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.

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.

Evidence for proton motive force dependent transport of selenite by Clostridium pasteurianum

1989 ◽  
Vol 35 (4) ◽  
pp. 481-486 ◽  
Author(s):  
Richard D. Bryant ◽  
Edward J. Laishley
Keyword(s):  
Energy Source ◽  
Proton Conductor ◽  
Anion Transport ◽  
Proton Motive Force ◽  
Motive Force ◽  
Clostridium Pasteurianum ◽  
Source Temperature ◽  
Carbonyl Cyanide ◽  
Proton Symport ◽  
Dependent Transport

The proton motive force mediated the transport of selenite [Formula: see text] in Clostridium pasteurianum cells by proton symport. The proton conductor, carbonyl cyanide m-chlorophenylhydrazone, inhibited [Formula: see text] uptake while N,N′-dicyclohexylcarbodiimide prevented [Formula: see text] uptake by presumably inhibiting the unidirectional ATPase. Acid pulse studies and antibiotic experiments with valinomycin suggest that the transmembrane ΔpH component of the proton motive force mediated the transport of [Formula: see text] into the cells. The [Formula: see text] porter system in C. pasteurianum was found to be dependent upon energy source, temperature, and medium pH.Key words: proton motive force, anion transport, selenite, Clostridium pasteurianum.

scholarly journals Mechanism of Bactericidal Activity of Microcin L inEscherichia coliandSalmonella enterica

2010 ◽  
Vol 55 (3) ◽  
pp. 997-1007 ◽  
Author(s):  
Natacha Morin ◽  
Isabelle Lanneluc ◽  
Nathalie Connil ◽  
Marie Cottenceau ◽  
Anne Marie Pons ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bactericidal Activity ◽  
Salmonella Enterica ◽  
Cytoplasmic Membrane ◽  
Genetic System ◽  
Proton Motive Force ◽  
Motive Force ◽  
Membrane Properties ◽  
Outer Membrane Receptor ◽  
E Coli

ABSTRACTFor the first time, the mechanism of action of microcin L (MccL) was investigated in live bacteria. MccL is a gene-encoded peptide produced byEscherichia coliLR05 that exhibits a strong antibacterial activity against relatedEnterobacteriaceae, includingSalmonella entericaserovars Typhimurium and Enteritidis. We first subcloned the MccL genetic system to remove the sequences not involved in MccL production. We then optimized the MccL purification procedure to obtain large amounts of purified microcin to investigate its antimicrobial and membrane properties. We showed that MccL did not induce outer membrane permeabilization, which indicated that MccL did not use this way to kill the sensitive cell or to enter into it. Using a set ofE. coliandSalmonella entericamutants lacking iron-siderophore receptors, we demonstrated that the MccL uptake required the outer membrane receptor Cir. Moreover, the MccL bactericidal activity was shown to depend on the TonB protein that transduces the proton-motive force of the cytoplasmic membrane to transport iron-siderophore complexes across the outer membrane. Using carbonyl cyanide 3-chlorophenylhydrazone, which is known to fully dissipate the proton-motive force, we proved that the proton-motive force was required for the bactericidal activity of MccL onE. coli. In addition, we showed that a primary target of MccL could be the cytoplasmic membrane: a high level of MccL disrupted the inner membrane potential ofE. colicells. However, no permeabilization of the membrane was detected.

scholarly journals Colicin Biology

2007 ◽  
Vol 71 (1) ◽  
pp. 158-229 ◽  
Author(s):  
Eric Cascales ◽  
Susan K. Buchanan ◽  
Denis Duché ◽  
Colin Kleanthous ◽  
Roland Lloubès ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Atomic Level ◽  
Fundamental Aspect ◽  
Genetic Determinants ◽  
Extracellular Medium ◽  
E Coli ◽  
Voltage Dependent ◽  
Tonb System ◽  
Specific Receptors

SUMMARY Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of colicins as laboratory or biotechnological tools and their mode of evolution are discussed.

The melibiose/Na + symporter of Escherichia coli : kinetic and molecular properties

1990 ◽  
Vol 326 (1236) ◽  
pp. 411-423 ◽  
Keyword(s):  
Escherichia Coli ◽  
Complex Cation ◽  
Expression System ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Coupling Mechanism ◽  
Facilitated Diffusion ◽  
Histidine Residues ◽  
Sugar Binding ◽  
Dependent Transport

The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H + , Na + or Li + -sugar symporter. The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar—carrier facing the cell interior. Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment. Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described. Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism. Only substitution of arginine for His94 greatly interferes with the carrier function. It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease.

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 Modification of Outer Membrane Protein Profile and Evidence Suggesting an Active Drug Pump in Enterobacter aerogenes Clinical Strains

2003 ◽  
Vol 47 (5) ◽  
pp. 1555-1559 ◽  
Author(s):  
Stéphane Gayet ◽  
Renaud Chollet ◽  
Gérard Molle ◽  
Jean-Marie Pagès ◽  
Jacqueline Chevalier
Keyword(s):  
Outer Membrane ◽  
Protein Profile ◽  
Outer Membrane Proteins ◽  
Enterobacter Aerogenes ◽  
Normal Structure ◽  
Clinical Strains ◽  
Resistance Strategies ◽  
Carbonyl Cyanide ◽  
Energy Dependent ◽  
Drug Pump

ABSTRACT Two clinical strains of Enterobacter aerogenes that exhibited phenotypes of multiresistance to β-lactam antibiotics, fluoroquinolones, chloramphenicol, tetracycline, and kanamycin were investigated. Both strains showed a porin pattern different from that of a susceptible strain, with a drastic reduction in the amount of the major porin but with an apparently conserved normal structure (size and immunogenicity), together with overproduction of two known outer membrane proteins, OmpX and LamB. In addition, the full-length O-polysaccharide phenotype was replaced by a semirough Ra phenotype. Moreover, in one isolate the intracellular accumulation of chloramphenicol was increased in the presence of the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone, suggesting an energy-dependent efflux of chloramphenicol in this strain. The resistance strategies used by these isolates appear to be similar to that induced by stress in Escherichia coli cells.

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