Research

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False colored SEM of Prosthecomicrobium hirschii. This bacterium produces remarkable cell morphologies.

My lab is interested in understanding the principles that govern bacterial morphology, a readily observable facet of microbial cell biology. One of the major unsolved questions in microbiology is how bacteria generate specific shapes. Bacteria exhibit an amazing diversity of shapes and sizes that are precisely reproduced at every generation, indicating that morphology plays an important role in the life of these bacteria. Impressive progress has been made in the past few years in understanding the mechanism of cell shape determination in a few model bacterial systems, including the discovery that bacteria possess a cytoskeleton, but we are still very far having a comprehensive understanding of how bacterial morphologies are generated. My lab takes advantage of recent technical advances in microscopy, construction of fluorescent fusion proteins, and high throughput sequencing, to make strides in understanding how bacteria generate specific morphologies.

In most bacteria, the cell wall is comprised of peptidoglycan and is a major determinant of cell shape. In a majority of rod-shaped bacteria, including E. coli, cells elongate by the lateral insertion of newly synthesized peptidoglycan along their sidewalls. (Click the link to see a movie of stained E. coli growth).

Dispersal of the red dye along the sidewalls in growing E. coli cells indicates lateral cell growth.

Dispersal of the red dye along the sidewalls in growing E. coli cells indicates lateral cell growth.

In contrast, some rod-shaped bacteria utilize precise targeting of peptidoglycan to specific polar locations to enable cell elongation.  Indeed, we have previously shown that constrained polar growth is responsible for elongation in many species of Rhizobiales. (Click the link to see a movie of stained A. tumefaciens growth.)

Retention of the red dye in growing A. tumefaciens cells indicates polar growth.

Retention of the red dye in growing A. tumefaciens cells indicates polar growth.

My lab uses Agrobacterium tumefaciens, a well-studied plant pathogen and causative agent of crown gall disease, as a model to understand how bacteria constrain peptidoglycan synthesis to specific cellular localizations. Presently, we are focused on addressing the following key question:

What is the mechanism underlying polar growth of A. tumefaciens?

Visualization of active peptidoglycan synthesis in A. tumefaciens cells.

Visualization of active peptidoglycan synthesis in A. tumefaciens cells.

We know that peptidoglycan synthesis occurs at the cell pole during elongation and at mid-cell during cell division.  Now we want to understand how and why bacteria in the Rhizobiales grow this way.

Why is this a challenging question?  Most of the known proteins involved in bacterial cell elongation are absent from the genome sequences of all bacteria belonging to the Rhizobiales. Thus, we cannot simply look for homologs of elongation proteins in other bacteria.  Instead we are using a number of approaches to identify candidate proteins involved in this process.

Project 1. Identification of candidate involved in polar elongation in A. tumefaciens.

Identification of essential genes following TnSeq

Identification of essential genes following TnSeq

We hypothesize that some of the proteins involved in elongation of A. tumefaciens must be essential for cell survival.  Thus, we are collaborating with Patrick Curtis at Ole Miss to identify all of the essential genes in A. tumefaciens using TnSeq. Bioinformatic analysis of this gene set has allowed us to generate a prioritized list of candidate genes involved in polar elongation.  We are constructing a variety of strains (including disruptions, overexpression, and fluorescent protein fusions) to test our hypothesis that these may be involved in the elongation of A. tumefaciens.

Project 2. Identification of mutants with cell wall defects.

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Measuring growth of A. tumefaciens cells under strain conditions

The spatial or temporal dynamics of peptidoglycan synthesis is likely to be highly regulated.  Furthermore, the composition of peptidoglycan can change in different environments.  We expect that identification of regulators of peptidoglycan synthesis and composition will be important for understanding the mechanism of A. tumefaciens cell growth.  We are using a high-throughput screen to identify these regulatory genes based on the assumption that disruption of the gene may lead to defects in the cell wall.  To identify mutants with cell wall defects, we are screening a library of transposon mutants by monitoring their growth when embedded in agarose (under physical strain) as compared to in liquid. Mutants of interest will have growth defects in agarose but not in liquid.

Project 3. Characterization of the role of LD-transpeptidation in A. tumefaciens peptidoglycan synthesis.  

Peptidoglycan cross-links

Peptidoglycan cross-links

Peptidoglycan contain peptide cross-links to link adjacent glycan strands.  In Gram-negative bacteria, the vast majority of cross-links are typically made by DD-transpeptidation, with only a small percentage of cross-links made by LD-transpeptidation.

In contrast, the peptidoglycan profile of A. tumefaciens reveals that only about half the cross-links are made by DD-transpeptidation.  The remaining half of the cross-links are made through LD-transpeptidation.  This observation has led us to hypothesize the LD-transpeptidation may play an important role in the synthesis of peptidoglycan in A. tumefaciens.     We have identified 4 putative LD-transpeptidases in the A. tumefaciens genome and are in the process of characterizing the role of these enzymes in peptidoglycan synthesis.

What is the big picture? Knowledge gained though these studies will be applied to enhance our understanding of the precisely targeted growth in other Alphaproteobacteria, including those that display remarkable morphologies. Our goal is to better understand the role of zonal peptidoglycan synthesis in generating both relatively simple and dramatically complex bacterial shapes.

Is there a practical application for this research? Yes, we expect that discovery of novel proteins involved in bacterial cell elongation may prove to be new targets for antimicrobial strategies.  This is particularly important given the presence of important animal and human pathogens (including Brucella, which is notoriously difficult to treat) with in the Rhizobiales.

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