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.

Various cell shapes are encountered in the prokaryotic world, but how they are achieved is poorly understood. Intermediate filaments (IFs) of the eukaryotic cytoskeleton play an important role in cell shape in higher organisms. No such filaments have been found in prokaryotes. Here, we describe a bacterial equivalent to IF proteins, named crescentin, whose cytoskeletal function is required for the vibrioid and helical shapes of Caulobacter crescentus. Without crescentin, the cells adopt a straight-rod morphology. Crescentin has characteristic features of IF proteins including the ability to assemble into filaments in vitro without energy or cofactor requirements. In vivo, crescentin forms a helical structure that colocalizes with the inner cell curvatures beneath the cytoplasmic membrane. We propose that IF-like filaments of crescentin assemble into a helical structure, which by applying its geometry to the cell, generates a vibrioid or helical cell shape depending on the length of the cell.

During infection, chemokines sequestered on endothelium induce recruitment of circulating leukocytes into the tissue where they chemotax along chemokine gradients toward the afflicted site. The aim of this in vivo study was to determine whether a chemokine gradient was formed intravascularly and influenced intraluminal neutrophil crawling and transmigration. A chemokine gradient was induced by placing a macrophage inflammatory protein-2 (MIP-2)-containing (CXCL2) gel on the cremaster muscle of anesthetized wild-type mice or heparanase-overexpressing transgenic mice (hpa-tg) with truncated heparan sulfate (HS) side chains. Neutrophil-endothelial interactions were visualized by intravital microscopy and chemokine gradients detected by confocal microscopy. Localized extravascular chemokine release (MIP-2 gel) induced directed neutrophil crawling along a chemotactic gradient immobilized on the endothelium and accelerated their recruitment into the target tissue compared with homogeneous extravascular chemokine concentration (MIP-2 superfusion). Endothelial chemokine sequestration occurred exclusively in venules and was HS-dependent, and neutrophils in hpa-tg mice exhibited random crawling. Despite similar numbers of adherent neutrophils in hpa-tg and wild-type mice, the altered crawling in hpa-tg mice was translated into decreased number of emigrated neutrophils and ultimately decreased the ability to clear bacterial infections. In conclusion, an intravascular chemokine gradient sequestered by endothelial HS effectively directs crawling leukocytes toward transmigration loci close to the infection site. IntroductionChemokine-induced recruitment of circulating leukocytes is fundamental in the immune response to bacterial infections. The leukocyte recruitment cascade is initiated by endothelial cell activation and presentation of chemokines to rolling leukocytes, which, by activating leukocyte integrins, results in leukocyte adhesion to and diapedesis through the vessel wall. [1][2][3] Recently, an additional step in the leukocyte recruitment cascade was detected bridging adhesion and diapedesis, namely, Mac-1-mediated intraluminal crawling. [4][5][6][7] In these studies, neutrophils were observed to crawl on endothelium in all directions before transmigration through endothelial junctions; and if crawling were disabled, diapedesis was delayed and occurred preferentially through the transcellular pathway. 4,5 However, chemokine presence on endothelium is not enough to initiate leukocyte diapedesis; a chemotactic gradient over the vessel wall with a higher extravascular concentration is required. 8 Outside the vasculature, leukocytes chemotax along a chemical gradient in the extracellular matrix toward the chemokine source. 9,10 During infection, a multitude of chemotactic factors are present simultaneously, and the ability of leukocytes to prioritize between end-target (bacterial peptides) and intermediate (eg, macrophage inflammatory protein-2 [MIP-2]) chemotactic cues is crucial for leukocytes to find the site of in...

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SummaryThe CtrA master transcriptional regulator is a central control element in Caulobacter cell cycle progression and polar morphogenesis. Because of its critical role, CtrA activity is temporally regulated by multiple mechanisms including phosphorylation and ClpXPdependent degradation of CtrA. The CckA histidine kinase is known to contribute to CtrA phosphorylation. We show here that genes differentially expressed in a ctrA temperature-sensitive (ts) mutant are similarly affected in a cckA ts mutant, that the phosphorylation of CckA coincides temporally with CtrA phosphorylation during the cell cycle, and that CckA is essential for viability because it is required for CtrA phosphorylation. Thus, it is the signal transduction pathway mediated by CckA that culminates in CtrA activation, which is temporally regulated and essential for cell cycle progression. CckA also positively regulates CtrA activity by a mechanism that is independent of CtrA phosphorylation. CtrA is more stable in the presence of CckA than it is absence, suggesting that CckA may also be involved, directly or indirectly, in the regulation of CtrA proteolysis.

Six genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii were identified using Tn5 mutagenesis. Four of them displayed homology to the previously cloned and sequenced Agrobacteriurn turnefaciens cellulose genes celA, celf3, ce/C and celE These genes are organized similarly in R. legurninosarum bv. trifolii. In addition, there were strong indications that two tandemly located genes, celRl and celR2, probably organized as one operon, are involved in the regulation of cellulose synthesis. The deduced amino acid sequences of these genes displayed a high degree of similarity to the Caulobacter crescentus DivK and PleD proteins that belong to the family of two-component response regulators. This is to our knowledge the first report of genes involved in the regulation of cellulose synthesis. Results from attachment assays and electron microscopic studies indicated that cellulose synthesis in R. leguminosarum bv. trifolii is induced upon close contact with plant roots during the attachment process.

A conserved domain, called GGDEF (referring to a conserved central sequence pattern), is detected in many procaryotic proteins, often in various combinations with putative sensory-regulatory components. Most sequenced bacterial genomes contain several different GGDEF proteins. The function of this domain has so far not been experimentally shown. Through genetic complementation using genes from three different bacteria encoding proteins with GGDEF domains as the only element in common, we present genetic data indicating (a) that the GGDEF domain is responsible for the diguanylate cyclase activity of these proteins, and (b) that the activity of cellulose synthase in Rhizobium leguminosarum bv. trifolii and Agrobacterium tumefaciens is regulated by cyclic di-GMP as in Acetobacter xylinum. ß

The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid-and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.A variety of prokaryotic organisms are capable of enzymatically reducing atmospheric nitrogen to ammonia. This process, known as biological nitrogen fixation, can be performed either by free-living bacteria (e.g., Klebsiella pneumoniae) or by symbiotic bacteria. Nitrogen-fixing symbioses between plants belonging to the family Leguminosae and soil bacteria collectively called rhizobia contribute substantially to plant productivity. Ultimately, these associations lead to the formation of specialized structures called nodules on the stems or roots of host plants, where infecting rhizobia differentiate into bacteroids that reduce atmospheric nitrogen to ammonia. The ammonia is incorporated into amino acids that are supplied to the host, which return the favor by supplying the microsymbionts with photosynthates. Nodulation, the process that leads to bacterial colonization of root or stem nodules, is highly selective; a continuous exchange of molecular signals between the two symbionts enables the host to distinguish compatible rhizobia from potential pathogens. Initially, lipochitooligosaccharidic nodulation factors (Nod factors) that are secreted by rhizobia in response to plant flavonoids are essential for infection. Later, additional bacterial signals, such as surface polysaccharides or secreted proteins, are also required for efficient nodulation of specific hosts (7,35).Among the known microsymbionts, Rhizobium sp. strain NGR234 has the rare ability to nodulate more than 112 genera of legumes (37). The closely related strain Rhizobium fredii USDA257 forms nodules on a smaller subset of plants (Ͼ79 genera), but it fixes nitrogen with Glycine max and Glycine soja, two hosts that fail to establish effective symbioses with NGR234. Early work showed that mutations in the cultivar specificity locus nolXWBTUV modified the host range of USDA257 so that it included the improved soybean variety McCall (14,28). Fla...