Chemoproteomic identification of a DPP4 homolog in Bacteroides thetaiotaomicron.

Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.

The impact of iron and heme availability on the healthy human gut microbiome in vivo and in vitro.

Responses of the indigenous human gut commensal microbiota to iron are poorly understood because of an emphasis on in vitro studies of pathogen iron sensitivity. In a study of iron supplementation in healthy humans, we identified gradual microbiota shifts in some participants correlated with bacterial iron internalization. To identify direct effects due to taxon-specific iron sensitivity, we used participant stool samples to derive diverse in vitro communities. Iron supplementation of these communities caused small compositional shifts, mimicking those in vivo, whereas iron deprivation dramatically inhibited growth with irreversible, cumulative reduction in diversity and replacement of dominant species. Sensitivity of individual species to iron deprivation in axenic culture generally predicted iron dependency in a community. Finally, exogenous heme acted as a source of inorganic iron to prevent depletion of some species. Our results highlight the complementarity of in vivo and in vitro studies in understanding how environmental factors affect gut microbiotas.

Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs.

The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.

Cell Size: Fat Makes Cells Fat.

Nutrients are required for the multiple biosynthetic pathways that result in cell growth, and faster growth due to increased nutrient supply results in larger cell volume. A new study demonstrates that fatty-acid availability limits growth rate and cell envelope capacity, revealing that fatty-acid synthesis is the primary determinant of cell size in bacteria and in budding yeast.

Design of High-Specificity Nanocarriers by Exploiting Non-Equilibrium Effects in Cancer Cell Targeting.

Although targeting of cancer cells using drug-delivering nanocarriers holds promise for improving therapeutic agent specificity, the strategy of maximizing ligand affinity for receptors overexpressed on cancer cells is suboptimal. To determine design principles that maximize nanocarrier specificity for cancer cells, we studied a generalized kinetics-based theoretical model of nanocarriers with one or more ligands that specifically bind these overexpressed receptors. We show that kinetics inherent to the system play an important role in determining specificity and can in fact be exploited to attain orders of magnitude improvement in specificity. In contrast to the current trend of therapeutic design, we show that these specificity increases can generally be achieved by a combination of low rates of endocytosis and nanocarriers with multiple low-affinity ligands. These results are broadly robust across endocytosis mechanisms and drug-delivery protocols, suggesting the need for a paradigm shift in receptor-targeted drug-delivery design.

Helical insertion of peptidoglycan produces chiral ordering of the bacterial cell wall.

The regulation of cell shape is a common challenge faced by organisms across all biological kingdoms. In nearly all bacteria, cell shape is determined by the architecture of the peptidoglycan cell wall, a macromolecule consisting of glycan strands crosslinked by peptides. In addition to shape, cell growth must also maintain the wall structural integrity to prevent lysis due to large turgor pressures. Robustness can be accomplished by establishing a globally ordered cell-wall network, although how a bacterium generates and maintains peptidoglycan order on the micron scale using nanometer-sized proteins remains a mystery. Here, we demonstrate that left-handed chirality of the MreB cytoskeleton in the rod-shaped bacterium Escherichia coli gives rise to a global, right-handed chiral ordering of the cell wall. Local, MreB-guided insertion of material into the peptidoglycan network naturally orders the glycan strands and causes cells to twist left-handedly during elongational growth. Through comparison with the right-handed twisting of Bacillus subtilis cells, our work supports a common mechanism linking helical insertion and chiral cell-wall ordering in rod-shaped bacteria. These physical principles of cell growth link the molecular structure of the bacterial cytoskeleton, mechanisms of wall synthesis, and the coordination of cell-wall architecture.

The bacterial actin MreB rotates, and rotation depends on cell-wall assembly.

Bacterial cells possess multiple cytoskeletal proteins involved in a wide range of cellular processes. These cytoskeletal proteins are dynamic, but the driving forces and cellular functions of these dynamics remain poorly understood. Eukaryotic cytoskeletal dynamics are often driven by motor proteins, but in bacteria no motors that drive cytoskeletal motion have been identified to date. Here, we quantitatively study the dynamics of the Escherichia coli actin homolog MreB, which is essential for the maintenance of rod-like cell shape in bacteria. We find that MreB rotates around the long axis of the cell in a persistent manner. Whereas previous studies have suggested that MreB dynamics are driven by its own polymerization, we show that MreB rotation does not depend on its own polymerization but rather requires the assembly of the peptidoglycan cell wall. The cell-wall synthesis machinery thus either constitutes a novel type of extracellular motor that exerts force on cytoplasmic MreB, or is indirectly required for an as-yet-unidentified motor. Biophysical simulations suggest that one function of MreB rotation is to ensure a uniform distribution of new peptidoglycan insertion sites, a necessary condition to maintain rod shape during growth. These findings both broaden the view of cytoskeletal motors and deepen our understanding of the physical basis of bacterial morphogenesis.

Cell shape and cell-wall organization in Gram-negative bacteria.

In bacterial cells, the peptidoglycan cell wall is the stress-bearing structure that dictates cell shape. Although many molecular details of the composition and assembly of cell-wall components are known, how the network of peptidoglycan subunits is organized to give the cell shape during normal growth and how it is reorganized in response to damage or environmental forces have been relatively unexplored. In this work, we introduce a quantitative physical model of the bacterial cell wall that predicts the mechanical response of cell shape to peptidoglycan damage and perturbation in the rod-shaped Gram-negative bacterium Escherichia coli. To test these predictions, we use time-lapse imaging experiments to show that damage often manifests as a bulge on the sidewall, coupled to large-scale bending of the cylindrical cell wall around the bulge. Our physical model also suggests a surprising robustness of cell shape to peptidoglycan defects, helping explain the observed porosity of the cell wall and the ability of cells to grow and maintain their shape even under conditions that limit peptide crosslinking. Finally, we show that many common bacterial cell shapes can be realized within the same model via simple spatial patterning of peptidoglycan defects, suggesting that minor patterning changes could underlie the great diversity of shapes observed in the bacterial kingdom.

Pattern formation within Escherichia coli: diffusion, membrane attachment, and self-interaction of MinD molecules.

In E. coli, accurate cell division depends upon the oscillation of Min proteins from pole to pole. We provide a model for the polar localization of MinD based only on diffusion, a delay for nucleotide exchange, and different rates of attachment to the bare membrane and the occupied membrane. We derive analytically the probability density, and correspondingly the length scale, for MinD attachment zones. Our simple analytical model illustrates the processes giving rise to the observed localization of cellular MinD zones.

Dynamic structures in Escherichia coli: spontaneous formation of MinE rings and MinD polar zones.

In Escherichia coli, division site selection is regulated in part by the Min-protein system. Oscillations of the Min proteins from pole to pole every approximately 40 sec have been revealed by in vivo studies of GFP fusions. The dynamic oscillatory structures produced by the Min proteins, including a ring of MinE protein, compact polar zones of MinD, and zebra-striped oscillations in filamentous cells, remain unexplained. We show that the Min oscillations, including mutant phenotypes, can be accounted for by in vitro-observed interactions involving MinD and MinE, with a crucial role played by the rate of nucleotide exchange. Recent discoveries suggest that protein oscillations may play a general role in proper chromosome and plasmid partitioning.