scholarly journals Identification, isolation, and structural studies of the outer membrane lipopolysaccharide of Caulobacter crescentus.

1992 ◽  
Vol 174 (23) ◽  
pp. 7595-7605 ◽  
Author(s):  
N Ravenscroft ◽  
S G Walker ◽  
G G Dutton ◽  
J Smith
Keyword(s):  
Outer Membrane ◽  
Caulobacter Crescentus ◽  
Structural Studies

scholarly journals Two Outer Membrane Proteins Are Required for Maximal Type I Secretion of the Caulobacter crescentus S-Layer Protein

2004 ◽  
Vol 186 (23) ◽  
pp. 8000-8009 ◽  
Author(s):  
Michael C. Toporowski ◽  
John F. Nomellini ◽  
Peter Awram ◽  
John Smit
Keyword(s):  
Outer Membrane ◽  
Caulobacter Crescentus ◽  
Outer Membrane Proteins ◽  
Functional System ◽  
Type I ◽  
Wild Type ◽  
Residual Level ◽  
The Individual ◽  
Multicopy Vector

ABSTRACT Transport of RsaA, the crystalline S-layer subunit protein of Caulobacter crescentus, is mediated by a type I secretion mechanism. Two proteins have been identified that play the role of the outer membrane protein (OMP) component in the RsaA secretion machinery. The genes rsaF a and rsaF b were identified by similarity to the Escherichia coli hemolysin secretion OMP TolC by using the C. crescentus genome sequence. The rsaF a gene is located several kilobases downstream of the other transporter genes, while rsaF b is completely unlinked. An rsaF a knockout had ∼56% secretion compared to wild-type levels, while the rsaF b knockout reduced secretion levels to ∼79%. When expression of both proteins was eliminated, there was no RsaA secretion, but a residual level of ∼9% remained inside the cell, suggesting posttranslational autoregulation. Complementation with either of the individual rsaF genes by use of a multicopy vector, which resulted in 8- to 10-fold overexpression of the proteins, did not restore RsaA secretion to wild-type levels, indicating that both rsaF genes were required for full-level secretion. However, overexpression of rsaFa (with normal rsaF b levels) in concert with overexpression of rsaA resulted in a 28% increase in RsaA secretion, indicating a potential for significantly increasing expression levels of an already highly expressing type I secretion system. This is the only known example of type I secretion requiring two OMPs to assemble a fully functional system.

scholarly journals Identification of Genes Required for Synthesis of the Adhesive Holdfast in Caulobacter crescentus

2003 ◽  
Vol 185 (4) ◽  
pp. 1432-1442 ◽  
Author(s):  
Chris S. Smith ◽  
Aaron Hinz ◽  
Diane Bodenmiller ◽  
David E. Larson ◽  
Yves V. Brun
Keyword(s):  
Outer Membrane ◽  
Gene Cluster ◽  
Xanthomonas Campestris ◽  
Caulobacter Crescentus ◽  
Sequence Similarity ◽  
Gram Negative ◽  
Transmission Electron ◽  
Polar Development ◽  
Transposon Insertions ◽  
Polar Organelle

ABSTRACT Adhesion to both abiotic and biotic surfaces by the gram-negative prothescate bacterium Caulobacter crescentus is mediated by a polar organelle called the “holdfast,” which enables the bacterium to form stable monolayer biofilms. The holdfast, a complex polysaccharide composed in part of N-acetylglucosamine, localizes to the tip of the stalk (a thin cylindrical extension of the cell wall and membranes). We report here the isolation of adhesion mutants with transposon insertions in an uncharacterized gene cluster involved in holdfast biogenesis (hfs) as well as in previously identified polar development genes (podJ and pleC), and the holdfast attachment genes (hfa). Clean deletions of three of the four genes in the hfs gene cluster (hfsDAB) resulted in a severe holdfast biogenesis phenotype. These mutants do not bind to surfaces or to a fluorescently labeled lectin, specific for N-acetylglucosamine. Transmission electron microscopy indicated that the hfsDAB mutants fail to synthesize a holdfast at the stalk tip. The predicted hfs gene products have significant sequence similarity to proteins necessary for exopolysaccharide export in gram-negative bacteria. HfsA has sequence similarity to GumC from Xanthomonas campestris, which is involved in exopolysaccharide export in the periplasm. HfsD has sequence similarity to Wza from Escherichia coli, an outer membrane protein involved in secretion of polysaccharide through the outer membrane. HfsB is a novel protein involved in holdfast biogenesis. These data suggest that the hfs genes play an important role in holdfast export.

scholarly journals Mechanisms of Resistance to the Contact-Dependent Bacteriocin CdzC/D inCaulobacter crescentus

2019 ◽  
Vol 201 (8) ◽  
Author(s):  
Leonor García-Bayona ◽  
Kevin Gozzi ◽  
Michael T. Laub
Keyword(s):  
Outer Membrane ◽  
Caulobacter Crescentus ◽  
Cell Envelope ◽  
Target Cells ◽  
Dependent Manner ◽  
Content Type ◽  
Repeat Protein ◽  
Outer Membranes ◽  
Oligotrophic Bacterium ◽  
Small Hydrophobic

ABSTRACTThe Cdz bacteriocin system allows the aquatic oligotrophic bacteriumCaulobacter crescentusto kill closely related species in a contact-dependent manner. The toxin, which aggregates on the surfaces of producer cells, is composed of two small hydrophobic proteins, CdzC and CdzD, each bearing an extended glycine-zipper motif, that together induce inner membrane depolarization and kill target cells. To further characterize the mechanism of Cdz delivery and toxicity, we screened for mutations that render a target strain resistant to Cdz-mediated killing. These mutations mapped to four loci, including a TonB-dependent receptor, a three-gene operon (namedzerRABforzipperenveloperesistance), andperA(forpentapeptideenveloperesistance). Mutations in thezerRABlocus led to its overproduction and to potential changes in cell envelope composition, which may diminish the susceptibility of cells to Cdz toxins. TheperAgene is also required to maintain a normal cell envelope, but our screen identified mutations that confer resistance to Cdz toxins without substantially affecting the cell envelope functions of PerA. We demonstrate that PerA, which encodes a pentapeptide repeat protein predicted to form a quadrilateral β-helix, localizes primarily to the outer membrane of cells, where it may serve as a receptor for the Cdz toxins. Collectively, these results provide new insights into the function and mechanisms of an atypical, contact-dependent bacteriocin system.IMPORTANCEBacteriocins are commonly used by bacteria to kill neighboring cells that compete for resources. Although most bacteriocins are secreted, the aquatic, oligotrophic bacteriumCaulobacter crescentusproduces a two-peptide bacteriocin, CdzC/D, that remains attached to the outer membranes of cells, enabling contact-dependent killing of cells lacking the immunity protein CdzI. The receptor for CdzC/D has not previously been reported. Here, we describe a genetic screen for mutations that confer resistance to CdzC/D. One locus identified,perA, encodes a pentapeptide repeat protein that resides in the outer membrane of target cells, where it may act as the direct receptor for CdzC/D. Collectively, our results provide new insight into bacteriocin function and diversity.

scholarly journals ExbBD-Dependent Transport of Maltodextrins through the Novel MalA Protein across the Outer Membrane of Caulobacter crescentus

2005 ◽  
Vol 187 (24) ◽  
pp. 8300-8311 ◽  
Author(s):  
Heidi Neugebauer ◽  
Christina Herrmann ◽  
Winfried Kammer ◽  
Gerold Schwarz ◽  
Alfred Nordheim ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Caulobacter Crescentus ◽  
Vitamin B ◽  
Wild Type ◽  
E Coli ◽  
Iron Source ◽  
Maltose Transport ◽  
Dependent Transport

ABSTRACT Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe3+ and vitamin B12—the substrates hitherto known to be transported by TonB-dependent transporters—the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [14C]maltodextrins from [14C]maltose to [14C]maltopentaose. [14C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a Kd of 0.2 μM, while the second transport had a Kd of 5 μM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 μM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [14C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe3+-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe3+-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with Kd values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B12 and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B12 has been demonstrated.

scholarly journals Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus

Microbiology ◽  
2014 ◽  
Vol 160 (3) ◽  
pp. 635-645 ◽  
Author(s):  
Gerald N. Presley ◽  
Matthew J. Payea ◽  
Logan R. Hurst ◽  
Annie E. Egan ◽  
Brandon S. Martin ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Organic Molecules ◽  
Caulobacter Crescentus ◽  
Nutrient Transport ◽  
Proton Motive Force ◽  
Facilitated Diffusion ◽  
Extracellular Medium ◽  
Oligotrophic Bacterium ◽  
Carbonyl Cyanide ◽  
Dependent Transport

The oligotrophic bacterium Caulobacter crescentus has the ability to metabolize various organic molecules, including plant structural carbohydrates, as a carbon source. The nature of β-glucosidase (BGL)-mediated gluco-oligosaccharide degradation and nutrient transport across the outer membrane in C. crescentus was investigated. All gluco-oligosaccharides tested (up to celloheptose) supported growth in M2 minimal media but not cellulose or CM-cellulose. The periplasmic and outer membrane fractions showed highest BGL activity, but no significant BGL activity was observed in the cytosol or extracellular medium. Cells grown in cellobiose showed expression of specific BGLs and TonB-dependent receptors (TBDRs). Carbonyl cyanide 3-chlorophenylhydrazone lowered the rate of cell growth in cellobiose but not in glucose, indicating potential cellobiose transport into the cell by a proton motive force-dependent process, such as TBDR-dependent transport, and facilitated diffusion of glucose across the outer membrane via specific porins. These results suggest that C. crescentus acquires carbon from cellulose-derived gluco-oligosaccharides found in the environment by extracellular and periplasmic BGL activity and TBDR-mediated transport. This report on extracellular degradation of gluco-oligosaccharides and methods of nutrient acquisition by C. crescentus supports a broader suite of carbohydrate metabolic capabilities suggested by the C. crescentus genome sequence that until now have not been reported.

Structural studies on the capsid of Caulobacter crescentus bacteriophage φCbK

1972 ◽  
Vol 71 (2) ◽  
pp. 201-216 ◽  
Author(s):  
K.R. Leonard ◽  
A.K. Kleinschmidt ◽  
Nina Agabian-Keshishian ◽  
Lucille Shapiro ◽  
J.V. Maizel
Keyword(s):  
Caulobacter Crescentus ◽  
Structural Studies

scholarly journals Establishment of a Protein Concentration Gradient in the Outer Membrane Requires Two Diffusion-Limiting Mechanisms

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Luis David Ginez ◽  
Aurora Osorio ◽  
Laura Camarena ◽  
Sebastian Poggio
Keyword(s):  
Cell Wall ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Concentration Gradient ◽  
Protein Concentration ◽  
Caulobacter Crescentus ◽  
Outer Membrane Proteins ◽  
Structural Domains ◽  
Content Type ◽  
Terminal Domain

ABSTRACTOmpA-like proteins are involved in the stabilization of the outer membrane, resistance to osmotic stress, and pathogenesis. InCaulobacter crescentus, OmpA2 forms a physiologically relevant concentration gradient that forms by an uncharacterized mechanism, in which the gradient orientation depends on the position of the gene locus. This suggests that OmpA2 is synthesized and translocated to the periplasm close to the position of the gene and that the gradient forms by diffusion of the protein from this point. To further understand how the OmpA2 gradient is established, we determined the localization and mobility of the full protein and of its two structural domains. We show that OmpA2 does not diffuse and that both domains are required for gradient formation. The C-terminal domain binds tightly to the cell wall and the immobility of the full protein depends on the binding of this domain to the peptidoglycan; in contrast, the N-terminal membrane β-barrel diffuses slowly. Our results support a model in which once OmpA2 is translocated to the periplasm, the N-terminal membrane β-barrel is required for an initial fast restriction of diffusion until the position of the protein is stabilized by the binding of the C-terminal domain to the cell wall. The implications of these results on outer membrane protein diffusion and organization are discussed.IMPORTANCEProtein concentration gradients play a relevant role in the organization of the bacterial cell. TheCaulobacter crescentusprotein OmpA2 forms an outer membrane polar concentration gradient. To understand the molecular mechanism that determines the formation of this gradient, we characterized the mobility and localization of the full protein and of its two structural domains an integral outer membrane β-barrel and a periplasmic peptidoglycan binding domain. Each domain has a different role in the formation of the OmpA2 gradient, which occurs in two steps. We also show that the OmpA2 outer membrane β-barrel can diffuse, which is in contrast to what has been reported previously for several integral outer membrane proteins inEscherichia coli, suggesting a different organization of the outer membrane proteins.

Sign in / Sign up

Export Citation Format

Share Document