Biomedical subjects

Yves V Brun

Publications and source records attributed to Yves V Brun.

At least 19 recordsLinked to original sources

PubMed2006

A nutrient uptake role for bacterial cell envelope extensions.

Bacteria exist in a variety of morphologies, but the relationship between cellular forms and biological functions remains poorly understood. We show that stalks (prosthecae), cylindrical extensions of the Caulobacter crescentus cell envelope, can take up and hydrolyze organic phosphate molecules and contain the high-affinity phosphate-binding protein PstS, but not PstA, a protein that is required for transport of phosphate into the cytoplasm. Therefore, uptake, hydrolysis, and periplasmic binding of a phosphate source can take place in the stalk, but high-affinity import must take place in the cell body. Furthermore, by using analytical modeling, we illustrate the biophysical advantage of the stalk as a morphological adaptation to the diffusion-limited, oligotrophic environments where C. crescentus thrives. This advantage is due to the fact that a stalk is long and thin, a favorable shape for maximizing contact with diffusing nutrients while minimizing increases in both surface area and cell volume.

Animals↗

PubMed2006

Adhesion of single bacterial cells in the micronewton range.

The adhesion of bacteria to surfaces plays critical roles in the environment, disease, and industry. In aquatic environments, Caulobacter crescentus is one of the first colonizers of submerged surfaces. Using a micromanipulation technique, we measured the adhesion force of single C. crescentus cells attached to borosilicate substrates through their adhesive holdfast. The detachment forces measured for 14 cells ranged over 0.11 to 2.26 microN, averaging 0.59 +/- 0.62 microN. Based on the calculation of stress distribution with the finite element analysis method (dividing an object into small grids and calculating relevant parameters for all of the elements), the adhesion strength between the holdfast and the substrate is >68 N/mm(2) in the central region of contact. To our knowledge, this strength of adhesion is the strongest ever measured for biological adhesives.

Bacterial Adhesion↗

PubMed2006

A molecular beacon defines bacterial cell asymmetry.

Many cells divide asymmetrically by generating two different cell ends or poles prior to cell division, but the mechanisms by which cells distinguish one pole from the other is poorly understood. In this issue of Cell, Huitema et al. (2006) and Lam et al. (2006) describe a protein that defines one specific pole of a bacterial cell by localizing to the site of cell division to be inherited by both progeny at the resulting new poles.

Bacterial Proteins↗

PubMed2006

Dissection of functional domains of the polar localization factor PodJ in Caulobacter crescentus.

The polar organelle development protein, PodJ, is important for proper establishment of polarity in Caulobacter crescentus. podJ null mutants are unable to form holdfast or pili, have reduced swarming motility, and have difficulty ejecting the flagellum during the swarmer to stalked cell transition. In this study, we create a series of truncation mutants to investigate functional domains of PodJ. We show that PodJ has a transmembrane domain between amino acids 600 and 670. We identify a periplasmic region important for pili production and a cytoplasmic region required for holdfast formation and swarming motility, and establish that PleC localization is not required for holdfast formation and motility in soft agar. Analysis of the mutants reveals that the last 54 amino acids of the protein negatively regulate processing of the full-length form of the protein, PodJ(L), to a shorter form, PodJ(S). Finally, we identify a cytoplasmic region of PodJ involved in targeting it to the flagellar pole, and a periplasmic region required for localization of PleC.

Bacterial Proteins↗

PubMed2006

Comparative genomic evidence for a close relationship between the dimorphic prosthecate bacteria Hyphomonas neptunium and Caulobacter crescentus.

The dimorphic prosthecate bacteria (DPB) are alpha-proteobacteria that reproduce in an asymmetric manner rather than by binary fission and are of interest as simple models of development. Prior to this work, the only member of this group for which genome sequence was available was the model freshwater organism Caulobacter crescentus. Here we describe the genome sequence of Hyphomonas neptunium, a marine member of the DPB that differs from C. crescentus in that H. neptunium uses its stalk as a reproductive structure. Genome analysis indicates that this organism shares more genes with C. crescentus than it does with Silicibacter pomeroyi (a closer relative according to 16S rRNA phylogeny), that it relies upon a heterotrophic strategy utilizing a wide range of substrates, that its cell cycle is likely to be regulated in a similar manner to that of C. crescentus, and that the outer membrane complements of H. neptunium and C. crescentus are remarkably similar. H. neptunium swarmer cells are highly motile via a single polar flagellum. With the exception of cheY and cheR, genes required for chemotaxis were absent in the H. neptunium genome. Consistent with this observation, H. neptunium swarmer cells did not respond to any chemotactic stimuli that were tested, which suggests that H. neptunium motility is a random dispersal mechanism for swarmer cells rather than a stimulus-controlled navigation system for locating specific environments. In addition to providing insights into bacterial development, the H. neptunium genome will provide an important resource for the study of other interesting biological processes including chromosome segregation, polar growth, and cell aging.

Alphaproteobacteria↗

PubMed2006

Mutations in DivL and CckA rescue a divJ null mutant of Caulobacter crescentus by reducing the activity of CtrA.

Polar development and cell division in Caulobacter crescentus are controlled and coordinated by multiple signal transduction proteins. divJ encodes a histidine kinase. A null mutation in divJ results in a reduced growth rate, cell filamentation, and mislocalized stalks. Suppressor analysis of divJ identified mutations in genes encoding the tyrosine kinase (divL) and the histidine kinase (cckA). The divL and cckA suppressor alleles all have single amino acid substitutions, some of which confer a temperature-sensitive phenotype, particularly in a wild-type background. Analysis of transcription levels from several positively regulated CtrA-dependent promoters reveals high expression in the divJ mutant, suggesting that DivJ normally serves to reduce CtrA activity. The divL and cckA suppressors reduce the amount of transcription from promoters positively regulated by CtrA, indicating that the mutations in divL and cckA are suppressing the defects of the divJ mutant by reducing the abnormally high level of CtrA activity. Immunoblotting showed no major perturbations in the CtrA protein level in any of these strains, suggesting that the high amount of CtrA activity seen in the divJ mutant and the reduced amount of activity in the suppressors are regulated at the level of activation and not transcription, translation, or degradation. In vivo phosphorylation assays confirmed that divJ mutants have elevated levels of CtrA phosphorylation and that this level is reduced in the suppressors with mutations in divL.

Bacterial Proteins↗

PubMed2005

Effects of tryptic peptide esterification in MALDI mass spectrometry.

The effect of esterification on MALDI ion yield is investigated by using alcohols having different aliphatic chain lengths. For peptides whose ionization yields increase with derivatization, more hydrophobic alcohols tend to yield greater peak enhancements. The completeness of the reaction increases from propanol to methanol. Undesired solvolysis of the amide group in the side chain of Asn or Gln leads to unexpected ester products. Ethanol is suggested as the optimal alcohol for esterification in proteomics experiments since it yields almost complete esterification without substantial solvolysis. Ethanol esterification was employed to facilitate the identification of gel-separated proteins.

Alcohols↗

PubMed2005

The elastic properties of the caulobacter crescentus adhesive holdfast are dependent on oligomers of N-acetylglucosamine.

The aquatic bacterium Caulobacter crescentus attaches to solid surfaces through an adhesive holdfast located at the tip of its polar stalk, a thin cylindrical extension of the cell membrane. In this paper, the elastic properties of the C. crescentus stalk and holdfast assembly were studied by using video light microscopy. In particular, the contribution of oligomers of N-acetylglucosamine (GlcNAc) to the elasticity of holdfast was examined by lysozyme digestion. C. crescentus cells attached to a surface undergo Brownian motion while confined effectively in a harmonic potential. Mathematical analysis of such motion enabled us to determine the force constant of the stalk-holdfast assembly, which quantifies its elastic properties. The measured force constant exhibits no dependence on stalk length, consistent with the theoretical estimate showing that the stalk can be treated as a rigid rod with respect to fluctuations of the attached cells. Therefore, the force constant of the stalk-holdfast assembly can be attributed to the elasticity of the holdfast. Motions of cells in a rosette were found to be correlated, consistent with the elastic characteristics of the holdfast. Atomic force microscopy analysis indicates that the height of a dried (in air) holdfast is approximately one-third of that of a wet (in water) holdfast, consistent with the gel-like nature of the holdfast. Lysozyme, which cleaves oligomers of GlcNAc, reduced the force constant to less than 10% of its original value, consistent with the polysaccharide gel-like nature of the holdfast. These results also indicate that GlcNAc polymers play an important role in the strength of the holdfast.

Acetylglucosamine↗

PubMed2005

Caulobacter crescentus requires RodA and MreB for stalk synthesis and prevention of ectopic pole formation.

Caulobacter crescentus cells treated with amdinocillin, an antibiotic which specifically inhibits the cell elongation transpeptidase penicillin binding protein 2 in Escherichia coli, exhibit defects in stalk elongation and morphology, indicating that stalk synthesis may be a specialized form of cell elongation. In order to investigate this possibility further, we examined the roles of two other proteins important for cell elongation, RodA and MreB. We show that, in C. crescentus, the rodA gene is essential and that RodA depletion leads to a loss of control over stalk and cell body diameter and a stalk elongation defect. In addition, we demonstrate that MreB depletion leads to a stalk elongation defect and conclude that stalk elongation is a more constrained form of cell elongation. Our results strongly suggest that MreB by itself does not determine the diameter of the cell body or stalk. Finally, we show that cells recovering from MreB depletion exhibit a strong budding and branching cell body phenotype and possess ectopic poles, as evidenced by the presence of multiple, misplaced, and sometimes highly branched stalks at the ends of these buds and branches. This phenotype is also seen to a lesser extent in cells recovering from RodA depletion and amdinocillin treatment. We conclude that MreB, RodA, and the target(s) of amdinocillin all contribute to the maintenance of cellular polarity in C. crescentus.

Amdinocillin↗

PubMed2004

Cell cycle-dependent abundance, stability and localization of FtsA and FtsQ in Caulobacter crescentus.

Coordination between cell division and DNA replication is ensured by checkpoints that act through proteins required for cell division. Following a block in DNA replication, transcription of the cell division progression genes ftsA and ftsQ is prevented in Caulobacter crescentus. One requirement for this checkpoint is that FtsA and/or FtsQ should be limiting for division in the next cell cycle. We show that the number of FtsA and FtsQ molecules fluctuates such that their concentration is low in swarmer and stalked cells, peaks in pre-divisional cells, and then dramatically decreases after cell division. Despite constitutive expression from an inducible promoter, FtsA and FtsQ levels still vary during the cell cycle, and the half-life of FtsA increases from 13 min in swarmer cells to 55 min in stalked cell types, confirming cell type-specific degradation. The post-division degradation of FtsA and FtsQ in swarmer cells reduces their concentration to 7% and 10% of their maximal level, respectively, strongly suggesting that de novo synthesis of both proteins is required for each division cycle. The localization of FtsA and FtsQ is also cell type-specific. FtsA and FtsQ are recruited to the midcell during a short period in late pre-divisional cells, consistent with the demonstrated requirement of FtsA for late stages of cell division. As previously reported for FtsZ, constitutive expression of FtsA causes cell division defects. These results indicate that the tight control of FtsA, and probably FtsQ, by cell cycle transcription, proteolysis, and localization are critical for optimal cell division and the coordination of cell division with the DNA replication cycle.

Amino Acid Sequence↗

PubMed2004

A temperature-sensitive mutation in the dnaE gene of Caulobacter crescentus that prevents initiation of DNA replication but not ongoing elongation of DNA.

A genetic screen for cell division cycle mutants of Caulobacter crescentus identified a temperature-sensitive DNA replication mutant. Genetic complementation experiments revealed a mutation within the dnaE gene, encoding the alpha-catalytic subunit of DNA polymerase III holoenzyme. Sequencing of the temperature-sensitive dnaE allele indicated a single base pair substitution resulting in a change from valine to glutamic acid within the C-terminal portion of the protein. This mutation lies in a region of the DnaE protein shown in Escherichia coli, to be important in interactions with other essential DNA replication proteins. Using DNA replication assays and fluorescence flow cytometry, we show that the observed block in DNA synthesis in the Caulobacter dnaE mutant strain occurs at the initiation stage of replication and that there is also a partial block of DNA elongation.

Caulobacter crescentus↗

PubMed2004

Development of surface adhesion in Caulobacter crescentus.

Caulobacter crescentus has a dimorphic life cycle composed of a motile stage and a sessile stage. In the sessile stage, C. crescentus is often found tightly attached to a surface through its adhesive holdfast. In this study, we examined the contribution of growth and external structures to the attachment of C. crescentus to abiotic surfaces. We show that the holdfast is essential but not sufficient for optimal attachment. Rather, adhesion in C. crescentus is a complex developmental process. We found that the attachment of C. crescentus to surfaces is cell cycle regulated and that growth or energy or both are essential for this process. The initial stage of attachment occurs in swarmer cells and is facilitated by flagellar motility and pili. Our results suggest that strong attachment is mediated by the synthesis of a holdfast as the swarmer cell differentiates into a stalked cell.

Adhesins, Bacterial↗

PubMed2003

Cell cycle timing and developmental checkpoints in Caulobacter crescentus.

Development in Caulobacter reflects a level of complexity once thought only to exist in eukaryotic cells. The cell cycle and development are not isolated from each other, but are interdependent processes. Checkpoints are in place to ensure that both cell cycle and developmental processes are completed accurately before the next stage is initiated. The timing of these processes is regulated by signal transduction networks that integrate signals from DNA replication, cell division and development. These signal transduction networks achieve precise timing of the cell cycle and development by regulating temporal gene expression, and protein activity by dynamic spatial localization within the cell and timed proteolysis.

Caulobacter crescentus↗

PubMed2003

The Caulobacter crescentus polar organelle development protein PodJ is differentially localized and is required for polar targeting of the PleC development regulator.

Regulation of polar development and cell division in Caulobacter crescentus relies on the dynamic localization of several proteins to cell poles at specific stages of the cell cycle. The polar organelle development protein, PodJ, is required for the synthesis of the adhesive holdfast and pili. Here we show the cell cycle localization of PodJ and describe a novel role for this protein in controlling the dynamic localization of the developmental regulator PleC. In swarmer cells, a short form of PodJ is localized at the flagellated pole. Upon differentiation of the swarmer cell into a stalked cell, full length PodJ is synthesized and localizes to the pole opposite the stalk. In late predivisional cells, full length PodJ is processed into a short form which remains localized at the flagellar pole after cell division and is degraded during swarmer to stalked cell differentiation. Polar localization of the developmental regulator PleC requires the presence of PodJ. In contrast, the polar localization of PodJ is not dependent on the presence of PleC. These results indicate that PodJ is an important determinant for the localization of a major regulator of cell differentiation. Thus, PodJ acts directly or indirectly to target PleC to the incipient swarmer pole, to establish the cellular asymmetry that leads to the synthesis of holdfasts and pili at their proper subcellular location.

Bacterial Proteins↗

PubMed2003

The HfaB and HfaD adhesion proteins of Caulobacter crescentus are localized in the stalk.

The differentiating bacterium Caulobacter crescentus produces two different cell types at each cell division, a motile swarmer cell and an adhesive stalked cell. The stalked cell harbours a stalk, a thin cylindrical extension of the cell surface. The tip of the stalk is decorated with a holdfast, an adhesive organelle composed at least in part of polysaccharides. The synthesis of the stalk and holdfast occur at the same pole during swarmer cell differentiation. Mutations in the hfaABDC gene cluster had been shown to disrupt the attachment of the holdfast to the tip of the stalk, but the role of individual genes was unknown. We used lacZ fusions of various DNA fragments from the hfaABDC region to show that these genes form an operon. In order to analyse the relative contribution of the different genes to holdfast attachment, mutations were constructed for each gene. hfaC was not required for holdfast attachment or binding to surfaces. The hfaA and hfaD mutants shed some holdfast material into the surrounding medium and were partially deficient in binding to surfaces. Unlike hfaA and hfaB mutants, hfaD mutants were still able to form rosettes efficiently. Cells with insertions in hfaB were unable to bind to surfaces, and lectin binding studies indicated that the hfaB mutants had the strongest holdfast shedding phenotype. We determined that HfaB and HfaD are membrane-associated proteins and that HfaB is a lipoprotein. Purification of stalks and cell bodies indicated that both HfaB and HfaD are enriched in the stalk as compared to the cell body. These results suggest that HfaB and HfaD, and probably HfaA, serve to anchor the holdfast to the tip of the stalk.

Adhesins, Bacterial↗

PubMed2003

Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus.

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.

Adhesins, Bacterial↗

PubMed2002

Artifacts and unassigned masses encountered in peptide mass mapping.

In peptide mass mapping of isolated proteins, a significant number of the observed mass spectral peaks are often uninterpreted. These peaks derive from a number of sources: errors in the genome that give rise to incorrect peptide mass predictions, undocumented post-translational modifications, sample handling-induced modifications, contaminants in the sample, non-standard protein cleavage sites, and non-protein components of the sample. In a study of the stalk organelle of Caulobacter crescentus, roughly one-third (782/2215) of all observed masses could not be assigned to the proteins identified in the gel spots (Karty et al., J. Proteome Res., 1 (2002) 325). By interpreting these masses, this work illuminates a number of phenomena that may arise in the course of peptide mass mapping of electrophoretically separated proteins and presents results from a number of related studies.

Amino Acid Sequence↗

PubMed2002

DNA replication initiation is required for mid-cell positioning of FtsZ rings in Caulobacter crescentus.

Polymerization of the GTPase FtsZ to form a structure called the Z-ring is the earliest known step in bacterial cell division. Mid-cell Z-ring assembly coincides with the beginning of the replication cycle in the differentiating bacterium Caulobacter crescentus. Z-ring disassembly occurs at the end of the division cycle, resulting in the complete degradation of FtsZ from both stalked and swarmer progeny cells. New Z-rings can only form in the replicative stalked cell. Conditional mutants in DNA replication were used to determine what role DNA replication events play in the process of Z-ring assembly at different stages in the cell cycle. Z-ring assembly occurred even when early stages of DNA replication were blocked; however, the Z-rings were localized at a subpolar region of the cell. Z-rings only assembled at the proper mid-cell location if DNA replication had initiated. Z-ring assembly coincided with areas containing little or no DNA, and Z-rings could not form over an unreplicated chromosome. Overexpressed FtsZ in the absence of DNA replication did not stimulate productive mid-cell Z-ring assembly but, instead, caused the ends of cells to constrict over an extended area away from the nucleoid. These results indicate that the state of chromosome replication is a major determinant of Z-ring localization in Caulobacter.

Bacterial Proteins↗