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7.20B: Caulobacter Differentiation - Biology

7.20B:  Caulobacter Differentiation - Biology


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A Caulobacter is used for studying the regulation of the cell cycle, asymmetric cell division, and cellular differentiation.

LEARNING OBJECTIVES

Explain how caulobacter serve as a model organism

Key Points

  • The Caulobacter cell cycle regulatory system controls many modular subsystems that organize the progression of cell growth and reproduction.
  • The central feature of the cell cycle regulation is a cyclical genetic circuit—a cell cycle engine –- that is centered around the successive interactions of four master regulatory proteins: DnaA, GcrA, CtrA, and CcrM.
  • The interactions of four master regulatory proteins: DnaA, GcrA, CtrA, and CcrM directly control the timing of expression of over 200 genes. The four master regulatory proteins are synthesized and then eliminated from the cell one after the other over the course of the cell cycle.

Key Terms

  • senescence: Ceasing to divide by mitosis because of shortening of telomeres or excessive DNA damage.
  • differentiation: In cellular differentiation, a less specialized cell becomes a more specialized cell.
  • modular: Consisting of separate modules; especially where each module performs or fulfills some specified function and could be replaced by a similar module for the same function, independently of the other modules.

Caulobacter crescentus is a Gram-negative, oligotrophic bacterium widely distributed in fresh water lakes and streams. Caulobacter is an important model organism for studying the regulation of the cell cycle, asymmetric cell division, and cellular differentiation. Caulobacter daughter cells have two very different forms. One daughter is a mobile “swarmer” cell that has a single flagellum at one cell pole that provides swimming motility for chemotaxis. The other daughter, called the “stalked” cell has a tubular stalk structure protruding from one pole that has an adhesive holdfast material on its end, with which the stalked cell can adhere to surfaces. Swarmer cells differentiate into stalked cells after a short period of motility. Chromosome replication and cell division only occurs in the stalked cell stage. Its name is due to the fact that it forms a crescent shape; crescentin is a protein that imparts this shape.

In the laboratory, researchers distinguish between C. crescentusstrain CB15 (the strain originally isolated from a freshwater lake) and NA1000 (the primary experimental strain). In strain NA1000, which was derived from CB15 in the 1970’s, the stalked and predivisional cells can be physically separated in the laboratory from new swarmer cells, while cell types from strain CB15 cannot be physically separated. The isolated swarmer cells can then be grown as a synchronized cell culture. Detailed study of the molecular development of these cells as they progress through the cell cycle has enabled researchers to understand Caulobacter cell cycle regulation in great detail. Due to this capacity to be physically synchronized, strain NA1000 has become the predominant experimental Caulobacter strain throughout the world. Additional phenotypic differences between the two strains have subsequently accumulated due to selective pressures on the NA1000 strain in the laboratory environment. The genetic basis of the phenotypic differences between the two strains results from coding, regulatory, and insertion/deletion polymorphisms at five chromosomal loci. “C. Crescentus” is synonymous with “Caulobacter Vibrioides. ”

The Caulobacter cell cycle regulatory system controls many modular subsystems that organize the progression of cell growth and reproduction. A control system constructed using biochemical and genetic logic circuitry organizes the timing of initiation of each of these subsystems. The central feature of the cell cycle regulation is a cyclical genetic circuit—a cell cycle engine –- that is centered around the successive interactions of four master regulatory proteins: DnaA, GcrA, CtrA, and CcrM. These four proteins directly control the timing of expression of over 200 genes. The four master regulatory proteins are synthesized and then eliminated from the cell one after the other over the course of the cell cycle. Several additional cell signaling pathways are also essential to the proper functioning of this cell cycle engine.

The principal role of these signaling pathways is to ensure reliable production and elimination of the CtrA protein from the cell at just the right times in the cell cycle. An essential feature of the Caulobacter cell cycle is that the chromosome is replicated once and only once per cell cycle. This is in contrast to the E. coli cell cycle where there can be overlapping rounds of chromosome replication simultaneously underway. The opposing roles of the Caulobacter DnaA and CtrA proteins are essential to the tight control of Caulobacter chromosome replication. The DnaA protein acts at the origin of replication to initiate the replication of the chromosome. The CtrA protein, in contrast, acts to block initiation of replication, so it must be removed from the cell before chromosome replication can begin. Multiple additional regulatory pathways integral to cell cycle regulation and involving both phospho signaling pathways and regulated control of protein proteolysis act to assure that DnaA and CtrA are present in the cell exactly when they are needed.

Caulobacter was the first asymmetric bacterium shown to age. Reproductive senescence was measured as the decline in the number of progeny produced over time. A similar phenomenon has since been described in the bacterium Escherichia coli, which gives rise to morphologically similar daughter cells.


Anatomy of a bacterial cell cycle

Two recent reports describe mRNA and protein expression patterns in the bacterium Caulobacter crescentus. The combined use of DNA microarray and proteomic analyses provides a powerful new perspective for unraveling the global regulatory networks of this complex bacterium.

The seemingly simple process of bacterial growth and division requires an impressive orchestration of functions. Cells monitor and coordinate DNA replication with cell division to ensure that each progeny cell receives an accurate copy of the genome. Optimal growth requires coordination of the cell cycle with metabolic processes. In bacteria that undergo cellular differentiation, developmental events are coordinated with growth and with the cell cycle [1,2]. Complex regulatory networks must be in place to ensure the proper coordination of these events, not only in bacteria but in all organisms. Our understanding of these networks is still limited, even in well-studied model organisms, because most studies have analyzed expression patterns of, at best, a few genes. The advent of complete genome sequence information, coupled with post-genomic high-throughput techniques, now allows the global analysis of gene expression and protein levels throughout the cell cycle. As with so many previous revolutions in the biological sciences, microbes are providing the benchmark with which to test the new technologies [3,4]. The recent publication of two articles analyzing the cell cycle of Caulobacter crescentus [5,6] illustrates the advantage that the combined use of DNA microarray and proteomic methods can provide in understanding global cellular processes.


Cell Structure and Metabolism

Caulobacter are Gram-negative, rod-like cells that can be flagellated in a polar manner or have a stalk. Caulobacter lack intracellular organelles. They are hereotrophic aerobes and can be found in aquatic environments attached to particulate matter, plant materials, or other microorganisms by its stalk. Caulobacter asymmetrically divides to produce two types of daughter cells that are functionally and structurally different. The cylindrical body of a stalk cell is approximately 0.7 micrometers in diameter and 2-3 micrometers in length. The swarmer cell is smaller than the stalk cell initially the different hydrodynamics due to the different morphologies between the swarmer and stalk cells allow swarmers to be isolated relatively easily (Stanford).


To Sing Fung and Ding Xiang Liu
Vol. 73, 2019

Abstract

Human coronavirus (HCoV) infection causes respiratory diseases with mild to severe outcomes. In the last 15 years, we have witnessed the emergence of two zoonotic, highly pathogenic HCoVs: severe acute respiratory syndrome coronavirus (SARS-CoV) and . Read More

Figure 1: Taxonomy of HCoVs: the updated classification scheme of HCoV and other coronaviruses. The six known HCoVs are in blue. Abbreviations: BtCoV, bat coronavirus BuCoV, bulbul coronavirus HCoV.

Figure 2: Genome structure of human coronaviruses (HCoVs). Schematic diagram showing the genome structure of six known HCoVs (not to scale). The 5′-cap structure (5′-C) and 3′-polyadenylation (AnAOH-3.

Figure 3: Replication cycle of human coronaviruses (HCoVs). Schematic diagram showing the general replication cycle of HCoVs. Infection starts with the attachment of HCoVs to the cognate cellular rece.

Figure 4: Induction and modulation of autophagy by HCoV infection. Schematic diagram showing the signaling pathway of autophagy and the modulatory mechanisms utilized by HCoV. Viruses and viral compon.

Figure 5: Apoptosis induced by HCoV infection and modulatory mechanisms. Schematic diagram showing the signaling pathway of intrinsic and extrinsic apoptosis induction and the modulatory mechanisms ut.

Figure 6: Induction and modulation of unfolded protein response by HCoV infection. Schematic diagram showing the three branches of UPR signaling pathway activated and regulated by HCoV infection. Viru.

Figure 7: Activation and modulation of MAPK signaling pathways by HCoV infection. Schematic diagram showing the activation and modulation of MAPK signaling pathway by HCoV infection. Viruses and viral.

Figure 8: Type I interferon induction and signaling during HCoV infection and modulatory mechanisms. Schematic diagram showing the induction and signaling pathways of type I interferon during HCoV inf.


Regulation of the Caulobacter flagellar gene hierarchy not just for motility

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.

Abstract

The Caulobacter crescentus flagellum serves not only as a motility apparatus, but also as a key landmark in the differentiation of this asymmetrically dividing bacterium. A distinctive aspect of flagellum biosynthesis is the periodic expression of the flagellar genes during the cell cycle in a sequence corresponding to the order of gene product assembly into the growing flagellum. This program of gene expression is achieved in part by the organization of flagellar genes into a four-tiered regulatory hierarchy that controls their expression at both the transcriptional and post-transcriptional levels. Because of the close interconnection of the developmental program to the asymmetric cell-division cycle in C. crescentus, studies of flagellar gene regulation and motility have also begun to reveal basic mechanisms responsible for control of the cell cycle itself. Here, we review recent work on regulation of the flagellar gene hierarchy in C. crescentus and consider regulatory mechanisms that are distinct from those described in Escherichia coli and Salmonella typhimurium.


Mitochondrial Machineries for Protein Import and Assembly

Nils Wiedemann and Nikolaus Pfanner
Vol. 86, 2017

Abstract

Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol and are imported into mitochondria by five transport . Read More

Figure 1: Overview of the five major protein import pathways of mitochondria. Presequence-carrying preproteins are imported by the translocase of the outer mitochondrial membrane (TOM) and the presequ.

Figure 2: The presequence pathway into the mitochondrial inner membrane (IM) and matrix. The translocase of the outer membrane (TOM) consists of three receptor proteins, the channel-forming protein To.

Figure 3: Role of the oxidase assembly (OXA) translocase in protein sorting. Proteins synthesized by mitochondrial ribosomes are exported into the inner membrane (IM) by the OXA translocase the ribos.

Figure 4: Carrier pathway into the inner membrane. The precursors of the hydrophobic metabolite carriers are synthesized without a cleavable presequence. The precursors are bound to cytosolic chaperon.

Figure 5: Mitochondrial intermembrane space import and assembly (MIA) machinery. Many intermembrane space (IMS) proteins contain characteristic cysteine motifs. The precursors are kept in a reduced an.

Figure 6: Biogenesis of β-barrel proteins of the outer mitochondrial membrane. The precursors of β-barrel proteins are initially imported by the translocase of the outer membrane (TOM), bind to small .

Figure 7: The dual role of mitochondrial distribution and morphology protein 10 (Mdm10) in protein assembly and organelle contact sites. Mdm10 associates with the sorting and assembly machinery (SAM) .

Figure 8: Multiple import pathways for integral α-helical proteins of the mitochondrial outer membrane. The precursors of proteins with an N-terminal signal anchor sequence are typically inserted into.

Figure 9: The mitochondrial contact site and cristae organizing system (MICOS) interacts with protein translocases. MICOS consists of two core subunits, Mic10 and Mic60. Mic10 forms large oligomers th.


Examples of differentiation in the following topics:

Selective and Differential Media

  • Mannitol salt agar (MSA) which is selective for Gram-positive bacteria and differential for mannitol.
  • Differential media or indicator media distinguish one microorganism type from another growing on the same media.
  • Examples of differential media include:
  • Streptococcuseosin methylene blue (EMB), which is differential for lactose and sucrose fermentation.
  • MacConkey (MCK), which is differential for lactose fermentationmannitol salt agar (MSA), which is differential for mannitol fermentation.

Development of the Dual Lymphocyte System

  • Mammalian stem cells differentiate into several kinds of blood cell within the bone marrow.
  • During this process, all lymphocytes originate from a common lymphoid progenitor before differentiating into their distinct lymphocyte types.
  • B and T cells) differentiate further after exposure to an antigen they form effector and memory lymphocytes.
  • Mammalian stem cells differentiate into several kinds of blood cell within the bone marrow.
  • All lymphocytes originate during this process from a common lymphoid progenitor before differentiating into their distinct lymphocyte types.

Tests That Differentiate Between T Cells and B cells

  • Methods used to differentiate T cells and B cells include staining cell surface receptors and functional assays like the T lymphocyte cytotoxicity assay.
  • The expression of different markers allows the separation/differentiation of T and B cells.
  • These techniques are based on staining B and T cells for unique cell surface markers known as cluster of differentiation (CD).
  • Describe how T cells and B cells can be differentiated using staining of cell surface receptors and functional assays like the T lymphocyte cytotoxicity assay

Interference Microscopy

  • There are three types of interference microscopy: classical, differential contrast, and fluorescence contrast.
  • Since its introduction in the late 1960s differential interference contrast microscopy (DIC) has been popular in biomedical research because it produces high-resolution images of fine structures by enhancing the contrasted interfaces.
  • Fluorescence differential interference contrast (FLIC) microscopy was developed by combining fluorescence microscopy with DIC to minimize the effects of photobleaching on fluorochromes bound to the stained specimen.

Caulobacter Differentiation

  • A Caulobacter is used for studying the regulation of the cell cycle, asymmetric cell division, and cellular differentiation.
  • Caulobacter is an important model organism for studying the regulation of the cell cycle, asymmetric cell division, and cellular differentiation.
  • Swarmer cells differentiate into stalked cells after a short period of motility.
  • Swarmer cells differentiate into stalked cells after a short period of motility.

Detecting Acid and Gas Production

  • Culture media can be used to differentiate between different kinds of bacteria by detecting acid or gas production.
  • Differential media, also known as indicator media, distinguish one microorganism type from another growing on the same media.
  • Differential media are used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria.
  • This raises the pH of the medium, allowing the O157:H7 strain to be differentiated from other E. coli strains through the action of the pH indicator in the medium.

Classes of T Cells

  • This activation results in the expansion of the antigen-specific lymphocyte pool and the differentiation of these cells into effector and memory cells.
  • In response to antigenic stimulation, helper T cells (characterized by the expression of CD4 marker on their surface) secrete proteins called cytokines, whose function is to stimulate the proliferation and differentiation of the T cells themselves, as well as other cells, including B cells, macrophages, and other leukocytes.
  • Memory T cells are an expanded population of T cells specific for antigens that can respond rapidly to subsequent encounter with that antigen and differentiate into effector cell to eliminate the antigen.

Clonal Selection of Antibody-Producing Cells

  • The differentiated effector cells derived from an activated lymphocyte will bear receptors of identical specificity as the parental cell.
  • All B cells derive from a particular cell, and as such, the antibodies and their differentiated progenies can recognize and/or bind the same specific surface components composed of biological macromolecules (epitope) of a given antigen.
  • Most of such B cells differentiate into plasma cells that secrete antibodies into blood that bind the same epitope that elicited proliferation in the first place.
  • B cells that have not been activated by antigen are known as naive lymphocytes those that have met their antigen, become activated, and have differentiated further into fully functional lymphocytes are known as effector B lymphocytes.
  • Clonal selection of lymphocytes: 1) A hematopoietic stem cell undergoes differentiation and genetic rearrangement to produce 2) immature lymphocytes with many different antigen receptors.

Morphologically Unusual Proteobacteria

  • Caulobacter is an important model organism for studying the regulation of the cell cycle, asymmetric cell division, and cellular differentiation.
  • Swarmer cells differentiate into stalked cells after a short period of motility.
  • Swarmer cells differentiate into stalked cells after a short period of motility.

Bacterial Differentiation

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Localization of Surface Structures During Procaryotic Differentiation: Role of Cell Division in Caulobacter crescentus

Asymmetric cell division in Caulobacter crescentus produces two cell types, a stalked cell and a new swarmer cell, with characteristics surface structures. We have examined the role of the cell cycle in the differentiation of these two cells using the adsorption of bacteriophage øLC72, the assembly of the polar flagellum, and stalk formation as assays for changes in surface morphology. Previous studies of this aquatic bacterium [17, 25] have suggested that the replicating chromosome acts as a ‘clock’ in timing the formation of the flagellar filament at one pole of the new swarmer cell. The analysis of conditional cell cycle mutants presented here extends these results by showing that DNA synthesis is also required for adsorption of phage øLC72 and, more importantly, they also suggest that a late cell division step is involved in determining the spatial pattern in which the phage receptors and flagella are assembled. We propose that this cell division step is required for formation of ‘organizational’ centers which direct the assembly of surface structures at the new cell poles, and for the polarity reversal in assembly that accompanies swarmer cell to stalked cell development.


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Supplementary Information

Supplementary Table 1, Supplementary Figures 1–10, Supplementary Note 1

Reporting Summary

Supplementary Video 1

Asymmetric cell division of YhjH-mChy-PopZ positive cells bearing plasmids cells bearing plasmid pBad-YmP. Same conditions as Fig. 1c. This experiment was performed three times and produced similar results.

Supplementary Videos 2

Examples of asymmetric cell division by YhjH-mChy-PopZ positive cells bearing plasmids pAC-YC-YmP-S and pBad-MrkA-rbs-GFP. Same conditions as Supplementary Video 1 and Fig. 3d. This experiment was performed three times and produced similar results.

Supplementary Videos 3

Examples of asymmetric cell division by YhjH-mChy-PopZ positive cells bearing plasmids pAC-YC-YmP-S and pBad-MrkA-rbs-GFP. Same conditions as Supplementary Video 1 and Fig. 3d. This experiment was performed three times and produced similar results.

Supplementary Video 4

Phase contrast movie of cells exposed to 4 h of pulsed green light, in presence of 0.2% of arabinose. Same conditions as Fig. 4f,g, left panels. This experiment was performed more than three times and produced similar results.

Supplementary Video 5

mChy fluorescence movie of cells exposed to 4 h of pulsed green light, in presence of 0.2% of arabinose. Same conditions as Fig. 4f,g, left panels. This experiment was performed three times and produced similar results.

Supplementary Video 6

Phase contrast movie of cells exposed to 4 h of constant red light, in presence of 0.2% of arabinose. Same conditions as Fig. 4f,g, right panels. This experiment was performed more than three times and produced similar results.

Supplementary Video 7

mChy fluorescence movie of cells exposed to 4 h of constant red light, in presence of 0.2% of arabinose. Same conditions as Figure 4f,g, right panels. This experiment was performed three times and produced similar results.

Supplementary Video 8

Phase contrast movie of wild-type (MG1655) cells. This experiment was performed more than three times and produced similar results.

Supplementary Video 9

Phase contrast movie of ∆motA-motB strain. This experiment was performed more than three times and produced similar results.