Caloric restriction causes symmetric cell division and delays aging in Escherichia coli

Aging is one of the most intriguing processes of biology and despite decades of research, many aspects of aging are poorly understood. Aging is known to occur in bacteria and yeast that divide with morphological asymmetry. Morphologically symmetrically dividing bacteria such as _Escherichia coli_ were assumed not to age until they were shown to divide with functional asymmetry leading to aging and death of some of the cells even in exponentially growing cultures. In asymmetrically dividing _E. coli_ the newly synthesized components are presumed to occupy one pole so that after division one of the daughter cells receives newly synthesized components whereas the other retains the older components. Mathematical models predicted that at the population level, asymmetric growth should result in higher growth rate and symmetric growth in higher growth yield. Therefore, arguably symmetric cell division should be selected in low nutrient environments and asymmetric division in nutrient rich environments. A further prediction was that lower substrate concentrations should strengthen repair mechanisms and suppress aging whereas higher substrate concentrations suppress repair and enhance aging. We show here that _E. coli_ divides more symmetrically under caloric restriction, that both genetic selection and phenotypic plasticity are important determinants of cell division symmetry and also that the proportion of cells that stop dividing and therefore are presumably dead is significantly lower in symmetrically dividing cultures. However, contrary to the prediction, symmetry was not always accompanied by reduced growth rate. These results demonstrate that asymmetry of division in _E. coli_ is not hardwired but responsive to the nutritional environment. This provides a new perspective on why caloric restriction increases lifespan in organisms ranging from microbes to mammals. Symmetry of division may be a mechanism spanning across the width of life forms but regulating aging in different ways in different forms

Ethanolamine regulates CqsR quorum-sensing signaling in Vibrio cholerae

The pathogen that causes cholera, Vibrio cholerae, uses the cell-cell communication process known as quorum sensing (QS) to regulate virulence factor production and biofilm formation in response to changes in population density and complexity. QS is mediated through the detection of extracellular chemical signals called autoinducers. Four histidine kinases, LuxPQ, CqsS, CqsR and VpsS, have been identified as receptors to activate the key QS regulator LuxO at low cell density. At high cell density, detection of autoinducers by these receptors leads to deactivation of LuxO, resulting in population-wide gene expression changes. While the cognate autoinducers that regulate the activity of CqsS and LuxQ are known, the signals that regulate CqsR have not been determined. Here we show that the common metabolite ethanolamine specifically interacts with the ligand-binding CACHE domain of CqsR in vitro and induces the high cell-density QS response through CqsR kinase inhibition in V. cholerae cells. We also identified residues in the CqsR CACHE domain important for ethanolamine detection and signal transduction. Moreover, mutations disrupting endogenous ethanolamine production in V. cholerae delay the onset of, but do not abolish, the high cell-density QS gene expression. Finally, we demonstrate that modulation of CqsR QS response by ethanolamine occurs inside animal hosts. Our findings suggest that V. cholerae uses CqsR as a dual-function receptor to integrate information from the self-made signals as well as exogenous ethanolamine as an environmental cue to modulate QS response

Genetics of type VI secretion and natural transformation in Vibrio cholerae

The facultative waterborne pathogen Vibrio cholerae transitions between its human host and the environment where it colonizes chitinous surfaces in aquatic settings. Growth on chitin coordinates the induction of sets of genes for 1) chitin utilization; 2) a type VI secretion system that allows contact-dependent killing of neighboring bacteria; and 3) DNA uptake by natural transformation, which is a mechanism for horizontal gene transfer. This thesis describes the regulatory network controlling these behaviors in V. cholerae and the consequences of their coordinate regulation. Results from high-throughput RNA sequencing (RNA-seq) show that transcription factor CytR is one of four positive regulators comprising the chitin-induced regulatory network. A combination of genetic and phenotypic assays reveal the four regulators TfoX, HapR, QstR and CytR control each behavior in a distinct manner in a commonly used clinical reference strain of V. cholerae. Whole genome sequencing and bioinformatics analyses of a set of strains isolated from diverse sources reveal novel type VI secretion system components present in environmental, but not clinical isolates. Finally, I show that chitin-induced natural transformation can facilitate horizontal gene transfer of distinct type VI secretion system genes between strains. Horizontally acquired effector-immunity proteins are functional in the new genetic background and can be employed in antibacterial antagonism against parental cells and simultaneously protect against attacks by the donor cells. This thesis sheds light on diverse behavioral adaptations that allow this important human pathogen to spread and persist in the environment.Ph.D

CytR Is a Global Positive Regulator of Competence, Type VI Secretion, and Chitinases in <i>Vibrio cholerae</i>

<div><p>The facultative pathogen <i>Vibrio cholerae</i> transitions between its human host and aquatic reservoirs where it colonizes chitinous surfaces. Growth on chitin induces expression of chitin utilization genes, genes involved in DNA uptake by natural transformation, and a type VI secretion system that allows contact-dependent killing of neighboring bacteria. We have previously shown that the transcription factor CytR, thought to primarily regulate the pyrimidine nucleoside scavenging response, is required for natural competence in <i>V</i>. <i>cholerae</i>. Through high-throughput RNA sequencing (RNA-seq), we show that CytR positively regulates the majority of competence genes, the three type VI secretion operons, and the four known or predicted chitinases. We used transcriptional reporters and phenotypic analysis to determine the individual contributions of quorum sensing, which is controlled by the transcription factors HapR and QstR; chitin utilization that is mediated by TfoX; and pyrimidine starvation that is orchestrated by CytR, toward each of these processes. We find that in <i>V</i>. <i>cholerae</i>, CytR is a global regulator of multiple behaviors affecting fitness and adaptability in the environment.</p></div

Expression of Type VI secretion system genes and T6SS-mediated killing are positively regulated by CytR, TfoX, HapR, and QstR.

<p><i>V</i>. <i>cholerae</i> C6706 with indicated alleles of <i>tfoX</i>, <i>cytR</i>, <i>hapR</i>, and <i>qstR</i> (+, native;-, deletion; *, constitutively expressed) were analyzed for expression of bioluminescence from a plasmid-encoded <i>lux</i> transcriptional reporter fusion to the promoter of first gene of a T6SS auxiliary cluster, <i>vca0017</i> (Panel A). Bioluminescence is defined as relative light production per OD₆₀₀ (RLU). All strains are deleted for <i>luxO</i> and are therefore constitutive for HapR expression (*) when the <i>hapR</i> gene is present. Data shown are mean values ± standard deviation for triplicates from one representative experiment of three performed. ‡ indicates a p-value < 0.01, † indicates a p-value <0.05. N.S. denotes not significant, calculated using a two-tailed Student’s t-test. Bars 2–5 are compared to bar 1 and bars 7–9 are compared to bar 6. Panel B: Chloramphenicol resistant <i>E</i>. <i>coli</i> prey were incubated with the indicated <i>V</i>. <i>cholerae</i> predator strains at a ratio of 1:10 on membrane filters to monitor contact-dependent killing. Total surviving prey cfus are represented in each case.</p

Expression of <i>V</i>. <i>cholerae</i> chitinases requires TfoX and CytR, but not HapR or QstR.

<p>Panel A: <i>V</i>. <i>cholerae</i> strains with indicated alleles of <i>tfoX</i>, <i>cytR</i>, <i>hapR</i> and <i>qstR</i> (+, native;-, deletion; *, constitutively expressed), were analyzed for expression of bioluminescence from a plasmid-encoded <i>lux</i> transcriptional reporter fusion to the promoter of the chitinase <i>chiA1</i>. All strains are deleted for <i>luxO</i> and are therefore constitutive for HapR expression (*) when the <i>hapR</i> gene is present. Bioluminescence is defined as relative light production per OD₆₀₀ (RLU). ‡ indicates a p-value < 0.01, † indicates a p-value <0.05. N.S. denotes not significant, calculated using a two-tailed Student’s t-test. Bars 2–5 are compared to bar 1. Panel B and C: Chitin agar plate assays. <i>V</i>. <i>cholerae</i> strains with indicated alleles of <i>tfoX</i>, <i>cytR</i>, <i>hapR</i>, and <i>qstR</i> were assayed for chitinase activity which results in a zone of clearing on LB plates containing 2% colloidal chitin (panel B). Strains constitutive for TfoX (*) and isogenic strains deleted for <i>cytR</i>, <i>tfoX</i> and the CytR-dependent chitinases <i>chiA1</i>, <i>chiA2</i>, <i>vc0769</i>, <i>vca0700</i>, a <i>chiA1 chiA2</i> double mutant and a strain deleted for all four chitinases were assayed for the contribution of individual chitinase genes to chitinase activity (panel C).</p

CytR and TfoX co-regulate natural competence, chitinase expression and the type VI secretion system.

<p>Panel A: <i>V</i>. <i>cholerae</i> C6706 is capable of natural transformation in LB medium lacking chitin if <i>tfoX</i> is constitutively expressed (TfoX*, bar 1) but not if <i>tfoX</i> is under control of its native promoter (TfoX⁺, bars 3 and 4). No transformants were detected in the absence of CytR (CytR^-, bars 2 and 4). Transformation frequency is expressed as the number of kanamycin resistant cfu mL^-1 divided by total cfu mL^-1. The limit of detection (d.l.) is 1 x 10⁻⁸. Data are shown as mean ± standard deviation from three independent biological replicates. Panel B: Heat map of genes differentially regulated by CytR in the absence (TfoX⁺, column 1) or presence (TfoX*, column 2) of TfoX induction, and genes differentially regulated by TfoX in the absence (CytR^-, column 3) or presence (CytR⁺, column 4) of a functional <i>cytR</i> gene. The majority of known competence genes are positively regulated by both TfoX and CytR and can be classified into four distinct regulatory classes (see text for details). CytR and TfoX positively regulate the three known T6SS gene clusters as well as four chitinase genes. CytR negatively regulates nucleoside uptake and catabolism genes in a TfoX-independent manner.</p

Competence genes are differentially regulated by TfoX, CytR, HapR and QstR.

<p><i>V</i>. <i>cholerae</i> C6706 derivatives with native alleles of <i>tfoX</i>, <i>cytR</i> and <i>qstR</i> (not constitutively expressed, denoted by +), alleles of <i>tfoX</i> or <i>qstR</i> made constitutive by replacing the chromosomal native promoter with a <i>ptac</i> promoter (indicated by *), or containing in-frame deletions of <i>tfoX</i>, <i>cytR</i>, <i>hapR</i> and <i>qstR</i> (-), were analyzed for expression of bioluminescence from plasmid-encoded <i>lux</i> transcriptional reporter fusions. Expression profiles are shown for the transcriptional regulator <i>qstR</i> (Panel A) and for a member of each regulatory class: class I, <i>comEA</i> (Panel B) class II, <i>pilM</i> (Panel C) class III, <i>pilF</i>, (Panel D), and class IV, <i>pilT</i> (Panel E). All strains are deleted for <i>luxO</i> and are therefore constitutive for HapR expression (*) when the <i>hapR</i> gene is present. Bioluminescence is represented as relative light production per OD₆₀₀ (RLU) and data shown are mean values ± standard deviation from three biological replicates of one representative experiment of three. Data are shown as mean values ± standard deviation. ‡ indicates a p-value < 0.01, † indicates a p-value <0.05. N.S. denotes not significant, calculated using a two-tailed Student’s t-test. In Panels A to E, bars 2–5 are compared to bar 1; in Panels A and B, bars 7–9 are compared to bar 6. Panel F: A TfoX* CytR⁺ HapR* QstR* strain is transformable in LB in the absence of chitin induction, but an isogenic strain carrying a <i>qstR</i> deletion was poorly transformable. The <i>hapR</i> deletion strain was partially restored for transformation by constitutive expression of QstR (*), but strains deleted for <i>cytR</i> or <i>tfoX</i> were not restored for competence by the QstR* allele. The limit of detection is 1 x 10⁻⁸ cfu. mL^-1 (d.l.).</p