Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Brzozowski, R; White, Maria L; Eswara, Prahathees J.

doi:10.3791/59905

Cited by 16 publications

(12 citation statements)

References 23 publications

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“…In the cultures that were treated with both, cells were pre-treated for 10 min with putrescine prior to the addition of phage. Cultures were then allowed to grow for an additional 2 h. Fluorescence microscopy was then performed as previously described (Brzozowski et al, 2019). Briefly, cells from culture aliquots of each strain in different treatment condition were stained with 1 µg/ml SynaptoRed (FM4-64) and 1 µg/ml DAPI to visualize the membrane and DNA respectively.…”

Section: Methodsmentioning

confidence: 99%

Bacterial threat assessment of bacteriophage infection is mediated by intracellular polyamine accumulation and Gac/Rsm signaling

Mattos

Nemudryi

Faith

et al. 2022

Preprint

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When eukaryotic cells are killed by pathogenic microorganisms, damage-associated and pathogen-associated signals are generated that alert other cells of nearby danger. Bacteria can detect the death of their kin; however, how bacteria make threat assessments of cellular injury is largely unexplored. Here we show that polyamines released by lysed bacteria serve as damage-associated molecules in Pseudomonas aeruginosa. In response to exogenous polyamines, Gac/Rsm and cyclic-di-GMP signaling is activated and intracellular polyamine levels increase. In the absence of a threat, polyamines are catabolized, and intracellular polyamines return to basal levels, but cells infected by bacteriophage increase and maintain intracellular polyamine levels, which inhibits phage replication. Phage species not inhibited by polyamines did not trigger polyamine accumulation by P. aeruginosa, suggesting polyamine accumulation and metabolism are targets in the phage-host arms-race. Our results suggest that like eukaryotic cells, bacteria can differentiate damage-associated and pathogen-associated signals to make threat assessments of cellular injury.

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Section: Methodsmentioning

confidence: 99%

Bacterial threat assessment of bacteriophage infection is mediated by intracellular polyamine accumulation and Gac/Rsm signaling

Mattos

Nemudryi

Faith

et al. 2022

Preprint

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“…Cells were then resuspended in 100 μl of PBS, and the red membrane stain FM4-64 was added at a final concentration of 1 μg/ml. The sample was prepared for microscopy by spotting 5 μl of the cell suspension onto the glass coverslip of a MatTek glass bottom dish and then covering it with a 1% agarose pad made with sterile water, as described previously ( 23 ). All imaging was completed at room temperature inside an environmental chamber using a GE Applied Precision DeltaVision Elite deconvolution fluorescence microscope.…”

Section: Methodsmentioning

confidence: 99%

Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

Brzozowski

Tomlinson

Sacco

et al. 2020

mSphere

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Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

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“…Microscopy was carried out as previously described (68). Briefly, 1 ml aliquots of B. subtilis cultures were pelleted and then washed with 1x phosphate buffer saline (PBS) via centrifugation.…”

Section: Microscopymentioning

confidence: 99%

“…As described previously, strain MW205 was grown to mid-log phase (OD 0.5-0.8) before 5 μl was aliquoted onto a MatTek dish and covered with 1% agarose pad made with LB (68). Samples were allowed to adjust to the microscope chamber at 30 °C for a period of 30 min before 10 μl of 10 mM IPTG was added to the agarose pad for the plus inducer conditions.…”

Section: Timelapse Microscopymentioning

confidence: 99%

MraZ is a transcriptional inhibitor of cell division in Bacillus subtilis

White

Hough-Neidig

Khan

et al. 2022

Preprint

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The bacterial division and cell wall (dcw) cluster is a highly conserved region of the genome which encodes several essential cell division factors including the central divisome protein FtsZ. Understanding the regulation of this region is key to our overall understanding of the division process. mraZ is found at the 5 prime end of the dcw cluster and previous studies have described MraZ as a sequence-specific DNA binding protein. In this article, we investigate MraZ to elucidate its role in Bacillus subtilis. Through our investigation, we demonstrate that increased levels of MraZ result in lethal filamentation due to repression of its own operon (mraZ-mraW-ftsL-pbpB). We observe rescue of filamentation upon decoupling ftsL expression, but not other genes in the operon, from MraZ control. Furthermore, through timelapse microscopy we were able to identify that overexpression of mraZ, results in de-condensation of the FtsZ ring (Z-ring). This is likely due to depletion of FtsL, and thus, we believe the precise role of FtsL is likely in Z-ring maturation and promotion of subsequent treadmilling. Our data suggests that regulation of the mra operon may be an alternative way for cells to quickly arrest cytokinesis potentially during entry into stationary phase and in the event of DNA replication arrest.

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Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Cited by 16 publications

References 23 publications

Bacterial threat assessment of bacteriophage infection is mediated by intracellular polyamine accumulation and Gac/Rsm signaling

Bacterial threat assessment of bacteriophage infection is mediated by intracellular polyamine accumulation and Gac/Rsm signaling

Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

MraZ is a transcriptional inhibitor of cell division in Bacillus subtilis

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