Maarten G. K. Ghequire

Ribosomally encoded antibacterial proteins and peptides from Pseudomonas

Members of the Pseudomonas genus produce diverse secondary metabolites affecting other bacteria, fungi or predating nematodes and protozoa but are also equipped with the capacity to secrete different types of ribosomally encoded toxic peptides and proteins, ranging from small microcins to large tailocins. Studies with the human pathogen Pseudomonas aeruginosa have revealed that effector proteins of type VI secretion systems are part of the antibacterial armamentarium deployed by pseudomonads. A novel class of antibacterial proteins with structural similarity to plant lectins was discovered by studying antagonism among plant-associated Pseudomonas strains. A genomic perspective on pseudomonad bacteriocinogeny shows that the modular architecture of S pyocins of P. aeruginosa is retained in a large diversified group of bacteriocins, most of which target DNA or RNA. Similar modularity is present in as yet poorly characterized Rhs (recombination hot spot) proteins and CDI (contact-dependent inhibition) proteins. Well-delimited domains for receptor recognition or cytotoxicity enable the design of chimeric toxins with novel functionalities, which has been applied successfully for S and R pyocins. Little is known regarding how these antibacterials are released and ultimately reach their targets. Other remaining issues concern the identification of environmental triggers activating these systems and assessment of their ecological impact in niches populated by pseudomonads.

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.

Flocculation gene variability in industrial brewer’s yeast strains

The brewer’s yeast genome encodes a ‘Flo’ flocculin family responsible for flocculation. Controlled floc formation or flocculation at the end of fermentation is of great importance in the brewing industry since it is a cost-effective and environmental-friendly technique to separate yeast cells from the final beer. FLO genes have the notable capacity to evolve and diverge many times faster than other genes. In actual practice, this genetic variability may directly alter the flocculin structure, which in turn may affect the flocculation onset and/or strength in an uncontrolled manner. Here, 16 ale and lager yeast strains from different breweries, one laboratory Saccharomyces cerevisiae and one reference Saccharomyces pastorianus strain, with divergent flocculation strengths, were selected and screened for characteristic FLO gene sequences. Most of the strains could be distinguished by a typical pattern of these FLO gene markers. The FLO1 and FLO10 markers were only present in five out of the 18 yeast strains, while the FLO9 marker was ubiquitous in all the tested strains. Surprisingly, three strongly flocculating ale yeast strains in this screening also share a typical ‘lager’ yeast FLO gene marker. Further analysis revealed that a complete Lg-FLO1 allele was present in these ale yeasts. Taken together, this explicit genetic variation between flocculation genes hampers attempts to understand and control the flocculation behavior in industrial brewer’s yeasts.

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.

Indexing the Pseudomonas specialized metabolome enabled the discovery of poaeamide B and the bananamides

Pseudomonads are cosmopolitan microorganisms able to produce a wide array of specialized metabolites. These molecules allow Pseudomonas to scavenge nutrients, sense population density and enhance or inhibit growth of competing microorganisms. However, these valuable metabolites are typically characterized one-molecule–one-microbe at a time, instead of being inventoried in large numbers. To index and map the diversity of molecules detected from these organisms, 260 strains of ecologically diverse origins were subjected to mass-spectrometry-based molecular networking. Molecular networking not only enables dereplication of molecules, but also sheds light on their structural relationships. Moreover, it accelerates the discovery of new molecules. Here, by indexing the Pseudomonas specialized metabolome, we report the molecular-networking-based discovery of four molecules and their evolutionary relationships: a poaeamide analogue and a molecular subfamily of cyclic lipopeptides, bananamides 1, 2 and 3. Analysis of their biosynthetic gene cluster shows that it constitutes a distinct evolutionary branch of the Pseudomonas cyclic lipopeptides. Through analysis of an additional 370 extracts of wheat-associated Pseudomonas, we demonstrate how the detailed knowledge from our reference index can be efficiently propagated to annotate complex metabolomic data from other studies, akin to the way in which newly generated genomic information can be compared to data from public databases.

Structural Determinants for Activity and Specificity of the Bacterial Toxin LlpA

Lectin-like bacteriotoxic proteins, identified in several plant-associated bacteria, are able to selectively kill closely related species, including several phytopathogens, such as Pseudomonas syringae and Xanthomonas species, but so far their mode of action remains unrevealed. The crystal structure of LlpABW, the prototype lectin-like bacteriocin from Pseudomonas putida, reveals an architecture of two monocot mannose-binding lectin (MMBL) domains and a C-terminal β-hairpin extension. The C-terminal MMBL domain (C-domain) adopts a fold very similar to MMBL domains from plant lectins and contains a binding site for mannose and oligomannosides. Mutational analysis indicates that an intact sugar-binding pocket in this domain is crucial for bactericidal activity. The N-terminal MMBL domain (N-domain) adopts the same fold but is structurally more divergent and lacks a functional mannose-binding site. Differential activity of engineered N/C-domain chimers derived from two LlpA homologues with different killing spectra, disclosed that the N-domain determines target specificity. Apparently this bacteriocin is assembled from two structurally similar domains that evolved separately towards dedicated functions in target recognition and bacteriotoxicity.

Plant lectin‐like antibacterial proteins from phytopathogens Pseudomonas syringae and Xanthomonas citri

The genomes of Pseudomonas syringae pv. syringae 642 and Xanthomonas citri pv. malvacearum LMG 761 each carry a putative homologue of the plant lectin-like bacteriocin (llpA) genes previously identified in the rhizosphere isolate Pseudomonas putida BW11M1 and the biocontrol strain Pseudomonas fluorescens Pf-5. The respective purified recombinant proteins, LlpAPss642 and LlpAXcm761 , display genus-specific antibacterial activity across species boundaries. The inhibitory spectrum of the P. syringae bacteriocin overlaps partially with those of the P. putida and P. fluorescens LlpAs. Notably, Xanthomonas axonopodis pv. citri str. 306 secretes a protein identical to LlpAXcm761 . The functional characterization of LlpA proteins from two different phytopathogenic γ-proteobacterial species expands the lectin-like bacteriocin family beyond the Pseudomonas genus and suggests its involvement in competition among closely related plant-associated bacteria with different lifestyles.

Different Ancestries of R Tailocins in Rhizospheric Pseudomonas Isolates

Bacterial genomes accommodate a variety of mobile genetic elements, including bacteriophage-related clusters that encode phage tail-like protein complexes playing a role in interactions with eukaryotic or prokaryotic cells. Such tailocins are unable to replicate inside target cells due to the lack of a phage head with associated DNA. A subset of tailocins mediate antagonistic activities with bacteriocin-like specificity. Functional characterization of bactericidal tailocins of two Pseudomonas putida rhizosphere isolates revealed not only extensive similarity with the tail assembly module of the Pseudomonas aeruginosa R-type pyocins but also differences in genomic integration site, regulatory genes, and lytic release modules. Conversely, these three features are quite similar between strains of the P. putida and Pseudomonas fluorescens clades, although phylogenetic analysis of tail genes suggests them to have evolved separately. Unlike P. aeruginosa R pyocin elements, the tailocin gene clusters of other pseudomonads frequently carry cargo genes, including bacteriocins. Compared with P. aeruginosa, the tailocin tail fiber sequences that act as specificity determinants have diverged much more extensively among the other pseudomonad species, mostly isolates from soil and plant environments. Activity of the P. putida antibacterial particles requires a functional lipopolysaccharide layer on target cells, but contrary to R pyocins from P. aeruginosa, strain susceptibilities surpass species boundaries.

Functional divergence of gene duplicates through ectopic recombination

Gene duplication stimulates evolutionary innovation as the resulting paralogs acquire mutations that lead to sub‐ or neofunctionalization. A comprehensive in silico analysis of paralogs in Saccharomyces cerevisiae reveals that duplicates of cell‐surface and subtelomeric genes also undergo ectopic recombination, which leads to new chimaeric alleles. Mimicking such intergenic recombination events in the FLO (flocculation) family of cell‐surface genes shows that chimaeric FLO alleles confer different adhesion phenotypes than the parental genes. Our results indicate that intergenic recombination between paralogs can generate a large set of new alleles, thereby providing the raw material for evolutionary adaptation and innovation.

Identification and functional analysis of a bacteriocin, pyocin S6, with ribonuclease activity from a Pseudomonas aeruginosa cystic fibrosis clinical isolate

S‐type pyocins are bacteriocins produced by Pseudomonas aeruginosa isolates to antagonize or kill other strains of the same species. They have a modular organization comprising a receptor‐binding domain recognizing a surface constituent of the target bacterium, a domain for translocation through the periplasm, and a killing or toxic domain with DNase, tRNase, or pore‐forming activity. Pyocins S2, S3, S4, and S5 recognize TonB‐dependent ferri‐siderophore receptors in the outer membrane. We here describe a new nuclease bacteriocin, pyocin S6, encoded in the genome of a P. aeruginosa cystic fibrosis (CF) clinical isolate, CF_PA39. Similarly to pyocins S1 and S2, the S6 toxin–immunity gene tandem was recruited to the genomic region encoding exotoxin A. The pyocin S6 receptor‐binding and translocation domains are identical to those of pyocin S1, whereas the killing domain is similar to the 16S ribonuclease domain of Escherichia coli colicin E3. The cytotoxic activity was abolished in pyocin S6 forms with a mutation in the colicin E3‐equivalent catalytic motif. The CF_PA39 S6 immunity gene displays a higher expression level than the gene encoding the killing protein, the latter being only detected when bacteria are grown under iron‐limiting conditions. In the S1‐pyocinogenic strain P. aeruginosa ATCC 25324 and pyocin S2 producer P. aeruginosa PAO1, a remnant of the pyocin S6 killing domain and an intact S6‐type immunity gene are located downstream of their respective pyocin operons. Strain PAO1 is insensitive for pyocin S6, and its S6‐type immunity gene provides protection against pyocin S6 activity. Purified pyocin S6 inhibits one‐fifth of 110 P. aeruginosa CF clinical isolates tested, showing clearer inhibition zones when the target cells are grown under iron limitation. In this panel, about half of the CF clinical isolates were found to host the S6 genes. The pyocin S6 locus is also present in the genome of some non‐CF clinical isolates.