certain genes and repression of others. Dual function regulatory proteins play a key role in the differential regulation of gene expression. While repression can be achieved by any DNA binding protein through steric occlusion of RNA polymerase in the promoter region, activation often requires a surface on the regulatory protein to contact RNAP and thus facilitate transcription initiation. RNAP itself is also a DNA binding protein, therefore it can function as a transcriptional repressor. Searching the Escherichia coli promoter database we found that ∼14% of the identified ‘forward’ promoters overlap with a promoter oriented in the opposite direction. In this article we combine a mathematical model with experimental analysis of synthetic regulatory regions to investigate interference of overlapping promoters. We find that promoter interference depends on the characteristics of overlapping promoters. The model predicts that promoter strength and interference can be regulated separately, which provides unique opportunities for regulation. Our experimental data suggest that in principle any DNA binding protein can be used for both activation and repression of promoter transcription, depending on the context. These findings can be exploited in the construction of synthetic networks

Commitment to lysogeny is preceded by a prolonged period of sensitivity to the late lytic regulator Q in bacteriophage Λ

A key event in development is the irreversible commitment to a particular cell fate, which may be concurrent with or delayed with respect to the initial cell fate decision. In this work, we use the paradigmatic bacteriophage λ lysis-lysogeny decision circuit to study the timing of commitment. The lysis-lysogeny decision is made based on the expression trajectory of CII. The chosen developmental strategy is manifested by repression of the pR and pL promoters by CI (lysogeny) or by antitermination of late gene expression by Q (lysis). We found that expression of Q in trans from a plasmid at the time of infection resulted in a uniform lytic decision. Furthermore, expression of Q up to 50 min after infection results in lysis of the majority of cells which initially chose lysogenic development. In contrast, expression of Q in cells containing a single chromosomal prophage had no effect on cell growth, indicating commitment to lysogeny. Notably, if the prophage was present in 10 plasmid-borne copies, Q expression resulted in lytic development, suggesting that the cellular phage chromosome number is the critical determinant of the timing of lysogenic commitment. Based on our results, we conclude that (i) the lysogenic decision made by the CI-Cro switch soon after infection can be overruled by ectopic Q expression at least for a time equivalent to one phage life cycle, (ii) the presence of multiple λ chromosomes is a prerequisite for a successful Q-mediated switch from lysogenic to lytic development, and (iii) phage chromosomes within the same cell can reach different decisions