| 1. |
- Daitch, Allison K., et al.
(författare)
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EstG is a novel esterase required for cell envelope integrity in Caulobacter
- 2023
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Ingår i: Current Biology. - : Cell Press. - 0960-9822 .- 1879-0445. ; 33:2, s. 228-240.e7
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Tidskriftsartikel (refereegranskat)abstract
- Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the ⍺-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.
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| 2. |
- Figueroa-Cuilan, Wanda M., et al.
(författare)
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Quantitative analysis of morphogenesis and growth dynamics in an obligate intracellular bacterium
- 2023
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Ingår i: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 34:7
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Tidskriftsartikel (refereegranskat)abstract
- Obligate intracellular bacteria of the order Rickettsiales include important human pathogens. However, our understanding of the biology of Rickettsia species is limited by challenges imposed by their obligate intracellular lifestyle. To overcome this roadblock, we developed methods to assess cell wall composition, growth, and morphology of Rickettsia parkeri, a human pathogen in the spotted fever group of the Rickettsia genus. Analysis of the cell wall of R. parkeri revealed unique features that distinguish it from free-living alphaproteobacteria. Using a novel fluorescence microscopy approach, we quantified R. parkeri morphology in live host cells and found that the fraction of the population undergoing cell division decreased over the course of infection. We further demonstrated the feasibility of localizing fluorescence fusions, for example, to the cell division protein ZapA, in live R. parkeri for the first time. To evaluate population growth kinetics, we developed an imaging-based assay that improves on the throughput and resolution of other methods. Finally, we applied these tools to quantitatively demonstrate that the actin homologue MreB is required for R. parkeri growth and rod shape. Collectively, a toolkit was developed of high-throughput, quantitative tools to understand growth and morphogenesis of R. parkeri that is translatable to other obligate intracellular bacteria.
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| 3. |
- Howell, Matthew, et al.
(författare)
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Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division
- 2019
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Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 111:4, s. 1074-1092
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Tidskriftsartikel (refereegranskat)abstract
- The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re-direct peptidoglycan biosynthesis to mid-cell during cell division in polar-growing Alphaproteobacteria are largely unknown. Here, we explore the role of early division proteins of Agrobacterium tumefaciens including three FtsZ homologs, FtsA and FtsW in the transition from polar growth to mid-cell growth and ultimately cell division. Although two of the three FtsZ homologs localize to mid-cell, exhibit GTPase activity and form co-polymers, only one, FtsZ(AT), is required for cell division. We find that FtsZ(AT) is required not only for constriction and cell separation, but also for initiation of peptidoglycan synthesis at mid-cell and cessation of polar peptidoglycan biosynthesis. Depletion of FtsZ(AT) in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage by depletion of FtsA or FtsW, polar growth is terminated and ectopic growth poles emerge from mid-cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.
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| 4. |
- Woldemeskel, Selamawit Abi, et al.
(författare)
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The conserved transcriptional regulator CdnL is required for metabolic homeostasis and morphogenesis in Caulobacter
- 2020
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Ingår i: PLOS Genetics. - : Public Library of Science (PLOS). - 1553-7390 .- 1553-7404. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial growth and division require regulated synthesis of the macromolecules used to expand and replicate components of the cell. Transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor sigma(70). The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where sigma(70) localizes and stabilizes the open promoter complex. However, the contributions of CdnL to metabolic homeostasis and bacterial physiology are not well understood. Here, we show that Caulobacter crescentus cells lacking CdnL have severe morphological and growth defects. Specifically, Delta cdnL cells grow slowly in both rich and defined media, and are wider, more curved, and have shorter stalks than WT cells. These defects arise from transcriptional downregulation of most major classes of biosynthetic genes, leading to significant decreases in the levels of critical metabolites, including pyruvate, alpha-ketoglutarate, ATP, NAD(+), UDP-N-acetyl-glucosamine, lipid II, and purine and pyrimidine precursors. Notably, we find that Delta cdnL cells are glutamate auxotrophs, and Delta cdnL is synthetic lethal with other genetic perturbations that limit glutamate synthesis and lipid II production. Our findings implicate CdnL as a direct and indirect regulator of genes required for metabolic homeostasis that impacts morphogenesis through availability of lipid II and other metabolites. Author summary To grow and divide, bacteria must accumulate precursor molecules to support duplication and expansion of cellular materials. One mechanism by which bacteria do this is by regulating the expression of genes whose products are important for production of these molecules. How gene expression is maintained or altered to support synthesis of appropriate molecules to balance growth with nutrient availability is not fully understood. In this paper, we describe the role of a regulator of gene expression called CdnL in maintaining levels of molecules required for bacterial growth and reproduction. CdnL broadly impacts the levels of genes required for most biosynthetic processes. CdnL's broad impact on transcription has downstream consequences on growth rate, cell shape, and nutrient requirements for growth. We report that CdnL is particularly important for maintaining levels of the amino acid glutamate and the cell wall precursor lipid II, each of which is critical for supporting proper growth and cell morphology. Our results implicate CdnL as a broadly conserved regulator of metabolic homeostasis, growth, and cell shape in bacteria.
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