Biology subjects

Rojas, E. R.

Publications and source records attributed to Rojas, E. R..

8 recordsLinked to original sources

bioRxiv2024

Non-linear stress-softening of the bacterial cell wall confers cell shape homeostasis

The bacillus - or rod - is a pervasive cellular morphology among bacteria. Rod-shaped bacteria elongate without widening by reinforcing their cell wall anisotropically, along the cells circumference, but it is unknown how cells adaptively tune anisotropy to homeostatically control cell width. Through super-resolution measurements of cell wall mechanical properties, we discovered that the Bacillus subtilis cell wall exhibits non-linear stress-softening exclusively in the circumferential direction. Furthermore, during steady-state growth the cell wall is inflated precisely to the acute non-linear transition. Physics-based theory correctly predicted that this transition underlies the negative feedback that governs cell width homeostasis. In other words, the cell wall is a "smart material" whose exotic mechanical properties are exquisitely adapted to execute cellular morphogenesis.

biophysics↗

bioRxiv2024

Wall teichoic acids regulate peptidoglycan synthesis by paving cell wall microstructure

The cell wall is a polymeric exoskeleton that defines the size and shape of bacteria; it is composed of peptidoglycan and, in Gram-positive bacteria, wall teichoic acid polymers. Two systems synthesize peptidoglycan in rod-shaped bacteria, which are found pervasively across bacterial clades: the multi-protein Rod complexes synthesize anisotropic peptidoglycan, which is required for rod shape because it reinforces the cell wall along its circumference1-3, whereas the non-essential enzyme PBP1 synthesizes isotropic peptidoglycan1,4 (Fig. 1A). In Gram-positive bacteria, rod shape also requires wall teichoic acids5 for unknown reasons. Here, we show that wall teichoic acids promote rod shape by preventing the formation of nanoscopic pores in the Bacillus subtilis cell wall, which lead to amorphous growth by activating PBP1 and inhibiting Rod complexes. Depleting wall teichoic acids resulted in pores within minutes, coinciding with a rapid increase in PBP1-mediated synthesis. PBP1s ability to sustain teichoic acid-less growth depended on its intrinsically disordered domain. In contrast to previous steady-state measurements6,7, we found that wall teichoic acid depletion caused the transient arrest of Rod complexes prior to the onset of amorphous growth. Finally, one of the two synthetically lethal cell wall hydrolases in B. subtilis8, LytE, became essential during wall teichoic acid depletion, meaning that PBP1 and LytE execute a novel, amorphous mode of growth. Collectively, our results identify the cell wall, via its molecular-scale structure, as a non-canonical auto-regulator of its own synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/610702v3_fig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@154996forg.highwire.dtl.DTLVardef@12561c3org.highwire.dtl.DTLVardef@1356cacorg.highwire.dtl.DTLVardef@719d22_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1:C_FLOATNO Inhibiting wall teichoic acid synthesis decreases Rod complex activity prior to cell shape loss.(A) Schematic of Gram-positive cell wall synthesis. (B) Cell length growth rate [Figure 1] for wild-type cells during 0.5 g/mL tunicamycin treatment. Blue line shows smoothed population median, error bars show standard deviation. Data shown for 2,048 discrete cell tracks from 3 biological replicates. Inset: micrographs taken (i) before and (ii) after 45 min tunicamycin treatment. Scale bars 5 m. (C) Time course for the percentage of processive Mbl filaments during 0.5 g/mL tunicamycin treatment. Error bars show 95% confidence intervals across biological replicates (bootstrap analysis). Analysis performed on 62,901 discrete filament tracks from 4 biological replicates. Inset shows representative fluorescent kymographs of Mbl motion across the cell waist in (i) LB and (ii) after 30 min tunicamycin treatment. Red dotted line follows a processive Mbl filament. Scale bars 1 m. (D) Mean fluorescent puncta per unit cell length for wild-type, {Delta}ponA and ponATP-cells during tunicamycin treatment, labeled with fluorescent D-amino acids. Error bars show 95% confidence intervals. Analysis performed over 2,017 segmentations from 4 biological replicates (wild-type), 857 segmentations from 3 biological replicates ({Delta}ponA) and 257 cells from 2 biological replicates (ponATP-). All timepoints show statistical significance for difference in mean between cell types (Students T-test, P<0.01). Dots show individual replicates. Measurements at 50 min tunicamycin treatment used a lower exposure time so are plotted separately. Inset shows micrographs of (i,iii,v) wild-type and (ii,iv,vi) {Delta}ponA cells at (i,ii) 0 min, (iii,iv) 30 min, and (v,vi) 50 min of treatment. Micrographs are identically saturated across cell types. Scale bars 5 m. (E) Percentage of peptidoglycan subunits in monomers, measured by HPLC (Methods). Two biological replicates per strain. Mean values calculated across biological replicates. Error bars show 95% confidence intervals. Individual datapoints shown in red. Statistical significance calculated using Students T-test, P<0.05. C_FIG

microbiology↗

bioRxiv2024

Beta-barrel proteins dictate the effect of core oligosaccharide composition on outer membranemechanics

The outer membrane is the defining structure of Gram-negative bacteria. We previously demonstrated that it is critical for the mechanical integrity of the cell envelope and therefore to the robustness of the bacterial cell as a whole. Here, to determine the key molecules and moieties within the outer membrane that underlie its contribution to cell envelope mechanics, we measured cell-envelope stiffness across several sets of mutants with altered outer-membrane sugar content, protein content, and electric charge. To decouple outer membrane stiffness from total cell envelope stiffness, we developed a novel microfluidics-based "osmotic force extension" assay. In tandem, we developed a simple method to increase throughput of microfluidics experiments by performing them on color-coded pools of mutants. Using Escherichia coli as a model Gram-negative bacterium, we found that truncating the core oligosaccharide, deleting the {beta}-barrel protein OmpA, or deleting lipoprotein outer membrane-cell wall linkers all had the same modest, convergent effect on total cell-envelope stiffness but had large, varying effects on the ability of the cell wall to transfer tension to the outer membrane during large hyperosmotic shocks. Surprisingly, altering lipid A charge had little effect on the mechanical properties of the envelope. Importantly, the presence or absence of OmpA determined whether truncating the core oligosaccharide decreased or increased envelope stiffness (respectively), revealing sign epistasis between these components. Based on these data we propose a specific structural model in which the chemical interactions between lipopolysaccharides, {beta}-barrel proteins, and phospholipids coordinately determine cell envelope stiffness, and the ability of the outer membrane to functionally share mechanical loads with the cell wall. Statement of SignificanceThe outer membrane is the defining cellular structure of Gram-negative bacteria, a group that contains many important pathogens like Escherichia coli, Vibrio cholerae, and Pseudomonas aeruginosa. One role of the outer membrane is to block the uptake of small molecules like antibiotics. However, it is becoming increasingly clear that it also functions as a structural exoskeleton that is critical for the cells ability to cope with internal and external mechanical forces. Here, we carefully dissect the molecular basis for the load-bearing capacity of the outer membrane by screening a set of mutants with a new cell biophysics assay.

biophysics↗

bioRxiv2023

A fitness landscape instability governs the morphological diversity of tip-growing cells

Cellular morphology affects many aspects of cellular and organismal physiology. This makes it challenging to understand the evolutionary basis for specific morphologies since the various facets of cellular physiology may exert competing selective pressures on this trait. The influence of these pressures, moreover, will depend on the mechanisms of cellular morphogenesis. To address this problem, we combined experimental cell biology with mechanics-based theory to analyze the morphological diversity of tip-growing cells from across the tree of life. We discovered that an instability in the convergent mechanism of "inflationary" growth shared by these cells leads directly to a bifurcation in their fitness landscape, which imposes a strong global constraint on their morphologies. Additionally, we found that co-selection for cell size and elongation rate explains variation among observable morphologies. This analysis rationalizes the morphology - and provides quantitative insight into the ecology - of an enormous diversity of important fungal, plant, protistan, and bacterial systems. Additionally, our study elucidates a fundamental principle of evolutionary-developmental biology that would be difficult to rigorously demonstrate in more complex systems.

cell biology↗

bioRxiv2022

Mechanical compression induces persistent bacterial growth during bacteriophage predation

Although the relationship between bacteria and lytic bacteriophage is fundamentally antagonistic, these microbes not only coexist but thrive side-by-side in myriad ecological environments. The mechanisms by which coexistence is achieved, however, are not fully understood. By examining Escherichia coli and bacteriophage T7 population dynamics at the single-cell and single-virion level using a novel microfluidics-based assay, we observed bacteria growing "persistently" when perfused with high-titer bacteriophage. Persistence occurred at a frequency five orders of magnitude higher than is expected from natural selection of bacteriophage-resistant mutants. Rather, the frequency of persistence was correlated with the degree to which the bacteria were mechanically compressed by the microfluidic perfusion chamber. Using a combination of mutagenesis and fluorescent imaging techniques, we found that compression induces persistence by activating the Rcs phosphorelay pathway, which results in the synthesis of extracellular capsule that sterically blocks bacteriophage adsorption. Other forms of mechanical stimulation also promoted Rcs activity and persistence. These findings have important implications for our understanding of microbial ecology in many important environments, including the gut and the soil, where bacteria grow in confinement. Significance StatementBacteria and bacteriophage form one of the most fundamental and important predator-prey relationships on earth, yet the factors that promote long-term stability of their populations are unknown. Here, we demonstrate that Escherichia coli is able to rapidly grow during bacteriophage predation if they are doing so in spatially confined environments. This discovery revises our understanding of bacteria-bacteriophage population dynamics in many real-world environments where bacteria grow in such environments, such as the gut and the soil. Additionally, this result has critical implications for the potential of antibacterial therapies to function during pathogenesis, when bacteria are also mechanically stimulated.

microbiology↗

bioRxiv2022

Phage genome cleavage enables resuscitation from Cas13-induced bacterial dormancy

CRISPR-Cas systems provide their prokaryotic hosts with sequence-specific immunity to foreign genetic elements, including bacteriophages and plasmids. While most interfere with phage infection though cleavage of viral DNA, type VI CRISPR systems use the RNA-guided nuclease Cas13 to recognize mRNA targets. Upon engaging with target RNA, Cas13 cleaves both phage and host transcripts nonspecifically, leading to a state of cell dormancy that is incompatible with phage propagation. However, whether and how infected cells recover from dormancy is not clear. Here we show that type VI CRISPR systems frequently co-occur with DNA-cleaving restriction modification (RM) systems. Using genetics and microscopy, we show that Cas13 and RM systems synergize in anti-phage defense in the natural type VI CRISPR host Listeria seeligeri. Cleavage of the phage genome by RM removes the source of phage transcripts, enabling cells to recover from Cas13-induced cellular dormancy. We find that Cas13 and RM systems operating simultaneously eliminate phage DNA and neutralize infection more effectively than either defense alone. Thus, cells harboring both defense systems exhibit robust anti-phage immunity and survive infection. Our work therefore reveals that type VI CRISPR immunity is cell-autonomous and non-abortive, if paired with RM or similar DNA-targeting defenses. The ability of an abortive response to be resolved by the actions of another anti-phage defense has implications for the roles of diverse host-directed immune systems in bacteria.

microbiology↗

bioRxiv2022

The cell wall polymer initiates plasma membrane partitioning in mycobacteria

Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced by interactions with other membranes and solid supports. While cellular membranes can departition in response to various perturbations, including disruption of bilayer-extrinsic structures, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis can be spatially and biochemically departitioned by the fluidizing agent benzyl alcohol. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth upon fluidizer washout. PonA2s role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. We find that the cell wall polymer, but not active cell wall polymerization, is critical for membrane partitioning. Our work highlights a key initiating role for bilayer-extrinsic structures in patterning cellular membranes.

molecular biology↗

bioRxiv2018

Decoupling of rates of protein synthesis from cell expansion leads to supergrowth

Cell growth is a complex process in which cells synthesize cellular components while they increase in size. It is generally assumed that the rate of biosynthesis must somehow be coordinated with the rate of growth in order to maintain intracellular concentrations. However, little is known about potential feedback mechanisms that could achieve proteome homeostasis, or the consequences when this homeostasis is perturbed. Here, we identified conditions in which fission yeast cells are prevented from volume expansion but nevertheless continue to synthesize biomass, leading to global accumulation of proteins and increased cytoplasmic density. Upon removal of these perturbations, this biomass accumulation drove cells to undergo a multi-generational period of \"supergrowth\" in which rapid volume growth outpaced biosynthesis, returning proteome concentrations back to normal within hours. These findings demonstrate a novel mechanism for global proteome homeostasis based on modulation of volume growth and dilution.

cell biology↗