CsrA and its regulators control the time-point of ColicinE2 release in Escherichia coli

The bacterial SOS response is a cellular reaction to DNA damage, that, among other actions, triggers the expression of colicin - toxic bacteriocins in Escherichia coli that are released to kill close relatives competing for resources. However, it is largely unknown, how the complex network regulating toxin expression controls the time-point of toxin release to prevent premature release of inefficient protein concentrations. Here, we study how different regulatory mechanisms affect production and release of the bacteriocin ColicinE2 in Escherichia coli. Combining experimental and theoretical approaches, we demonstrate that the global carbon storage regulator CsrA controls the duration of the delay between toxin production and release and emphasize the importance of CsrA sequestering elements for the timing of ColicinE2 release. In particular, we show that ssDNA originating from rolling-circle replication of the toxin-producing plasmid represents a yet unknown additional CsrA sequestering element, which is essential in the ColicinE2-producing strain to enable toxin release by reducing the amount of free CsrA molecules in the bacterial cell. Taken together, our findings show that CsrA times ColicinE2 release and reveal a dual function for CsrA as an ssDNA and mRNA-binding protein, introducing ssDNA as an important post-transcriptional gene regulatory element

On noise and single-cell expression dynamics in toxin-driven bacterial competition

Complex microbial communities are composed of a multitude of bacterial strains that interact with each other in many different ways. Stability of such systems is crucial for their long-term survival, especially in fluctuating environments. It is still largely unknown what factors influence bacterial interaction dynamics and how they affect bacterial competition. But, the interaction of strains can be driven by the production of toxins or public goods. Therefore, it is crucial to get further insight into the gene expression dynamics of these compounds in order to understand the development of such complex ecosystems. Factors affecting bacterial competition such as the timing of release of interacting components and the amount being released into the environment have to be studied in order to determine their influence on competition outcome. Additionally, it is unknown how noise in gene expression dynamics of interacting compounds and the resulting release distributions influence bacterial competition. In this study, the plasmid encoded toxin producing ColicinE2 system of the well-known organism Escherichia coli was used as a model system. Bacterial interactions involving this strain are driven by the production and release of a toxin called colicin which kills closely related competitors. Therefore, in this combined experimental and theoretical study, toxin expression dynamics were investigated and how they determine the timing and amount of toxin being released. Additionally, mechanisms of noise control of both, toxin production and release in the ColicinE2 expression system were analyzed. Finally, the influence of stochasticity in single-cell expression dynamics and toxin production on bacterial competition outcome between a colicin producing strain and a toxin sensitive strain were investigated. Using this analysis, I was able to show that both toxin expression dynamics and noise in the ColicinE2 system are mainly controlled by globally acting regulatory proteins such as LexA and CsrA. Regarding CsrA, factors affecting the availability of free CsrA play an important role. Furthermore, I was able to identify single-stranded DNA produced by replication of the toxin producing plasmid as a new, previously unknown regulatory component influencing CsrA abundance in the cell. In addition, I could show that the metabolism of the bacterial cell influences the timing of toxin release, which is in turn correlated to the actual amount of released toxin. Finally, I could show how these toxin expression dynamics affect competition outcome for colicin driven bacterial interaction and could determine the importance of high toxin amounts as well as heterogeneity in toxin release timing for the competitive success of the colicin producing population.Komplexe mikrobielle Gemeinschaften bestehen aus vielen verschiedenen Bakterienstämmen die eine Vielzahl an Interaktionsmöglichkeiten miteinander besitzen. Vor allem in Umgebungen die vielen Schwankungen ausgesetzt sind, ist die Stabilität eines solchen Ökosystems ein wichtiges Überlebenskriterium. Es ist jedoch noch kaum bekannt welche Faktoren die dynamischen Prozesse der bakteriellen Interaktion beeinflussen und wie sich die dadurch veränderten Prozesse auf den bakteriellen Wettbewerb auswirken. Die Interaktion von verschiedenen Bakterienstämmen kann z.B. durch die Produktion und Abgabe von allgemein nutzbaren Substanzen (z.B. Proteine,...) erfolgen. Daher ist es wichtig die Produktionsdynamiken solcher Substanzen in einzelnen Zellen (mikroskopische Interaktionsebene) zu untersuchen um ihren Einfluss auf die Zusammensetzung komplexer Ökosysteme (makroskopische Interaktionsebene) verstehen zu können. Dabei ist eine quantitative Analyse spezifischer Interaktionsparameter von besonderem Interesse, wie z.B. ihre Produktionsmenge und ihr Abgabezeitpunkt, um zu verstehen wie sich Änderungen dieser Parameter auf das Wettbewerbsergebnis zwischen den Interaktionspartnern auswirken. Ein weiterer wichtiger Faktor, der diese Parameter und damit den bakteriellen Wettbewerb beeinflussen kann ist stochastisches Rauschen. In dieser Arbeit wird das plasmidkodierte ColicinE2 System von Escherichia coli als Modellsystem genutzt um oben genannte Aspekte zu studieren. Ein wichtiger Faktor der Interaktionen bei denen ein solcher Stamm beteiligt ist, ist die Produktion und Abgabe eines Toxins (Colicin genannt), das nahe verwandte Bakterien tötet. Daher wird in dieser Arbeit in einer Kombination aus experimenteller und theoretischer Analyse untersucht welchen Einfluss Einzelzellparameter wie der Zeitpunkt der Toxinabgabe und die Menge des abgegebenen Toxins auf den makroskopischen bakteriellen Wettbewerb (Populationsebene) haben. Des Weiteren wird analysiert welche regulatorische Mechanismen des ColicinE2 Systems das Rauschen von Toxinproduktionsmenge und Abgabezeitpunkt des Toxins kontrollieren. Abschließend wird derWettbewerb zwischen einem toxinproduzierenden C-Stamm und einem toxinsensitiven S-Stamm untersucht und wie sich die zuvor untersuchten Expressionsdynamiken der einzelnen Zellen und Stochastizität der Genexpression auf den Wettbewerb zwischen dem C-Stamm und dem S-Stamm auswirken. Anhand dieser Untersuchungen konnte ich zeigen, dass die Toxinexpressionsdynamik und deren Rauschen im ColicinE2 System hauptsächlich durch globale Regulatoren wie die Proteine LexA oder CsrA kontrolliert werden. Im Bezug auf CsrA sind vor allem die Verfügbarkeit von freiem CsrA und welche Regulationskomponenten diese Verfügbarkeit steuern wichtig. Dabei konnte ich einzelsträngige DNA, die bei der Replikation des Colicinplasmids entsteht, als neuen Regulationsfaktor für freies CsrA identifizieren. Außerdem konnte ich zeigen, dass sich der Metabolismus der Bakterienzelle auf die Dynamiken der Toxinproduktion auswirkt und der Abgabezeitpunkt des Toxins mit der abgegebenen Colicinmenge korreliert. Des Weiteren konnte ich zeigen, dass sich die Toxinexpressionsdynamiken auf das Resultat des bakteriellen Wettbewerbs auswirken und dass sowohl die abgegebene Toxinmenge als auch eine zeitlich heterogene Toxinabgabe wichtig für den Wettbewerbserfolg der colicinproduzierenden Population sind

Gene expression noise in a complex artificial toxin expression system

Gene expression is an intrinsically stochastic process. Fluctuations in transcription and translation lead to cell-to-cell variations in mRNA and protein levels affecting cellular function and cell fate. Here, using fluorescence time-lapse microscopy, we quantify noise dynamics in an artificial operon in Escherichia coli, which is based on the native operon of ColicinE2, a toxin. In the natural system, toxin expression is controlled by a complex regulatory network;upon induction of the bacterial SOS response, ColicinE2 is produced (cea gene) and released (cel gene) by cell lysis. Using this ColicinE2-based operon, we demonstrate that upon induction of the SOS response noise of cells expressing the operon is significantly lower for the (mainly) transcriptionally regulated gene cea compared to the additionally post-transcriptionally regulated gene cel. Likewise, we find that mutations affecting the transcriptional regulation by the repressor LexA do not significantly alter the population noise, whereas specific mutations to post-transcriptionally regulating units, strongly influence noise levels of both genes. Furthermore, our data indicate that global factors, such as the plasmid copy number of the operon encoding plasmid, affect gene expression noise of the entire operon. Taken together, our results provide insights on how noise in a native toxin-producing operon is controlled and underline the importance of post-transcriptional regulation for noise control in this system

Lysis cassette-mediated exoprotein release in Yersinia entomophaga is controlled by a PhoB-like regulator

Secretion of exoproteins is a key component of bacterial virulence, and is tightly regulated in response to environmental stimuli and host-dependent signals. The entomopathogenic bacterium Yersinia entomophaga MH96 produces a wide range of exoproteins including its main virulence factor, the 2.46 MDa insecticidal Yen-Tc toxin complex. Previously, a high-throughput transposon-based screening assay identified the region of exoprotein release (YeRER) as essential to exoprotein release in MH96. This study defines the role of the YeRER associated ambiguous holin/endolysin-based lysis cluster (ALC) and the novel RoeA regulator in the regulation and release of exoproteins in MH96. A mutation in the ambiguous lysis cassette (ALC) region abolished exoprotein release and caused cell elongation, a phenotype able to be restored through trans-complementation with an intact ALC region. Endogenous ALC did not impact cell growth of the wild type, while artificial expression of an optimized ALC caused cell lysis. Using HolA-sfGFP and Rz1-sfGFP reporters, Rz1 expression was observed in all cells while HolA expression was limited to a small proportion of cells, which increased over time. Transcriptomic assessments found expression of the genes encoding the prominent exoproteins, including the Yen-Tc, was reduced in the roeA mutant and identified a 220 ncRNA of the YeRER intergenic region that, when trans complemented in the wildtype, abolished exoprotein release. A model for Y. entomophaga mediated exoprotein regulation and release is proposed. IMPORTANCE While theoretical models exist, there is not yet any empirical data that links ALC phage-like lysis cassettes with the release of large macro-molecular toxin complexes, such as Yen-Tc in Gram-negative bacteria. In this study, we demonstrate that the novel Y. entomophaga RoeA activates the production of exoproteins (including Yen-Tc) and the ALC at the transcriptional level. The translation of the ALC holin is confined to a subpopulation of cells that then lyse over time, indicative of a complex hierarchical regulatory network. The presence of an orthologous RoeA and a HolA like holin 59 of an eCIS Afp element in Pseudomonas chlororaphis, combined with the presented data, suggests a shared mechanism is required for the release of some large macromolecular protein assemblies, such as the Yen-Tc, and further supports classification of phage-like lysis clusters as type 10 secretion systems

大腸菌二成分制御系情報伝達ネットワークの研究

For survival, organisms adapt to environmental changes by switch the gene expression. Simple unicellular organisms that possess only thousands of genes such as bacteria shows the amazing adaptation ability against a variety of environment. Therefore, in bacteria, limited gene regulation mechanism may forms the transduction network with others for multiply the gene regulation pathway. Two component system which is the major signal transduction pathway employed in wide varieties of bacteria, is generally composed of the sensor kinase (SK) that monitors an external signal(s) and the response regulator (RR) that controls physiological activities for response against external signals. A total of about 30 unique TCS pairs of SK and RR have been identified in E. coli based on the gene organization and the further genetic and/or biochemical data. In addition to these, there is the several RRs which pairing partner SK are not found and its function are unkown in E.coli. The process of TCS signal transduction generally show a high level of specificity, while a certain level of cross-regulation has been identified at the signal transduction pathways in E. coli: cross talk in recognition of signals by the sensor SK (stage 1); cross talk in phosphorylation of RRs by SKs (stage 2); and cross talk in recognition of regulation target promoters between RRs (stage 3). Cross talk between TCS pairs has been established at three stages of the signal transduction pathways. Network formation between the TCSs may contribute for the bacterial adaptation to various environment. However, the perspective of the TCS network does not yet become clear. Especially, there are few reports for the cross recognition of the promoter by RRs. And E. coli possesses function unknown orphan RRs. In this study, for the elucidation of the entire signal transduction network of E. coli, I performed the comprehensive analysis of the stage 3 cross talk among RRs. And I investigated the role and activation mechanism of uncharacterized orphan RR. The study of the stage 3 cross talk between NarL-family RRs is described in the chapter 2. In the same line study, the cross talk between OmpR family RRs were also analyzed and described in chapter 3. The chapter 4 focuses on the uncharacterized RR YgeK. Taking all the chapter together, my thesis presents the specific and complicated promoter recognition by RRs and function of atypical RR YgeK that plays the role of growth in acetate medium and biofilm formation. These findings provide the insight into the perspective of TCS signal transduction network and contribute for understanding the mechanism how bacteria adapt and survive againt to environment change.博士(生命科学)法政大学 (Hosei University

Salmonella and Multidirectional Communication in the Gut

Salmonella enterica serovar Typhymurium (S. Typhimurium) is a bacterial pathogen which is a cause of over a million cases of gastrointestinal illness worldwide. The GI tract is a large and complex environment influenced by both the host and microbes which inhabit the host’s gut. In the gut S. Typhimurium employs various virulence factors such as SPI-1 and SPI-2 to attach to the epithelial cells and persist in the body. It also initiates host inflammatory responses by inducing production of reactive oxygen species and inflammatory cytokines. Additionally, S. Typhimurium uses unique metabolic pathways to compete for limited nutrients under inflammatory conditions and during the initial colonization stage. Some members of the resident microbiota can exacerbate S. Typhimurium-induced pathology by providing necessary substrates to the pathogen and by degrading host defense mechanisms. The dense and diverse gut microbiota utilizes a variety of signaling molecules for intra- and inter-species communication to coordinate its members. Resident microbiota can also communicate with the central and enteric nervous system through neural, endocrine, immune and humoral pathways. This brain-gut communication is involved in the regulation of host and microbiota and is greatly affected by stress. While S. Typhimurium regulates gene expression by self-produced quorum sensing molecules, such as AI-2 and AI-3, it also recognize signals produced by other microbes and the host in order to regulate its growth and virulence, and in some cases, antimicrobial resistance. In the healthy gut, resident microbiota provides colonization resistance, however inflammation shifts the balance between the pathogen and microbiota thus contributing to the S. Typhimurium blooms. In summary, S. Typhimurium employs multiple tactics to establish itself in the gut; however, the microbial composition, and existing inflammatory and neural-hormonal processes also play roles in the development of the S. Typhimurium infection. This dissertation discusses the multidirectional interactions of S. Typhimurium, host and microbiota