| 1. |
- Wirebrand, Lisa, 1986-
(författare)
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Global regulatory factors that impact metabolic and lifestyle choices in Pseudomonas putida
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pseudomonas putida strains have a broad metabolic capacity and are innately resistant to many harmful substances – properties that make them of interest for a number of industrial and biotechnological application. They can rapidly adapt to changes in physico-chemical parameters in the soil and water environments they naturally inhabit. Like other bacteria, they have evolved both specific and cross-acting global regulatory circuits to control endurance traits and life style choices in order to survive. Three such survival tactics are 1) the ability to control flagella-mediated motility to search for metabolically favourable locations, 2) to produce protective biofilm structures to resist environmental insults, and 3) to distinguish the energetically most favourable carbon source amongst an array on offer. These processes are often co-ordinated regulated by intersecting networks that are controlled by global signalling molecules (second messengers) such as the nucleotides ppGpp and c-di-GMP, and globally acting proteins.In the first part of my thesis I present evidence that the PP4397 protein of P. putida is responsible for slowing down flagella-driven motility in response to c-di-GMP signalling from a dual-functional c-di-GMP turnover protein termed PP2258. This connection is expanded upon to present a potential signal transduction pathway from a surface located receptor to PP2258 and the c-di-GMP responsive PP4397 protein, and from there to the flagella motors to determine flagella performance. The transcriptional regulatory studies that accompany this work suggest a means by which transcriptional control may serve to initiate a co-ordinated blocking of de novo flagella biogenesis and slowing-down flagella rotation – two processes needed to enter the biofilm mode of growth. Exiting from a biofilm matrix is also a c-di-GMP elicited behaviour, prompted when nutrients become scarce. In my second piece of work I present evidence that hunger-signals in the form of ppGpp directly control transcription to elevate the levels of a c-di-GMP degrading protein – BifA – which lies at the heart of programed biofilm dispersal. The final part of my thesis, concerns how the global regulatory proteins Hfq and Crc act at multiple levels to subvert catabolism of phenolics to favour other preferred sources of carbon. Evidence is presented that this involves a two-tiered translational repression – one at the level of the master regulator of the system, and another at the level of the catabolic enzymes. This study also revealed a hitherto unsuspected role of Crc in maintenance of an IncP-2 plasmid within a bacterial population. This latter finding has implications for a wide variety of processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids.
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| 2. |
- Holmfeldt, Linda, 1979-
(författare)
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On the role of small regulatory molecules in the interplay between σ54- and σ70-dependent transcription
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Signal responsive transcriptional control in bacteria is mediated through both specific and global regulatory circuits to attune promoter output to prevailing conditions. Divergent transcription of a regulatory gene and a cognate promoter under its control provides an opportunity for interplay between transcription dependent on RNA polymerases utilizing various σ-factors, each of which programs the holoenzyme to recognize different classes of promoters. The work presented in this thesis analyses the consequences and mechanisms behind interplay between σ54- and σ70-dependent transcription within the dmp-system of Pseudomonas sp. CF600. The dmp-system confers the ability to grow at the expense of (methyl)phenols and is controlled by two promoters that drive non-overlapping divergent transcription from a common intergenic region: i) the σ54-Po promoter, which controls an operon encoding a suit of specialized catabolic enzymes, and ii) the σ70-Pr promoter, which controls production of the aromatic sensor DmpR - a mechano-activator whose transcription-promoting activity is obligatory for activity of the σ54-Po promoter. The σ54-Po promoter and its dependence on two non-classical transcriptional regulators - the alarmone ppGpp and its co-factor DksA that directly target RNA polymerase - are the focus of the first part of the thesis. These studies utilized ppGpp and DksA deficient strains, mutant RNA polymerases that bypass the need for ppGpp and DksA, reconstituted in vitro transcription systems, and a series of DmpR-regulated hybrid σ54-promoters with different affinities for σ54-RNA polymerase, together with analysis of protein levels of key transcriptional components. Collectively with previous work, these studies provide the experimental support for a robust but purely passive mechanism for ppGpp and DksA global regulation of σ54-transcription, which is likely to also be pertinent for transcription mediated via any alternative σ-factor (Papers I-III). The second part of the thesis focuses on additional roles of ppGpp and DksA through their direct and indirect effects on the activity of the σ70-Pr promoter. These studies unexpectedly revealed that the σ70-Pr promoter is regulated by a novel mechanism in which σ54-RNA polymerase occupancy and activity at the σ54-Po promoter stimulates σ70-Pr output. Evidence is presented that ppGpp and DksA, through DmpR levels, control a feed forward loop to reinforce silence of the σ54-Po promoter under high energy conditions with robust transcription from σ54-Po when the catabolic enzymes are needed. The interplay outlined above effectively places a σ70-dependent promoter under dual control of two forms of RNA polymerases, and also makes it subservient to regulatory signals that elicit activity of σ54-RNA polymerase. The possibility that such dual sensitivity may be a prevalent, but previously unappreciated, mechanism by which bacteria integrate diverse and/or conflicting signals to gain appropriate transcriptional control is discussed.
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| 3. |
- Madhushani, W. K. Anjana, 1983-
(författare)
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Multiple regulatory inputs for hierarchical control of phenol catabolism by Pseudomonas putida
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer.Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial.I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence for a model in which Crc and Hfq co-target multiple RNA motifs to bring about a two-tiered regulation to subvert catabolism of phenols in the face of preferred substrates – one at the level of the regulator DmpR and another at the level of translation of the catabolic enzymes.
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| 4. |
- Saha, Chayan Kumar, 1988-
(författare)
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Bioinformatics, evolution and revolution in our understanding of toxin-antitoxin systems
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacteria experience a wide range of natural challenges during their life cycles, to which they must respond and adapt to live. Under stressed conditions such as amino acid starvation, bacteria slow down their growth mechanism by producing small alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp. Accumulation of (p)ppGpp results in a comprehensive alteration in cellular metabolism. The alarmone (p)ppGpp is produced and degraded by enzymes belonging to the RelA/SpoT Homologue (RSH) protein family, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli. The members of the RSH protein family can be classified into long multi-domain RSHs and short single-domain RSHs. Long RSH enzymes such as RelA and SpoT, carry both the (p)ppGpp hydrolysis (HD) domain and (p)ppGpp synthesis (SYNTH) domain in the N-terminal domain enzymatic region (NTD), in combination with additional domains in the C-terminal domain regulatory region (CTD). Short single-domain RSHs can be divided into small alarmone hydrolases (SAHs) carrying the HD domain, and small alarmone synthetases (SASs) that only have the SYNTH domain. At the beginning of my PhD, I studied the diversity of RSH proteins across the tree of life. I identified 35,615 RSH proteins from analyses of 24,072 genomes. I used large-scale phylogenetic analyses to classify the RSH proteins into 13 long RSHs, 11 SAHs and 30 SASs subfamilies. To address why bacteria often carry multiple SASs in the same genome and predict new functions, I developed a computational tool called FlaGs – standing for Flanking Genes – to analyse the conservation of genomic neighbourhoods in large datasets. I also developed a web-based version of FlaGs, called webFlaGs, that is publicly accessible and is used by biologists all over the world. The application of FlaGs to SAS RSHs led to the discovery that multiple SAS subfamilies are encoded in conserved and frequently overlapping two or three-gene architecture, reminiscent of toxin−antitoxin (TA) systems. Five SAS representatives from the FaRel, FaRel2, PhRel, PhRel2 and CapRel subfamilies were experimentally validated as toxins (toxSASs) that are neutralised by the products of six neighbouring antitoxin genes. The toxSAS enzyme FaRel from Cellulomonas marina is encoded as the central gene of a conserved three-gene architecture and acts through the production of nucleotide alarmone ppGpp and its unusual toxic analogue ppApp, causing a significant depletion of ATP and GTP. FaRel toxicity can be countered by both downstream and upstream cognate antitoxins. The latter contains a SAH domain that neutralises toxicity through degradation of ppGpp as well as ppApp. Combining phylogenetic and FlaGs analyses we have discovered that the DUF4065 domain of unknown function is a widely distributed antitoxin domain in putative TA-like operons with dozens of distinct toxic domains. Nine DUF4065-containing antitoxins and their cognate toxins were experimentally validated as TA pairs using toxicity neutralisation assays. These antitoxins form complexes with their diverse cognate toxins. Given the versatility of DUF4065, we have renamed this domain Panacea. We hypothesise that there are multiple hyperpromiscuous antitoxins like Panacea that can be associated with many non-homologous toxin domains, which may also be hyperpromiscuous. Thus, TA systems across all bacteria can be represented as a network of toxin and antitoxin domain combinations, with hyperpromiscuous domains being hubs in the network. To test this and compute a network, I developed a new, iterative version of FlaGs, called NetFlax (standing for Network-FlaGs for toxins and antitoxins), that can identify TA-like gene architectures in an unsupervised manner and generate a toxin-antitoxin domain interaction network. The results from NetFlax verify our strategy since we have rediscovered multiple previously characterised TAs as well as brand new ones. We find that Panacea is unusual but not unique in its hyperpromiscuity and our network indicates the presence of novel hyperpromiscuous domains still to be explored. Our findings also demonstrate how a core network of TAs is evolutionarily linked to various accessory genome systems, including conjugative transfer and phage defence mechanisms. The existing network can potentially be a framework on which future discoveries of the biological role of TAs can be mapped.
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| 5. |
- Seibt, Henrik, 1987-
(författare)
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Deciphering control of Mechano-Transcription Activators of σ54-RNA polymerase
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- To survive and proliferate, bacteria have to respond to a plethora of fluctuating signals within their habitats. Transcriptional control is one crucial entry point for such signal-responsive adaption responses. In this thesis I present new insights into the signal-responsive control of two specific transcriptional regulators that belong to a specialized class of mechano-transcriptional regulators. These regulators employ ATP-hydrolysis to engage and remodel σ54-RNA polymerase, which allows transcriptional initiation from the promoters they control. In the first part of my thesis I present findings on DmpR – the obligate activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR is a sensory-regulator that can only transition to its active multimeric form upon binding a phenolic compound and ATP. Previous work has established that binding of phenolic effectors by the N-terminal domain of DmpR relieves inter-domain repression of its central ATPase domain and further that a structured inter-domain linker between the phenolic- and ATP-binding domains is involved in coupling these processes. However, the mechanism underlying this coupling remained enigmatic. Here I present evidence that a tyrosine residue of the inter-domain linker (Y233) serves as a gatekeeper to constrain ATP-hydrolysis and phenolic-responsive transcriptional activation by DmpR. A model is presented in which binding of phenolics relocates Y233 from the ATP-binding site to synchronise signal-reception with multimerisation to provide appropriate sensitivity of the transcriptional response. Given that Y233 counterparts are present in many ligand-responsive mechano-transcriptional regulators, the model is likely to be pertinent for numerous members of this family. The finding that an alanine substitution of Y233 enhances transcriptional responses adds a new approach to manipulating the sensitivity of this class of proteins and thereby generate hyper-sensitive detectors of aromatic pollutants for use in safe guarding the environment.The second part of my thesis concerns VCA0117 – a master regulator of the type VI contractile nanomachinery of Vibrio cholerae, which it utilizes to introduce toxic proteins into both bacterial and eukaryotic cells. These type VI-mediated properties enable V. cholerae to establish infections and to thrive in niches co-occupied by predators and competing bacteria. VCA0117 is strictly required for functionality of the type VI system through its role in controlling production of a key type VI structural protein called Hcp, which is encoded within two small s54-dependent operons. This regulatory role is conserved in both pandemic and non-pandemic V. cholerae strains. However, while some strains come pre-equipped with a functional system, others do not, and require specific growth conditions of low temperature and high osmolarity for type VI expression. Within this work, integration of these regulatory growth signals was traced to the activity of the promoter controlling a large operon in which many components of the machinery and VCA0117 is itself encoded. This in turn elevates the levels of VCA0117, which is all that is required to overcome the need for the specialized growth conditions of low temperature and/or high osmolarity. A model is presented in which signal integration via the activity of the large operon promoter to elevate levels of VCA0117 ultimately dictates a sufficient supply of the missing Hcp component required for completion of a functional type VI machine. Repercussions of the proposed quantity-based regulatory circuit of VCA0117 for generating bacterial sub-populations that are differentially “fit” for different environmental eventualities are discussed.
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