Hijacking the hijackers: Escherichia coli pathogenicity islands redirect helper phage packaging for their own benefit

Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution

Electrostatic Effects in the Folding of the SH3 Domain of the c-Src Tyrosine Kinase: pH-Dependence in 3D-Domain Swapping and Amyloid Formation

The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6522 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P212121, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging β-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation.This research was funded by the Spanish Ministry of Science and Innovation and Ministry of Economy and Competitiveness and FEDER (EU): BIO2009-13261-C02-01/02 (ACA); BIO2012-39922-C02-01/02 (ACA); CTQ2013-4493 (JLN) and CSD2008-00005 (JLN); Andalusian Regional Government (Spain) and FEDER (EU): P09-CVI-5063 (ACA); and Valentian Regional Government (Spain) and FEDER (EU): Prometeo 2013/018 (JLN). Data collection was supported by European Synchrotron Radiation Facility (ESRF), Grenoble, France: BAG proposals MX-1406 (ACA) and MX-1541 (ACA); and ALBA (Barcelona, Spain) proposals 2012010072 (ACA) and 2012100378 (ACA)

Molecular insights on SaPI inducing mechanism

Abstract del póster presentado al 8th Congress of European Microbiologist. FEMS 2019. Glasgow, Scotland. 7-11 July 2019(Póster. PM296) Background: S. aureus pathogenicity islands (SaPIs) are parasitic mobile genetic elements that exploit phages for induction and transfer. SaPIs integrate to the host chromosome and are repressed by Stl, the SaPI master regulator. SaPI induction is occurred when the SaPI Stl/DNA complex is disrupted via a specific protein encoded by helper phage. The inducer for SaPI1 is a phage protein called Sri. Interestingly, and in addition to SaPI1 de-repression, Sri blocks bacterial DNA replication by binding to the helicase loader protein (DnaI). Objectives: The fact that a small protein like Sri (52aa) interacts with two unrelated proteins raises several interesting questions; are DnaI and Stl sharing similar structural conformations being recognized by the same interacting residues on Sri, or by contrast, Sri has two interacting faces, one to interact with DnaI and the other to interact with Stl? Unraveling those questions would define SaPIs nature. If the DnaI and Stl share a conserved domain or similar fold, SaPIs would be then considered as phage parasites. By contrast, if Sri has two different interacting regions, this would imply in somehow that SaPIs can provide unrecognized advantages for the phage. Methods: To solve the above questions, in vivo and in vitro methods have been used including molecular, biochemical and protein crystallography techniques. Results: Our results provide insights on the mechanism that is used by SaPI1 to interact with Sri protein, highlighting these elements as one of the most fascinating mobiles genetic elements i

Hijacking the hijackers: Escherichia coli phage-inducible chromosomal islands redirect lambda phage packaging for their own benefit

Abstract de la comunicación oral presentada al 8th Congress of European Microbiologist. FEMS 2019. Glasgow, Scotland. 7-11 July 2019(Oral 098) Background: Phage-inducible chromosomal islands (PICIs) are clinically relevant mobile genetic elements widespread among Gram-positive and Gram-negative bacteria. Understanding the different PICI packaging mechanisms is essential to comprehend their intra- and inter-generic transfer, which contribute to bacterial evolution, host adaptation and pathogenesis. Objectives: Hijacking and blocking phage functions is vital for the PICI molecular pirates. They usually encode a battery of genetic resources to interfere with the phage. The objective of this study was to characterise the mechanism employed by EcCICFT073 to hijack the phage TerS, redirecting its affinity to package the PICI dsDNA. Methods: We identified by in vivo (phage evolution, genetic scarless mutations, competition assays) and in vitro (Two-hybrid assays, X-ray crystallography) experiments the molecular mechanism by which EcCICFT073 recruits the phage packaging machinery. Results: This study identified a novel and elegant one-shot strategy used by some cos lambda E. coli PICIs like EcCICFT073. Unlike other cos PICIs that carry the same phage cos sequence, EcCICFT073 carries the lambda phage cosQ and cosN, but a different cosB region (region involved in packaging initiation). Hence, these PICIs have developed a sophisticated strategy by encoding a protein, renamed as Rpp (Redirecting phage packaging) which forms a heterodimer with the phage TerS to perform dual roles: i) forming a new functional hetero DNA-binding (DBD) region that will be used to recognise the PICI cosB site; and ii) this new DBD would be unable to recognise the phage cos site, blocking phage packaging. This novel strategy highlights PICIs as sophisticated parasites in nature

The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability

Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine, the first step in the synthesis of catecholamine neurotransmitters. The protein contains a 159-residue regulatory domain (RD) at its N-terminus that forms dimers in solution; the N-terminal region of RDTyrH (residues 1–71) is absent in the solution structure of the domain. We have characterized the conformational stability of two species of RDTyrH (one containing the N-terminal region and another lacking the first 64 residues) to clarify how that N-terminal region modulates the conformational stability of RD. Under the conditions used in this study, the RD species lacking the first 64 residues is a monomer at pH 7.0, with a small conformational stability at 25 °C (4.7 ± 0.8 kcal mol^–1). On the other hand, the entire RDTyrH is dimeric at physiological pH, with an estimated dissociation constant of 1.6 μM, as determined by zonal gel filtration chromatography; dimer dissociation was spectroscopically silent to circular dichroism but not to fluoresecence. Both RD species were disordered below physiological pH, but the acquisition of secondary native-like structure occurs at pHs lower than those measured for the attainment of tertiary native- and compactness-like arrangements

Non-canonical Staphylococcus aureus pathogenicity island repression

19 páginas, 9 figuras, 1 tabla.Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here, we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterize a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.This work was supported by grants MR/V000772/1, MR/M003876/1 and MR/S00940X/1 from the Medical Research Council (UK), BB/N002873/1, BB/S003835/1 and BB/V002376/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), Wellcome Trust201531/Z/16/Z, and ERC-ADG-2014 Proposal n◦670932 Dut-signal from EU to J.R.P.; grants PID2019-108541GB-I00 from Spanish Government (Ministerio de Econom´ıa y Competitividad y Ministerio de Ciencia e Innovacion) and PROMETEO ´ /2020/012 from Valencian Government to A.M.; grants MOE2017-T2-2-163 and MOE2019-T2-2-162 from the Ministry of Education to J.C.; and grant NIHR01 AI083255 to G.C. J.T. was supported by NIH IRACDA Grant K12GM093857 to Virginia Commonwealth University. We acknowledge Diamond Light Source for time on Beamline I03 for X-ray crystallography and B21 for SEC-SAXS under Proposal 16258. L.M.-R. was the recipient of a Spanish postdoctoral fellowship from Fundacion Ram ´ on Areces (2018–2020). J.R.P. is ´thankful to the Royal Society and the Wolfson Foundation for providing him support through a Royal Society Wolfson Fellowship. Funding for open access charge: University funds.Peer reviewe

Overall fold of the monomeric structure of the c-Src-SH3 domain.

<p>Overall fold of the monomeric species of the WT c-Src-SH3 domain (WT_M, PDB code 4JZ4). The AU is composed by two chains of the SH3 domain; both chains are represented as a cartoon (white). The n-Src loop residues in chains A and B are shown in red. In chain B, the poor electronic density in the difference maps does not allow to model residues 114-115. Both chains show a nickel-binding site at the N-terminal formed by the residues His83-Ser82-Gly81, with slight differences in the conformation and in the axial ligand (nickel ion is represented with a green sphere). All the figures were performed using the program Pymol 1.7 (distributed by Schrödinger).</p

Nucleation site of the WT c-Src SH3 domain.

<p>Hydrogen-bond interactions among the residues belonging to the diverging β-turn and those of the distal loop are shown in green dotted lines. WT_M (PDB code 4JZ4) chains A (panel A) and B (panel B) are shown in blue and cyan, respectively. (C) Intertwined dimer structure of the WT c-Src SH3 domain (PDB code 4JZ3), residues at chain A are shown in white sticks and those belonging to the symmetry related molecule (chain B) are in magenta sticks.</p

The histidine-phosphocarrier protein of the phosphoenolpyruvate: sugar phosphotransferase system of bacillus sphaericus self-associates

15 pags, 7 figs, 1 tabThe phosphotransferase system (PTS) is involved in the use of carbon sources in bacteria. Bacillus sphaericus, a bacterium with the ability to produce insecticidal proteins, is unable to use hexoses and pentoses as the sole carbon source, but it has ptsHI genes encoding the two general proteins of the PTS: enzyme I (EI) and the histidine phosphocarrier (HPr). In this work, we describe the biophysical and structural properties of HPr from B. sphaericus, HPrbs, and its affinity towards EI of other species to find out whether there is inter-species binding. Conversely to what happens to other members of the HPr family, HPrbs forms several self-associated species. The conformational stability of the protein is low, and it unfolds irreversibly during heating. The protein binds to the N-terminal domain of EI from Streptomyces coelicolor, EINsc, with a higher affinity than that of the natural partner of EINsc, HPrsc. Modelling of the complex between EINsc and HPrbs suggests that binding occurs similarly to that observed in other HPr species. We discuss the functional implications of the oligomeric states of HPrbs for the glycolytic activity of B. sphaericus, as well as a strategy to inhibit binding between HPrsc and EINsc. © 2013 Doménech et al.This work was supported by the Spanish Ministerio de Ciencia e Innovación (MCINN) (CTQ2011-24393, and CSD2008-00005 to JLN; BIO2009-13261-C02- 01/02 and P09-CVI-5063, with Fondo Social Europeo (ESF) to ACA; and BFU2010-19451 to AVC), Diputación General de Aragón (PI044/09 to AVC), intramural BIFI 2011 projects (to AVC and JLN), Junta de Andalucía (BIO-328 to ACA), and by grants from the Agencia Nacional de Promoción Científica y Tecnológica Argentina (ANPCyT; PICT-2011-2778) (to CNC). SMR was supported by the CSD2008-00005. The stays of RD in the laboratory of AVC were supported by the Spanish Ministerio de Ciencia e Innovación (BFU2008-02302-BMC)