Bacterial Swarming Reduces Proteus mirabilis and Vibrio parahaemolyticus Cell Stiffness and Increases ß-Lactam Susceptibility.

Swarmer cells of the Gram-negative uropathogenic bacteria Proteus mirabilis and Vibrio parahaemolyticus become long (>10 to 100 µm) and multinucleate during their growth and motility on polymer surfaces. We demonstrated that the increasing cell length is accompanied by a large increase in flexibility. Using a microfluidic assay to measure single-cell mechanics, we identified large differences in the swarmer cell stiffness (bending rigidity) of P. mirabilis (5.5 × 10-22 N m2) and V. parahaemolyticus (1.0 × 10-22 N m2) compared to vegetative cells (1.4 × 10-20 N m2 and 2.2 × 10-22 N m2, respectively). The reduction in bending rigidity (∼2-fold to ∼26-fold) was accompanied by a decrease in the average polysaccharide strand length of the peptidoglycan layer of the cell wall from 28 to 30 disaccharides to 19 to 22 disaccharides. Atomic force microscopy revealed a reduction in P. mirabilis peptidoglycan thickness from 1.5 nm (vegetative cells) to 1.0 nm (swarmer cells), and electron cryotomography indicated changes in swarmer cell wall morphology. P. mirabilis and V. parahaemolyticus swarmer cells became increasingly sensitive to osmotic pressure and susceptible to cell wall-modifying antibiotics (compared to vegetative cells)-they were ∼30% more likely to die after 3 h of treatment with MICs of the ß-lactams cephalexin and penicillin G. The adaptive cost of "swarming" was offset by the increase in cell susceptibility to physical and chemical changes in their environment, thereby suggesting the development of new chemotherapies for bacteria that leverage swarming for the colonization of hosts and for survival.IMPORTANCEProteus mirabilis and Vibrio parahaemolyticus are bacteria that infect humans. To adapt to environmental changes, these bacteria alter their cell morphology and move collectively to access new sources of nutrients in a process referred to as "swarming." We found that changes in the composition and thickness of the peptidoglycan layer of the cell wall make swarmer cells of P. mirabilis and V. parahaemolyticus more flexible (i.e., reduce cell stiffness) and that they become more sensitive to osmotic pressure and cell wall-targeting antibiotics (e.g., ß-lactams). These results highlight the importance of assessing the extracellular environment in determining antibiotic doses and the use of ß-lactam antibiotics for treating infections caused by swarmer cells of P. mirabilis and V. parahaemolyticus.

Cardiolipin Alters Rhodobacter sphaeroides Cell Shape by Affecting Peptidoglycan Precursor Biosynthesis.

Cardiolipin (CL) is an anionic phospholipid that plays an important role in regulating protein biochemistry in bacteria and mitochondria. Deleting the CL synthase gene (Δcls) in Rhodobacter sphaeroides depletes CL and decreases cell length by 20%. Using a chemical biology approach, we found that a CL deficiency does not impair the function of the cell wall elongasome in R. sphaeroides; instead, biosynthesis of the peptidoglycan (PG) precursor lipid II is decreased. Treating R. sphaeroides cells with fosfomycin and d-cycloserine inhibits lipid II biosynthesis and creates phenotypes in cell shape, PG composition, and spatial PG assembly that are strikingly similar to those seen with R. sphaeroides Δcls cells, suggesting that CL deficiency alters the elongation of R. sphaeroides cells by reducing lipid II biosynthesis. We found that MurG-a glycosyltransferase that performs the last step of lipid II biosynthesis-interacts with anionic phospholipids in native (i.e., R. sphaeroides) and artificial membranes. Lipid II production decreases 25% in R. sphaeroides Δcls cells compared to wild-type cells, and overexpression of MurG in R. sphaeroides Δcls cells restores their rod shape, indicating that CL deficiency decreases MurG activity and alters cell shape. The R. sphaeroides Δcls mutant is more sensitive than the wild-type strain to antibiotics targeting PG synthesis, including fosfomycin, d-cycloserine, S-(3,4-dichlorobenzyl)isothiourea (A22), mecillinam, and ampicillin, suggesting that CL biosynthesis may be a potential target for combination chemotherapies that block the bacterial cell wall.IMPORTANCE The phospholipid composition of the cell membrane influences the spatial and temporal biochemistry of cells. We studied molecular mechanisms connecting membrane composition to cell morphology in the model bacterium Rhodobacter sphaeroides The peptidoglycan (PG) layer of the cell wall is a dominant component of cell mechanical properties; consequently, it has been an important antibiotic target. We found that the anionic phospholipid cardiolipin (CL) plays a role in determination of the shape of R. sphaeroides cells by affecting PG precursor biosynthesis. Removing CL in R. sphaeroides alters cell morphology and increases its sensitivity to antibiotics targeting proteins synthesizing PG. These studies provide a connection to spatial biochemical control in mitochondria, which contain an inner membrane with topological features in common with R. sphaeroides.

Mechanical Genomic Studies Reveal the Role of d-Alanine Metabolism in Pseudomonas aeruginosa Cell Stiffness.

The stiffness of bacteria prevents cells from bursting due to the large osmotic pressure across the cell wall. Many successful antibiotic chemotherapies target elements that alter mechanical properties of bacteria, and yet a global view of the biochemistry underlying the regulation of bacterial cell stiffness is still emerging. This connection is particularly interesting in opportunistic human pathogens such as Pseudomonas aeruginosa that have a large (80%) proportion of genes of unknown function and low susceptibility to different families of antibiotics, including beta-lactams, aminoglycosides, and quinolones. We used a high-throughput technique to study a library of 5,790 loss-of-function mutants covering ~80% of the nonessential genes and correlated P. aeruginosa individual genes with cell stiffness. We identified 42 genes coding for proteins with diverse functions that, when deleted individually, decreased cell stiffness by >20%. This approach enabled us to construct a "mechanical genome" for P. aeruginosa d-Alanine dehydrogenase (DadA) is an enzyme that converts d-Ala to pyruvate that was included among the hits; when DadA was deleted, cell stiffness decreased by 18% (using multiple assays to measure mechanics). An increase in the concentration of d-Ala in cells downregulated the expression of genes in peptidoglycan (PG) biosynthesis, including the peptidoglycan-cross-linking transpeptidase genes ponA and dacC Consistent with this observation, ultraperformance liquid chromatography-mass spectrometry analysis of murein from P. aeruginosa cells revealed that dadA deletion mutants contained PG with reduced cross-linking and altered composition compared to wild-type cells.IMPORTANCE The mechanical properties of bacteria are important for protecting cells against physical stress. The cell wall is the best-characterized cellular element contributing to bacterial cell mechanics; however, the biochemistry underlying its regulation and assembly is still not completely understood. Using a unique high-throughput biophysical assay, we identified genes coding proteins that modulate cell stiffness in the opportunistic human pathogen Pseudomonas aeruginosa This approach enabled us to discover proteins with roles in a diverse range of biochemical pathways that influence the stiffness of P. aeruginosa cells. We demonstrate that d-Ala-a component of the peptidoglycan-is tightly regulated in cells and that its accumulation reduces expression of machinery that cross-links this material and decreases cell stiffness. This research demonstrates that there is much to learn about mechanical regulation in bacteria, and these studies revealed new nonessential P. aeruginosa targets that may enhance antibacterial chemotherapies or lead to new approaches.

The outer membrane is an essential load-bearing element in Gram-negative bacteria.

Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.

Bacterial Cell Mechanics.

Cellular mechanical properties play an integral role in bacterial survival and adaptation. Historically, the bacterial cell wall and, in particular, the layer of polymeric material called the peptidoglycan were the elements to which cell mechanics could be primarily attributed. Disrupting the biochemical machinery that assembles the peptidoglycan (e.g., using the ß-lactam family of antibiotics) alters the structure of this material, leads to mechanical defects, and results in cell lysis. Decades after the discovery of peptidoglycan-synthesizing enzymes, the mechanisms that underlie their positioning and regulation are still not entirely understood. In addition, recent evidence suggests a diverse group of other biochemical elements influence bacterial cell mechanics, may be regulated by new cellular mechanisms, and may be triggered in different environmental contexts to enable cell adaptation and survival. This review summarizes the contributions that different biomolecular components of the cell wall (e.g., lipopolysaccharides, wall and lipoteichoic acids, lipid bilayers, peptidoglycan, and proteins) make to Gram-negative and Gram-positive bacterial cell mechanics. We discuss the contribution of individual proteins and macromolecular complexes in cell mechanics and the tools that make it possible to quantitatively decipher the biochemical machinery that contributes to bacterial cell mechanics. Advances in this area may provide insight into new biology and influence the development of antibacterial chemotherapies.

Mechanical Genomics Identifies Diverse Modulators of Bacterial Cell Stiffness.

Bacteria must maintain mechanical integrity to withstand the large osmotic pressure differential across the cell membrane and wall. Although maintaining mechanical integrity is critical for proper cellular function, a fact exploited by prominent cell-wall-targeting antibiotics, the proteins that contribute to cellular mechanics remain unidentified. Here, we describe a high-throughput optical method for quantifying cell stiffness and apply this technique to a genome-wide collection of ∼4,000 Escherichia coli mutants. We identify genes with roles in diverse functional processes spanning cell-wall synthesis, energy production, and DNA replication and repair that significantly change cell stiffness when deleted. We observe that proteins with biochemically redundant roles in cell-wall synthesis exhibit different stiffness defects when deleted. Correlating our data with chemical screens reveals that reducing membrane potential generally increases cell stiffness. In total, our work demonstrates that bacterial cell stiffness is a property of both the cell wall and broader cell physiology and lays the groundwork for future systematic studies of mechanoregulation.

Anionic Phospholipids Stabilize RecA Filament Bundles in Escherichia coli.

We characterize the interaction of RecA with membranes in vivo and in vitro and demonstrate that RecA binds tightly to the anionic phospholipids cardiolipin (CL) and phosphatidylglycerol (PG). Using computational models, we identify two regions of RecA that interact with PG and CL: (1) the N-terminal helix and (2) loop L2. Mutating these regions decreased the affinity of RecA to PG and CL in vitro. Using 3D super-resolution microscopy, we demonstrate that depleting Escherichia coli PG and CL altered the localization of RecA foci and hindered the formation of RecA filament bundles. Consequently, E. coli cells lacking aPLs fail to initiate a robust SOS response after DNA damage, indicating that the membrane acts as a scaffold for nucleating the formation of RecA filament bundles and plays an important role in the SOS response.

Bacterial cellulose as a substrate for microbial cell culture.

Bacterial cellulose (BC) has a range of structural and physicochemical properties that make it a particularly useful material for the culture of bacteria. We studied the growth of 14 genera of bacteria on BC substrates produced by Acetobacter xylinum and compared the results to growth on the commercially available biopolymers agar, gellan, and xanthan. We demonstrate that BC produces rates of bacterial cell growth that typically exceed those on the commercial biopolymers and yields cultures with higher titers of cells at stationary phase. The morphology of the cells did not change during growth on BC. The rates of nutrient diffusion in BC being higher than those in other biopolymers is likely a primary factor that leads to higher growth rates. Collectively, our results suggest that the use of BC may open new avenues in microbiology by facilitating bacterial cell culture and isolation.

Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity.

Although bacterial cells are known to experience large forces from osmotic pressure differences and their local microenvironment, quantitative measurements of the mechanical properties of growing bacterial cells have been limited. We provide an experimental approach and theoretical framework for measuring the mechanical properties of live bacteria. We encapsulated bacteria in agarose with a user-defined stiffness, measured the growth rate of individual cells and fit data to a thin-shell mechanical model to extract the effective longitudinal Young's modulus of the cell envelope of Escherichia coli (50-150 MPa), Bacillus subtilis (100-200 MPa) and Pseudomonas aeruginosa (100-200 MPa). Our data provide estimates of cell wall stiffness similar to values obtained via the more labour-intensive technique of atomic force microscopy. To address physiological perturbations that produce changes in cellular mechanical properties, we tested the effect of A22-induced MreB depolymerization on the stiffness of E. coli. The effective longitudinal Young's modulus was not significantly affected by A22 treatment at short time scales, supporting a model in which the interactions between MreB and the cell wall persist on the same time scale as growth. Our technique therefore enables the rapid determination of how changes in genotype and biochemistry affect the mechanical properties of the bacterial envelope.

Microcellular extrusion foaming of poly(lactide)/poly(butylene adipate-co-terephthalate) blends.

Foamed poly(lactide) (PLA)/poly(butylene adipate-co-terephthalate) (PBAT) blends were processed via the microcellular extrusion process using CO2 as a blowing agent. Talc has been added to promote heterogeneous nucleation. Two types of PLA/PBAT blend systems were investigated: Ecovio, which is a commercially available compatibilized PLA/PBAT blend; and a non-compatibilized PLA/PBAT blend at the same PLA/PBAT ratio (i.e., 45:55 by weight percent). Six different formulations were investigated: pure PLA, PLA-talc, Ecovio, Ecovio-talc, non-compatibilized PLA/PBAT blend, and non-compatibilized PLA/PBAT-talc. The effects of various processing parameters such as die temperature, talc and compatibilization on various foaming properties such as cell morphology, volume expansion ratio (VER), open cell content (OCC) and crystallinity were investigated. As per the DSC thermograms, it was observed that compatibilization has merged the two distinctive melting peaks of PLA and PBAT into a single peak while lowering the peak temperature. In general, the addition of talc has decreased the average cell size and VER and increased the cell density and crystallinity; however, it has varying effects on the open cell content. Compatibilization has reduced the average cell size and volume expansion but increased the cell density and had varying and no effects on the OCC and crystallinity, respectively. Similar to compatibilization, the die temperature was found to have varying and no effects on the OCC and crystallinity, respectively. Except for PLA and non-compatibilized PLA/PBAT blend, the cell size and VER of all other formulations did not vary much throughout the entire temperature range (130-150°C). The cell density was found to be insensitive to die temperatures except for Ecovio and Ecovio-talc.