Bypassing the need for subcellular localization of a polysaccharide export‐anchor complex by overexpressing its protein subunits

Javens, June W.; Wan, Zhe; Hardy, Gail G.; Brun, Yves V.

doi:10.1111/mmi.12281

Cited by 15 publications

(23 citation statements)

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“…Lectin blots were performed as described previously (Javens et al, ), with a few modifications. One millliter of M2G‐grown cells in mid‐log phase (OD 600 of 0.4–0.8) was centrifuged for 5 min at 7,500 g .…”

Section: Methodsmentioning

confidence: 99%

“…Lectin blots were performed as described previously (Javens et al, 2013), with a few modifications. One millliter of M2G-grown cells in mid-log phase (OD 600 of 0.4-0.8) was centrifuged for 5 min at 7,500 g. Cells were washed with 10 mM Tris HCl buffer, centrifuged for 5 min at 7,500 g and resuspended in 100 µl of 10 mM Tris HCl buffer.…”

Section: Lectin Blot For Quantification Of Total Holdfast Producedmentioning

confidence: 99%

“…The swarmer cell differentiates into a replication-competent stalked cell by shedding its flagellum, retracting its pili, and synthesizing a holdfast and a stalk at the same pole of the cell. The holdfast mediates permanent attachment (Ong et al, 1990) and is synthesized by a Wzy-dependent pathway similar to Escherichia coli group 1 capsular polysaccharide biosynthesis (Smith et al, 2003;Toh et al, 2008;Hardy et al, 2010;Javens et al, 2013;Wan et al, 2013;Hardy et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA

Berne

Ellison

Agarwal

et al. 2018

Molecular Microbiology

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To permanently attach to surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by surface sensing, in which swarmer cells rapidly synthesize holdfast in direct response to surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus surface sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions.

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Section: Methodsmentioning

confidence: 99%

Section: Lectin Blot For Quantification Of Total Holdfast Producedmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA

Berne

Ellison

Agarwal

et al. 2018

Molecular Microbiology

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“…While it is known that at least some of the holdfast synthesis genes display changes in transcription activity during the cell cycle [32], and microarray experiments have shown that holdfast genes have altered transcription in a ctrA mutant [7,33], it has also been shown that holdfast synthesis can be stimulated in swarmer cells when they contact a surface [34], and that developmental holdfast synthesis is also likely regulated by cyclic-di-GMP levels [35]. We have recently shown that the holdfast synthesis and anchoring machineries are synthesized and polarly localized in predivisional cells in preparation for holdfast synthesis in the next cell cycle [36,37]. Therefore, it is likely that CtrA regulates the synthesis of the holdfast synthesis-anchoring machinery in predivisional cells, but that the activation of this machinery is regulated by surface contact and developmental signals.…”

Section: Resultsmentioning

confidence: 99%

Effect of a ctrA promoter mutation, causing a reduction in CtrA abundance, on the cell cycle and development of Caulobacter crescentus

2013

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BackgroundPolar development during the alphaproteobacterium Caulobacter crescentus cell cycle is integrated to the point that individual mutations can have pleiotropic effects on the synthesis of polar organelles. Disruption of the genes encoding the histidine kinase PleC, or its localization factor PodJ, disrupts synthesis or functionality of pili, flagella and adhesive holdfast. However, the mechanism by which these mutations affect polar development is not well understood. The aim of this study was to identify new regulators that control multiple aspects of polar organelle development.ResultsTo identify mutants with pleiotropic polar organelle synthesis defects, transposon mutagenesis was performed and mutants were selected based resistance to the pili-tropic bacteriophage ΦCbK. Mutants were then screened for defects in motility and holdfast production. Only a single podJ/pleC-independent mutant was isolated which had defects in all three phenotypes. Directed phage assays confirmed the phage resistance phenotype, while the strain demonstrated a similar dispersal radius as a podJ mutant in swarm agar, and treatment with a fluorescent lectin that labels the holdfast showed no staining for this mutant. The transposon had inserted into the promoter region of ctrA, a gene encoding a master transcriptional regulator of the cell cycle, disrupting native transcription but still allowing reduced transcriptional activity and protein production of this essential protein. Transcriptional fusions showed that essential genes controlled by CtrA exhibited minor to moderate changes in expression in the ctrA promoter mutant, while the pilA gene, encoding the subunit of the pilus filament, had a drastic decrease in gene expression. Introduction of a plasmid-born copy of ctrA under its native promoter complemented the phage resistance and holdfast defects, as well as a moderate cell morphology defect, but not the swarming defect.ConclusionsA mutation was identified that caused pleiotropic defects in polar organelle synthesis, and revealed the surprising result that some CtrA-dependent promoters are more sensitive to changes in CtrA concentration than others. However, the fact that no pleiotropic mutations were found in new regulators suggests that downstream signaling of PleC/PodJ is either essential, redundant, or branching such that all three phenotypes were not simultaneously affected.

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“…Additional sugar residues are then added to form a repeat unit on the lipid carrier by three glycosyltransferases HfsG, HfsJ (17) and HfsL (18). The acetyltransferase HfsK (32) and the polysaccharide deacetylase HfsH (21) modify one or more sugar residue. The lipid carrier with the repeat units is transported across the inner membrane into the periplasm by a flippase (HfsF) (17, 19).…”

Section: Introductionmentioning

confidence: 99%

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

Chepkwony

Berne

Brun

2019

Preprint

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17Bacterial adhesion is affected by environmental factors, such as ionic strength, pH, 18 temperature, and shear forces, and therefore marine bacteria must have developed holdfasts 19 with different composition and structures than their freshwater counterparts to adapt to their 20 natural environment. The dimorphic a-proteobacterium Hirschia baltica is a marine budding 21 bacterium in the Caulobacterales clade. H. baltica uses a polar adhesin, the holdfast, located at 22 the cell pole opposite the reproductive stalk for surface attachment and cell-cell adhesion. The 23 holdfast adhesin has been best characterized in Caulobacter crescentus, a freshwater member 24 of the Caulobacterales, and little is known about holdfast composition and properties in marine 25 Caulobacterales. Here we use H. baltica as a model to characterize holdfast properties in 26 marine Caulobacterales. We show that freshwater and marine Caulobacterales use similar 27 genes in holdfast biogenesis and that these genes are highly conserved among the two genera. 28We also determine that H. baltica produces larger holdfast than C. crescentus and that those 29 holdfasts have a different chemical composition, as they contain N-acetylglucosamine and 30 galactose monosaccharide residues and proteins, but lack DNA. Finally, we show that H. baltica 31 holdfasts tolerate higher ionic strength than those of C. crescentus. We conclude that marine 32 Caulobacterales holdfasts have physicochemical properties that maximize binding in high ionic 33 strength environments. 34 35 IMPORTANCE 36 Most bacteria spend a large amount of their lifespan attached to surfaces, forming 37 complex multicellular communities called biofilms. Bacteria can colonize virtually any surface, 38 therefore they have adapted to bind efficiently in very different environments. In this study, we 39 compare the adhesive holdfasts produced by the freshwater bacterium C. crescentus and a 40 relative, the marine bacterium H. baltica. We show that H. baltica holdfasts have a different 41 3 morphology and chemical composition, and tolerate high ionic strength. Our results show that H. 42 baltica holdfast is an excellent model to study the effect of ionic strength on adhesion and 43 providing insights on the physicochemical properties required for adhesion in the marine 44 environment. 45 46 48 communities, known as biofilms (1). To irreversibly adhere to surfaces and form these complex 49 mutil-cellular communities, bacteria produce strong adhesins, mainly composed of proteins or 50 polysaccharides (2, 3). Bacterial adhesion is affected by different environmental conditions such 51 as pH, temperature, shear forces, and ionic strength (2,(4)(5)(6). In marine environments, bacteria 52 face 500 times higher ionic strength than in freshwater (7), therefore, marine bacteria have 53 evolved ways to overcome the effect of ionic strength and bind permanently to surfaces in high 54 salt environments such as seas and oceans. 55Caulobacterales are Alphaproteobacteria found in various habitats, from olig...

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Bypassing the need for subcellular localization of a polysaccharide export‐anchor complex by overexpressing its protein subunits

Cited by 15 publications

References 64 publications

Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA

Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA

Effect of a ctrA promoter mutation, causing a reduction in CtrA abundance, on the cell cycle and development of Caulobacter crescentus

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

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