Analysis of an Uncharacterized Gene in Caulobacter Crescentus and Its Novel Connections with Cell Cycle Regulatory Machinery
Analysis of an Uncharacterized Gene in Caulobacter Crescentus and Its Novel Connections with Cell Cycle Regulatory Machinery

Author: Haibi Wang

Publisher:

Published: 2019

Total Pages: 149

ISBN-13: 9781658425261

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Cell division and differentiation are complex biological phenomena that occur in all kingdoms of life. Understanding the molecular mechanisms that underlie these complex processes often requires the study of experimentally tractable model organisms. As a Gram-negative bacteria with less than four thousand genes, Caulobacter crescentus exhibits cell differentiation, highly regulated chromosome replication and segregation, and asymmetric cell division with every turn of the cell cycle. To achieve these behaviors, Caulobacter utilizes spatial control mechanisms such as sub-cellular compartmentalization and protein localization. The cell poles are particularly enriched for cell cycle regulatory proteins. Many of these proteins are localized by the hub protein PopZ, which forms a three-dimensional scaffold that also aids in chromosome segregation. The PopZ scaffold also includes proteolysis activity, which regulates cell cycle progression in a manner that is analogous to well-known eukaryotic systems. In this dissertation, I characterized an evolutionarily conserved protein of unknown function, which is now named SpbR (Swarmer pole blocking factoR). SpbR is a pole-localized protein that has co-evolved with PopZ and other polar proteins. Strikingly, SpbR over-production exhibited a severe chromosome segregation phenotype, in which the newly replicated centromere failed to travel across the cell to its normal destination at the opposite pole. SpbR overproduction results in its accumulation at the old pole, where it physically interacts with PopZ. This prevents the relocation of PopZ to the new pole, thereby eliminating a positional cue for centromere translocation. Consistent with this, the centromere translocation phenotype of SpbR overproducing cells is further enhanced in genetic backgrounds that accumulate higher SpbR or reduce chromosome segregation activity. We find that pole-localized SpbR is normally cleared by proteolysis before the time of chromosome segregation, indicating that SpbR turnover is part of the cell cycle-dependent program of polar development.

Novel Genetic Elements and Features of the Caulobacter Crescentus System
Novel Genetic Elements and Features of the Caulobacter Crescentus System

Author: Eduardo Abeliuk Acuna

Publisher:

Published: 2011

Total Pages:

ISBN-13:

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In the last decade remarkable technologies, such as high-density tiling arrays and high-throughput DNA sequencing have enabled whole-genome studies at an unprecedented scale and resolution. In this work I describe how we used these technologies to map novel genetic elements as well as regulatory features of the bacterium Caulobacter crescentus, a model species used for understanding the prokaryotic cell-cycle. I describe a dynamic programming algorithm I used to determine the location of 27 novel small RNA transcripts (sRNAs), several novel antisense RNA transcripts, as well as the accurate mapping of mRNA transcripts. Many of these predictions were validated biochemically. In addition, I describe how we coupled high-throughput transposon mutagenesis with statistical analysis to map all the essential genetic elements in the Caulobacter crescentus genome to 8 base-pair accuracy. We found that 480 out of the 3960 genes contained in Caulobacter's genome are essential for cell viability, as well as 3 out of the 27 novel sRNA that we had identified. Interestingly, there are 90 essential small intergenic DNA segments of unknown function. Finally, I describe the operon organization of Caulobacter's genome.

Investigating the Genetic Linkage Between Chromosomal Replication and Cell Division in Caulobacter Crescentus
Investigating the Genetic Linkage Between Chromosomal Replication and Cell Division in Caulobacter Crescentus

Author: Duha AlAwad

Publisher:

Published: 2022

Total Pages: 0

ISBN-13:

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"Caulobacter crescentus is a well-established model for studying the bacterial cell cycle, a complex process where the stages of growth, chromosome replication, and cell division often overlap, highlighting the presence of a highly coordinated regulatory network that remains partially understood. To search for novel regulators of the cell cycle, specifically of chromosomal replication, we developed a novel molecular screen to isolate dipM-like mutants. DipM is an endopeptidase and a cell division protein that was implicated in the coordination of DNA replication and cell division by our group. We believe that by generating Caulobacter mutants and selecting dipM-like phenotypes, it is possible to target defects in the pathway that regulate the progression from chromosomal replication to cell wall division, where DipM seems to be a key player. Out of nearly a hundred mutants, three dipM-like mutants were selected for genome sequencing, MUT1, MUT2, and MUT3. Bioinformatics analysis of these mutants allowed the identification of six gene candidates that could be linked to the regulation of the bacterial cell cycle, specifically chromosomal replication. We further investigated MUT1 and MUT3 by complementing them with the wildtype (WT) counterparts of the mutated genes to test for WT phenotype restoration. Our findings suggest that the identified gene candidates contribute to the cell cycle progression, specifically to chromosomal replication, by maintaining protein homeostasis. We speculate that DipM works with other regulatory proteins to sense and react to disturbances in protein homeostasis. Overall, our findings provide evidence for the effectiveness of our genetic screening technique and its capacity to detect cell cycle regulators coordinating between chromosome replication and cell wall division"--

Regulatory Pathways Controlling Cell Division After DNA Damage in Caulobacter Crescentus
Regulatory Pathways Controlling Cell Division After DNA Damage in Caulobacter Crescentus

Author: Joshua Wexler Modell

Publisher:

Published: 2013

Total Pages: 145

ISBN-13:

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All cells must coordinate DNA replication with cell division in order to faithfully propagate whole chromosomes to daughter cells. During episodes of DNA damage, cells often delay division until the lesions have been repaired and replication has completed. The paradigm for the bacterial response to DNA damage is the transcriptional induction of "SOS" genes, and many organisms encode an SOS-induced cell division inhibitor. However, the mechanistic details of division inhibition are understood only in the y-proteobacterium E. coli, and it is unclear whether there are SOS-independent modes of division inhibition. I have studied the DNA damage response in the [alpha]-proteobacterium Caulobacter crescentus and identified two damage-induced cell division inhibitors. sidA is an SOS-induced division inhibitor whereas didA is induced in an SOS-independent fashion. Unlike most division inhibitors, SidA and DidA do not disrupt the localization of the cell division scaffold FtsZ or any other component of the cell division machinery or "divisome". Instead, SidA and DidA target the lateacting division proteins FtsW, FtsI, and FtsN to prevent divisome constriction, demonstrating that divisome components other than FtsZ can serve as regulatory targets. I have characterized mutations infts W andftsI which suppress the activities of both inhibitors, likely by causing cells to divide hyperactively. These results suggest that the FtsW/FtsI/FtsN subcomplex serves as an important regulatory node and may play an unexpected role in triggering divisome constriction in Caulobacter. I show that cells require at least one inhibitor to properly delay division following DNA damage, as cells lacking both inhibitors divide prematurely and suffer a viability defect in the presence of the DNA damaging agent mitomycin C (MMC). This finding suggests that some degree of redundancy exists within the Caulobacter response to MMC. Finally, I describe ongoing experiments which explore the origins of the SOS-independent induction of didA.

Molecular Microbiology of Heavy Metals
Molecular Microbiology of Heavy Metals

Author: Dietrich H. Nies

Publisher: Springer Science & Business Media

Published: 2007-03-24

Total Pages: 455

ISBN-13: 3540697713

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This book covers allocation of metals in cells, metal transporter, storage and metalloregulatory proteins, cellular responses to metal ion stress, transcription of genes involved in metal ion homeostasis, uptake of essential metals, metal efflux and other detoxification mechanisms. The book also discusses metal bioreporters for the nanomolar range of concentration and tools to address the metallome. In addition, coverage details specific metals.

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria
Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria

Author: Frans J. de Bruijn

Publisher: John Wiley & Sons

Published: 2016-07-13

Total Pages: 1472

ISBN-13: 1119004896

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Bacteria in various habitats are subject to continuously changing environmental conditions, such as nutrient deprivation, heat and cold stress, UV radiation, oxidative stress, dessication, acid stress, nitrosative stress, cell envelope stress, heavy metal exposure, osmotic stress, and others. In order to survive, they have to respond to these conditions by adapting their physiology through sometimes drastic changes in gene expression. In addition they may adapt by changing their morphology, forming biofilms, fruiting bodies or spores, filaments, Viable But Not Culturable (VBNC) cells or moving away from stress compounds via chemotaxis. Changes in gene expression constitute the main component of the bacterial response to stress and environmental changes, and involve a myriad of different mechanisms, including (alternative) sigma factors, bi- or tri-component regulatory systems, small non-coding RNA’s, chaperones, CHRIS-Cas systems, DNA repair, toxin-antitoxin systems, the stringent response, efflux pumps, alarmones, and modulation of the cell envelope or membranes, to name a few. Many regulatory elements are conserved in different bacteria; however there are endless variations on the theme and novel elements of gene regulation in bacteria inhabiting particular environments are constantly being discovered. Especially in (pathogenic) bacteria colonizing the human body a plethora of bacterial responses to innate stresses such as pH, reactive nitrogen and oxygen species and antibiotic stress are being described. An attempt is made to not only cover model systems but give a broad overview of the stress-responsive regulatory systems in a variety of bacteria, including medically important bacteria, where elucidation of certain aspects of these systems could lead to treatment strategies of the pathogens. Many of the regulatory systems being uncovered are specific, but there is also considerable “cross-talk” between different circuits. Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria is a comprehensive two-volume work bringing together both review and original research articles on key topics in stress and environmental control of gene expression in bacteria. Volume One contains key overview chapters, as well as content on one/two/three component regulatory systems and stress responses, sigma factors and stress responses, small non-coding RNAs and stress responses, toxin-antitoxin systems and stress responses, stringent response to stress, responses to UV irradiation, SOS and double stranded systems repair systems and stress, adaptation to both oxidative and osmotic stress, and desiccation tolerance and drought stress. Volume Two covers heat shock responses, chaperonins and stress, cold shock responses, adaptation to acid stress, nitrosative stress, and envelope stress, as well as iron homeostasis, metal resistance, quorum sensing, chemotaxis and biofilm formation, and viable but not culturable (VBNC) cells. Covering the full breadth of current stress and environmental control of gene expression studies and expanding it towards future advances in the field, these two volumes are a one-stop reference for (non) medical molecular geneticists interested in gene regulation under stress.

Lasso Peptides
Lasso Peptides

Author: Yanyan Li

Publisher: Springer

Published: 2014-10-21

Total Pages: 113

ISBN-13: 1493910108

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Lasso peptides form a growing family of fascinating ribosomally-synthesized and post-translationally modified peptides produced by bacteria. They contain 15 to 24 residues and share a unique interlocked topology that involves an N-terminal 7 to 9-residue macrolactam ring where the C-terminal tail is threaded and irreversibly trapped. The ring results from the condensation of the N-terminal amino group with a side-chain carboxylate of a glutamate at position 8 or 9, or an aspartate at position 7, 8 or 9. The trapping of the tail involves bulky amino acids located in the tail below and above the ring and/or disulfide bridges connecting the ring and the tail. Lasso peptides are subdivided into three subtypes depending on the absence (class II) or presence of one (class III) or two (class I) disulfide bridges. The lasso topology results in highly compact structures that give to lasso peptides an extraordinary stability towards both protease degradation and denaturing conditions. Lasso peptides are generally receptor antagonists, enzyme inhibitors and/or antibacterial or antiviral (anti-HIV) agents. The lasso scaffold and the associated biological activities shown by lasso peptides on different key targets make them promising molecules with high therapeutic potential. Their application in drug design has been exemplified by the development of an integrin antagonist based on a lasso peptide scaffold. The biosynthesis machinery of lasso peptides is therefore of high biotechnological interest, especially since such highly compact and stable structures have to date revealed inaccessible by peptide synthesis. Lasso peptides are produced from a linear precursor LasA, which undergoes a maturation process involving several steps, in particular cleavage of the leader peptide and cyclization. The post-translational modifications are ensured by a dedicated enzymatic machinery, which is composed of an ATP-dependent cysteine protease (LasB) and a lactam synthetase (LasC) that form an enzymatic complex called lasso synthetase. Microcin J25, produced by Escherichia coli AY25, is the archetype of lasso peptides and the most extensively studied. To date only around forty lasso peptides have been isolated, but genome mining approaches have revealed that they are widely distributed among Proteobacteria and Actinobacteria, particularly in Streptomyces, making available a rich resource of novel lasso peptides and enzyme machineries towards lasso topologies.