scholarly journals Composition of the Holdfast Polysaccharide fromCaulobacter crescentus

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
David M. Hershey ◽  
Sara Porfírio ◽  
Ian Black ◽  
Bernhard Jaehrig ◽  
Christian Heiss ◽  
...  

ABSTRACTSurface colonization is central to the lifestyles of many bacteria. Exploiting surface niches requires sophisticated systems for sensing and attaching to solid materials.Caulobacter crescentussynthesizes a polysaccharide-based adhesin known as the holdfast at one of its cell poles, which enables tight attachment to exogenous surfaces. The genes required for holdfast biosynthesis have been analyzed in detail, but difficulties in isolating analytical quantities of the adhesin have limited efforts to characterize its chemical structure. In this report, we describe a method to extract the holdfast fromC. crescentuscultures and present a survey of its carbohydrate content. Glucose, 3-O-methylglucose, mannose,N-acetylglucosamine, and xylose were detected in our extracts. Our results provide evidence that the holdfast contains a 1,4-linked backbone of glucose, mannose,N-acetylglucosamine, and xylose that is decorated with branches at the C-6 positions of glucose and mannose. By defining the monosaccharide components in the polysaccharide, our work establishes a framework for characterizing enzymes in the holdfast pathway and provides a broader understanding of how polysaccharide adhesins are built.IMPORTANCETo colonize solid substrates, bacteria often deploy dedicated adhesins that facilitate attachment to surfaces.Caulobacter crescentusinitiates surface colonization by secreting a carbohydrate-based adhesin called the holdfast. Because little is known about the chemical makeup of the holdfast, the pathway for its biosynthesis and the physical basis for its unique adhesive properties are poorly understood. This study outlines a method to extract theC. crescentusholdfast and describes the monosaccharide components contained within the adhesive matrix. The composition analysis adds to our understanding of the chemical basis for holdfast attachment and provides missing information needed to characterize enzymes in the biosynthetic pathway.

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David M. Hershey ◽  
Aretha Fiebig ◽  
Sean Crosson

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.


mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
David M. Hershey ◽  
Aretha Fiebig ◽  
Sean Crosson

ABSTRACTDue to their intimate physical interactions with the environment, surface polysaccharides are critical determinants of fitness for bacteria.Caulobacter crescentusproduces a specialized structure at one of its cell poles called the holdfast that enables attachment to surfaces. Previous studies have shown that the holdfast is composed of carbohydrate-based material and identified a number of genes required for holdfast development. However, incomplete information about its chemical structure, biosynthetic genes, and regulatory principles has limited progress in understanding the mechanism of holdfast synthesis. We leveraged the adhesive properties of the holdfast to perform a saturating screen for genes affecting attachment to cheesecloth over a multiday time course. Using similarities in the temporal profiles of mutants in a transposon library, we defined discrete clusters of genes with related effects on cheesecloth colonization. Holdfast synthesis, flagellar motility, type IV pilus assembly, and smooth lipopolysaccharide (SLPS) production represented key classes of adhesion determinants. Examining these clusters in detail allowed us to predict and experimentally define the functions of multiple uncharacterized genes in both the holdfast and SLPS pathways. In addition, we showed that the pilus and the flagellum control holdfast synthesis separately by modulating the holdfast inhibitorhfiA.This report defines a set of genes contributing to adhesion that includes newly discovered genes required for holdfast biosynthesis and attachment. Our data provide evidence that the holdfast contains a complex polysaccharide with at least four monosaccharides in the repeating unit and underscore the central role of cell polarity in mediating attachment ofC. crescentusto surfaces.IMPORTANCEBacteria routinely encounter biotic and abiotic materials in their surrounding environments, and they often enlist specific behavioral programs to colonize these materials. Adhesion is an early step in colonizing a surface.Caulobacter crescentusproduces a structure called the holdfast which allows this organism to attach to and colonize surfaces. To understand how the holdfast is produced, we performed a genome-wide search for genes that contribute to adhesion by selecting for mutants that could not attach to cheesecloth. We discovered complex interactions between genes that mediate surface contact and genes that contribute to holdfast development. Our genetic selection identified what likely represents a comprehensive set of genes required to generate a holdfast, laying the groundwork for a detailed characterization of the enzymes that build this specialized adhesin.


mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Matteo Sangermani ◽  
Isabelle Hug ◽  
Nora Sauter ◽  
Thomas Pfohl ◽  
Urs Jenal

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.


2017 ◽  
Vol 199 (13) ◽  
Author(s):  
Angel A. Aguirre ◽  
Alexandre M. Vicente ◽  
Steven W. Hardwick ◽  
Daniela M. Alvelos ◽  
Ricardo R. Mazzon ◽  
...  

ABSTRACT In diverse bacterial lineages, multienzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic Gram-negative bacterium Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below those optimum for growth. Phenotype analyses of rhlE, rhlB, and rhlE rhlB mutant strains show that RhlE is important for cell fitness at low temperature and its role may not be substituted by RhlB. Transcriptional and translational fusions of rhlE to the lacZ reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease, mainly through posttranscriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase, and ribosomal protein S1, also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins. IMPORTANCE DEAD-box RNA helicases are often present in the RNA degradosome complex, helping unwind secondary structures to facilitate degradation. Caulobacter crescentus is an interesting organism to investigate degradosome remodeling with change in temperature, because it thrives in freshwater bodies and withstands low temperature. In this study, we show that at low temperature, the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not be complemented by RhlB, although RhlE is able to complement for rhlB loss. These results suggest that RhlE has a specific role in the degradosome at low temperature, potentially improving adaptation to this condition.


2012 ◽  
Vol 86 (18) ◽  
pp. 10234-10235 ◽  
Author(s):  
Gaël Panis ◽  
Christophe Lambert ◽  
Patrick H. Viollier

φCbK is a B3 morphotype bacteriophage of theSiphoviridaefamily that infectsCaulobacter crescentus, the preeminent model system for bacterial cell cycle studies. The last 4 decades of research with φCbK as a genetic and cytological tool to study the biology of the host warrant an investigation of the phage genome composition. Herein, we report the complete genome sequence of φCbK and highlight unusual features that emerged from its annotation. The complete genome analysis of the φCbK phage provides new insight into its characteristics and potential interactions with itsCaulobacter crescentushost, setting the stage for future functional studies with φCbK.


2014 ◽  
Vol 61 (4) ◽  
pp. 224-231 ◽  
Author(s):  
Ruben Suarez-Hernandez ◽  
Jose G. Gonzalez-Rodriguez ◽  
Gloria F. Dominguez-Patiño ◽  
Alberto Martinez-Villafañe

Purpose – The purpose of this investigation is to study the corrosion inhibition of carbon steel (CS) using a “green” inhibitor, Opuntia ficus-indica, in an aerated, 0.5 M H2SO4 solution at different concentrations and temperatures. Design/methodology/approach – Weight loss determinations, surface studies, electrochemical impedance spectroscopy and potentiodynamic polarization were applied during the investigation. Findings – It was observed that Opuntia ficus-indica extract can decrease the corrosion rate of CS, and its efficiency increases with increasing concentration up to 1,000 ppm and with time, but decreases with increasing the temperature from 25 to 600C. The inhibitory activity is due to the presence of phenolic compounds in its chemical structure. Research limitations/implications – The work was done under static conditions, whereas in acid cleaning conditions, there is a dynamic system. However, the findings may apply to both the systems. Practical implications – CS is used in acidic environments in the acid cleaning industry. Social implications – Results of this work show that it is possible to reduce the cost of repair of equipment and the environmental impact of corrosion. Originality/value – There are very few investigations on the study of Opuntia ficus-indica leaf extract as a green inhibitor in an acidic environment.


2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Alexander B. Westbye ◽  
Lukas Kater ◽  
Christina Wiesmann ◽  
Hao Ding ◽  
Calvin K. Yip ◽  
...  

ABSTRACTSeveral members of theRhodobacterales(Alphaproteobacteria) produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is theRhodobacter capsulatusGTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in theAlphaproteobacteriaand is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production inR. capsulatus. We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels inR. capsulatusand identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in theRhodobacterales.IMPORTANCEMembers of theRhodobacteralesare abundant in ocean and freshwater environments. The conserved GTA produced by manyRhodobacteralesmay have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA inR. capsulatusextends the model of CtrA regulation fromCaulobacter crescentusto a member of theRhodobacterales. We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.


2019 ◽  
Vol 201 (24) ◽  
Author(s):  
Ali Farrokhi ◽  
Hua Liu ◽  
George Szatmari

ABSTRACT Chromosome dimers occur in bacterial cells as a result of the recombinational repair of DNA. In most bacteria, chromosome dimers are resolved by XerCD site-specific recombination at the dif (deletion-induced filamentation) site located in the terminus region of the chromosome. Caulobacter crescentus, a Gram-negative oligotrophic bacterium, also possesses Xer recombinases, called CcXerC and CcXerD, which have been shown to interact with the Escherichia coli dif site in vitro. Previous studies on Caulobacter have suggested the presence of a dif site (referred to in this paper as dif1CC), but no in vitro data have shown any association with this site and the CcXer proteins. Using recursive hidden Markov modeling, another group has proposed a second dif site, which we call dif2CC, which shows more similarity to the dif consensus sequence. Here, by using a combination of in vitro experiments, we compare the affinities and the cleavage abilities of CcXerCD recombinases for both dif sites. Our results show that dif2CC displays a higher affinity for CcXerC and CcXerD and is bound cooperatively by these proteins, which is not the case for the original dif1CC site. Furthermore, dif2CC nicked substrates are more efficiently cleaved by CcXerCD, and deletion of the site results in about 5 to 10% of cells showing an altered cellular morphology. IMPORTANCE Bacteria utilize site-specific recombination for a variety of purposes, including the control of gene expression, acquisition of genetic elements, and the resolution of dimeric chromosomes. Failure to resolve dimeric chromosomes can lead to cell division defects in a percentage of the cell population. The work presented here shows the existence of a chromosomal resolution system in C. crescentus. Defects in this resolution system result in the formation of chains of cells. Further understanding of how these cells remain linked together will help in the understanding of how chromosome segregation and cell division are linked in C. crescentus.


mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Ludovic Pelosi ◽  
Chau-Duy-Tam Vo ◽  
Sophie Saphia Abby ◽  
Laurent Loiseau ◽  
Bérengère Rascalou ◽  
...  

ABSTRACTMost bacteria can generate ATP by respiratory metabolism, in which electrons are shuttled from reduced substrates to terminal electron acceptors, via quinone molecules like ubiquinone. Dioxygen (O2) is the terminal electron acceptor of aerobic respiration and serves as a co-substrate in the biosynthesis of ubiquinone. Here, we characterize a novel, O2-independent pathway for the biosynthesis of ubiquinone. This pathway relies on three proteins, UbiT (YhbT), UbiU (YhbU), and UbiV (YhbV). UbiT contains an SCP2 lipid-binding domain and is likely an accessory factor of the biosynthetic pathway, while UbiU and UbiV (UbiU-UbiV) are involved in hydroxylation reactions and represent a novel class of O2-independent hydroxylases. We demonstrate that UbiU-UbiV form a heterodimer, wherein each protein binds a 4Fe-4S cluster via conserved cysteines that are essential for activity. The UbiT, -U, and -V proteins are found in alpha-, beta-, and gammaproteobacterial clades, including several human pathogens, supporting the widespread distribution of a previously unrecognized capacity to synthesize ubiquinone in the absence of O2. Together, the O2-dependent and O2-independent ubiquinone biosynthesis pathways contribute to optimizing bacterial metabolism over the entire O2range.IMPORTANCEIn order to colonize environments with large O2gradients or fluctuating O2levels, bacteria have developed metabolic responses that remain incompletely understood. Such adaptations have been recently linked to antibiotic resistance, virulence, and the capacity to develop in complex ecosystems like the microbiota. Here, we identify a novel pathway for the biosynthesis of ubiquinone, a molecule with a key role in cellular bioenergetics. We link three uncharacterized genes ofEscherichia colito this pathway and show that the pathway functions independently from O2. In contrast, the long-described pathway for ubiquinone biosynthesis requires O2as a substrate. In fact, we find that many proteobacteria are equipped with the O2-dependent and O2-independent pathways, supporting that they are able to synthesize ubiquinone over the entire O2range. Overall, we propose that the novel O2-independent pathway is part of the metabolic plasticity developed by proteobacteria to face various environmental O2levels.


2019 ◽  
Vol 50 (2) ◽  
pp. 280-287 ◽  
Author(s):  
Farzane Jahantigh

Purpose The inclusion of natural antioxidants and nanoparticles into edible coatings can improve their structure, functionality and applicability in foods. This study aims to determine the antibacterial effect of new nano-composite based on bean pod shell gum (BPSG), silver nanoparticle (SNP) and herbal essential oil (EO). Design/methodology/approach For this purpose, BPSG (4 per cent w/v), SNP (0.5-1 per cent w/v) and Helichrysum arenarium EO (1-2 per cent v/v) were mixed. After preparation the edible film, the antibacterial activity was tested on five food-borne pathogenic bacteria in two categories including two-gram positives and three-gram negatives bacteria. The EO composition was determined by GC–mass spectrometry and the antibacterial activity was tested using disk diffusion test. Findings Results showed that gram-positive bacteria were more susceptible than gram-negative bacteria. Increasing Helichrysum arenarium EO and SNP content increased antimicrobial activity of the edible film based on BPSG, so that the treatment containing 2 per cent v/v Helichrysum arenarium EO and 1 per cent w/v SNP led to the highest inhibition zone (8.1-13.1 mm) compared to treatment containing 1 per cent v/v Helichrysum arenarium EO and 0.5 per cent w/v SNP with inhibition zone range of 5.4-9.9 mm and the difference was statistically significant (p = 0.41). Also, the chemical composition analysis of the EO identified a total of 38 compounds in which a-pinene (32 per cent), 1,8-cineole (16 per cent), α-humulene (15 per cent) and ß-caryophyllene (8 per cent) were the main fractions. Other separated components accounted less than 29 per cent of the oil. Originality/value In general, SNP and Helichrysum arenarium EO improved the functional properties including the antimicrobial activity of the edible film based on BPSG, which increases the potential to be used as active packaging for fresh products.


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