12 research outputs found
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Metabolic Strategies to Cope with Overcrowding in a Pseudomonas aeruginosa Biofilm
Bacteria, while traditionally studied in liquid suspensions, are often found in nature as biofilms, aggregates of cells enclosed in self-produced matrices. Cells in biofilms have a fitness advantage over those that are free-living, as the biofilm lifestyle is correlated with increased resistance to various assaults, including antimicrobials, UV exposure, and dehydration. These biofilm-associated characteristics have important clinical implications, as biofilm-based bacterial infections are a major cause of morbidity in immunocompromised individuals. With this increased resiliency, however, comes a major challenge that arises during biofilm growth: the formation of resource gradients. My thesis work focused on one particular gradient, that of oxygen, which is established in biofilms formed by Pseudomonas aeruginosa. This bacterium has multiple mechanisms for coping with limited access to oxygen, including a highly-branched respiratory system for optimal oxygen scavenging and production and utilization of redox-active molecules called phenazines. The purpose of this thesis has been to investigate the different strategies used by P. aeruginosa to deal with the oxygen limitation precipitated by the biofilm lifestyle.
In Chapter 1, I will provide the necessary background for understanding the principles of redox balancing, metabolism, respiration, biofilm physiology, and phenazine utilization in P. aeruginosa. The work described in Chapter 2 provides evidence for the formation of a novel terminal oxidase complex that plays a biofilm-specific role in P. aeruginosa growth. The results in this chapter also suggest that specific terminal oxidase complexes differ in the timing of their contributions to biofilm growth and implicate the novel complex in mediating reduction of phenazines in biofilms.
Chapter 3 expands upon the principle of metabolic versatility exemplified by the results discussed in Chapter 2. The research presented in this chapter looks at how varying the source of electrons that feed into the respiratory chain influences downstream electron transfer steps, including terminal oxidase activities and phenazine production and utilization. The data presented in Chapters 2 and 3 add to the growing body of evidence that bacterial growth in liquid culture is distinct from that in biofilms and underscores the need for more biofilm-based research that can inform treatment strategies for P. aeruginosa infections.
The results described in Chapter 4 take an even broader look at the strategies used by P. aeruginosa to sustain efficient metabolism under conditions of potential stress. An important node of central metabolism is pyruvate, which can be transformed in a number of ways. In this chapter, I will consider two pathways of pyruvate metabolism: fermentation to lactate and carboxylation to oxaloacetate. I will present data indicating that a previously-uncharacterized lactate dehydrogenase contributes to P. aeruginosa growth under specific growth conditions and that pyruvate carboxylation contributes to optimal progress through central metabolic pathways. I will also describe experiments that characterize the contributions of another carboxylase, previously thought to function as the pyruvate carboxylase, to P. aeruginosa’s ability to grow on selected nutrient sources. Finally, I will discuss how redox state informs biofilm formation in a phylogenetically distinct bacterium, Bacillus subtilis, highlighting the universality of redox reactions in driving metabolic processes.
In sum, the research presented in this thesis broadens our understanding of the immense respiratory and metabolic flexibility of P. aeruginosa and serves as an important reminder of the discrete factors that govern liquid culture and biofilm growth
An Aerobic Exercise: Defining the Roles of Pseudomonas aeruginosa Terminal Oxidases
The opportunistic pathogen Pseudomonas aeruginosa encodes a large and diverse complement of aerobic terminal oxidases, which is thought to contribute to its ability to thrive in settings with low oxygen availability. In this issue, Arai et al. (J. Bacteriol. 196:4206 – 4215, 2014, doi:http://dx.doi.org/10.1128/JB.02176-14) present a thorough characterization of these five complexes, enabling a more detailed understanding of aerobic respiration in this organism
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An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence
Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e. ‘orphan’) operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen Pseudomonas aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets
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Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase
Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds 1–6. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase (ACC) is a target for drug discovery against diabetes, cancer and other diseases 7–9. We report here the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kD), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active toward short- and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase (LCC). The holoenzyme is a homo-hexamer with molecular weight of 720 kD. The 3.0 Å crystal structure of Mycobacterium avium subspecies paratuberculosis LCC (MapLCC) holoenzyme revealed an architecture that is strikingly different compared to those of related biotin-dependent carboxylases 10,11. In addition, the domains of each monomer have no direct contacts with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources
Spatial alanine metabolism determines local growth dynamics of textitEscherichia coli colonies
Bacteria commonly live in spatially structured biofilm assemblages, which are encased by an extracellular matrix. Metabolic activity of the cells inside biofilms causes gradients in local environmental conditions, which leads to the emergence of physiologically differentiated subpopulations. Information about the properties and spatial arrangement of such metabolic subpopulations, as well as their interaction strength and interaction length scales are lacking, even for model systems like textitEscherichia coli colony biofilms grown on agar-solidified media. Here, we use an unbiased approach, based on temporal and spatial transcriptome and metabolome data acquired during textitE. coli colony biofilm growth, to study the spatial organization of metabolism. We discovered that alanine displays a unique pattern among amino acids and that alanine metabolism is spatially and temporally heterogeneous. At the anoxic base of the colony, where carbon and nitrogen sources are abundant, cells secrete alanine textitvia the transporter AlaE. In contrast, cells utilize alanine as a carbon and nitrogen source in the oxic nutrient-deprived region at the colony mid-height, textitvia the enzymes DadA and DadX. This spatially structured alanine cross-feeding influences cellular viability and growth in the cross-feeding-dependent region, which shapes the overall colony morphology. More generally, our results on this precisely controllable biofilm model system demonstrate a remarkable spatiotemporal complexity of metabolism in biofilms. A better characterization of the spatiotemporal metabolic heterogeneities and dependencies is essential for understanding the physiology, architecture, and function of biofilms
Bifunctionality of a biofilm matrix protein controlled by redox state
Significance
The biofilm matrix is a critical target in the hunt for novel strategies to destabilize or stabilize biofilms. Knowledge of the processes controlling matrix assembly is therefore an essential prerequisite to exploitation. Here, we highlight that the complexity of the biofilm matrix is even higher than anticipated, with one matrix component making two independent functional contributions to the community. The influence the protein exerts is dependent on the local environmental properties, providing another dimension to consider during analysis. These findings add to the evidence that bacteria can evolve multifunctional uses for the extracellular matrix components.</jats:p
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The Pseudomonas aeruginosa Complement of Lactate Dehydrogenases Enables Use of d- and l-Lactate and Metabolic Cross-Feeding
Lactate is thought to serve as a carbon and energy source during chronic infections. Sites of bacterial colonization can contain two enantiomers of lactate: the l-form, generally produced by the host, and the d-form, which is usually produced by bacteria, including the pulmonary pathogen Pseudomonas aeruginosa. Here, we characterize P. aeruginosa’s set of four enzymes that it can use to interconvert pyruvate and lactate, the functions of which depend on the availability of oxygen and specific enantiomers of lactate. We also show that anaerobic pyruvate fermentation triggers production of the aerobic d-lactate dehydrogenase in both liquid cultures and biofilms, thereby enabling metabolic cross-feeding of lactate over time and space between subpopulations of cells. These metabolic pathways might contribute to P. aeruginosa growth and survival in the lung.Pseudomonas aeruginosa is the most common cause of chronic, biofilm-based lung infections in patients with cystic fibrosis (CF). Sputum from patients with CF has been shown to contain oxic and hypoxic subzones as well as millimolar concentrations of lactate. Here, we describe the physiological roles and expression patterns of P. aeruginosa lactate dehydrogenases in the contexts of different growth regimes. P. aeruginosa produces four enzymes annotated as lactate dehydrogenases, three of which are known to contribute to anaerobic or aerobic metabolism in liquid cultures. These three are LdhA, which reduces pyruvate to d-lactate during anaerobic survival, and LldE and LldD, which oxidize d-lactate and l-lactate, respectively, during aerobic growth. We demonstrate that the fourth enzyme, LldA, performs redundant l-lactate oxidation during growth in aerobic cultures in both a defined MOPS (morpholinepropanesulfonic acid)-based medium and synthetic CF sputum media. However, LldA differs from LldD in that its expression is induced specifically by the l-enantiomer of lactate. We also show that the P. aeruginosa lactate dehydrogenases perform functions in colony biofilms that are similar to their functions in liquid cultures. Finally, we provide evidence that the enzymes LdhA and LldE have the potential to support metabolic cross-feeding in biofilms, where LdhA can catalyze the production of d-lactate in the anaerobic zone, which is then used as a substrate in the aerobic zone. Together, these observations further our understanding of the metabolic pathways that can contribute to P. aeruginosa growth and survival during CF lung infection
Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence
R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes. PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection