Publications by authors named "Yves V Brun"

113 Publications

Competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake.

Mol Microbiol 2021 Mar 23. Epub 2021 Mar 23.

Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA.

The competence pili of transformable Gram-positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram-negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram-positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) leave transient cavities in the cell wall that facilitate DNA passage, (2) form static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via dynamic activity. Here, we use a recently described pilus labeling approach to demonstrate that competence pili in Streptococcus pneumoniae are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling the principal pilus monomer, ComGC, with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results suggest that Gram-positive competence pili in other species may also be dynamic and retractile structures that play an active role in DNA uptake.
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http://dx.doi.org/10.1111/mmi.14718DOI Listing
March 2021

Roadmap on emerging concepts in the physical biology of bacterial biofilms: from surface sensing to community formation.

Phys Biol 2021 Jun 23;18(5). Epub 2021 Jun 23.

Department of Physics and Astronomy, University of Southern California, Los Angeles, California, CA 90089, United States of America.

Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor.
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http://dx.doi.org/10.1088/1478-3975/abdc0eDOI Listing
June 2021

Special Sections for the 8th Biennial International Conference on the Biology of Vibrios.

Authors:
Yves V Brun

J Bacteriol 2020 11 19;202(24). Epub 2020 Nov 19.

Department of Microbiology, Infectious Diseases, and Immunology, Université de Montréal, Montreal, Quebec, Canada

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http://dx.doi.org/10.1128/JB.00543-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685546PMC
November 2020

A Division of Labor in the Recruitment and Topological Organization of a Bacterial Morphogenic Complex.

Curr Biol 2020 10 13;30(20):3908-3922.e4. Epub 2020 Aug 13.

Department of Biology, Indiana University, 1001 E. 3rd Street, Bloomington, IN 47405, USA; Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Pavillon Roger-Gaudry, C.P. 6128, Succursale Centreville, Montréal, Canada. Electronic address:

Bacteria come in an array of shapes and sizes, but the mechanisms underlying diverse morphologies are poorly understood. The peptidoglycan (PG) cell wall is the primary determinant of cell shape. At the molecular level, morphological variation often results from the regulation of enzymes involved in cell elongation and division. These enzymes are spatially controlled by cytoskeletal scaffolding proteins, which both recruit and organize the PG synthesis complex. How then do cells define alternative morphogenic processes that are distinct from cell elongation and division? To address this, we have turned to the specific morphotype of Alphaproteobacterial stalks. Stalk synthesis is a specialized form of zonal growth, which requires PG synthesis in a spatially constrained zone to extend a thin cylindrical projection of the cell envelope. The morphogen SpmX defines the site of stalk PG synthesis, but SpmX is a PG hydrolase. How then does a non-cytoskeletal protein, SpmX, define and constrain PG synthesis to form stalks? Here, we report that SpmX and the bactofilin BacA act in concert to regulate stalk synthesis in Asticcacaulis biprosthecum. We show that SpmX recruits BacA to the site of stalk synthesis. BacA then serves as a stalk-specific topological organizer for PG synthesis activity, including its recruiter SpmX, at the base of the stalk. In the absence of BacA, cells produce "pseudostalks" that are the result of unconstrained PG synthesis. Therefore, the protein responsible for recruitment of a morphogenic PG remodeling complex, SpmX, is distinct from the protein that topologically organizes the complex, BacA.
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http://dx.doi.org/10.1016/j.cub.2020.07.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578058PMC
October 2020

Surface sensing stimulates cellular differentiation in .

Proc Natl Acad Sci U S A 2020 07 13;117(30):17984-17991. Epub 2020 Jul 13.

Department of Biology, Indiana University, Bloomington, IN 47405;

Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium employs a specialized dimorphic life cycle consisting of two differentiated cell types. How environmental cues, including mechanical inputs such as contact with a surface, regulate this cell cycle remain unclear. Here, we find that surface sensing by the physical perturbation of retracting extracellular pilus filaments accelerates cell-cycle progression and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer-membrane pilus secretin CpaC stimulates early initiation of chromosome replication. In addition, we find that surface contact stimulates cell-cycle progression by demonstrating that surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell-cycle regulator cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory histidine kinases that control cell fate and differentiation. Together, these results demonstrate that surface contact and sensing by alterations in pilus activity stimulate to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle.
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http://dx.doi.org/10.1073/pnas.1920291117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395532PMC
July 2020

c-di-GMP modulates type IV MSHA pilus retraction and surface attachment in Vibrio cholerae.

Nat Commun 2020 03 25;11(1):1549. Epub 2020 Mar 25.

Department of Microbiology and Environmental Toxicology, University of California - Santa Cruz, 1156 High St., BioMed 245, Santa Cruz, CA, 95064, USA.

Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3',5'-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization.
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http://dx.doi.org/10.1038/s41467-020-15331-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096442PMC
March 2020

Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling.

Nature 2020 02 12;578(7796):582-587. Epub 2020 Feb 12.

M. G. DeGroote Institute for Infectious Disease Research, David Braley Centre for Antibiotic Discovery, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.

Addressing the ongoing antibiotic crisis requires the discovery of compounds with novel mechanisms of action that are capable of treating drug-resistant infections. Many antibiotics are sourced from specialized metabolites produced by bacteria, particularly those of the Actinomycetes family. Although actinomycete extracts have traditionally been screened using activity-based platforms, this approach has become unfavourable owing to the frequent rediscovery of known compounds. Genome sequencing of actinomycetes reveals an untapped reservoir of biosynthetic gene clusters, but prioritization is required to predict which gene clusters may yield promising new chemical matter. Here we make use of the phylogeny of biosynthetic genes along with the lack of known resistance determinants to predict divergent members of the glycopeptide family of antibiotics that are likely to possess new biological activities. Using these predictions, we uncovered two members of a new functional class of glycopeptide antibiotics-the known glycopeptide antibiotic complestatin and a newly discovered compound we call corbomycin-that have a novel mode of action. We show that by binding to peptidoglycan, complestatin and corbomycin block the action of autolysins-essential peptidoglycan hydrolases that are required for remodelling of the cell wall during growth. Corbomycin and complestatin have low levels of resistance development and are effective in reducing bacterial burden in a mouse model of skin MRSA infection.
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http://dx.doi.org/10.1038/s41586-020-1990-9DOI Listing
February 2020

A bifunctional ATPase drives tad pilus extension and retraction.

Sci Adv 2019 12 18;5(12):eaay2591. Epub 2019 Dec 18.

Department of Biology, Indiana University, 1001 E. 3rd Street, Bloomington, IN 47405, USA.

A widespread class of prokaryotic motors powered by secretion motor adenosine triphosphatases (ATPases) drives the dynamic extension and retraction of extracellular fibers, such as type IV pili (T4P). Among these, the tight adherence (tad) pili are critical for surface sensing and biofilm formation. As for most other motors belonging to this class, how tad pili retract despite lacking a dedicated retraction motor ATPase has remained a mystery. Here, we find that a bifunctional pilus motor ATPase, CpaF, drives both activities through adenosine 5'-triphosphate (ATP) hydrolysis. We show that mutations within CpaF result in a correlated reduction in the rates of extension and retraction that directly scales with decreased ATP hydrolysis and retraction force. Thus, a single motor ATPase drives the bidirectional processes of pilus fiber extension and retraction.
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http://dx.doi.org/10.1126/sciadv.aay2591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920026PMC
December 2019

In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer.

Cell 2020 01 26;180(2):348-358.e15. Epub 2019 Dec 26.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom; Central Oxford Structural Microscopy and Imaging Centre, South Parks Road, Oxford OX1 3RE, United Kingdom. Electronic address:

Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.
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http://dx.doi.org/10.1016/j.cell.2019.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978808PMC
January 2020

The Two Chemotaxis Clusters in Caulobacter crescentus Play Different Roles in Chemotaxis and Biofilm Regulation.

J Bacteriol 2019 09 22;201(18). Epub 2019 Aug 22.

Department of Biology, Indiana University, Bloomington, Indiana, USA

The holdfast polysaccharide adhesin is crucial for irreversible cell adhesion and biofilm formation in 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 of and showed that only the previously characterized major cluster is involved in the chemotactic response toward different carbon sources. However, both chemotaxis clusters encoded in the genome play a role in biofilm formation and holdfast production by regulating the expression of , 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 regulate expression, the CheB proteins are positive regulators, thus providing a modulation of holdfast synthesis and surface attachment. Chemosensory 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. The genome 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 modulate adhesion 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.
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http://dx.doi.org/10.1128/JB.00071-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707910PMC
September 2019

Origin of a Core Bacterial Gene via Co-option and Detoxification of a Phage Lysin.

Curr Biol 2019 05 9;29(10):1634-1646.e6. Epub 2019 May 9.

Department of Biology, Indiana University, 1001 E. 3rd Street, Bloomington, IN 47405, USA; Faculté de Médecine, Département de Microbiologie, infectiologie et immunologie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, QC H3C 3J7, Canada. Electronic address:

Temperate phages constitute a potentially beneficial genetic reservoir for bacterial innovation despite being selfish entities encoding an infection cycle inherently at odds with bacterial fitness. These phages integrate their genomes into the bacterial host during infection, donating new but deleterious genetic material: the phage genome encodes toxic genes, such as lysins, that kill the bacterium during the phage infection cycle. Remarkably, some bacteria have exploited the destructive properties of phage genes for their own benefit by co-opting them as toxins for functions related to bacterial warfare, virulence, and secretion. However, do toxic phage genes ever become raw material for functional innovation? Here, we report on a toxic phage gene whose product has lost its toxicity and has become a domain of a core cellular factor, SpmX, throughout the bacterial order Caulobacterales. Using a combination of phylogenetics, bioinformatics, structural biology, cell biology, and biochemistry, we have investigated the origin and function of SpmX and determined that its occurrence is the result of the detoxification of a phage peptidoglycan hydrolase gene. We show that the retained, attenuated activity of the phage-derived domain plays an important role in proper cell morphology and developmental regulation in representatives of this large bacterial clade. To our knowledge, this is the first observation of a phage gene domestication event in which a toxic phage gene has been co-opted for core cellular function at the root of a large bacterial clade.
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http://dx.doi.org/10.1016/j.cub.2019.04.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594375PMC
May 2019

A Multiprotein Complex Anchors Adhesive Holdfast at the Outer Membrane of Caulobacter crescentus.

J Bacteriol 2019 09 22;201(18). Epub 2019 Aug 22.

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

Adhesion allows microbes to colonize surfaces and is the first stage in biofilm formation. Stable attachment of the freshwater alphaproteobacterium to surfaces requires an adhesive polysaccharide called holdfast, which is synthesized at a specific cell pole and ultimately found at the tip of cylindrical extensions of the cell envelope called stalks. Secretion and anchoring of holdfast to the cell surface are governed by proteins HfsDAB and HfaABD, respectively. The arrangement and organization of these proteins with respect to each other and the cell envelope, and the mechanism by which the holdfast is anchored on cells, are unknown. In this study, we have imaged a series of mutants using electron cryotomography, revealing the architecture and arrangement of the molecular machinery involved in holdfast anchoring in cells. We found that the holdfast is anchored to cells by a defined complex made up of the HfaABD proteins and that the HfsDAB secretion proteins are essential for proper assembly and localization of the HfaABD anchor. Subtomogram averaging of cell stalk tips showed that the HfaABD complex spans the outer membrane. The anchor protein HfaB is the major component of the anchor complex located on the periplasmic side of the outer membrane, while HfaA and HfaD are located on the cell surface. HfaB is the critical component of the complex, without which no HfaABD complex was observed in cells. These results allow us to propose a working model of holdfast anchoring, laying the groundwork for further structural and cell biological investigations. Adhesion and biofilm formation are fundamental processes that accompany bacterial colonization of surfaces, which are of critical importance in many infections. biofilm formation proceeds via irreversible adhesion mediated by a polar polysaccharide called holdfast. Mechanistic and structural details of how the holdfast is secreted and anchored on cells are still lacking. Here, we have assigned the location and described the arrangement of the holdfast anchor complex. This work increases our knowledge of the relatively underexplored field of polysaccharide-mediated adhesion by identifying structural elements that anchor polysaccharides to the cell envelope, which is important in a variety of bacterial species.
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http://dx.doi.org/10.1128/JB.00112-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707917PMC
September 2019

Real-time microscopy and physical perturbation of bacterial pili using maleimide-conjugated molecules.

Nat Protoc 2019 06 26;14(6):1803-1819. Epub 2019 Apr 26.

Department of Biology, Indiana University, Bloomington, IN, USA.

Bacteria use surface-exposed, proteinaceous fibers called pili for diverse behaviors, including horizontal gene transfer, surface sensing, motility, and pathogenicity. Visualization of these filamentous nanomachines and their activity in live cells has proven challenging, largely due to their small size. Here, we describe a broadly applicable method for labeling and imaging pili and other surface-exposed nanomachines in live cells. This technique uses a combination of genetics and maleimide-based click chemistry in which a cysteine substitution is made in the major pilin subunit for subsequent labeling with thiol-reactive maleimide dyes. Large maleimide-conjugated molecules can also be used to physically interfere with the dynamic activity of filamentous nanomachines. We describe parameters for selecting cysteine substitution positions, optimized labeling conditions for epifluorescence imaging of pilus fibers, and methods for impeding pilus activity. After cysteine knock-in strains have been generated, this protocol can be completed within 30 min to a few hours, depending on the species and the experiment of choice. Visualization of extracellular nanomachines such as pili using this approach can provide a more comprehensive understanding of the role played by these structures in distinct bacterial behaviors.
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http://dx.doi.org/10.1038/s41596-019-0162-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461830PMC
June 2019

Comparative Analysis of Ionic Strength Tolerance between Freshwater and Marine Adhesins.

J Bacteriol 2019 09 22;201(18). Epub 2019 Aug 22.

Department of Biology, Indiana University, Bloomington, Indiana, USA

Bacterial 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 alphaproteobacterium is a marine budding bacterium in the clade 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 , a freshwater member of the , and little is known about holdfast compositions and properties in marine Here, we use as a model to characterize holdfast properties in marine We show that freshwater and marine use similar genes in holdfast biogenesis and that these genes are highly conserved among the species in the two genera. We determine that produces a larger holdfast than and that the holdfasts have different chemical compositions, as they contain -acetylglucosamine and galactose monosaccharide residues and proteins but lack DNA. Finally, we show that holdfasts tolerate higher ionic strength than those of We conclude that marine holdfasts have physicochemical properties that maximize binding in high-ionic-strength environments. Most 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 bacterium and a relative, the marine bacterium We show that holdfasts have a different morphology and chemical composition and tolerate high ionic strength. Our results show that the holdfast 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.
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http://dx.doi.org/10.1128/JB.00061-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707932PMC
September 2019

Flagellar Mutants Have Reduced Pilus Synthesis in .

J Bacteriol 2019 09 22;201(18). Epub 2019 Aug 22.

Department of Biology, Indiana University, Bloomington, Indiana, USA

Surface appendages, such as flagella and type IV pili, mediate a broad range of bacterial behaviors, including motility, attachment, and surface sensing. While many species harbor both flagella and type IV pili, little is known about how or if their syntheses are coupled. Here, we show that deletions of genes encoding different flagellum machinery components result in a reduction of pilus synthesis in First, we show that different flagellar mutants exhibit different levels of sensitivity to a pilus-dependent phage and that fewer cells within populations of flagellar mutants make pili. Furthermore, we find that single cells within flagellar mutant populations produce fewer pili per cell. We demonstrate that these gene deletions result in reduced transcription of pilus-associated genes and have a slight but significant effect on general transcription profiles. Finally, we show that the decrease in pilus production is due to a reduction in the pool of pilin subunits that are polymerized into pilus fibers. These data demonstrate that mutations in flagellar gene components not only affect motility but also can have considerable and unexpected consequences for other aspects of cell biology. Most bacterial species synthesize surface-exposed appendages that are important for environmental interactions and survival under diverse conditions. It is often assumed that these appendages act independently of each other and that mutations in either system can be used to assess functionality in specific processes. However, we show that mutations in flagellar genes can impact the production of type IV pili, as well as alter general RNA transcriptional profiles compared to a wild-type strain. These data demonstrate that seemingly simple mutations can broadly affect cell-regulatory networks.
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http://dx.doi.org/10.1128/JB.00031-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707913PMC
September 2019

Fluorogenic D-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays.

Nat Chem 2019 04 25;11(4):335-341. Epub 2019 Feb 25.

Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA.

Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic D-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D- and L,D-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.
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http://dx.doi.org/10.1038/s41557-019-0217-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444347PMC
April 2019

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

Mol Microbiol 2018 10 5;110(2):219-238. Epub 2018 Oct 5.

Department of Biology, Indiana University, 1001 E. 3rd Street, Bloomington, IN, 47405, USA.

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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http://dx.doi.org/10.1111/mmi.14099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195837PMC
October 2018

Bacterial adhesion at the single-cell level.

Nat Rev Microbiol 2018 10;16(10):616-627

Department of Biology, Indiana University, Bloomington, IN, USA.

The formation of multicellular microbial communities, called biofilms, starts from the adhesion of a few planktonic cells to the surface. The transition from a free-living planktonic lifestyle to a sessile, attached state is a multifactorial process that is determined by biological, chemical and physical properties of the environment, the surface and the bacterial cell. The initial weak, reversible interactions between a bacterium and a surface strengthen to yield irreversible adhesion. In this Review, we summarize our understanding of the mechanisms governing bacterial adhesion at the single-cell level, including the physical forces experienced by a cell before reaching the surface, the first contact with a surface and the transition from reversible to permanent adhesion.
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http://dx.doi.org/10.1038/s41579-018-0057-5DOI Listing
October 2018

Restricted Localization of Photosynthetic Intracytoplasmic Membranes (ICMs) in Multiple Genera of Purple Nonsulfur Bacteria.

mBio 2018 07 3;9(4). Epub 2018 Jul 3.

Department of Biology, Indiana University, Bloomington, Indiana, USA

In bacteria and eukaryotes alike, proper cellular physiology relies on robust subcellular organization. For the phototrophic purple nonsulfur bacteria (PNSB), this organization entails the use of a light-harvesting, membrane-bound compartment known as the intracytoplasmic membrane (ICM). Here we show that ICMs are spatially and temporally localized in diverse patterns among PNSB. We visualized ICMs in live cells of 14 PNSB species across nine genera by exploiting the natural autofluorescence of the photosynthetic pigment bacteriochlorophyll (BChl). We then quantitatively characterized ICM localization using automated computational analysis of BChl fluorescence patterns within single cells across the population. We revealed that while many PNSB elaborate ICMs along the entirety of the cell, species across as least two genera restrict ICMs to discrete, nonrandom sites near cell poles in a manner coordinated with cell growth and division. Phylogenetic and phenotypic comparisons established that ICM localization and ICM architecture are not strictly interdependent and that neither trait fully correlates with the evolutionary relatedness of the species. The natural diversity of ICM localization revealed herein has implications for both the evolution of phototrophic organisms and their light-harvesting compartments and the mechanisms underpinning spatial organization of bacterial compartments. Many bacteria organize their cellular space by constructing subcellular compartments that are arranged in specific, physiologically relevant patterns. The purple nonsulfur bacteria (PNSB) utilize a membrane-bound compartment known as the intracytoplasmic membrane (ICM) to harvest light for photosynthesis. It was previously unknown whether ICM localization within cells is systematic or irregular and if ICM localization is conserved among PNSB. Here we surveyed ICM localization in diverse PNSB and show that ICMs are spatially organized in species-specific patterns. Most strikingly, several PNSB resolutely restrict ICMs to regions near the cell poles, leaving much of the cell devoid of light-harvesting machinery. Our results demonstrate that bacteria of a common lifestyle utilize unequal portions of their intracellular space to harvest light, despite light harvesting being a process that is intuitively influenced by surface area. Our findings therefore raise fundamental questions about ICM biology and evolution.
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http://dx.doi.org/10.1128/mBio.00780-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030561PMC
July 2018

Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae.

Nat Microbiol 2018 07 11;3(7):773-780. Epub 2018 Jun 11.

Department of Biology, Indiana University, Bloomington, IN, USA.

Natural transformation is a broadly conserved mechanism of horizontal gene transfer in bacterial species that can shape evolution and foster the spread of antibiotic resistance determinants, promote antigenic variation and lead to the acquisition of novel virulence factors. Surface appendages called competence pili promote DNA uptake during the first step of natural transformation ; however, their mechanism of action has remained unclear owing to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus-labelling method, we define the mechanism for type IV competence pilus-mediated DNA uptake during natural transformation. First, we show that type IV competence pili bind to extracellular double-stranded DNA via their tip and demonstrate that this binding is critical for DNA uptake. Next, we show that type IV competence pili are dynamic structures and that pilus retraction brings tip-bound DNA to the cell surface. Finally, we show that pilus retraction is spatiotemporally coupled to DNA internalization and that sterically obstructing pilus retraction prevents DNA uptake. Together, these results indicate that type IV competence pili directly bind to DNA via their tip and mediate DNA internalization through retraction during this conserved mechanism of horizontal gene transfer.
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http://dx.doi.org/10.1038/s41564-018-0174-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582970PMC
July 2018

The cell wall hydrolase Pmp23 is important for assembly and stability of the division ring in Streptococcus pneumoniae.

Sci Rep 2018 05 15;8(1):7591. Epub 2018 May 15.

Institut de Biologie Structurale (IBS), University Grenoble Alpes, CEA, CNRS, 38000, Grenoble, France.

Bacterial division is intimately linked to synthesis and remodeling of the peptidoglycan, a cage-like polymer that surrounds the bacterial cell, providing shape and mechanical resistance. The bacterial division machinery, which is scaffolded by the cytoskeleton protein FtsZ, includes proteins with enzymatic, structural or regulatory functions. These proteins establish a complex network of transient functional and/or physical interactions which preserve cell shape and cell integrity. Cell wall hydrolases required for peptidoglycan remodeling are major contributors to this mechanism. Consistent with this, their deletion or depletion often results in morphological and/or division defects. However, the exact function of most of them remains elusive. In this work, we show that the putative lysozyme activity of the cell wall hydrolase Pmp23 is important for proper morphology and cell division in the opportunistic human pathogen Streptococcus pneumoniae. Our data indicate that active Pmp23 is required for proper localization of the Z-ring and the FtsZ-positioning protein MapZ. In addition, Pmp23 localizes to the division site and interacts directly with the essential peptidoglycan synthase PBP2x. Altogether, our data reveal a new regulatory function for peptidoglycan hydrolases.
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http://dx.doi.org/10.1038/s41598-018-25882-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954120PMC
May 2018

Host-Polarized Cell Growth in Animal Symbionts.

Curr Biol 2018 04 22;28(7):1039-1051.e5. Epub 2018 Mar 22.

University of Vienna, Department of Ecogenomics and Systems Biology, Archaeal Biology and Ecogenomics Division, Althanstrasse 14, 1090 Vienna, Austria. Electronic address:

To determine the fundamentals of cell growth, we must extend cell biological studies to non-model organisms. Here, we investigated the growth modes of the only two rods known to widen instead of elongating, Candidatus Thiosymbion oneisti and Thiosymbion hypermnestrae. These bacteria are attached by one pole to the surface of their respective nematode hosts. By incubating live Ca. T. oneisti and T. hypermnestrae with a peptidoglycan metabolic probe, we observed that the insertion of new cell wall starts at the poles and proceeds inward, concomitantly with FtsZ-based membrane constriction. Remarkably, in Ca. T. hypermnestrae, the proximal, animal-attached pole grows before the distal, free pole, indicating that the peptidoglycan synthesis machinery is host oriented. Immunostaining of the symbionts with an antibody against the actin homolog MreB revealed that it was arranged medially-that is, parallel to the cell long axis-throughout the symbiont life cycle. Given that depolymerization of MreB abolished newly synthesized peptidoglycan insertion and impaired divisome assembly, we conclude that MreB function is required for symbiont widening and division. In conclusion, our data invoke a reassessment of the localization and function of the bacterial actin homolog.
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http://dx.doi.org/10.1016/j.cub.2018.02.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611161PMC
April 2018

Layered Structure and Complex Mechanochemistry Underlie Strength and Versatility in a Bacterial Adhesive.

mBio 2018 02 6;9(1). Epub 2018 Feb 6.

Department of Biology, Indiana University, Bloomington, Indiana, USA

While designing synthetic adhesives that perform in aqueous environments has proven challenging, microorganisms commonly produce bioadhesives that efficiently attach to a variety of substrates, including wet surfaces. The aquatic bacterium uses a discrete polysaccharide complex, the holdfast, to strongly attach to surfaces and resist flow. The holdfast is extremely versatile and has impressive adhesive strength. Here, we used atomic force microscopy in conjunction with superresolution microscopy and enzymatic assays to unravel the complex structure of the holdfast and to characterize its chemical constituents and their role in adhesion. Our data support a model whereby the holdfast is a heterogeneous material organized as two layers: a stiffer nanoscopic core layer wrapped into a sparse, far-reaching, flexible brush layer. Moreover, we found that the elastic response of the holdfast evolves after surface contact from initially heterogeneous to more homogeneous. From a composition point of view, besides -acetyl--glucosamine (NAG), the only component that had been identified to date, our data show that the holdfast contains peptides and DNA. We hypothesize that, while polypeptides are the most important components for adhesive force, the presence of DNA mainly impacts the brush layer and the strength of initial adhesion, with NAG playing a primarily structural role within the core. The unanticipated complexity of both the structure and composition of the holdfast likely underlies its versatility as a wet adhesive and its distinctive strength. Continued improvements in understanding of the mechanochemistry of this bioadhesive could provide new insights into how bacteria attach to surfaces and could inform the development of new adhesives. There is an urgent need for strong, biocompatible bioadhesives that perform underwater. To strongly adhere to surfaces and resist flow underwater, the bacterium produces an adhesive called the holdfast, the mechanochemistry of which remains undefined. We show that the holdfast is a layered structure with a stiff core layer and a polymeric brush layer and consists of polysaccharides, polypeptides, and DNA. The DNA appears to play a role in the structure of the brush layer and initial adhesion, the peptides in adhesive strength, and the polysaccharides in the structure of the core. The complex, multilayer organization and diverse chemistry described here underlie the distinctive adhesive properties of the holdfast and will provide important insights into the mechanisms of bacterial adhesion and bioadhesive applications.
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http://dx.doi.org/10.1128/mBio.02359-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801468PMC
February 2018

Author Correction: Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation.

Nat Microbiol 2018 Feb;3(2):254

Department of Biology, Indiana University Bloomington, Bloomington, IN, 47405, USA.

In the original version of this Article, a grant number and acknowledgement were omitted. The Acknowledgements section should have stated that one of the 3D SIM microscopes used for this research was supported by Medical Research Council UK grant (MR/K015753/1) to S. Foster, University of Sheffield, UK, and that the authors thank C. Walther and S. Foster for the access and their kind help with this. This has now been corrected in all versions of the Article.
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http://dx.doi.org/10.1038/s41564-017-0087-1DOI Listing
February 2018

Evolutionary determinants of genome-wide nucleotide composition.

Nat Ecol Evol 2018 02 1;2(2):237-240. Epub 2018 Jan 1.

Center for Mechanisms of Evolution, Arizona State University, PO Box 877701, Tempe, AZ, USA.

One of the long-standing mysteries of evolutionary genomics is the source of the wide phylogenetic diversity in genome nucleotide composition (G + C versus A + T), which must be a consequence of interspecific differences in mutation bias, the efficiency of selection for different nucleotides or a combination of the two. We demonstrate that although genomic G + C composition is strongly driven by mutation bias, it is also substantially modified by direct selection and/or as a by-product of biased gene conversion. Moreover, G + C composition at fourfold redundant sites is consistently elevated above the neutral expectation-more so than for any other class of sites.
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http://dx.doi.org/10.1038/s41559-017-0425-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855595PMC
February 2018

A programmed cell division delay preserves genome integrity during natural genetic transformation in Streptococcus pneumoniae.

Nat Commun 2017 11 20;8(1):1621. Epub 2017 Nov 20.

Laboratoire de Microbiologie et Génétique Moléculaires, Centre de Biologie Intégrative (CBI), Centre National de la Recherche Scientifique (CNRS), Université de Toulouse, UPS, 31062, Toulouse, France.

Competence for genetic transformation is a differentiation program during which exogenous DNA is imported into the cell and integrated into the chromosome. In Streptococcus pneumoniae, competence develops transiently and synchronously in all cells during exponential phase, and is accompanied by a pause in growth. Here, we reveal that this pause is linked to the cell cycle. At least two parallel pathways impair peptidoglycan synthesis in competent cells. Single-cell analyses demonstrate that ComM, a membrane protein induced during competence, inhibits both initiation of cell division and final constriction of the cytokinetic ring. Competence also interferes with the activity of the serine/threonine kinase StkP, the central regulator of pneumococcal cell division. We further present evidence that the ComM-mediated delay in division preserves genomic integrity during transformation. We propose that cell division arrest is programmed in competent pneumococcal cells to ensure that transformation is complete before resumption of cell division, to provide this pathogen with the maximum potential for genetic diversity and adaptation.
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http://dx.doi.org/10.1038/s41467-017-01716-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696345PMC
November 2017

Mutations in Sugar-Nucleotide Synthesis Genes Restore Holdfast Polysaccharide Anchoring to Caulobacter crescentus Holdfast Anchor Mutants.

J Bacteriol 2018 02 10;200(3). Epub 2018 Jan 10.

Department of Biology, Indiana University, Bloomington, Indiana, USA

Attachment is essential for microorganisms to establish interactions with both biotic and abiotic surfaces. Stable attachment of to surfaces requires an adhesive polysaccharide holdfast, but the exact composition of the holdfast is unknown. The holdfast is anchored to the cell envelope by outer membrane proteins HfaA, HfaB, and HfaD. oldast nchor gene mutations result in holdfast shedding and reduced cell adherence. Translocation of HfaA and HfaD to the cell surface requires HfaB. The Wzx homolog HfsF is predicted to be a bacterial polysaccharide flippase. An deletion significantly reduced the amount of holdfast produced per cell and slightly reduced adherence. A Δ Δ double mutant was completely deficient in adherence. A suppressor screen that restored adhesion in the Δ Δ mutant identified mutations in three genes: , , and Both WbqV and RfbB belong to a family of nucleoside-diphosphate epimerases, and RmlA has similarity to nucleotidyltransferases. The loss of or in the Δ Δ mutant reduced holdfast shedding but did not restore holdfast synthesis to parental levels. Loss of or did not restore adherence to a Δ mutant but did restore adherence and holdfast anchoring to a Δ mutant, confirming that suppression occurs through restoration of holdfast anchoring. The adherence and holdfast anchoring of a Δ mutant could be restored by or mutation, but such mutations could not suppress these phenotypes in the Δ mutant. We hypothesize that HfaB plays an additional role in holdfast anchoring or helps to translocate an unknown factor that is important for holdfast anchoring. Biofilm formation results in increased resistance to both environmental stresses and antibiotics. requires an adhesive holdfast for permanent attachment and biofilm formation, but the exact mechanism of polysaccharide anchoring to the cell and the holdfast composition are unknown. Here we identify novel polysaccharide genes that affect holdfast anchoring to the cell. We identify a new role for the holdfast anchor protein HfaB. This work increases our specific knowledge of the polysaccharide adhesin involved in attachment and the general knowledge regarding production and anchoring of polysaccharide adhesins by bacteria. This work also explores the interactions between different polysaccharide biosynthesis and secretion systems in bacteria.
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http://dx.doi.org/10.1128/JB.00597-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763047PMC
February 2018

Obstruction of pilus retraction stimulates bacterial surface sensing.

Science 2017 10;358(6362):535-538

Department of Biology, Indiana University, 1001 East 3rd Street, Bloomington, IN 47405, USA.

It is critical for bacteria to recognize surface contact and initiate physiological changes required for surface-associated lifestyles. Ubiquitous microbial appendages called pili are involved in sensing surfaces and facilitating downstream behaviors, but the mechanism by which pili mediate surface sensing has been unclear. We visualized pili undergoing dynamic cycles of extension and retraction. Within seconds of surface contact, these cycles ceased, which coincided with synthesis of the adhesive holdfast required for attachment. Physically blocking pili imposed resistance to pilus retraction, which was sufficient to stimulate holdfast synthesis without surface contact. Thus, to sense surfaces, bacteria use the resistance on retracting, surface-bound pili that occurs upon surface contact.
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http://dx.doi.org/10.1126/science.aan5706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805138PMC
October 2017

The Molecular Basis of Noncanonical Bacterial Morphology.

Trends Microbiol 2018 03 19;26(3):191-208. Epub 2017 Oct 19.

Department of Biology, Indiana University, 1001 E. 3rd St, Bloomington, IN 47405, USA. Electronic address:

Bacteria come in a wide variety of shapes and sizes. The true picture of bacterial morphological diversity is likely skewed due to an experimental focus on pathogens and industrially relevant organisms. Indeed, most of the work elucidating the genes and molecular processes involved in maintaining bacterial morphology has been limited to rod- or coccal-shaped model systems. The mechanisms of shape evolution, the molecular processes underlying diverse shapes and growth modes, and how individual cells can dynamically modulate their shape are just beginning to be revealed. Here we discuss recent work aimed at advancing our knowledge of shape diversity and uncovering the molecular basis for shape generation in noncanonical and morphologically complex bacteria.
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http://dx.doi.org/10.1016/j.tim.2017.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834356PMC
March 2018

Full color palette of fluorescent d-amino acids for labeling of bacterial cell walls.

Chem Sci 2017 Sep 7;8(9):6313-6321. Epub 2017 Jul 7.

Department of Molecular and Cellular Biochemistry , Indiana University , Bloomington , IN 47405 , USA . Email:

Fluorescent d-amino acids (FDAAs) enable efficient labeling of peptidoglycan in diverse bacterial species. Conducted by enzymes involved in peptidoglycan biosynthesis, FDAA labeling allows specific probing of cell wall formation/remodeling activity, bacterial growth and cell morphology. Their broad application and high biocompatibility have made FDAAs an important and effective tool for studies of peptidoglycan synthesis and dynamics, which, in turn, has created a demand for the development of new FDAA probes. Here, we report the synthesis of new FDAAs, with emission wavelengths that span the entire visible spectrum. We also provide data to characterize their photochemical and physical properties, and we demonstrate their utility for visualizing peptidoglycan synthesis in Gram-negative and Gram-positive bacterial species. Finally, we show the permeability of FDAAs toward the outer-membrane of Gram-negative organisms, pinpointing the probes available for effective labeling in these species. This improved FDAA toolkit will enable numerous applications for the study of peptidoglycan biosynthesis and dynamics.
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http://dx.doi.org/10.1039/c7sc01800bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628581PMC
September 2017