scholarly journals Comparative Analysis of Ionic Strength Tolerance between Freshwater and MarineCaulobacteralesAdhesins

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Nelson K. Chepkwony ◽  
Cécile Berne ◽  
Yves V. Brun
Keyword(s):  
Ionic Strength ◽  
Physicochemical Properties ◽  
Bacterial Adhesion ◽  
Marine Bacterium ◽  
Caulobacter Crescentus ◽  
High Ionic Strength ◽  
Chemical Compositions ◽  
Shear Forces ◽  
Surface Attachment ◽  
Content Type

ABSTRACTBacterial adhesion is affected by environmental factors, such as ionic strength, pH, temperature, and shear forces. Therefore, marine bacteria must have developed adhesins with different compositions and structures than those of their freshwater counterparts to adapt to their natural environment. The dimorphic alphaproteobacteriumHirschia balticais a marine budding bacterium in the cladeCaulobacterales.H. balticauses a polar adhesin, the holdfast, located at the cell pole opposite the reproductive stalk, for surface attachment and cell-cell adhesion. The holdfast adhesin has been best characterized inCaulobacter crescentus, a freshwater member of theCaulobacterales, and little is known about holdfast compositions and properties in marineCaulobacterales. Here, we useH. balticaas a model to characterize holdfast properties in marineCaulobacterales. We show that freshwater and marineCaulobacteralesuse similar genes in holdfast biogenesis and that these genes are highly conserved among the species in the two genera. We determine thatH. balticaproduces a larger holdfast thanC. crescentusand that the holdfasts have different chemical compositions, as they containN-acetylglucosamine and galactose monosaccharide residues and proteins but lack DNA. Finally, we show thatH. balticaholdfasts tolerate higher ionic strength than those ofC. crescentus. We conclude that marineCaulobacteralesholdfasts have physicochemical properties that maximize binding in high-ionic-strength environments.IMPORTANCEMost bacteria spend a large part of their life spans attached to surfaces, forming complex multicellular communities called biofilms. Bacteria can colonize virtually any surface, and therefore, they have adapted to bind efficiently in very different environments. In this study, we compare the adhesive holdfasts produced by the freshwater bacteriumC. crescentusand a relative, the marine bacteriumH. baltica. We show thatH. balticaholdfasts have a different morphology and chemical composition and tolerate high ionic strength. Our results show that theH. balticaholdfast is an excellent model to study the effect of ionic strength on adhesion and provides insights into the physicochemical properties required for adhesion in the marine environment.

scholarly journals Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins

2019 ◽  
Author(s):  
Nelson K. Chepkwony ◽  
Cécile Berne ◽  
Yves V. Brun
Keyword(s):  
Comparative Analysis ◽  
Chemical Composition ◽  
Ionic Strength ◽  
Physicochemical Properties ◽  
Bacterial Adhesion ◽  
Marine Bacterium ◽  
Caulobacter Crescentus ◽  
High Ionic Strength ◽  
Shear Forces ◽  
Surface Attachment

ABSTRACTBacterial adhesion is affected by environmental factors, such as ionic strength, pH, temperature, and shear forces, and therefore marine bacteria must have developed holdfasts with different composition and structures than their freshwater counterparts to adapt to their natural environment. The dimorphic α-proteobacterium Hirschia baltica is a marine budding bacterium in the Caulobacterales clade. H. baltica uses a polar adhesin, the holdfast, located at the cell pole opposite the reproductive stalk for surface attachment and cell-cell adhesion. The holdfast adhesin has been best characterized in Caulobacter crescentus, a freshwater member of the Caulobacterales, and little is known about holdfast composition and properties in marine Caulobacterales. Here we use H. baltica as a model to characterize holdfast properties in marine Caulobacterales. We show that freshwater and marine Caulobacterales use similar genes in holdfast biogenesis and that these genes are highly conserved among the two genera. We also determine that H. baltica produces larger holdfast than C. crescentus and that those holdfasts have a different chemical composition, as they contain N-acetylglucosamine and galactose monosaccharide residues and proteins, but lack DNA. Finally, we show that H. baltica holdfasts tolerate higher ionic strength than those of C. crescentus. We conclude that marine Caulobacterales holdfasts have physicochemical properties that maximize binding in high ionic strength environments.IMPORTANCEMost bacteria spend a large amount of their lifespan attached to surfaces, forming complex multicellular communities called biofilms. Bacteria can colonize virtually any surface, therefore they have adapted to bind efficiently in very different environments. In this study, we compare the adhesive holdfasts produced by the freshwater bacterium C. crescentus and a relative, the marine bacterium H. baltica. We show that H. baltica holdfasts have a different morphology and chemical composition, and tolerate high ionic strength. Our results show that H. baltica holdfast is an excellent model to study the effect of ionic strength on adhesion and providing insights on the physicochemical properties required for adhesion in the marine environment.

scholarly journals A polysaccharide deacetylase enhances bacterial adhesion in high ionic strength environments

2021 ◽  
Author(s):  
Nelson K Chepkwony ◽  
Yves V Brun
Keyword(s):  
Ionic Strength ◽  
Bacterial Adhesion ◽  
Caulobacter Crescentus ◽  
High Ionic Strength ◽  
Aquatic Environments ◽  
Binding Properties ◽  
Surface Attachment ◽  
Physico Chemical ◽  
Temperature Shear ◽  
Chemical Conditions

The adhesion of organisms to surfaces in aquatic environments provides a diversity of benefits such as better access to nutrients or protection from the elements or from predation. Differences in ionic strength, pH, temperature, shear forces, and other environmental factors impact adhesion and organisms have evolved various strategies to optimize their adhesins for their specific environmental conditions. We know essentially nothing about how bacteria evolved their adhesive mechanisms to attach efficiently in environments with different physico-chemical conditions. Many species of Alphaproteobacteria, including members of the order Caulobacterales, use a polar adhesin, called holdfast, for surface attachment and subsequent biofilm formation in both freshwater and marine environments. Hirschia baltica, a marine member of Caulobacterales, produces a holdfast adhesin that tolerates a drastically higher ionic strength than the holdfast produced by its freshwater relative, Caulobacter crescentus. In this work, we show that the holdfast polysaccharide deacetylase HfsH plays an important role in adherence in high ionic strength environments. We show that deletion of hfsH in H. baltica disrupts holdfast binding properties and structure. Increasing expression of HfsH in C. crescentus improved holdfast binding in high salinity, whereas lowering HfsH expression in H. baltica reduced holdfast binding at high ionic strength. We conclude that HfsH plays a role in modulating holdfast binding at high ionic strength and hypothesize that this modulation occurs through varied deacetylation of holdfast polysaccharides.

scholarly journals Flagellar Perturbations Activate Adhesion through Two Distinct Pathways in Caulobacter crescentus

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David M. Hershey ◽  
Aretha Fiebig ◽  
Sean Crosson
Keyword(s):  
Caulobacter Crescentus ◽  
Mechanical Stimuli ◽  
Flagellar Motility ◽  
Surface Attachment ◽  
Content Type ◽  
Surface Colonization ◽  
Epistasis Analysis ◽  
Flagellar Assembly ◽  
Ecological Importance ◽  
Late Stages

ABSTRACT Bacteria carry out sophisticated developmental programs to colonize exogenous surfaces. The rotary flagellum, a dynamic machine that drives motility, is a key regulator of surface colonization. The specific signals recognized by flagella and the pathways by which those signals are transduced to coordinate adhesion remain subjects of debate. Mutations that disrupt flagellar assembly in the dimorphic bacterium Caulobacter crescentus stimulate the production of a polysaccharide adhesin called the holdfast. Using a genomewide phenotyping approach, we compared surface adhesion profiles in wild-type and flagellar mutant backgrounds of C. crescentus. We identified a diverse set of flagellar mutations that enhance adhesion by inducing a hyperholdfast phenotype and discovered a second set of mutations that suppress this phenotype. Epistasis analysis of the flagellar signaling suppressor (fss) mutations demonstrated that the flagellum stimulates holdfast production via two genetically distinct pathways. The developmental regulator PleD contributes to holdfast induction in mutants disrupted at both early and late stages of flagellar assembly. Mutants disrupted at late stages of flagellar assembly, which assemble an intact rotor complex, induce holdfast production through an additional process that requires the MotAB stator and its associated diguanylate cyclase, DgcB. We have assigned a subset of the fss genes to either the stator- or pleD-dependent networks and characterized two previously unidentified motility genes that regulate holdfast production via the stator complex. We propose a model through which the flagellum integrates mechanical stimuli into the C. crescentus developmental program to coordinate adhesion. IMPORTANCE Understanding how bacteria colonize solid surfaces is of significant clinical, industrial and ecological importance. In this study, we identified genes that are required for Caulobacter crescentus to activate surface attachment in response to signals from a macromolecular machine called the flagellum. Genes involved in transmitting information from the flagellum can be grouped into separate pathways, those that control the C. crescentus morphogenic program and those that are required for flagellar motility. Our results support a model in which a developmental and a mechanical signaling pathway operate in parallel downstream of the flagellum and converge to regulate adhesion. We conclude that the flagellum serves as a signaling hub by integrating internal and external cues to coordinate surface colonization and emphasize the role of signal integration in linking complex sets of environmental stimuli to individual behaviors.

scholarly journals Tad Pili Play a Dynamic Role in Caulobacter crescentus Surface Colonization

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Matteo Sangermani ◽  
Isabelle Hug ◽  
Nora Sauter ◽  
Thomas Pfohl ◽  
Urs Jenal
Keyword(s):  
Caulobacter Crescentus ◽  
Optical Trap ◽  
Model Organism ◽  
Natural Environments ◽  
Surface Attachment ◽  
Content Type ◽  
Surface Colonization ◽  
Bacterial Surface ◽  
Solid Media ◽  
Resilient Communities

ABSTRACT Bacterial surface attachment is mediated by filamentous appendages called pili. Here, we describe the role of Tad pili during surface colonization of Caulobacter crescentus. Using an optical trap and microfluidic controlled flow conditions to mimic natural environments, we demonstrated that Tad pili undergo repeated dynamic cycles of extension and retraction. Within seconds after establishing surface contact, pilus retraction reorients cells into an upright position, promoting walking-like movements against the medium flow. Pilus-mediated positioning of the flagellate pole close to the surface facilitates motor-mediated mechanical sensing and promotes anchoring of the holdfast, an adhesive substance that affords long-term attachment. We present evidence that the second messenger c-di-GMP regulates pilus dynamics during surface encounter in distinct ways, promoting increased activity at intermediate levels and retraction of pili at peak concentrations. We propose a model in which flagellum and Tad pili functionally interact and together impose a ratchet-like mechanism that progressively drives C. crescentus cells toward permanent surface attachment. IMPORTANCE Bacteria are able to colonize surfaces in environmental, industrial, and medical settings, where they form resilient communities called biofilms. In order to control bacterial surface colonization, microbiologists need to gain a detailed understanding of the processes that bacteria use to live at the liquid-surface interface and that allow them to adhere to and move on surfaces and eventually grow and persist on solid media. To facilitate these processes, bacteria are equipped with adhesive structures such as flagella and pili and with matrix components such as exopolysaccharides. How these cellular organelles are coordinated to optimize surface processes is currently subject to intense investigations. Here we used the model organism Caulobacter crescentus to demonstrate that polar pili are highly dynamic structures that are functionally interconnected with the flagellar motor to mediate surface sensing, thereby enforcing rapid and permanent surface attachment. These studies provide an entry point for an in-depth molecular analysis of bacterial surface colonization.

scholarly journals Proper Control of Caulobacter crescentus Cell Surface Adhesion Requires the General Protein Chaperone DnaK

2016 ◽  
Vol 198 (19) ◽  
pp. 2631-2642 ◽  
Author(s):  
Daniel S. Eaton ◽  
Sean Crosson ◽  
Aretha Fiebig
Keyword(s):  
Cell Adhesion ◽  
Caulobacter Crescentus ◽  
Surface Adhesion ◽  
Surface Attachment ◽  
Small Protein ◽  
Content Type ◽  
Chaperone Dnak ◽  
Protein Chaperone ◽  
General Protein ◽  
Lid Domain

ABSTRACTGrowth in a surface-attached bacterial community, or biofilm, confers a number of advantages. However, as a biofilm matures, high-density growth imposes stresses on individual cells, and it can become less advantageous for progeny to remain in the community. Thus, bacteria employ a variety of mechanisms to control attachment to and dispersal from surfaces in response to the state of the environment. The freshwater oligotrophCaulobacter crescentuscan elaborate a polysaccharide-rich polar organelle, known as the holdfast, which enables permanent surface attachment. Holdfast development is strongly inhibited by the small protein HfiA; mechanisms that control HfiA levels in the cell are not well understood. We have discovered a connection between the essential general protein chaperone, DnaK, and control ofC. crescentusholdfast development.C. crescentusmutants partially or completely lacking the C-terminal substrate binding “lid” domain of DnaK exhibit enhanced bulk surface attachment. Partial or complete truncation of the DnaK lid domain increases the probability that any single cell will develop a holdfast by 3- to 10-fold. These results are consistent with the observation that steady-state levels of an HfiA fusion protein are significantly diminished in strains that lack the entire lid domain of DnaK. While dispensable for growth, the lid domain ofC. crescentusDnaK is required for proper chaperone function, as evidenced by observed dysregulation of HfiA and holdfast development in strains expressing lidless DnaK mutants. We conclude that DnaK is an important molecular determinant of HfiA stability and surface adhesion control.IMPORTANCERegulatory control of cell adhesion ensures that bacterial cells can transition between free-living and surface-attached states. We define a role for the essential protein chaperone, DnaK, in the control ofCaulobacter crescentuscell adhesion.C. crescentussurface adhesion is mediated by an envelope-attached organelle known as the holdfast. Holdfast development is tightly controlled by HfiA, a small protein inhibitor that directly interacts with a WecG/TagA-family glycosyltransferase required for holdfast biosynthesis. We demonstrate that the C-terminal lid domain of DnaK is not essential for growth but is necessary for proper control of HfiA levels in the cell and for control of holdfast adhesin development.

scholarly journals DrpB (YedR) Is a Nonessential Cell Division Protein in Escherichia coli

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Atsushi Yahashiri ◽  
Jill T. Babor ◽  
Ariel L. Anwar ◽  
Ryan P. Bezy ◽  
Evan W. Piette ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Ionic Strength ◽  
Bacterial Cell ◽  
Enteric Bacteria ◽  
High Ionic Strength ◽  
Bacterial Cell Division ◽  
Content Type ◽  
E Coli ◽  
Low Ionic Strength

ABSTRACT We report that the small Escherichia coli membrane protein DrpB (formerly YedR) is involved in cell division. We discovered DrpB in a screen for multicopy suppressors of a ΔftsEX mutation that prevents divisome assembly when cells are plated on low ionic strength medium, such as lysogeny broth without NaCl. Characterization of DrpB revealed that (i) translation initiates at an ATG annotated as codon 22 rather than the GTG annotated as codon 1, (ii) DrpB localizes to the septal ring when cells are grown in medium of low ionic strength but localization is greatly reduced in medium of high ionic strength, (iii) overproduction of DrpB in a ΔftsEX mutant background improves recruitment of the septal peptidoglycan synthase FtsI, implying multicopy suppression works by rescuing septal ring assembly, (iv) a ΔdrpB mutant divides quite normally, but a ΔdrpB ΔdedD double mutant has a strong division and viability defect, albeit only in medium of high ionic strength, and (v) DrpB homologs are found in E. coli and a few closely related enteric bacteria, but not outside this group. In sum, DrpB is a poorly conserved nonessential division protein that improves the efficiency of cytokinesis under suboptimal conditions. Proteins like DrpB are likely to be a widespread feature of the bacterial cell division apparatus, but they are easily overlooked because mutants lack obvious shape defects. IMPORTANCE A thorough understanding of bacterial cell division requires identifying and characterizing all of the proteins that participate in this process. Our discovery of DrpB brings us one step closer to this goal in E. coli.

scholarly journals The Two Chemotaxis Clusters inCaulobacter crescentusPlay Different Roles in Chemotaxis and Biofilm Regulation

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Cécile Berne ◽  
Yves V. Brun
Keyword(s):  
Biofilm Formation ◽  
Caulobacter Crescentus ◽  
Carbon Sources ◽  
Synthesis Inhibitor ◽  
Gene Encoding ◽  
Surface Attachment ◽  
Content Type ◽  
Major Cluster ◽  
Chemotaxis Proteins ◽  
Chemosensory Systems

ABSTRACTThe holdfast polysaccharide adhesin is crucial for irreversible cell adhesion and biofilm formation inCaulobacter crescentus. Holdfast production is tightly controlled via developmental regulators, as well as via environmental and physical signals. Here, we identify a novel mode of regulation of holdfast synthesis that involves chemotaxis proteins. We characterized the two identified chemotaxis clusters ofC. crescentusand showed that only the previously characterized major cluster is involved in the chemotactic response toward different carbon sources. However, both chemotaxis clusters encoded in theC. crescentusgenome play a role in biofilm formation and holdfast production by regulating the expression ofhfiA, the gene encoding the holdfast inhibitor HfiA. We show that CheA and CheB proteins act in an antagonistic manner, as follows: while the two CheA proteins negatively regulatehfiAexpression, the CheB proteins are positive regulators, thus providing a modulation of holdfast synthesis and surface attachment.IMPORTANCEChemosensory systems constitute major signal transduction pathways in bacteria. These systems are involved in chemotaxis and other cell responses to environment conditions, such as the production of adhesins to enable irreversible adhesion to a surface and surface colonization. TheC. crescentusgenome encodes two complete chemotaxis clusters. Here, we characterized the second novel chemotaxis-like cluster. While only the major chemotaxis cluster is involved in chemotaxis, both chemotaxis systems modulateC. crescentusadhesion by controlling expression of the holdfast synthesis inhibitor HfiA. Here, we identify a new level in holdfast regulation, providing new insights into the control of adhesin production that leads to the formation of biofilms in response to the environment.

Importance of Protease Inhibition in Studies on Purified Factor VIII (Antihaemophilic Factor)

1976 ◽  
Vol 35 (01) ◽  
pp. 186-190 ◽  
Author(s):  
Eugen A. Beck ◽  
Peter Bachmann ◽  
Peter Barbier ◽  
Miha Furlan
Keyword(s):  
Ionic Strength ◽  
Factor Viii ◽  
Gel Filtration ◽  
High Ionic Strength ◽  
Procoagulant Activity ◽  
Carrier Protein ◽  
Protease Inhibition ◽  
Functional Sites ◽  
Molecular Weight Peak ◽  
Von Willebrand

SummaryAccording to some authors factor VIII procoagulant activity may be dissociable from carrier protein (MW~ 2 × 106) by agarose gel filtration, e.g. at high ionic strength. We were able to reproduce this phenomenon. However, addition of protease inhibitor (Trasylol) prevented the appearance of low molecular weight peak of factor VIII procoagulant activity both at high ionic strength and elevated temperature (37°C). We conclude from our results that procoagulant activity and carrier protein (von Willebrand factor, factor VIII antigen) are closely associated functional sites of native factor VIII macro molecule. Consequently, proteolytic degradation should be avoided in functional and structural studies on factor VIII and especially in preparing factor VIII concentrate for therapeutic use.

THE RELATIVE DISTRIBUTION OF THYROXINE, TRIIODOTHYRONINE AND 3,3′,5′-(REVERSE)-TRIIODOTHYRONINE IN VARIOUS FRACTIONS OF THYROGLOBULIN

1978 ◽  
Vol 88 (2) ◽  
pp. 298-305 ◽  
Author(s):  
Peter Laurberg
Keyword(s):  
Ionic Strength ◽  
Guinea Pig ◽  
Sucrose Gradient ◽  
Deae Cellulose ◽  
High Ionic Strength ◽  
Relative Distribution ◽  
Thyroid Extract ◽  
Reverse Triiodothyronine ◽  
Thyroid Secretion ◽  
Stable Iodine

ABSTRACT Thyroglobulin fractions rich and poor in new thyroglobulin were separated by means of DEAE-cellulose chromatography of dog thyroid extracts and by zonal ultracentrifugation in a sucrose gradient of guinea pig thyroid extract incubated at low temperature. The distribution of thyroxine, triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine in hydrolysates of the different fractions was estimated by radioimmunoassays. Following DEAE-cellulose chromatography there was a small but statistically significant increase in the T4/T3 ratio in thyroglobulin fractions eluted at high ionic strength - that is fractions relatively rich in stable iodine but poor in fresh thyroglobulin. There were no differences in the T4/rT3 ratios between the different fractions. The ratios between iodothyronines were almost identical in the various thyroglobulin fractions following zonal ultracentrifugation in a sucrose gradient of cold treated guinea pig thyroid extract. These findings lend no support to the possibility that a relatively high content of triiodothyronines in freshly synthesized thyroglobulin modulates the thyroid secretion towards a preferential secretion of triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine at the expense of the secretion of thyroxine.

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