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1.
Curr Opin Microbiol ; 79: 102476, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38688160

ABSTRACT

Bacterial biofilms are a prevalent multicellular life form in which individual members can undergo significant functional differentiation and are typically embedded in a complex extracellular matrix of proteinaceous fimbriae, extracellular DNA, and exopolysaccharides (EPS). Bacteria have evolved at least four major mechanisms for EPS biosynthesis, of which the synthase-dependent systems for bacterial cellulose secretion (Bcs) represent not only key biofilm determinants in a wide array of environmental and host-associated microbes, but also an important model system for the studies of processive glycan polymerization, cyclic diguanylate (c-di-GMP)-dependent synthase regulation, and biotechnological polymer applications. The secreted cellulosic chains can be decorated with additional chemical groups or can pack with various degrees of crystallinity depending on dedicated enzymatic complexes and/or cytoskeletal scaffolds. Here, I review recent progress in our understanding of synthase-dependent EPS biogenesis with a focus on common and idiosyncratic molecular mechanisms across diverse cellulose secretion systems.


Subject(s)
Bacteria , Bacterial Proteins , Biofilms , Cellulose , Polysaccharides, Bacterial , Cellulose/metabolism , Polysaccharides, Bacterial/metabolism , Bacteria/metabolism , Bacteria/genetics , Bacteria/enzymology , Biofilms/growth & development , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Cyclic GMP/metabolism , Cyclic GMP/analogs & derivatives , Glucosyltransferases
2.
Curr Biol ; 34(1): 106-116.e6, 2024 01 08.
Article in English | MEDLINE | ID: mdl-38141614

ABSTRACT

Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19th century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing ß- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.


Subject(s)
Cellulose , Gluconacetobacter , Proteobacteria/metabolism , Gluconacetobacter/chemistry , Gluconacetobacter/genetics , Gluconacetobacter/metabolism , Bacteria/metabolism , Biofilms
3.
Sci Adv ; 8(50): eadd1170, 2022 Dec 16.
Article in English | MEDLINE | ID: mdl-36525496

ABSTRACT

Cellulose, the most abundant biopolymer on Earth, is not only the predominant constituent of plants but also a key extracellular polysaccharide in the biofilms of many bacterial species. Depending on the producers, chemical modifications, and three-dimensional assemblies, bacterial cellulose (BC) can present diverse degrees of crystallinity. Highly ordered, or crystalline, cellulose presents great economical relevance due to its ever-growing number of biotechnological applications. Even if some acetic acid bacteria have long been identified as BC superproducers, the molecular mechanisms determining the secretion of crystalline versus amorphous cellulose remain largely unknown. Here, we present structural and mechanistic insights into the role of the accessory subunits BcsH (CcpAx) and BcsD (CesD) that determine crystalline BC secretion in the Gluconacetobacter lineage. We show that oligomeric BcsH drives the assembly of BcsD into a supramolecular cytoskeletal scaffold that likely stabilizes the cellulose-extruding synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly.

4.
Methods Mol Biol ; 1657: 403-416, 2017.
Article in English | MEDLINE | ID: mdl-28889310

ABSTRACT

Isothermal titration calorimetry (ITC) is a commonly used biophysical technique that enables the quantitative characterization of intermolecular interactions in solution. Based on enthalpy changes (ΔH) upon titration of the binding partner (e.g., a small-molecule ligand such as c-di-GMP) to the molecule of interest (e.g., a receptor protein), the resulting binding isotherms provide information on the equilibrium association/dissociation constants (K a, K d) and stoichiometry of binding (n), as well as on changes in the Gibbs free energy (ΔG) and entropy (ΔS) along the interaction. Here we present ITC experiments used for the characterization of c-di-GMP binding proteins and discuss advantages and potential caveats in the interpretation of results.


Subject(s)
Calorimetry , Cyclic GMP/analogs & derivatives , DNA-Binding Proteins/chemistry , Bacterial Proteins , Calorimetry/methods , Chromatography, Gel/methods , Cyclic GMP/chemistry , Cyclic GMP/metabolism , DNA-Binding Proteins/metabolism , Kinetics , Ligands , Models, Molecular , Molecular Conformation , Protein Binding , Structure-Activity Relationship
5.
Mol Microbiol ; 105(5): 741-754, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28618091

ABSTRACT

Pneumococcal natural transformation contributes to genomic plasticity, antibiotic resistance development and vaccine escape. Streptococcus pneumoniae, like many other naturally transformable species, has evolved sophisticated protein machinery for the binding and uptake of DNA. Two proteins encoded by the comF operon, ComFA and ComFC, are involved in transformation but their exact molecular roles remain unknown. In this study, we provide experimental evidence that ComFA binds to single stranded DNA (ssDNA) and has ssDNA-dependent ATPase activity. We show that both ComFA and ComFC are essential for the transformation process in pneumococci. Moreover, we show that these proteins interact with each other and with other proteins involved in homologous recombination, such as DprA, thus placing the ComFA-ComFC duo at the interface between DNA uptake and DNA recombination during transformation.


Subject(s)
Adenosine Triphosphatases/metabolism , DNA-Binding Proteins/metabolism , Transformation, Bacterial/physiology , Adenosine Triphosphatases/genetics , Bacterial Proteins/metabolism , DNA/metabolism , DNA, Single-Stranded/metabolism , Homologous Recombination , Membrane Proteins/metabolism , Protein Binding , Rec A Recombinases/metabolism , Recombination, Genetic , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/metabolism , Transformation, Bacterial/genetics
6.
Proc Natl Acad Sci U S A ; 113(2): E209-18, 2016 Jan 12.
Article in English | MEDLINE | ID: mdl-26712005

ABSTRACT

Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ(54)-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP-complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.


Subject(s)
Bacterial Proteins/metabolism , Biofilms/drug effects , Cyclic GMP/analogs & derivatives , Pseudomonas aeruginosa/physiology , Trans-Activators/metabolism , Amino Acid Motifs , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Sequence , Binding Sites , Calorimetry , Conserved Sequence , Cross-Linking Reagents , Crystallography, X-Ray , Cyclic GMP/pharmacology , DNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial/drug effects , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Promoter Regions, Genetic/genetics , Protein Multimerization/drug effects , Protein Stability , Protein Structure, Quaternary , Protein Structure, Tertiary , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Sequence Alignment , Solutions , Temperature , Trans-Activators/chemistry , Transcription, Genetic
7.
PLoS Pathog ; 11(4): e1004835, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25876066

ABSTRACT

The success of S. pneumoniae as a major human pathogen is largely due to its remarkable genomic plasticity, allowing efficient escape from antimicrobials action and host immune response. Natural transformation, or the active uptake and chromosomal integration of exogenous DNA during the transitory differentiated state competence, is the main mechanism for horizontal gene transfer and genomic makeover in pneumococci. Although transforming DNA has been proposed to be captured by Type 4 pili (T4P) in Gram-negative bacteria, and a competence-inducible comG operon encoding proteins homologous to T4P-biogenesis components is present in transformable Gram-positive bacteria, a prevailing hypothesis has been that S. pneumoniae assembles only short pseudopili to destabilize the cell wall for DNA entry. We recently identified a micrometer-sized T4P-like pilus on competent pneumococci, which likely serves as initial DNA receptor. A subsequent study, however, visualized a different structure--short, 'plaited' polymers--released in the medium of competent S. pneumoniae. Biochemical observation of concurrent pilin secretion led the authors to propose that the 'plaited' structures correspond to transformation pili acting as peptidoglycan drills that leave DNA entry pores upon secretion. Here we show that the 'plaited' filaments are not related to natural transformation as they are released by non-competent pneumococci, as well as by cells with disrupted pilus biogenesis components. Combining electron microscopy visualization with structural, biochemical and proteomic analyses, we further identify the 'plaited' polymers as spirosomes: macromolecular assemblies of the fermentative acetaldehyde-alcohol dehydrogenase enzyme AdhE that is well conserved in a broad range of Gram-positive and Gram-negative bacteria.


Subject(s)
Fimbriae, Bacterial/ultrastructure , Streptococcus pneumoniae/ultrastructure , Gene Transfer, Horizontal , Macromolecular Substances/ultrastructure , Microscopy, Electron, Transmission , Polymerase Chain Reaction , Proteomics , Streptococcus pneumoniae/genetics , Transformation, Bacterial/genetics
8.
Nature ; 516(7530): 250-3, 2014 Dec 11.
Article in English | MEDLINE | ID: mdl-25219853

ABSTRACT

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded ß-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å(3) pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism.


Subject(s)
Amyloid/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/chemistry , Lipoproteins/chemistry , Lipoproteins/metabolism , Biofilms , Cell Membrane , Crystallography, X-Ray , Diffusion , Entropy , Membrane Transport Proteins/metabolism , Models, Biological , Models, Molecular , Periplasm/metabolism , Protein Conformation , Protein Transport
11.
Nat Methods ; 9(7): 640-1, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22930829
12.
Nat Methods ; 8(11): 898, 2011 Nov.
Article in English | MEDLINE | ID: mdl-22148157
13.
Nat Methods ; 8(10): 793, 2011.
Article in English | MEDLINE | ID: mdl-22053346
14.
Nat Methods ; 8(9): 714, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21985006

ABSTRACT

An engineered fluorescent protein reveals electrical spikes in intact bacterial cells.

15.
Nat Methods ; 8(8): 622, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21916038
16.
PLoS Biol ; 9(2): e1000588, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-21304926

ABSTRACT

The bacterial second messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a central regulator for biofilm formation. Increased cellular c-di-GMP levels lead to stable cell attachment, which in Pseudomonas fluorescens requires the transmembrane receptor LapD. LapD exhibits a conserved and widely used modular architecture containing a HAMP domain and degenerate diguanylate cyclase and phosphodiesterase domains. c-di-GMP binding to the LapD degenerate phosphodiesterase domain is communicated via the HAMP relay to the periplasmic domain, triggering sequestration of the protease LapG, thus preventing cleavage of the surface adhesin LapA. Here, we elucidate the molecular mechanism of autoinhibition and activation of LapD based on structure-function analyses and crystal structures of the entire periplasmic domain and the intracellular signaling unit in two different states. In the absence of c-di-GMP, the intracellular module assumes an inactive conformation. Binding of c-di-GMP to the phosphodiesterase domain disrupts the inactive state, permitting the formation of a trans-subunit dimer interface between adjacent phosphodiesterase domains via interactions conserved in c-di-GMP-degrading enzymes. Efficient mechanical coupling of the conformational changes across the membrane is realized through an extensively domain-swapped, unique periplasmic fold. Our structural and functional analyses identified a conserved system for the regulation of periplasmic proteases in a wide variety of bacteria, including many free-living and pathogenic species.


Subject(s)
Cyclic GMP/analogs & derivatives , Periplasm/metabolism , Pseudomonas fluorescens/metabolism , Signal Transduction , Bacterial Adhesion , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Binding Sites , Biofilms , Crystallography, X-Ray , Cyclic GMP/metabolism , Cyclic GMP/physiology , Dimerization , Peptide Hydrolases/metabolism , Peptide Hydrolases/physiology , Phosphoric Diester Hydrolases/metabolism , Protein Interaction Mapping , Protein Structure, Tertiary , Pseudomonas fluorescens/genetics , Pseudomonas fluorescens/physiology , Structure-Activity Relationship
17.
Nat Methods ; 8(12): 1002, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22238779
18.
Methods Enzymol ; 471: 161-84, 2010.
Article in English | MEDLINE | ID: mdl-20946848

ABSTRACT

Many signal transduction and regulatory events are mediated by a change in oligomeric state upon posttranslational modification or ligand binding. Hence, the characterization of proteins and protein complexes with respect to their size and shape is crucial for elucidating the molecular mechanisms that control their activities. Commonly used methods for the determination of molecular weights of biological polymers such as standard size-exclusion chromatography or analytical ultracentrifugation have been applied successfully but have some limitations. Static multiangle light scattering presents an attractive alternative approach for absolute molecular weight measurements in solution. We review the biophysical principles, advantages, and pitfalls of some popular methods for determining the quaternary structure of proteins, using the response regulator diguanylate cyclase WspR from Pseudomonas and FimX, a protein involved in Pseudomonas aeruginosa twitching motility, as examples.


Subject(s)
Bacterial Proteins/metabolism , Biofilms/growth & development , Bacterial Proteins/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Phosphorus-Oxygen Lyases/genetics , Phosphorus-Oxygen Lyases/metabolism , Protein Binding/genetics , Protein Binding/physiology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Signal Transduction/genetics , Signal Transduction/physiology
19.
Science ; 327(5967): 866-8, 2010 Feb 12.
Article in English | MEDLINE | ID: mdl-20150502

ABSTRACT

Microorganisms can switch from a planktonic, free-swimming life-style to a sessile, colonial state, called a biofilm, which confers resistance to environmental stress. Conversion between the motile and biofilm life-styles has been attributed to increased levels of the prokaryotic second messenger cyclic di-guanosine monophosphate (c-di-GMP), yet the signaling mechanisms mediating such a global switch are poorly understood. Here we show that the transcriptional regulator VpsT from Vibrio cholerae directly senses c-di-GMP to inversely control extracellular matrix production and motility, which identifies VpsT as a master regulator for biofilm formation. Rather than being regulated by phosphorylation, VpsT undergoes a change in oligomerization on c-di-GMP binding.


Subject(s)
Bacterial Proteins/metabolism , Biofilms/growth & development , Cyclic GMP/analogs & derivatives , Extracellular Matrix/metabolism , Transcription Factors/metabolism , Vibrio cholerae O1/physiology , Amino Acid Motifs , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Crystallography, X-Ray , Cyclic GMP/metabolism , DNA, Bacterial/metabolism , Dimerization , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Models, Molecular , Movement , Point Mutation , Polysaccharides, Bacterial/genetics , Polysaccharides, Bacterial/metabolism , Protein Folding , Protein Multimerization , Protein Structure, Tertiary , Signal Transduction , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription, Genetic , Vibrio cholerae O1/cytology , Vibrio cholerae O1/genetics
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