N. Tschowri1, M.A. Schumacher2, S. Schlimpert3, N.B. Chinnam2, R.G. Brennan2, M.J. Buttner3
1Molecular Microbiology, Humboldt University Berlin, Berlin, Germany 2Biochemistry, Duke University School of Medicine, Durham, USA 3Molecular Microbiology, John Innes Centre, Norwich, United Kingdom
Background
Streptomyces are non-motile soil bacteria with a unique mycelial, sporulating life cycle and the ability to produce highly valuable secondary metabolites. Streptomyces venezuelae encodes for 10 putative cyclic di-GMP-metabolizing enzymes but the cellular processes controlled by the cyclic di-nucleotide remained elusive.
Objectives
Aiming to gain more knowledge about the regulation of the developmental processes in S.venezuelae, we found that elevated levels of c-di-GMP lead to a pronounced delay of the developmental program. On the other hand, depletion of c-di-GMP results in an enhanced sporulation phenotype due to bypassing the aerial mycelium stage.
Methods
We applied a global pull-down approach using the c-di-GMP Capture Compound (Caprotec, Berlin) and found a c-di-GMP-responsive regulator that connects c-di- GMP signalling pathways and developmental processes in S. venezuelae. Using structural and biochemical analyses we identified a previously unseen oligomeric form of c-di-GMP, which enables the effector protein to dimerize by binding to a heretofore unknown binding motif.
Conclusions
Altogether, we demonstrate that the signalling molecule c-di-GMP controls the hypha- to-spore transition in filamentous bacteria.
FEMS-2876
Bacterial persistence and Toxin - Antitoxins
Magic Spot Controls Bacterial Persister Cell Formation by Activating Toxin - Antitoxins
K. Gerdes1, E. Maisonneuve1, K. Winther1, E. Germain1, M. Roghanian1,
R. Jørgensen Bager1, M. Fazli1
1Department of Biology, University of Copenhagen, Copenhagen, Denmark
Background
We showed previously that multidrug tolerance (persistence) of the model
organism Escherichia coli K-12, depends stochastic induction of Toxin - Antitoxin (TA) activity by (p)ppGpp in a regulatory cascade that included also Lon protease and polyphosphate (Maisonneuve et al., Cell, 154, 1140-50, 2013). The TAs involved all encode inhibitors of translation, such as RelE, MazF, HicA etc. Remarkably, all antitoxins of E. coli K-12 encoded by type II TA loci are degraded by Lon that, in turn, is activated by polyphosphate. The model explaining stochastic induction of
persistence is shown in Figure 1. Sophie Helaine and David Holden (Imperial College London, UK) showed that a similar mechanism allows cells of Salmonella to survive antibiotic treatment within macrophages (Science 343, 204–208, 2014).
Objectives
Many bacterial pathogens contain multiple type II TA genes and rely on (p)ppGpp to be virulent (Figure 2). We will determine the role(s) that (p)ppGpp and TAs play in persistence and virulence of different bacterial pathogens, including, Mycobacterium tuberculosis, Burkholderia cenocepacia and Photorhabdus luminescence.
Methods
In this work, we will employ physiological, genetic, cytological and biochemical methods to reach our aim.
Conclusions
Preliminary results indicate that indeed (p)ppGpp is required for persistence of bacterial pathogens.
FEMS-3162
Bacterial persistence and Toxin - Antitoxins
FIC domains in Bartonella: Evolution of diversified host effectors from a widely-spread bacterial toxin/antitoxin system
C. Dehio1
1Biozentrum, University of Basel, Basel, Switzerland
The FIC domain being present in thousands of proteins and found in all domains of life mediates post-translational modification of target proteins, typically by transfer of an AMP moiety from ATP onto a target tyrosyl or threonyl side chain by a process called AMPylation or adenylylation. The alpha-proteobacterial genus Bartonella compises facultative intracellular pathogens that utilize a VirB type IV secretion system to translocate a cocktail of bacterial effectors protein into host cells in order to subvert cellular function to the benefit to the bacterial intruder. Diversified copies of the FIC domain are prominently presented in these effector sets, indicating that the various FIC domain variants may mediate diverse effector functions during infection. I will discuss the evolutionary origin of the FIC domain as a bacterial toxin-antitoxin system, its fusion with a type IV secretion signal to constitute an ancestral
translocateable effector, as well as the duplication and diversification of the ancestral effector gene in the course of the evolution of the effector cocktails that facilitate host cell infection by bartonellae in diverse mammals.
FEMS-0655
Bacterial persistence and Toxin - Antitoxins
Novel regulatory mechanisms in toxin-antitoxin modules R. Loris1
1Structural Biology Brussels, Vrije universiteit brussel, Brussel, Belgium
Prokaryotic toxin-antitoxin modules are involved in the establishment of persister cells. The latter involves complex regulatory mechanisms that link the regulation of protein activity to regulation of transcription. Recent structural and biochemical data shows a plethora of molecular mechanisms to achieve this goal. In the phd/doc module, conditional co-operativity is established via a combination of negative co- operativity through entropic exclusion of and IDP domain combined with a low to high affinity switch for the interaction between toxin and antitoxin. While Doc stabilizes the DNA binding conformation of Phd, Phd likewise inhibits the kinase activity of Doc and simultaneously prevents misfolding of Doc. The same phenomenon of conditional co- operativity is observed for mazEF and ccdAB, but with a different structural basis, although intrinsic disorder in the antitoxin is a common theme. Other TA modules such as higBA are regulated through a seemingly more simple mechanism where the antitoxin acts as the sole repressor, while toxin significantly weakens operator
binding. Such a mechanism is observed in several higBA modules as well as in mqsRA and allows for transcription regulation without the specific need of intrinsic disorder.
FEMS-2376
Bacterial persistence and Toxin - Antitoxins Chaperone-mediated control of toxin-antitoxins
P. Bordes1, A. Sala1, N. Slama1, P. Texier1, A.M. Cirinesi1, P. Genevaux1
1Laboratoire de Microbiologie et Génétique Moléculaires, CNRS Université Paul-
Sabatier, Toulouse, France
Bacterial toxin-antitoxin systems are stress-responsive elements generally composed of a stable toxin that forms an inactive complex with its less stable cognate antitoxin. In response to specific stress conditions the antitoxin is degraded by stress proteases and the free active toxin subsequently targets important cellular processes such as DNA replication or protein synthesis. It is believed that the resulting growth inhibition facilitates adaptation to stress and persistence. Mycobacterium tuberculosis, the causing agent of human tuberculosis, encodes 79 putative toxin-antitoxin systems and it has been proposed that persistence induced by active toxins might be relevant for its pathogenesis. The tripartite toxin–antitoxin–chaperone (TAC) system of M. tuberculosis is an atypical toxin-antitoxin system strongly induced in persisters and tightly controlled by the molecular chaperone Rv1957, which is related to the canonical SecB chaperone involved in Sec-dependent protein export in Gram
negative bacteria. Indeed, in spite of very little sequence similarity, Rv1957 is able to efficiently replace SecB during protein export in E. coli, and to specifically control the functional HigB1-HigA1 toxin-antitoxin system of M. tuberculosis. How does the mycobacterial SecB-like chaperone respond to stress and control the toxin activation cascade, and to what extent such activation is important for M. tuberculosis
persistence and virulence are so far unresolved questions.
In this work, we will present recent data concerning the molecular mechanism of TAC complex formation and toxin activation, with emphasis on the role played by the SecB-like chaperone.
FEMS-1011
CRISPR - biological and technological advances
Type II CRISPR-Cas9 systems: mechanisms, evolution and applications