Archaeal cells share common size control with bacteria despite noisier growth and division.

In nature, microorganisms exhibit different volumes spanning six orders of magnitude 1 . Despite their capability to create different sizes, a clonal population in a given environment maintains a uniform size across individual cells. Recent studies in eukaryotic and bacterial organisms showed that this homogeneity in cell size can be accomplished by growing a constant size between two cell cycle events (that is, the adder model 2-6 ). Demonstration of the adder model led to the hypothesis that this phenomenon is a consequence of convergent evolution. Given that archaeal cells share characteristics with both bacteria and eukaryotes, we investigated whether and how archaeal cells exhibit control over cell size. To this end, we developed a soft-lithography method of growing the archaeal cells to enable quantitative time-lapse imaging and single-cell analysis, which would be useful for other microorganisms. Using this method, we demonstrated that Halobacterium salinarum, a hypersaline-adapted archaeal organism, grows exponentially at the single-cell level and maintains a narrow-size distribution by adding a constant length between cell division events. Interestingly, the archaeal cells exhibited greater variability in cell division placement and exponential growth rate across individual cells in a population relative to those observed in Escherichia coli 6-9 . Here, we present a theoretical framework that explains how these larger fluctuations in archaeal cell cycle events contribute to cell size variability and control.

Bacterial Filament Systems: Toward Understanding Their Emergent Behavior and Cellular Functions.

Bacteria use homologs of eukaryotic cytoskeletal filaments to conduct many different tasks, controlling cell shape, division, and DNA segregation. These filaments, combined with factors that regulate their polymerization, create emergent self-organizing machines. Here, we summarize the current understanding of the assembly of these polymers and their spatial regulation by accessory factors, framing them in the context of being dynamical systems. We highlight how comparing the in vivo dynamics of the filaments with those measured in vitro has provided insight into the regulation, emergent behavior, and cellular functions of these polymeric systems.

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Structure-activity studies of divin: an inhibitor of bacterial cell division.

We describe the synthesis and SAR studies of divin-a small molecule that blocks bacterial division by perturbing the assembly of proteins at the site of cell septation. The bacteriostatic mechanism of action of divin is distinct from other reported inhibitors of bacterial cell division and provides an opportunity for assessing the therapeutic value of a new class of antimicrobial agents. We demonstrate a convenient synthetic route to divin and its analogs, and describe compounds with a 10-fold increase in solubility and a 4-fold improvement in potency. Divin analogs produce a phenotype that is identical to divin, suggesting that their biological activity comes from a similar mechanism of action. Our studies indicate that the 2-hydroxynaphthalenyl hydrazide portion of divin is essential for its activity and that alterations and substitution to the benzimidazole ring can increase its potency. The SAR study provides a critical opportunity to isolate drug resistant mutants and synthesize photoaffinity probes to determine the cellular target and biomolecular mechanism of divin.

Divin: a small molecule inhibitor of bacterial divisome assembly.

Bacterial cell division involves the dynamic assembly of division proteins and coordinated constriction of the cell envelope. A wide range of factors regulates cell division--including growth and environmental stresses--and the targeting of the division machinery has been a widely discussed approach for antimicrobial therapies. This paper introduces divin, a small molecule inhibitor of bacterial cell division that may facilitate mechanistic studies of this process. Divin disrupts the assembly of late division proteins, reduces peptidoglycan remodeling at the division site, and blocks compartmentalization of the cytoplasm. In contrast to other division inhibitors, divin does not interact with the tubulin homologue FtsZ, affect chromosome segregation, or activate regulatory mechanisms that inhibit cell division indirectly. Our studies of bacterial cell division using divin as a probe suggest that dividing bacteria proceed through several morphological stages of the cell envelope, and FtsZ is required but not sufficient to compartmentalize the cytoplasmic membrane at the division site. Divin is only moderately toxic to mammalian cells at concentrations that inhibit the growth of clinical pathogens. These characteristics make divin a useful probe for studying bacterial cell division and a starting point for the development of new classes of therapeutic agents.

Inhibitors of bacterial tubulin target bacterial membranes in vivo.

FtsZ is a homolog of eukaryotic tubulin that is widely conserved among bacteria and coordinates the assembly of the cell division machinery. FtsZ plays a central role in cell replication and is a target of interest for antibiotic development. Several FtsZ inhibitors have been reported. We characterized the mechanism of these compounds in bacteria and found that many of them disrupt the localization of membrane-associated proteins, including FtsZ, by reducing the transmembrane potential or perturbing membrane permeability. We tested whether the reported phenotypes of a broad collection of FtsZ inhibitors disrupt the transmembrane potential in Bacillus subtilis strain 168. Using a combination of flow cytometry and microscopy, we found that zantrin Z1, cinnamaldehyde, totarol, sanguinarine, and viriditoxin decreased the B. subtilis transmembrane potential or perturbed membrane permeability, and influenced the localization of the membrane-associated, division protein MinD. These studies demonstrate that small molecules that disrupt membrane function in bacterial cells produce phenotypes that are similar to the inhibition of proteins associated with membranes in vivo, including bacterial cytoskeleton homologs, such as FtsZ. The results provide a new dimension for consideration in the design and testing of inhibitors of bacterial targets that are membrane-associated and provide additional insight into the structural characteristics of antibiotics that disrupt the membrane.

DCAP: a broad-spectrum antibiotic that targets the cytoplasmic membrane of bacteria.

Persistent infections are frequently caused by dormant and biofilm-associated bacteria, which often display characteristically slow growth. Antibiotics that require rapid cell growth may be ineffective against these organisms and thus fail to prevent reoccurring infections. In contrast to growth-based antimicrobial agents, membrane-targeting drugs effectively kill slow-growing bacteria. Herein we introduce 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP), a potent broad-spectrum antibiotic that reduces the transmembrane potential of Gram-positive and Gram-negative bacteria and causes mislocalization of essential membrane-associated proteins, including MinD and FtsA. Importantly, DCAP kills nutrient-deprived microbes and sterilizes bacterial biofilms. DCAP is lethal against bacterial cells, has no effect on red blood cell membranes, and only decreases the viability of mammalian cells after ≥6 h. We conclude that membrane-active compounds are a promising solution for treating persistent infections. DCAP expands the limited number of compounds in this class of therapeutic small molecules and provides new opportunities for the development of potent broad-spectrum antimicrobial agents.

Chemical-biological studies of subcellular organization in bacteria.

The subcellular organization of biological molecules is a critical determinant of many bacterial processes, including growth, replication of the genome, and division, yet the details of many mechanisms that control intracellular organization remain unknown. Decoding this information will impact the field of bacterial physiology and can provide insight into eukaryotic biology, including related processes in mitochondria and chloroplasts. Small molecule probes provide unique advantages in studying these mechanisms and manipulating the organization of biomolecules in live bacterial cells. In this review, we describe small molecules that are available for investigating subcellular organization in bacteria, specifically targeting FtsZ, MreB, peptidoglycan, and lipid bilayers. We discuss how these probes have been used to study microbiological questions and conclude by providing suggestions about important areas in which chemical-biological approaches will have a revolutionary impact on the study of bacterial physiology.

Quorum sensing between Pseudomonas aeruginosa biofilms accelerates cell growth.

This manuscript describes the fabrication of arrays of spatially confined chambers embossed in a layer of poly(ethylene glycol) diacrylate (PEGDA) and their application to studying quorum sensing between communities of Pseudomonas aeruginosa. We hypothesized that biofilms may produce stable chemical signaling gradients in close proximity to surfaces, which influence the growth and development of nearby microcolonies into biofilms. To test this hypothesis, we embossed a layer of PEGDA with 1.5-mm wide chambers in which P. aeruginosa biofilms grew, secreted homoserine lactones (HSLs, small molecule regulators of quorum sensing), and formed spatial and temporal gradients of these compounds. In static growth conditions (i.e., no flow), nascent biofilms secreted N-(3-oxododecanoyl) HSL that formed a gradient in the hydrogel and was detected by P. aeruginosa cells that were ≤8 mm away. Diffusing HSLs increased the growth rate of cells in communities that were <3 mm away from the biofilm, where the concentration of HSL was >1 µM, and had little effect on communities farther away. The HSL gradient had no observable influence on biofilm structure. Surprisingly, 0.1-10 µM of N-(3-oxododecanoyl) HSL had no effect on cell growth in liquid culture. The results suggest that the secretion of HSLs from a biofilm enhances the growth of neighboring cells in contact with surfaces into communities and may influence their composition, organization, and diversity.

Encapsulating bacteria in agarose microparticles using microfluidics for high-throughput cell analysis and isolation.

The high-throughput analysis and isolation of bacterial cells encapsulated in agarose microparticles using fluorescence-activated cell sorting (FACS) is described. Flow-focusing microfluidic systems were used to create monodisperse microparticles that were ∼30 µm in diameter. The dimensions of these particles made them compatible with flow cytometry and FACS, and the sensitivity of these techniques reduced the incubation time for cell replication before analyses were carried out. The small volume of the microparticles (∼1-50 pL) minimized the quantity of reagents needed for bacterial studies. This platform made it possible to screen and isolate bacteria and apply a combination of techniques to rapidly determine the target of biologically active small molecules. As a pilot study, Escherichia coli cells were encapsulated in agarose microparticles, incubated in the presence of varying concentrations of rifampicin, and analyzed using FACS. The minimum inhibitory concentration of rifampicin was determined, and spontaneous mutants that had developed resistance to the antibiotic were isolated via FACS and characterized by DNA sequencing. The ß-subunit of RNA polymerase, RpoB, was confirmed as the target of rifampicin, and Q513L was the mutation most frequently observed. Using this approach, the time and quantity of antibiotics required for the isolation of mutants was reduced by 8- and 150-fold, respectively, compared to conventional microbiological techniques using nutrient agar plates. We envision that this technique will have an important impact on research in chemical biology, natural products chemistry, and the discovery and characterization of biologically active secondary metabolites.

Contribution of long-range interactions to the secondary structure of an unfolded globin.

This work explores the effect of long-range tertiary contacts on the distribution of residual secondary structure in the unfolded state of an alpha-helical protein. N-terminal fragments of increasing length, in conjunction with multidimensional nuclear magnetic resonance, were employed. A protein representative of the ubiquitous globin fold was chosen as the model system. We found that, while most of the detectable alpha-helical population in the unfolded ensemble does not depend on the presence of the C-terminal region (corresponding to the native G and H helices), specific N-to-C long-range contacts between the H and A-B-C regions enhance the helical secondary structure content of the N terminus (A-B-C regions). The simple approach introduced here, based on the evaluation of N-terminal polypeptide fragments of increasing length, is of general applicability to identify the influence of long-range interactions in unfolded proteins.

Fabrication of microbial biofilm arrays by geometric control of cell adhesion.

This paper presents a technique for patterning arrays of microbial biofilms on a wide range of different substrates using thin polymer stencils. The stencils function as "scaffolds" that provide geometric control over cell adhesion on surfaces and confine biofilm growth to specific regions of a substrate. We demonstrate the fabrication of biofilm arrays with features (e.g., individual biofilms) as small as 50 microm in diameter with physiological characteristics that are reproducible. Biofilm arrays of a range of microorganisms can be produced using this technique, including: P. aeruginosa, B. subtilis, S. epidermidis, V. fischeri, E. coli, and C. albicans. This approach provides a simple, user-configurable, and relatively inexpensive method for growing biofilms in both static and flow conditions. The method described in this paper makes it possible to study the chemical, physical, and environmental factors that affect biofilm development in a statistically relevant and reproducible format.

Dissecting microbiological systems using materials science.

Materials science offers microbiologists a wide variety of organic and inorganic materials with chemical and physical properties that can be precisely controlled. These materials present new capabilities for isolating, manipulating and studying bacteria and other microorganisms and are poised to transform microbiology. This review summarizes three classes of materials that span a range of length scales (nano, micro and meso) and describes a variety of fundamental questions in microbiology that can be studied by leveraging their properties.

Thermodynamic and kinetic characterization of apoHmpH, a fast-folding bacterial globin.

Despite the widespread presence of the globin fold in most living organisms, only eukaryotic globins have been employed as model proteins in folding/stability studies so far. This work introduces the first thermodynamic and kinetic characterization of a prokaryotic globin, that is, the apo form of the heme-binding domain of flavohemoglobin (apoHmpH) from Escherichia coli. This bacterial globin has a widely different sequence but nearly identical structure to its eukaryotic analogues. We show that apoHmpH is a well-folded monomeric protein with moderate stability at room temperature [apparent Delta G degrees (UN(w))=-3.1+/-0.3 kcal mol(-1); m(UN)=-1.7 kcal mol(-1) M(-1)] and predominant alpha-helical structure. Remarkably, apoHmpH is the fastest-folding globin known to date, as it refolds about 4- to 16-fold more rapidly than its eukaryotic analogues (e.g., sperm whale apomyoglobin and soybean apoleghemoglobin), populating a compact kinetic intermediate (beta(I)=0.9+/-0.2) with significant helical content. Additionally, the single Trp120 (located in the native H helix) becomes locked into a fully native-like environment within 6 ms, suggesting that this residue and its closest spatial neighbors complete their folding at ultrafast (submillisecond) speed. In summary, apoHmpH is a bacterial globin that shares the general folding scheme (i.e., a rapid burst phase followed by slower rate-determining phases) of its eukaryotic analogues but displays an overall faster folding and a kinetic intermediate with some fully native-like traits. This study supports the view that the general folding features of bacterial and eukaryotic globins are preserved through evolution while kinetic details differ.