The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament-like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology.

Helical cell shape of the gastric pathogen Helicobacter pylori has been suggested to promote virulence through viscosity-dependent enhancement of swimming velocity. However, H. pylori csd1 mutants, which are curved but lack helical twist, show normal velocity in viscous polymer solutions and the reason for their deficiency in stomach colonization has remained unclear. Characterization of new rod shaped mutants identified Csd4, a DL-carboxypeptidase of peptidoglycan (PG) tripeptide monomers and Csd5, a putative scaffolding protein. Morphological and biochemical studies indicated Csd4 tripeptide cleavage and Csd1 crosslinking relaxation modify the PG sacculus through independent networks that coordinately generate helical shape. csd4 mutants show attenuation of stomach colonization, but no change in proinflammatory cytokine induction, despite four-fold higher levels of Nod1-agonist tripeptides in the PG sacculus. Motility analysis of similarly shaped mutants bearing distinct alterations in PG modifications revealed deficits associated with shape, but only in gel-like media and not viscous solutions. As gastric mucus displays viscoelastic gel-like properties, our results suggest enhanced penetration of the mucus barrier underlies the fitness advantage conferred by H. pylori's characteristic shape.

The penicillin-binding protein (PBP) 1A is a major murein (peptidoglycan) synthase in Escherichia coli. The murein synthesis activity of PBP1A was studied in vitro with radioactive lipid II substrate. PBP1A produced murein glycan strands by transglycosylation and formed peptide cross-links by transpeptidation. Time course experiments revealed that PBP1A, unlike PBP1B, required the presence of polymerized glycan strands carrying monomeric peptides for cross-linking activity. PBP1A was capable of attaching nascent murein synthesized from radioactive lipid II to nonlabeled murein sacculi. The attachment of the new material occurred by transpeptidation reactions in which monomeric tri-and tetrapeptides in the sacculi were the acceptors.Most bacteria have a murein (peptidoglycan) sacculus that forms an uninterrupted net-like structure around the cytoplasmic membrane. The sacculus is essential for the osmotic stability of the cell (1) and consists of glycan strands that are crosslinked by short peptides (2). During growth and division, the sacculus is enlarged by the incorporation of lipid II precursor by a yet unknown mechanism, which involves the coordinated action of murein synthases, murein hydrolases, and probably regulatory proteins (3).Escherichia coli contains six known murein synthases (4 -8). However, it is not known what particular reaction these enzymes catalyze during the enlargement of the sacculus and, hence, which precise function they have in cell wall growth. Furthermore, most of the murein synthases from E. coli and other species are poorly or not characterized so far, perhaps because of difficulties in obtaining pure enzyme and lipid II substrate in sufficient quantities.The penicillin-binding proteins (PBPs) 2 PBP1A and PBP1B are the major bifunctional murein synthases in E. coli catalyzing both the oligomerization of the glycan strands and the formation of the peptide cross-links (9 -11). Both PBPs form independent dimers in vivo (12). PBP1A and PBP1B are not essential for cell growth, but cells lacking both enzymes are not viable, indicating that both have a similar, essential function that cannot be taken over by other murein synthases (5, 13). Yet, mutants lacking either PBP1A or PBP1B show particular phenotypes, indicating that these synthases may play distinct roles during cell growth and division. For example, mutants without PBP1B are more sensitive to ␤-lactam antibiotics than mutants without PBP1A (14). Furthermore, PBP1B-deficient mutants, but not PBP1A-deficient mutants, lose cell integrity upon inactivation of PBP2, PBP3, or the cell division protein FtsQ (15).The enzymatic activities of PBP1B with lipid II or artificial substrates have been studied previously (16 -19). However, apart from two early studies from Matsuhashi and colleagues (20,21), the activities of PBP1A remain uncharacterized. In this article, we report that PBP1A is capable of producing in vitro a cross-linked murein with glycan strands of (on average) almost 20 disaccharide units and with 18 -26% of the peptides bei...

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We present a method for the systematic identification of picogram quantities of new lipids in total extracts of tissues and fluids. It relies on the modularity of lipid structures and applies all-ions fragmentation LC-MS/MS and Arcadiate software to recognize individual modules originating from the same lipid precursor of known or assumed structure. In this way it alleviates the need to recognize and fragment very low abundant precursors of novel molecules in complex lipid extracts. In a single analysis of rat kidney extract the method identified 58 known and discovered 74 novel endogenous endocannabinoids and endocannabinoid-related molecules, including a novel class of N-acylaspartates that inhibit Hedgehog signaling while having no impact on endocannabinoid receptors.