ABSTRACT
Cyanobacteria are photosynthetic prokaryotes that are influential in global geochemistry and are promising candidates for industrial applications. Because the livelihood of cyanobacteria is directly dependent upon light, a comprehensive understanding of metabolism in these organisms requires taking into account the effects of day-night transitions and circadian regulation. These events synchronize intracellular processes with the solar day. Accordingly, metabolism is controlled and structured differently in cyanobacteria than in heterotrophic bacteria. Thus, the approaches applied to engineering heterotrophic bacteria will need to be revised for the cyanobacterial chassis. Here, we summarize important findings related to diurnal metabolism in cyanobacteria and present open questions in the field.
Subject(s)
Cyanobacteria/physiology , Photoperiod , Photosynthesis , Biotechnology , Circadian Clocks , Cyanobacteria/genetics , Gene Expression Regulation, Bacterial , NADP/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Specialized secretion systems are infamous for their contribution to host-pathogen interactions. Our discovery that the type VI secretion system delivers toxins between bacterial cells has broadened our understanding of how both pathogens and non-pathogens interact with one another, whether within or outside of the host.
Subject(s)
Antibiosis , Bacteria/metabolism , Bacterial Toxins/metabolism , Type VI Secretion Systems/metabolism , Protein TransportABSTRACT
The cyanobacterium Synechococcus elongatus relies upon photosynthesis to drive metabolism and growth. During darkness, Synechococcus stops growing, derives energy from its glycogen stores, and greatly decreases rates of macromolecular synthesis via unknown mechanisms. Here, we show that the stringent response, a stress response pathway whose genes are conserved across bacteria and plant plastids, contributes to this dark adaptation. Levels of the stringent response alarmone guanosine 3'-diphosphate 5'-diphosphate (ppGpp) rise after a shift from light to dark, indicating that darkness triggers the same response in cyanobacteria as starvation in heterotrophic bacteria. High levels of ppGpp are sufficient to stop growth and dramatically alter many aspects of cellular physiology, including levels of photosynthetic pigments and polyphosphate, DNA content, and the rate of translation. Cells unable to synthesize ppGpp display pronounced growth defects after exposure to darkness. The stringent response regulates expression of a number of genes in Synechococcus, including ribosomal hibernation promoting factor (hpf), which causes ribosomes to dimerize in the dark and may contribute to decreased translation. Although the metabolism of Synechococcus differentiates it from other model bacterial systems, the logic of the stringent response remains remarkably conserved, while at the same time having adapted to the unique stresses of the photosynthetic lifestyle.
Subject(s)
Adaptation, Physiological , Synechococcus/physiology , Acclimatization , DNA Replication , Darkness , Gene Expression Regulation, Bacterial , Guanosine Tetraphosphate/analysis , Guanosine Tetraphosphate/physiology , PhotosynthesisABSTRACT
Many carbon-fixing bacteria rely on a CO2 concentrating mechanism (CCM) to elevate the CO2 concentration around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The CCM is postulated to simultaneously enhance the rate of carboxylation and minimize oxygenation, a competitive reaction with O2 also catalyzed by RuBisCO. To achieve this effect, the CCM combines two features: active transport of inorganic carbon into the cell and colocalization of carbonic anhydrase and RuBisCO inside proteinaceous microcompartments called carboxysomes. Understanding the significance of the various CCM components requires reconciling biochemical intuition with a quantitative description of the system. To this end, we have developed a mathematical model of the CCM to analyze its energetic costs and the inherent intertwining of physiology and pH. We find that intracellular pH greatly affects the cost of inorganic carbon accumulation. At low pH the inorganic carbon pool contains more of the highly cell-permeable H2CO3, necessitating a substantial expenditure of energy on transport to maintain internal inorganic carbon levels. An intracellular pH ≈8 reduces leakage, making the CCM significantly more energetically efficient. This pH prediction coincides well with our measurement of intracellular pH in a model cyanobacterium. We also demonstrate that CO2 retention in the carboxysome is necessary, whereas selective uptake of HCO3 (-) into the carboxysome would not appreciably enhance energetic efficiency. Altogether, integration of pH produces a model that is quantitatively consistent with cyanobacterial physiology, emphasizing that pH cannot be neglected when describing biological systems interacting with inorganic carbon pools.
Subject(s)
Carbon Dioxide/metabolism , Cyanobacteria/metabolism , Energy Metabolism , Photosynthesis/genetics , Biological Transport/genetics , Carbon/metabolism , Carbon Cycle/genetics , Carbon Cycle/physiology , Carbonic Anhydrases/genetics , Carbonic Anhydrases/metabolism , Cyanobacteria/genetics , Cyanobacteria/growth & development , Hydrogen-Ion Concentration , Ribulose-Bisphosphate Carboxylase/genetics , Ribulose-Bisphosphate Carboxylase/metabolismABSTRACT
Cyanobacteria are a diverse bacterial phylum whose members possess a high degree of ultrastructural organization and unique gene regulatory mechanisms. Unraveling this complexity will require the use of live-cell fluorescence microscopy, but is impeded by the inherent fluorescent background associated with light-harvesting pigments and the need to feed photosynthetic cells light. Here, we outline a roadmap for overcoming these challenges. Specifically, we show that although basic cyanobacterial biology creates challenging experimental constraints, these restrictions can be mitigated by the careful choice of fluorophores and microscope instrumentation. Many of these choices are motivated by recent successful live-cell studies. We therefore also highlight how live-cell imaging has advanced our understanding of bacterial microcompartments, circadian rhythm, and the organization and segregation of the bacterial nucleoid.
Subject(s)
Cyanobacteria/physiology , Microscopy/methods , Bacteriological Techniques , Circadian Rhythm , Cyanobacteria/cytology , Equipment Design , Fluorescent Dyes/analysis , Image Processing, Computer-Assisted/methods , Microscopy/instrumentation , Microscopy, Fluorescence , Photosynthesis , Pigments, Biological/chemistry , Pigments, Biological/metabolism , PolyploidyABSTRACT
Interbacterial interaction pathways play an important role in defining the structure and complexity of bacterial associations. A quantitative description of such pathways offers promise for understanding the forces that contribute to community composition. We developed time-lapse fluorescence microscopy methods for quantitation of interbacterial interactions and applied these to the characterization of type VI secretion (T6S) in Pseudomonas aeruginosa. Our analyses allowed a direct determination of the efficiency of recipient cell lysis catalyzed by this intercellular toxin delivery pathway and provided evidence that its arsenal extends beyond known effector proteins. Measurement of T6S apparatus localization revealed correlated activation among neighboring cells, which, taken together with genetic data, implicate the elaboration of a functional T6S apparatus with a marked increase in susceptibility to intoxication. This possibility was supported by the identification of T6S-inactivating mutations in a genome-wide screen for resistance to T6S-mediated intoxication and by time-lapse fluorescence microscopy analyses showing a decreased lysis rate of recipient cells lacking T6S function. Our discoveries highlight the utility of single-cell approaches for measuring interbacterial phenomena and provide a foundation for studying the contribution of a widespread bacterial interaction pathway to community structure.
Subject(s)
Pseudomonas aeruginosa/physiology , Microscopy, FluorescenceABSTRACT
Sophisticated mechanisms are employed to facilitate information exchange between interfacing bacteria. A type VI secretion system (T6SS) of Pseudomonas aeruginosa was shown to deliver cell wall-targeting effectors to neighboring cells. However, the generality of bacteriolytic effectors and, moreover, of antibacterial T6S remained unknown. Using parameters derived from experimentally validated bacterial T6SS effectors we identified a phylogenetically disperse superfamily of T6SS-associated peptidoglycan-degrading effectors. The effectors separate into four families composed of peptidoglycan amidase enzymes of differing specificities. Effectors strictly co-occur with cognate immunity proteins, indicating that self-intoxication is a general property of antibacterial T6SSs and effector delivery by the system exerts a strong selective pressure in nature. The presence of antibacterial effectors in a plethora of organisms, including many that inhabit or infect polymicrobial niches in the human body, suggests that the system could mediate interbacterial interactions of both environmental and clinical significance.
Subject(s)
Amidohydrolases/genetics , Bacteria/genetics , Bacterial Secretion Systems/genetics , Amidohydrolases/metabolism , Antibiosis , Bacteria/metabolism , Bacterial Physiological Phenomena , Peptidoglycan/metabolismABSTRACT
Productive intercellular delivery of cargo by secretory systems requires exquisite temporal and spatial choreography. Our laboratory has demonstrated that the haemolysin co-regulated secretion island I (HSI-I)-encoded type VI secretion system (H1-T6SS) of Pseudomonas aeruginosa transfers effector proteins to other bacterial cells. The activity of these effectors requires cell contact-dependent delivery by the secretion apparatus, and thus their export is highly repressed under planktonic growth conditions. Here we define regulatory pathways that orchestrate efficient secretion by this system. We identified a T6S-associated protein, TagF, as a posttranslational repressor of the H1-T6SS. Strains activated by TagF derepression or stimulated through a previously identified threonine phosphorylation pathway (TPP) share the property of secretory ATPase recruitment to the T6S apparatus, yet display different effector output levels and genetic requirements for their export. We also found that these two pathways respond to distinct stimuli; we identified surface growth as a physiological cue that activates the H1-T6SS exclusively through the TPP. Coordination of posttranslational triggering with cell contact-promoting growth conditions provides a mechanism for the T6SS to avoid wasteful release of effectors.
Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Membrane Transport Proteins/metabolism , Pseudomonas aeruginosa/physiology , Genomic Islands , Phosphorylation , Protein Transport , Pseudomonas aeruginosa/geneticsABSTRACT
Peptidoglycan is the major structural constituent of the bacterial cell wall, forming a meshwork outside the cytoplasmic membrane that maintains cell shape and prevents lysis. In Gram-negative bacteria, peptidoglycan is located in the periplasm, where it is protected from exogenous lytic enzymes by the outer membrane. Here we show that the type VI secretion system of Pseudomonas aeruginosa breaches this barrier to deliver two effector proteins, Tse1 and Tse3, to the periplasm of recipient cells. In this compartment, the effectors hydrolyse peptidoglycan, thereby providing a fitness advantage for P. aeruginosa cells in competition with other bacteria. To protect itself from lysis by Tse1 and Tse3, P. aeruginosa uses specific periplasmically localized immunity proteins. The requirement for these immunity proteins depends on intercellular self-intoxication through an active type VI secretion system, indicating a mechanism for export whereby effectors do not access donor cell periplasm in transit.
Subject(s)
Bacterial Proteins/metabolism , Bacterial Secretion Systems , Bacteriolysis , Gram-Negative Bacteria/cytology , Gram-Negative Bacteria/metabolism , Microbial Interactions , Pseudomonas aeruginosa/metabolism , Amidohydrolases/chemistry , Amidohydrolases/genetics , Amidohydrolases/metabolism , Amino Acid Sequence , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/metabolism , Hydrolysis , Muramidase/chemistry , Muramidase/genetics , Muramidase/metabolism , Peptidoglycan/metabolism , Periplasm/metabolism , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/growth & development , Pseudomonas putida/growth & development , Pseudomonas putida/metabolism , Substrate SpecificityABSTRACT
The identification of bacterial secretion systems capable of translocating substrates into eukaryotic cells via needle-like appendages has opened fruitful and exciting areas of microbial pathogenesis research. The recent discovery of the type VI secretion system (T6SS) was met with early speculation that it too acts as a 'needle' that pathogens aim at host cells. New reports demonstrate that certain T6SSs are potent mediators of interbacterial interactions. In light of these findings, we examined earlier data indicating its role in pathogenesis. We conclude that although T6S can, in rare instances, directly influence interactions with higher organisms, the broader physiological significance of the system is likely to provide defense against simple eukaryotic cells and other bacteria in the environment. The crucial role of T6S in bacterial interactions, along with its presence in many organisms relevant to disease, suggests that it might be a key determinant in the progression and outcome of certain human polymicrobial infections.
Subject(s)
Bacteria/metabolism , Bacterial Secretion Systems , Bacteria/pathogenicity , Bacterial Infections/microbiology , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Host-Pathogen Interactions , HumansABSTRACT
Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.
Subject(s)
Bacterial Proteins/immunology , Bacterial Secretion Systems/immunology , Burkholderia/immunology , Burkholderia/pathogenicity , Host-Parasite Interactions/immunology , Animals , Bacteria/genetics , Bacteria/growth & development , Bacteria/metabolism , Bacterial Proteins/genetics , Bacterial Secretion Systems/genetics , Biofilms/growth & development , Burkholderia/genetics , Burkholderia Infections/genetics , Burkholderia Infections/immunology , Host-Parasite Interactions/genetics , Mice , Phylogeny , Virulence/genetics , Virulence/immunologyABSTRACT
The functional spectrum of a secretion system is defined by its substrates. Here we analyzed the secretomes of Pseudomonas aeruginosa mutants altered in regulation of the Hcp Secretion Island-I-encoded type VI secretion system (H1-T6SS). We identified three substrates of this system, proteins Tse1-3 (type six exported 1-3), which are coregulated with the secretory apparatus and secreted under tight posttranslational control. The Tse2 protein was found to be the toxin component of a toxin-immunity system and to arrest the growth of prokaryotic and eukaryotic cells when expressed intracellularly. In contrast, secreted Tse2 had no effect on eukaryotic cells; however, it provided a major growth advantage for P. aeruginosa strains, relative to those lacking immunity, in a manner dependent on cell contact and the H1-T6SS. This demonstration that the T6SS targets a toxin to bacteria helps reconcile the structural and evolutionary relationship between the T6SS and the bacteriophage tail and spike.