Skin and bones: the bacterial cytoskeleton, cell wall, and cell morphogenesis

The bacterial world is full of varying cell shapes and sizes, and individual species perpetuate a defined morphology generation after generation. We review recent findings and ideas about how bacteria use the cytoskeleton and other strategies to regulate cell growth in time and space to produce different shapes and sizes

Different RsbR paralogs in Bacillus subtilis affect cell viability when exposed to environmental stress

Stress is a universal phenomenon experienced by all living organisms. Bacteria have to react to stress quickly in order to survive in their environment. Environmental stress can be caused by a variety of factors, including acid, alcohol, salt, and heat. We are studying the model bacterium Bacillus subtilis, because its stress response resembles that of human pathogens such as Listeria. B. subtilis senses stress using a stressosome, a complex of 80 proteins that includes four variant RsbR protein paralogs, each of which produce a distinct stress response pattern to an identical stressor. We know that these four RsbR proteins work together to aid in survival in the presence of environmental stress in wild-type cells (WT), which contain all four RsbR proteins; however, we do not know how each RsbR protein affects cell fitness. To test how each individual RsbR protein affects survival, we performed a competition assay pairing strains containing individual RsbR variants against each other to determine if one protein aided in cell survival more than the other. To do this, we engineered strains of B. subtilis cells to only contain one of the four RsbR proteins. This allowed us to compete the RsbR proteins against one another on an individual basis. Our preliminary results indicate that the wild-type strain substantially outcompeted the other strains in every competition assay performed under acid stress except when competed against RC. These results show that cells containing only RC or all four RsbR proteins have a higher fitness in acid stress than cells containing only RA, RB, or RD.Lew Wentz FoundationMicrobiolog

Identification of new signaling components that govern biofilm formation by P. aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that is often associated with severe forms of many infections, including bronchiectasis and infections in the gut. Mortality is increased in patients who become infected with P. aeruginosa. P. aeruginosa poses a special treatment challenge due to its propensity to form biofilms, in which cells are surrounded by a self-produced extracellular matrix of proteins, DNA, and polysaccharides. Biofilms can help bacteria evade the host immune response, and the matrix represents a barrier that protects bacteria against antibiotic therapy. Because biofilms are difficult to treat once established in an infection, new strategies to prevent biofilm formation are critical to combat these infections. However, effective prevention depends on a fuller understanding of the signaling pathways that control biofilm formation. To identify new control points in the pathways governing biofilm formation by P. aeruginosa, we use transposon mutagenesis in conjunction with a visual assay for colony morphology, in which colony wrinkling indicates biofilm formation. Using these screens, we have been able to identify many promising candidates. For example, we have identified mutations in the genes fdnG and PA14_42090 as having smoother colony morphology, suggesting that these genes are involved in biofilm formation. Conversely, we have found that mutations in the purU2 and trxB1 genes show increased colony wrinkling, suggesting that these genes normally suppress biofilm formation. We will continue characterizing these genes by deleting them from the genome to confirm their roles in biofilm production and then testing how their absence or presence affects other known biofilm signaling molecules such as cyclic-di-GMP. Our detailed characterization of these candidate genes will provide fundamental knowledge that can then be used to devise future treatments to prevent biofilm formation in patients infected with P. aeruginosa.Lew Wentz FoundationMicrobiology and Molecular Genetic

Mutations in the Lipopolysaccharide Biosynthesis Pathway Interfere with Crescentin-Mediated Cell Curvature in Caulobacter crescentus

Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant Opolysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis

Stress response of RsbR protein paralogs in Bacillus subtilis under 1M sodium stress

Stress is a universal phenomenon, and all organisms need a way to cope with it. The Model bacterium Bacillus subtilis has a complex of proteins known as a stressosome that is responsible for sensing stressors in the environment and later promoting a stress response. The protein the we believe to be responsible for sensing stress is known as RsbR. The RsbR protein has 4 variants or paralogs that we know, when given identical stressors, promote different responses. However we did not know which paralog promoted the best overall fitness. We found that in 1M sodium stress, RsbRD showed the highest overall fitness. As we learn more about the stressosome we will be able to determine new and better ways to treat bacterial infections with this mechanism within stressful environments like the human body.Lew Wentz FoundationMicrobiology and Molecular Genetic

The Bacterial Cytoplasm Has Glass-like Properties and Is Fluidized by Metabolic Activity

SummaryThe physical nature of the bacterial cytoplasm is poorly understood even though it determines cytoplasmic dynamics and hence cellular physiology and behavior. Through single-particle tracking of protein filaments, plasmids, storage granules, and foreign particles of different sizes, we find that the bacterial cytoplasm displays properties that are characteristic of glass-forming liquids and changes from liquid-like to solid-like in a component size-dependent fashion. As a result, the motion of cytoplasmic components becomes disproportionally constrained with increasing size. Remarkably, cellular metabolism fluidizes the cytoplasm, allowing larger components to escape their local environment and explore larger regions of the cytoplasm. Consequently, cytoplasmic fluidity and dynamics dramatically change as cells shift between metabolically active and dormant states in response to fluctuating environments. Our findings provide insight into bacterial dormancy and have broad implications to our understanding of bacterial physiology, as the glassy behavior of the cytoplasm impacts all intracellular processes involving large components

7_20161004_WT_1EtOH_temp

Response of wild-type cells to the onset of 1% ethanol. Representative MTC1801 cell lineages are shown, with the mother cells oriented toward the bottom of the frame and the feeding/waste channel toward the top of the frame. This experiment corresponds to Movie 7

10_20161006_WT_4EtOH_temp

Response of wild-type cells to the onset of 4% ethanol. Representative MTC1801 cell lineages are shown, with the mother cells oriented toward the bottom of the frame and the feeding/waste channel toward the top of the frame. This experiment corresponds to Movie 10

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Response of RsbRA-only cells to the onset of 2% ethanol. Representative MTC1761 cell lineages are shown, with the mother cells oriented toward the bottom of the frame and the feeding/waste channel toward the top of the frame. This experiment corresponds to Movie 11

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Response of RsbRC-only cells to the onset of 1% ethanol. Representative MTC1765 cell lineages are shown, with the mother cells oriented toward the bottom of the frame and the feeding/waste channel toward the top of the frame. This experiment corresponds to Movie 16