Lectin-Like Bacteriocins

Bacteria produce a diverse array of antagonistic compounds to restrict growth of microbial rivals. Contributing to this warfare are bacteriocins: secreted antibacterial peptides, proteins and multi-protein complexes. These compounds typically eliminate competitors closely related to the producer. Lectin-like bacteriocins (LlpAs) constitute a distinct class of such proteins, produced by Pseudomonas as well as some other proteobacterial genera. LlpAs share a common architecture consisting of two B-lectin domains, followed by a short carboxy-terminal extension. Two surface-exposed moieties on susceptible Pseudomonas cells are targeted by the respective lectin modules. The carboxy-terminal domain binds D-rhamnose residues present in the lipopolysaccharide layer, whereas the amino-terminal domain interacts with a polymorphic external loop of the outer-membrane protein insertase BamA, hence determining selectivity. The absence of a toxin-immunity module as found in modular bacteriocins and other polymorphic toxin systems, hints toward a novel mode of killing initiated at the cellular surface, not requiring bacteriocin import. Despite significant progress in understanding the function of LlpAs, outstanding questions include the secretion machinery recruited by lectin-like bacteriocins for their release, as well as a better understanding of the environmental signals initiating their expression

A Natural Chimeric Pseudomonas Bacteriocin with Novel Pore-Forming Activity Parasitizes the Ferrichrome Transporter

Modular bacteriocins represent a major group of secreted protein toxins with a narrow spectrum of activity, involved in interference competition between Gram-negative bacteria. These antibacterial proteins include a domain for binding to the target cell and a toxin module at the carboxy terminus. Self-inhibition of producers is provided by coexpression of linked immunity genes that transiently inhibit the toxin''s activity through formation of bacteriocin-immunity complexes or by insertion in the inner membrane, depending on the type of toxin module. We demonstrate strain-specific inhibitory activity for PmnH, a Pseudomonas bacteriocin with an unprecedented dual-toxin architecture, hosting both a colicin M domain, potentially interfering with peptidoglycan synthesis, and a novel colicin N-type domain, a pore-forming module distinct from the colicin Ia-type domain in Pseudomonas aeruginosa pyocin S5. A downstream-linked gene product confers PmnH immunity upon susceptible strains. This protein, ImnH, has a transmembrane topology similar to that of Pseudomonas colicin M-like and pore-forming immunity proteins, although homology with either of these is essentially absent. The enhanced killing activity of PmnH under iron-limited growth conditions reflects parasitism of the ferrichrome-type transporter for entry into target cells, a strategy shown here to be used as well by monodomain colicin M-like bacteriocins from pseudomonads. The integration of a second type of toxin module in a bacteriocin gene could offer a competitive advantage against bacteria displaying immunity against only one of both toxic activities

The antimicrobial compound xantholysin defines a new group of Pseudomonas cyclic lipopeptides

The rhizosphere isolate Pseudomonas putida BW11M1 produces a mixture of cyclic lipopeptide congeners, designated xantholysins. Properties of the major compound xantholysin A, shared with several other Pseudomonas lipopeptides, include antifungal activity and toxicity to Gram-positive bacteria, a supportive role in biofilm formation, and facilitation of surface colonization through swarming. Atypical is the lipopeptide’s capacity to inhibit some Gram-negative bacteria, including several xanthomonads. The lipotetradecadepsipeptides are assembled by XtlA, XtlB and XtlC, three co-linearly operating non-ribosomal peptide synthetases (NRPSs) displaying similarity in modular architecture with the entolysin-producing enzymes of the entomopathogenic Pseudomonas entomophila L48. A shifted serine-incorporating unit in the eight-module enzyme XtlB elongating the central peptide moiety not only generates an amino acid sequence differing at several equivalent positions from entolysin, but also directs xantholysin’s macrocyclization into an octacyclic structure, distinct from the pentacyclic closure in entolysin. Relaxed fatty acid specificity during lipoinitiation by XtlA (acylation with 3-hydroxydodec-5-enoate instead of 3-hydroxydecanoate) and for incorporation of the ultimate amino acid by XtlC (valine instead of isoleucine) account for the production of the minor structural variants xantholysin C and B, respectively. Remarkably, the genetic backbones of the xantholysin and entolysin NRPS systems also bear pronounced phylogenetic similarity to those of the P. putida strains PCL1445 and RW10S2, albeit generating the seemingly structurally unrelated cyclic lipopeptides putisolvin (undecapeptide containing a cyclotetrapeptide) and WLIP (nonapeptide containing a cycloheptapeptide), respectively. This similarity includes the linked genes encoding the cognate LuxR-family regulator and tripartite export system components in addition to individual modules of the NRPS enzymes, and probably reflects a common evolutionary origin. Phylogenetic scrutiny of the modules used for selective amino acid activation by these synthetases indicates that bacteria such as pseudomonads recruit and reshuffle individual biosynthetic units and blocks thereof to engineer reorganized or novel NRPS assembly lines for diversified synthesis of lipopeptides

Protein-mediated killing among bacteria: structure and function of prokaryotic MMBL lectins

Production of bacteriocins represents a highly diversified defense mechanism, identified in virtually all lineages of bacteria. These proteinaceous toxins are specifically directed against related bacteria and their production confers to a eco-evolutionary advantage for the producing organism. In this work, the objective was to further investigate the LlpA family of lectin-like bacteriocins, originally identified in the banana rhizosphere isolate P. putida BW11M1.The solved crystal structure of LlpA, the prototype lectin-like bacteriocin, unequivocally assigned this protein to the class of the so-called monocot mannose-binding lectin (MMBL) proteins, also designated B-lectins. LlpA is built from two tightly interacting ß-prism-folded MMBL modules, stabilized by a C-terminal ß-hairpin extension. The MMBL modules of LlpA display pseudo-threefold symmetry, domain stabilization by a central tryptophan triad, and a ß-strand swap interconnecting these two domains, hallmarks of B-lectins. Each MMBL domain contains three putative mannose-binding motifs exhibiting different levels of sequence degeneracy. Of these six sites, only three retain their potential carbohydrate-binding capacity. Binding of methyl-D-alfa-mannopyranoside and oligomannosides could only be demonstrated for one of these sites, located in the C-terminal MMBL-domain. LlpA mutant proteins with a sterically occluded mannose-binding site displayed a reduced antibacterial function, clearly coupling the ability to bind carbohydrates with bacteriotoxicity. Differential activity of engineered domain chimers derived from two LlpA homologues with different killing spectra, revealed that the N-domain is the main determinant of target strain recognition. Plant MMBL proteins display a diverse range of antagonistic activities, including antifungal, insecticidal, nematicidal or antiviral function, but antibacterial activity could not be demonstrated however. Hence, assigning a bacteriocin to the MMBL family further expands its range of antagonistic functions.The MMBL domain is commonly found in eukaryotic lectins, being particularly widespread among plants but also recently identified in several fish and fungal species. In the proteomes of prokaryotes, this MMBL module may appear as a single unit, in tandem organization, or as a hybrid fused to at least one other domain. The former domain architectures are typically found in Gram-negative bacteria, particularly abundant among Pseudomonas and Burkholderia, while the latter are more often found in Gram-positive bacteria. So far, only one such hybrid protein, composed of a MMBL module fused to a putative peptidase domain, has been characterized. This protein, albusin B, equally acts as a bacteriocin. Phylogenetic analysis of the different MMBL domains from LlpAs demonstrated that N- and C-modules evolved separately: N-domains tend to cluster together, distant from equally clustered C-domains. This observation is in line with the functional segregation described previously. The antibacterial potentialof tandem-MMBL proteins retrieved from proteomes of different bacterial species from Pseudomonas, Xanthomonas and Burkholderia was explored. Using recombinantly-purified proteins, we were able to demonstrate narrow-spectrum, genus-specific activity for all these LlpAs. As different human and phytopathogenic strains are susceptible to these LlpAs, these results may provide interesting perspectives for future antibacterial strategies.An atypical tandem-MMBL protein was identified in an Arthrobacter strain. Having two highly homologous B-lectin modules, six perfectly conserved mannose-binding motifs, a virtually absent ß-hairpin and an oligomeric organization, it represents the sole LlpA-like protein identified in a Gram-positive bacterium so far. We were not able to identify an antibacterial function for this protein, but instead demonstrated anti-retroviral activity. Hence, this protein was called arthrohivin.In several pseudomonad genomes, a single-MMBL-domain protein, termed LlpB, is encoded. This protein displays antibacterial activity as well, which stands in contrast with the task division between domains in LlpA. LlpBs display pronounced similarity with the N-domains of LlpAs, which enabled to construct a model of LlpB based on the crystal structure of the LlpA from P. putida. Based on this structural prediction, LlpB can be assigned to the class of the MMBL proteins as well. This novel subtype lectin-like bacteriocin may represent a kind of minimal LlpA, with target strain recognition and carbohydrate-binding function, if present, condensed into only a single domain. Conceivably, domain duplication within an ancestral LlpB protein, may have given rise to the tandem architecture of LlpA proteins, as a way to diversify target recognition, uncoupled from actual killing activity.SUMMARY I SAMENVATTING III LIST OF ABBREVIATIONS V CHAPTER 1 ANTIBACTERIAL PEPTIDES AND PROTEINS IN GRAM-NEGATIVE BACTERIA 1 1.1 MICROCINS 2 1.2 COLICINS 4 1.2.1 Colicinogeny 4 1.2.2 Biological activities of colicins 4 1.2.3 Modular structure of colicins and self-protection 5 1.2.4 Colicin import 7 1.2.5 Regulation of colicin production 7 1.2.6 Colicins as promoters of microbial diversity? 9 1.2.7 Colicin-like bacteriocins from other γ-proteobacteria 10 1.3 S-TYPE PYOCINS 11 1.3.1 Modular architecture 11 1.3.2 S-pyocin uptake and immunity 12 1.3.3 Pyocin regulation 12 1.3.4 Pyocinogeny 12 1.4 PHAGE TAIL-LIKE BACTERIOCINS 13 1.4.1 R-type pyocins from Pseudomonas 13 1.4.2 F-type pyocins from Pseudomonas 14 1.4.3 Phage ancestry of R- and F-pyocins? 15 1.4.4 Phage tail-like bacteriocins in plant-associated bacteria 15 1.5 CONTACT-DEPENDENT GROWTH INHIBITION 16 1.5.1 Two-partner secretion system CDI 16 1.5.1.1 Structural organization of CdiA 16 1.5.1.2 Functionality of CdiI as a protector from auto-inhibition 17 1.5.1.3 Interaction with sensitive cells 17 1.5.1.4 CDI in other bacteria 18 1.5.1.5 Rhs elements as mediators of intercellular competition 19 1.6 INHIBITION MEDIATED BY TYPE VI SECRETION 19 1.7 MORE HIDDEN BACTERIOCIN DIVERSITY? 21 1.7.1 Ferredoxin-containing bacteriocins from Pectobacterium 21 1.7.2 Glycinecin A from Xanthomonas 21 1.7.3 Triple-subunit bacteriocin from Pseudomonas syringae 22 1.8 LECTIN-LIKE BACTERIOCINS: MEMBERS OF THE MMBL FAMILY 22 1.8.1 MMBL (monocot mannose-binding lectin)-type lectins: what is in a name? 22 1.8.2 Eukaryotic MMBLs revisited 24 1.8.3 Prokaryotic MMBLs: in search of a function 24 1.8.4 Bacterial killer MMBLs 26 1.8.5 Bacterial chimeric MMBLs: toxic proteins as well? 30 1.8.6 Bacteriocins with a novel mode of action? 32 1.9 SUPPORTING INFORMATION 33 1.10 SCOPE AND OUTLINE OF THE THESIS 34 1.11 BIBLIOGRAPHY 36 CHAPTER 2 ANTAGONISTIC ACTIVITIES OF PROKARTYOIC TANDEM-MMBL PROTEINS 45 ABSTRACT 45 2.1 PLANT LECTIN-LIKE ANTIBACTERIAL PROTEINS FROM PHYTOPATHOGENS PSEUDOMONAS SYRINGAE AND XANTHOMONAS CITRI 47 2.1.1 Introduction 47 2.1.2 Results 47 2.1.2.1 New candidate lectin-like bacteriocins 47 2.1.2.2 Expression of recombinant tandem MMBL lectins 48 2.1.2.3 Purification of recombinant proteins 50 2.1.2.4 Bacteriocin activity of novel LlpAs 50 2.1.3 Discussion 51 2.2 ANTIBACTERIAL ACTIVITY OF A LECTIN-LIKE BURKHOLDERIA CENOCEPACIA PROTEIN 54 2.2.1 Introduction 54 2.2.2 Results 55 2.2.2.1 Genes encoding LlpA-like proteins in Burkholderia genomes 55 2.2.2.2 Bacteriocin activity of B. cenocepacia LlpA6 56 2.2.2.3 Anti-biofilm activity of B. cenocepacia LlpA6 57 2.2.3 Discussion 59 2.3 ARTHROHIVIN, A TANDEM MONOCOT MANNOSE-BINDING LECTIN FROM AN ARTHROBACTER SOIL ISOLATE, INHIBITS HIV-1 INFECTION 60 2.3.1 Introduction 60 2.3.2 Results 60 2.3.2.1 Identification, recombinant production and purification of LlpA5 60 2.3.2.2 LlpA5 is an atypical tetrameric LlpA, lacking antibacterial activity 61 2.3.2.3 LlpA5 displays antiretroviral activity 61 2.3.3 Discussion 63 2.4 MATERIALS AND METHODS 64 2.4.1 Strains and culture conditions 64 2.4.2 Recombinant DNA methods 65 2.4.3 Overexpression and purification of recombinant LlpAs 65 2.4.4 Glycan array 66 2.4.5 Bacteriocin assay 66 2.4.6 Antifungal assays 67 2.4.7 Antiviral assay 67 2.4.8 Co-cultivation assay 68 2.4.9 Surface plasmon resonance (SPR) analysis 68 2.4.10 Determination of the MIC of LlpA6 68 2.4.11 Determination of the inhibition and eradication of Burkholderia biofilms 69 2.5 SUPPORTING INFORMATION 70 2.6 BIBLIOGRAPHY 70 CHAPTER 3 STRUCTURAL DETERMINANTS FOR ACTIVITY AND SPECIFICITY OF THE BACTERIAL TOXIN LLPA 75 ABSTRACT 75 3.1 INTRODUCTION 76 3.2 RESULTS 77 3.2.1 LlpA forms a rigid MMBL tandem 77 3.2.2 Domains of LlpABW are shaped by differential evolutionary pressure 79 3.2.3 LlpA is capable of binding mannose-containing carbohydrates 82 3.2.4 Carbohydrate-binding capacity is required for LlpA toxicity 86 3.2.5 All domains are necessary for LlpABW functionality 90 3.2.6 Target specificity of LlpA is hosted by the N-domain 91 3.3 DISCUSSION 93 3.4 MATERIALS AND METHODS 94 3.4.1 Strains and culture conditions 94 3.4.2 Recombinant DNA methods 94 3.4.3 Recombinant protein expression and purification 95 3.4.4 Antibacterial assays 96 3.4.5 Glycan array 96 3.4.6 Circular dichroism 97 3.4.7 Isothermal titration calorimetry 97 3.4.8 X-ray data collection and structure determination 97 3.4.9 Carbohydrate soaks 98 3.4.10 Flow cytometry 98 3.5 SUPPORTING INFORMATION 100 3.6 BIBLIOGRAPHY 100 CHAPTER 4 THE MONO-MMBL-DOMAIN PROTEIN LLPB FROM PSEUDOMONAS DEFINES A NEW CLASS OF LECTIN-LIKE BACTERIOCINS 103 ABSTRACT 103 4.1 INTRODUCTION 104 4.2 RESULTS 104 4.2.1 Identification of mono-MMBL-domain proteins in Pseudomonas 104 4.2.2 LlpBs display antibacterial but no antifungal activity 106 4.2.3 Heterologous expression of LlpB in Pseudomonas spp. 108 4.2.4 Monomeric nature of LlpB 108 4.2.5 LlpB as a member of the MMBL family 109 4.2.5.1 Structural similarities between LlpB and LlpA 109 4.2.5.2 Potential carbohydrate-binding capacity of LlpB 111 4.3 DISCUSSION 113 4.4 MATERIALS AND METHODS 114 4.4.1 Strains and culture conditions 114 4.4.2 DNA and cellular manipulations 115 4.4.3 Recombinant expression and purification of LlpBs 115 4.4.4 Point mutation analysis of LlpB and construction of chimeric tandem lectins 115 4.4.5 Antagonistic assays 116 4.4.6 Phylogenetic analysis 116 4.4.7 LlpB modeling 116 4.5 SUPPORTING INFORMATION 116 4.6 BIBLIOGRAPHY 116 CHAPTER 5 GENERAL DISCUSSION, CONCLUSIONS AND PERSPECTIVES 119 5.1 LLPA: A NATURAL CHILD OF THE MMBL FAMILY DISCLOSED 119 5.2 THE Β-HAIRPIN TAIL: IN CAUDA VENENUM? 121 5.3 LECTIN-BASED KILLING: TOWARDS A NEW MODE OF ACTION? 121 5.4 BEYOND MMBL BACTERIOCINS: ARTHROHIVIN AND MORE 123 5.5 BIBLIOGRAPHY 124 APPENDIX 125 LIST OF PUBLICATIONS 127nrpages: 127status: publishe

The bacteriocin armamentarium of Pseudomonas

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The bacteriocin arsenal of Pseudomonas

Pseudomonas is a metabolically versatile genus that has acquired several antagonism-mediating mechanisms in order to gain ground in competitive niches. Besides antibiotics, Pseudomonas also secretes ribosomally encoded antibacterial proteins, designated bacteriocins. In Pseudomonas aeruginosa, large phage tail-like bacteriocins (R and F) and smaller modular bacteriocins (S pyocins) have previously been studied. More recently, M-type and L-type pyocins have been added to its antibacterial complement. We performed a comprehensive analysis on available full and draft genomes of pseudomonads to explore their arsenal of bacteriocin genes. In addition to underlining the highly strain-specific nature of bacteriocinogeny, this genome mining revealed that the functional diversity of pseudomonad bacteriocins is much broader than estimated from the currently characterized bacteriocins. For S-type bacteriocins, the modular domain architecture apparently has driven extensive diversification. Toxin-immunity modules are combined with various target-specifying receptor-binding domains, suggesting that recombination events have taken place. In addition, these modules also appear in antagonism-mediating complexes of a different nature such as Rhs and CDI proteins. The dynamic nature of the pseudomonad bacteriocins is further illustrated by the occurrence of novel bacteriocin architectures in which two toxin modules are integrated, for example two DNase domains. In another group of bacteriocins, the L-type or lectin-like bacteriocins, a second functional type of antibacterial protein was identified. Contrary to classical L-type bacteriocins consisting of a lectin tandem, this novel subclass only hosts a single lectin domain, hence representing a novel type in which the strain-specific killing function is essentially condensed into a single lectin domain.status: publishe

The tailocin tale: peeling off phage tails

Bacteria produce a variety of particles resembling phage tails that are functional without an associated phage head. Acquired from diverse bacteriophage sources, these stand-alone units were sculpted to serve different ecological roles. Such tailocins mediate antagonism between related bacteria as well as interactions with eukaryotic cells.status: publishe

Distinct colicin M-like bacteriocin-immunity pairs in Burkholderia

The Escherichia coli bacteriocin colicin M (ColM) acts via degradation of the cell wall precursor lipid II in target cells. ColM producers avoid self-inhibition by a periplasmic immunity protein anchored in the inner membrane. In this study, we identified colM-like bacteriocin genes in genomes of several β-proteobacterial strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. Two selected Burkholderia ambifaria proteins, designated burkhocins M1 and M2, were produced recombinantly and showed antagonistic activity against Bcc strains. In their considerably sequence-diverged catalytic domain, a conserved aspartate residue equally proved pivotal for cytotoxicity. Immunity to M-type burkhocins is conferred upon susceptible strains by heterologous expression of a cognate gene located either upstream or downstream of the toxin gene. These genes lack homology with currently known ColM immunity genes and encode inner membrane-associated proteins of two distinct types, differing in predicted transmembrane topology and moiety exposed to the periplasm. The addition of burkhocins to the bacteriocin complement of Burkholderia reveals a wider phylogenetic distribution of ColM-like bacteriotoxins, beyond the γ-proteobacterial genera Escherichia, Pectobacterium and Pseudomonas, and illuminates the diversified nature of immunity-providing proteins.status: publishe

LlpB represents a second subclass of lectin-like bacteriocins

Bacteriocins are secreted bacterial proteins that selectively kill related strains. Lectin-like bacteriocins are atypical bacteriocins not requiring a cognate immunity factor and have been primarily studied in Pseudomonas. These so-called LlpAs are composed of a tandem of B-lectin domains. One domain interacts with d-rhamnose residues in the common polysaccharide antigen of Pseudomonas lipopolysaccharide (LPS). The other lectin domain is crucial for interference with the outer membrane protein assembly machinery by interacting with surface-exposed loops of its central component BamA. Via genome mining, we identified a second subclass of Pseudomonas lectin-like proteins, termed LlpB, consisting of a single B-lectin domain. We show that these proteins also display bactericidal activity. Among LlpB-resistant transposon mutants of an LlpB-susceptible Pseudomonas strain, a major subset was hit in an acyltransferase gene, predicted to be involved in LPS core modification, hereby suggesting that LlpBs equally attach to LPS for surface anchoring. This indicates that LPS binding and target strain specificity are condensed in a single B-lectin domain. The identification of this second subclass of lectin-like bacteriocins further expands the toolbox of antibacterial warfare deployed by bacteria and holds potential for their integration in biotechnological applications.status: publishe

MMBL proteins: from lectin to bacteriocin

Arguably, bacteriocins deployed in warfare among related bacteria are among the most diverse proteinacous compounds with respect to structure and mode of action. Identification of the first prokaryotic member of the so-called MMBLs (monocot mannose-binding lectins) or GNA (Galanthus nivalis agglutinin) lectin family and discovery of its genus-specific killer activity in the Gram-negative bacteria Pseudomonas and Xanthomonas has added yet another kind of toxin to this group of allelopathic molecules. This novel feature is reminiscent of the protective function, on the basis of antifungal, insecticidal, nematicidal or antiviral activity, assigned to or proposed for several of the eukaryotic MMBL proteins that are ubiquitously distributed among monocot plants, but also occur in some other plants, fish, sponges, amoebae and fungi. Direct bactericidal activity can also be effected by a C-type lectin, but this is a mammalian protein that limits mucosal colonization by Gram-positive bacteria. The presence of two divergent MMBL domains in the novel bacteriocins raises questions about task distribution between modules and the possible role of carbohydrate binding in the specificity of target strain recognition and killing. Notably, bacteriocin activity was also demonstrated for a hybrid MMBL protein with an accessory protease-like domain. This association with one or more additional modules, often with predicted peptide-hydrolysing or -binding activity, suggests that additional bacteriotoxic proteins may be found among the diverse chimaeric MMBL proteins encoded in prokaryotic genomes. A phylogenetic survey of the bacterial MMBL modules reveals a mosaic pattern of strongly diverged sequences, mainly occurring in soil-dwelling and rhizosphere bacteria, which may reflect a trans-kingdom acquisition of the ancestral genes.status: publishe