live-cell-fluorescence-microscopy-to-observe-essential-processes

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

University of Missouri

Mechanistic studies of many processes in Agrobacterium tumefaciens have been hampered by a lack of genetic tools for characterization of essential genes. In this study, we used a Tn7-based method for inducible control of transcription from an engineered site on the chromosome. We demonstrate that this method enables tighter control of inducible promoters than plasmid-based systems and can be used for depletion studies. The method enables the construction of depletion strains to characterize the roles of essential genes in A. tumefaciens Here, we used the strategy to deplete the alphaproteobacterial master regulator CtrA and found that depletion of this essential gene results in dramatic rounding of cells, which become nonviable.

Mini-Tn7 Insertion in an Artificial attTn7 Site Enables Depletion of the Essential Master Regulator CtrA in the Phytopathogen Agrobacterium tumefaciens.

Polar growth is the predominant mode of cell wall extension in the Actinobacteria and the alphaproteobacterial clade Rhizobiales. The observation of polar elongation in taxonomically diverse bacteria suggests that polar growth may have evolved independently. Indeed, the regulatory mechanisms governing the assembly of cell wall biosynthesis machinery at the pole are distinct in the Actinobacteria and Rhizobiales. Here we highlight recent advances in our understanding of polar growth mechanisms in bacteria, with an emphasis on Streptomyces and Agrobacterium. This review illustrates that common themes are emerging in the regulation of polar growth in diverse bacteria. Emerging themes include the use of landmark proteins to direct growth to the pole and coordination of polar growth with cell-cycle progression.

Building the bacterial cell wall at the pole.

Agrobacterium tumefaciens is a rod-shaped bacterium that grows by polar insertion of new peptidoglycan during cell elongation. As the cell cycle progresses, peptidoglycan synthesis at the pole ceases prior to insertion of new peptidoglycan at midcell to enable cell division. The A. tumefaciens homolog of the Caulobacter crescentus polar organelle development protein PopZ has been identified as a growth pole marker and a candidate polar growth-promoting factor. Here, we characterize the function of PopZ in cell growth and division of A. tumefaciens Consistent with previous observations, we observe that PopZ localizes specifically to the growth pole in wild-type cells. Despite the striking localization pattern of PopZ, we find the absence of the protein does not impair polar elongation or cause major changes in the peptidoglycan composition. Instead, we observe an atypical cell length distribution, including minicells, elongated cells, and cells with ectopic poles. Most minicells lack DNA, suggesting a defect in chromosome segregation. Furthermore, the canonical cell division proteins FtsZ and FtsA are misplaced, leading to asymmetric sites of cell constriction. Together, these data suggest that PopZ plays an important role in the regulation of chromosome segregation and cell division.IMPORTANCEA. tumefaciens is a bacterial plant pathogen and a natural genetic engineer. However, very little is known about the spatial and temporal regulation of cell wall biogenesis that leads to polar growth in this bacterium. Understanding the molecular basis of A. tumefaciens growth may allow for the development of innovations to prevent disease or to promote growth during biotechnology applications. Finally, since many closely related plant and animal pathogens exhibit polar growth, discoveries in A. tumefaciens may be broadly applicable for devising antimicrobial strategies.

Article	Year
Mini-Tn7 Insertion in an Artificial attTn7 Site Enables Depletion of the Essential Master Regulator CtrA in the Phytopathogen Agrobacterium tumefaciens. Applied and environmental microbiology\| PubMed ID: 27287320	2016
Building the bacterial cell wall at the pole. Current opinion in microbiology\| PubMed ID: 27504539	2016
Absence of the Polar Organizing Protein PopZ Results in Reduced and Asymmetric Cell Division in Agrobacterium tumefaciens. Journal of bacteriology\| PubMed ID: 28630123	2017

Matthew Howell

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