Jacob G. Malone

The YfiBNR Signal Transduction Mechanism Reveals Novel Targets for the Evolution of Persistent Pseudomonas aeruginosa in Cystic Fibrosis Airways

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs

Pseudomonas aeruginosa is an opportunistic pathogen that predominates during the later stages of cystic fibrosis (CF) lung infections. Over many years of chronic lung colonization, P. aeruginosa undergoes extensive adaptation to the lung environment, evolving both toward a persistent, low virulence state and simultaneously diversifying to produce a number of phenotypically distinct morphs. These lung-adapted P. aeruginosa strains include the small colony variants (SCVs), small, autoaggregative isolates that show enhanced biofilm formation, strong attachment to surfaces, and increased production of exopolysaccharides. Their appearance in the sputum of CF patients correlates with increased resistance to antibiotics, poor lung function, and prolonged persistence of infection, increasing their relevance as a subject for clinical investigation. The evolution of SCVs in the CF lung is associated with overproduction of the ubiquitous bacterial signaling molecule cyclic-di-GMP, with increased cyclic-di-GMP levels shown to be responsible for the SCV phenotype in a number of different CF lung isolates. Here, we review the current state of research in clinical P. aeruginosa SCVs. We will discuss the phenotypic characteristics underpinning the SCV morphotype, the clinical implications of lung colonization with SCVs, and the molecular basis and clinical evolution of the SCV phenotype in the CF lung environment.

Bacterial Rotary Export ATPases Are Allosterically Regulated by the Nucleotide Second Messenger Cyclic-di-GMP

Background: AAA+ ATPase proteins play integral roles in the export apparatus of many bacterial organelles. Results: The second messenger cyclic di-GMP binds specifically to multiple export ATPases at a highly conserved binding site. Conclusion: Cyclic di-GMP binding is central to the function of many different bacterial export complexes. Significance: This profoundly affects our understanding of numerous important bacterial organelles, including flagella, type III, and type VI secretion systems. The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.

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.

Adaptive Remodeling of the Bacterial Proteome by Specific Ribosomal Modification Regulates Pseudomonas Infection and Niche Colonisation.

Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome.

Bacterial pathogenesis of plants: future challenges from a microbial perspective: Challenges in Bacterial Molecular Plant Pathology.

Summary Future Challenges in Plant Pathology Plant infection is a complicated process. On encountering a plant, pathogenic microorganisms must first adapt to life on the epiphytic surface, and survive long enough to initiate an infection. Responsiveness to the environment is critical throughout infection, with intracellular and community‐level signal transduction pathways integrating environmental signals and triggering appropriate responses in the bacterial population. Ultimately, phytopathogens must migrate from the epiphytic surface into the plant tissue using motility and chemotaxis pathways. This migration is coupled with overcoming the physical and chemical barriers to entry into the plant apoplast. Once inside the plant, bacteria use an array of secretion systems to release phytotoxins and protein effectors that fulfil diverse pathogenic functions (Fig. 1) (Melotto and Kunkel, 2013; Phan Tran et al., 2011). As our understanding of the pathways and mechanisms underpinning plant pathogenicity increases, a number of central research challenges are emerging that will profoundly shape the direction of research in the future. We need to understand the bacterial phenotypes that promote epiphytic survival and surface adaptation in pathogenic bacteria. How do these pathways function in the context of the plant‐associated microbiome, and what impact does this complex microbial community have on the onset and severity of plant infections? The huge importance of bacterial signal transduction to every stage of plant infection is becoming increasingly clear. However, there is a great deal to learn about how these signalling pathways function in phytopathogenic bacteria, and the contribution they make to various aspects of plant pathogenicity. We are increasingly able to explore the structural and functional diversity of small‐molecule natural products from plant pathogens. We need to acquire a much better understanding of the production, deployment, functional redundancy and physiological roles of these molecules. Type III secretion systems (T3SSs) are important and well‐studied contributors to bacterial disease. Several key unanswered questions will shape future investigations of these systems. We need to define the mechanism of hierarchical and temporal control of effector secretion. For successful infection, effectors need to interact with host components to exert their function. Advanced biochemical, proteomic and cell biological techniques will enable us to study the function of effectors inside the host cell in more detail and on a broader scale. Population genomics analyses provide insight into evolutionary adaptation processes of phytopathogens. The determination of the diversity and distribution of type III effectors (T3Es) and other virulence genes within and across pathogenic species, pathovars and strains will allow us to understand how pathogens adapt to specific hosts, the evolutionary pathways available to them, and the possible future directions of the evolutionary arms race between effectors and molecular plant targets. Although pathogenic bacteria employ a host of different virulence and proliferation strategies, as a result of the space constraints, this review focuses mainly on the hemibiotrophic pathogens. We discuss the process of plant infection from the perspective of these important phytopathogens, and highlight new approaches to address the outstanding challenges in this important and fast‐moving field.

High levels of cyclic-di-GMP in plant-associated Pseudomonas correlate with evasion of plant immunity

Summary The plant innate immune system employs plasma membrane‐localized receptors that specifically perceive pathogen/microbe‐associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern‐triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant‐associated bacteria. Here, we show that cyclic‐di‐GMP [bis‐(3′‐5′)‐cyclic di‐guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic‐di‐GMP levels in the pathogen P seudomonas syringae pv. tomato (P to) DC3000, the opportunist P . aeruginosa  PAO1 and the commensal P . protegens  Pf‐5 inhibit flagellin synthesis and help the bacteria to evade FLS2‐mediated signalling in N icotiana benthamiana and A rabidopsis thaliana. Despite this, high cellular cyclic‐di‐GMP concentrations were shown to drastically reduce the virulence of P to  DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic‐di‐GMP signalling on bacterial behaviour.

A Minimal Threshold of c-di-GMP Is Essential for Fruiting Body Formation and Sporulation in Myxococcus xanthus

Generally, the second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-di-GMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be—at least partially—functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus.

An analysis of Pseudomonas genomic diversity in take-all infected wheat fields reveals the lasting impact of wheat cultivars on the soil microbiota

Summary Manipulation of the soil microbiota associated with crop plants has huge promise for the control of crop pathogens. However, to fully realize this potential we need a better understanding of the relationship between the soil environment and the genes and phenotypes that enable microbes to colonize plants and contribute to biocontrol. A recent 2 years of investigation into the effect of wheat variety on second year crop yield in the context of take‐all fungal infection presented the opportunity to examine soil microbiomes under closely defined field conditions. Amplicon sequencing of second year soil samples showed that P seudomonas spp. were particularly affected by the wheat cultivar grown in year one. Consequently, 318 rhizosphere‐associated P seudomonas fluorescens strains were isolated and characterized across a variety of genetic and phenotypic traits. Again, the wheat variety grown in the first year of the study was shown to exert considerable selective pressure on both the extent and nature of P seudomonas genomic diversity. Furthermore, multiple significant correlations were identified within the phenotypic/genetic structure of the Pseudomonas population, and between individual genotypes and the external wheat field environment. The approach outlined here has considerable future potential for our understanding of plant–microbe interactions, and for the broader analysis of complex microbial communities.

Life in earth – the root microbiome to the rescue?

Manipulation of the soil microbiome holds great promise for contributing to more environmentally benign agriculture, with soil microbes such as Pseudomonas promoting plant growth and effectively suppressing pathogenic microorganisms. Next-generation sequencing has enabled a new generation of research into soil microbiomes, presenting the opportunity to better understand and exploit these valuable resources. Soil bacterial communities are both highly complex and variable, and contain vast interspecies and intraspecies diversity, both of which respond to environmental variation. Therefore, we propose that a combination of whole microbiome analyses with in-depth examination of key microbial taxa will likely prove the most effective approach to understanding rhizosphere microbial interactions. This review highlights recent efforts in this direction, based around the important biocontrol bacterium Pseudomonas fluorescens.