Pyocin S5 import into Pseudomonas aeruginosa reveals a generic mode of bacteriocin transport

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria

Pyochelin, a siderophore of Pseudomonas aeruginosa: Physicochemical characterization of the iron(III), copper(II) and zinc(II) complexes:

Pseudomonas aeruginosa is an opportunistic pathogen which synthesizes two major siderophores, pyoverdine (Pvd) and pyochelin (Pch), to cover its needs in iron(III). Although the high affinity and specificity of Pvd toward iron(III) (pFe = 27.0) is well described in the literature, the physicochem. and coordination properties of Pch toward biol. relevant metals (Fe(III), Cu(II) or Zn(II)) have been scarcely investigated. We report a thorough physico-chem. investigation of Pch (potentiometry, spectrophotometries, ESI/MS) that highlighted its moderate but significantly higher affinity for Fe3+ (pFe = 16.0 at p[H] 7.4) than reported previously. We also demonstrated that Pch strongly chelates divalent metals such as Zn(II) (pZn = 11.8 at p[H] 7.4) and Cu(II) (pCu = 14.9 at p[H] 7.4) and forms predominantly 1:2 (M2+/Pch) complexes. Kinetic studies revealed that the formation of the ferric Pch complexes proceeds through a Eigen-Wilkins dissociative ligand interchange mechanism involving two protonated species of Pch and the Fe(OH)2+ species of Fe(III). Our physico-chem. parameters supports the previous biochem. studies which proposed that siderophores are not only devoted to iron(III) shuttling but most likely display other specific biol. role in the subtle metals homeostasis in microorganisms. This work also represents a step toward deciphering the role of siderophores throughout evolution. [on SciFinder(R)

Biosynthesis of the pyoverdine siderophore of Pseudomonas aeruginosa involves precursors with a myristic or a myristoleic acid chain

Pyoverdine I (PVDI) is the major siderophore produced by Pseudomonas aeruginosa to import iron. Biosynthesis of this chelator involves non‐ribosomal peptide synthetases and other enzymes. PvdQ is a periplasmic enzyme from the NTN hydrolase family and is involved in the final steps of PVDI biosynthesis. A pvdQ mutant produces two non‐fluorescent PVDI precursors with a higher molecular mass than PVDI. In the present study, we describe the use of mass spectrometry to determine the structure of these PVDI precursors and show that they both contain a unformed chromophore like ferribactin, and either a myristic or myristoleic chain that must be removed before PVDI is secreted into the extracellular medium

ACS Chem Biol

Pyoverdine I is the main siderophore secreted byPseudomonas aeruginosa PAO1 to obtain access to iron. After extracellular iron chelation, pyoverdine-Fe uptake into the bacteria involves a specific outer-membrane transporter, FpvA. Iron is then released in the periplasm by a mechanism involving no siderophore modification but probably iron reduction. The proteins involved in this dissociation step are currently unknown. The pyoverdine locus contains the fpvCDEF operon, which contains four genes. These genes encode an ABC transporter of unknown function with the distinguishing characteristic of encompassing two periplasmic binding proteins, FpvC and FpvF, associated with the ATPase, FpvE, and the permease, FpvD. Deletion of these four genes partially inhibited cytoplasmic uptake of (55)Fe in the presence of pyoverdine and markedly slowed down the in vivo kinetics of iron release from the siderophore. This transporter is therefore involved in iron acquisition by pyoverdine in P. aeruginosa. Sequence alignments clearly showed that FpvC and FpvF belong to two different subgroups of periplasmic binding proteins. FpvC appears to be a metal-binding protein, whereas FpvF has homology with ferrisiderophore binding proteins. In vivo cross-linking assays and incubation of purified FpvC and FpvF proteins showed formation of complexes between both proteins. These complexes were able to bind in vitro PVDI-Fe, PVDI-Ga, or apo PVDI. This is the first example of an ABC transporter involved in iron acquisition via siderophores, with two periplasmic binding proteins interacting with the ferrisiderophore. The possible roles of FpvCDEF in iron uptake by the PVDI pathway are discussed

The outer membrane porin OmpW of Acinetobacter baumannii is involved in iron uptake and colistin binding.

This study was undertaken to characterize functions of the outer membrane protein OmpW, which potentially contributes to the development of colistin- and imipenem-resistance in Acinetobacter baumannii. Reconstitution of OmpW in artificial lipid bilayers showed that it forms small channels (23 pS in 1 m KCl) and markedly interacts with iron and colistin, but not with imipenem. In vivo, (55) Fe uptake assays comparing the behaviours of ΔompW mutant and wild-type strains confirmed a role for OmpW in A. baumannii iron homeostasis. However, the loss of OmpW expression did not have an impact on A. baumannii susceptibilities to colistin or imipenem.journal articleresearch support, non-u.s. gov't2016 Jan2016 01 11importe

Multilayer design of crN/MoN superhard protective coatings and their characterisation

Multilayer CrN/MoN transition metal nitride coatings were studied in this research. Films were deposited by vacuum arc deposition (Arc-PVD) from Cr and Mo cathodes in nitrogen atmosphere p = 0.4 Pa. Three series of samples with different values of negative bias voltage (−20, −150, and −300 V) applied to the surface were fabricated. Each series has samples with 11, 22, 44, 88, 180 and 354 layers while total thickness was maintained with the same value. Samples were studied by scanning electron microscopy (SEM) on cross-sections and coatings surface, energy-dispersive X-ray spectroscopy (EDS), electron backscatter diffraction (EBSD), high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), micro-indentation. Two main cubic phases of γ-Mo N and cubic CrN were detected. It was observed that the crystal growth orientation changes while the negative bias voltage of the substrate decreases. The maximum values of hardness (38–42 GPa) among the studied samples were obtained for coatings with a minimal individual layer thickness of 20 nm deposited at U = −20 V. N 2 bFCT - Sumy State University(0116U002621). Foundation of Science and Technology (FCT) of Portugal [references NORTE-01- 0145-FEDER-022096, SFRH/BD/129614/2017], Network of Extreme Conditions Laboratories (NECL) and by Ukrainian state budget programs [No. 0116U006816, 0118U003579 and 0116U002621]. Partial support by COST Action CA15102 is also greatly appreciate