Revisiting the genus Photobacterium: taxonomy, ecology and pathogenesis

The genus Photobacterium, one of the eight genera included in the family Vibrionaceae, contains 27 species with valid names and it has received attention because of the bioluminescence and pathogenesis mechanisms that some of its species exhibit. However, the taxonomy and phylogeny of this genus are not completely elucidated; for example, P. logei and P. fischeri are now considered members of the genus Aliivibrio, and previously were included in the genus Vibrio. In addition, P. damselae subsp. piscicida was formed as a new combination for former Vibrio damsela and Pasteurella piscicida. Moreover, P. damselae subsp. damselae is an earlier heterotypic synonym of P. histaminum. To avoid these incovenences draft and complete genomic sequences of members of Photobacterium are increasingly becoming available and their use is now routine for many research laboratories to address diverse goals: species delineation with overall genomic indexes, phylogenetic analyses, comparative genomics, and phenotypic inference. The habitats and isolation source of the Photobacterium species include seawater, sea sediments, saline lake waters, and a variety of marine organisms with which the photobacteria establish different relationships, from symbiosis to pathogenic interactions. Several species of this genus contain bioluminescent strains in symbiosis with marine fish and cephalopods; in addition, other species enhance its growth at pressures above 1 atmosphere, by means of several high-pressure adaptation mechanisms and for this, they may be considered as piezophilic (former barophilic) bacteria. Until now, only P. jeanii, P. rosenbergii, P. sanctipauli, and the two subspecies of P. damselae have been reported as responsible agents of several pathologies on animal hosts, such as corals, sponges, fish and homeothermic animals. In this review we have revised and updated the taxonomy, ecology and pathogenicity of several members of this genus. [Int Microbiol 20(1): 1-10 (2017)]Keywords: Photobacterium · taxonomy · symbiosis · pathogenesis · virulence factor

Discovering the colours of industrial heritage characterisation of paint coatings from the powerplant at the Levada de Tomar

The paint coatings of three energy generators from the 20th-century powerplant at Levada de Tomar, Portugal, were investigated using micro-Raman and micro-X-ray fluorescence spectroscopies and scanning electron microscopy with energy dispersive spectroscopy. This multi-analytical approach was used to identify the colouring agents, thus providing a chronological chromatic pallet and allowing to infer on the use of the three energy generators. Together with traditional pigments like Prussian blue, red iron oxide, and carbon black, pigments used in industrial areas like copper phthalocyanine and toluidine red were identified as colouring agents. Complex paint systems of the oldest equipment (1924) were revealed as well as maintenance procedures of the equipment that worked during a longer time (1944–1990). Powdery carbon black layers, resulting from incomplete hydrocarbon combustion and present between metallic substrates and coating layers, suggested the inexistence of paint coatings replacement after the powerplant shutdown. The identification of magnetite as a corrosion product of iron alloy substrate revealed that corrosion developed after the engine shutdown and not during the operation period. The results obtained highlight the potentialities of scientific-based approach and Raman spectroscopy to the industrial heritage study, an emergent cultural area.publishersversionpublishe

Identification of Key Functions Required for Production and Utilization of the Siderophore Piscibactin Encoded by the High-Pathogenicity Island irp-HPI in Vibrionaceae

This article belongs to the Collection Microbial Virulence Factors[Abstract] Piscibactin is a widespread siderophore system present in many different bacteria, especially within the Vibrionaceae family. Previous works showed that most functions required for biosynthesis and transport of this siderophore are encoded by the high-pathogenicity island irp-HPI. In the present work, using Vibrio anguillarum as a model, we could identify additional key functions encoded by irp-HPI that are necessary for piscibactin production and transport and that have remained unknown. Allelic exchange mutagenesis, combined with cross-feeding bioassays and LC-MS analysis, were used to demonstrate that Irp4 protein is an essential component for piscibactin synthesis since it is the thioesterase required for nascent piscibactin be released from the NRPS Irp1. We also show that Irp8 is a MFS-type protein essential for piscibactin secretion. In addition, after passage through the outer membrane transporter FrpA, the completion of ferri-piscibactin internalization through the inner membrane would be achieved by the ABC-type transporter FrpBC. The expression of this transporter is coordinated with the expression of FrpA and with the genes encoding biosynthetic functions. Since piscibactin is a major virulence factor of some pathogenic vibrios, the elements of biosynthesis and transport described here could be additional interesting targets for the design of novel antimicrobials against these bacterial pathogens.M.B. was supported by grant PID2019-103891RJ-100 from MCIN/AEI/10.13039/501100011033 (Spain). The work was also supported by grants RTI2018-093634-B-C21/C22 from MCIN/AEI/10.13039/501100011033/FEDER “A way to make Europe”. Work in University of Santiago de Compostela and University of A Coruña was also supported by grants GRC2018/018 and GRC2018/039, respectively, from Xunta de Galicia and BLUEBIOLAB (0474_BLUEBIOLAB_1_E), Programme INTERREG V A of Spain-Portugal (POCTEP). L.A. also thanks Xunta de Galicia for a predoctoral fellowship co-funded with the European Social Fund (ED481A-2019/081)Xunta de Galicia; GRC2018/018Xunta de Galicia; GRC2018/039Xunta de Galicia; ED481A-2019/08

Revisiting the genus Photobacterium: taxonomy, ecology and pathogenesis

The genus Photobacterium, one of the eight genera included in the family Vibrionaceae, contains 27 species with valid names and it has received attention because of the bioluminescence and pathogenesis mechanisms that some of its species exhibit. However, the taxonomy and phylogeny of this genus are not completely elucidated; for example, P. logei and P. fischeri are now considered members of the genus Aliivibrio, and previously were included in the genus Vibrio. In addition, P. damselae subsp. piscicida was formed as a new combination for former Vibrio damsela and Pasteurella piscicida. Moreover, P. damselae subsp. damselae is an earlier heterotypic synonym of P. histaminum. To avoid these incovenences draft and complete genomic sequences of members of Photobacterium are increasingly becoming available and their use is now routine for many research laboratories to address diverse goals: species delineation with overall genomic indexes, phylogenetic analyses, comparative genomics, and phenotypic inference. The habitats and isolation source of the Photobacterium species include seawater, sea sediments, saline lake waters, and a variety of marine organisms with which the photobacteria establish different relationships, from symbiosis to pathogenic interactions. Several species of this genus contain bioluminescent strains in symbiosis with marine fish and cephalopods; in addition, other species enhance its growth at pressures above 1 atmosphere, by means of several high-pressure adaptation mechanisms and for this, they may be considered as piezophilic (former barophilic) bacteria. Until now, only P. jeanii, P. rosenbergii, P. sanctipauli, and the two subspecies of P. damselae have been reported as responsible agents of several pathologies on animal hosts, such as corals, sponges, fish and homeothermic animals. In this review we have revised and updated the taxonomy, ecology and pathogenicity of several members of this genus

Vibrio neptunius Produces Piscibactin and Amphibactin and Both Siderophores Contribute Significantly to Virulence for Clams

[Abstract] Vibrio neptunius is an inhabitant of mollusc microbiota and an opportunistic pathogen causing disease outbreaks in marine bivalve mollusc species including oysters and clams. Virulence of mollusc pathogenic vibrios is mainly associated with the production of extracellular products. However, siderophore production is a common feature in pathogenic marine bacteria but its role in fitness and virulence of mollusc pathogens remains unknown. We previously found that V. neptunius produces amphibactin, one of the most abundant siderophores in marine microbes. In this work, synthesis of the siderophore piscibactin was identified as the second siderophore produced by V. neptunius. Single and double mutants in biosynthetic genes of each siderophore system, piscibactin and amphibactin, were constructed in V. neptunius and their role in growth ability and virulence was characterized. Although the High Pathogenicity Island encoding piscibactin is a major virulence factor in vibrios pathogenic for fish, the V. neptunius wild type did not cause mortality in turbot. The results showed that amphibactin contributes more than piscibactin to bacterial fitness in vitro. However, infection challenges showed that each siderophore system contributes equally to virulence for molluscs. The V. neptunius strain unable to produce any siderophore was severely impaired to cause vibriosis in clams. Although the inactivation of one of the two siderophore systems (either amphibactin or piscibactin) significantly reduced virulence compared to the wild type strain, the ability to produce both siderophores simultaneously maximised the degree of virulence. Evaluation of the gene expression pattern of each siderophore system showed that they are simultaneously expressed when V. neptunius is cultivated under low iron availability in vitro and ex vivo. Finally, the analysis of the distribution of siderophore systems in genomes of Vibrio spp. pathogenic for molluscs showed that the gene clusters encoding amphibactin and piscibactin are widespread in the Coralliilyticus clade. Thus, siderophore production would constitute a key virulence factor for bivalve molluscs pathogenic vibrios.This work was supported by grants AGL2017-86183-R (AEI/FEDER, EU), RTI2018-093634-B-C21/C22 (AEI/FEDER, EU) and PID2019-103891RJ-100 (AEI) from the State Agency for Research (AEI) of Spain. AGL2017-86183-R and RTI2018-093634-B-C21/C22 were co-funded by the FEDER Programme from the European Union. Work in University of Santiago de Compostela was also supported by grants GRC2018/018 and 2021-CP112, and in University of A Coruña by grant GRC2018/039 from Xunta de Galicia (Spain). FG was financed with a fellowship ‘Programa de formación de recurso humano de alto nivel doctorado en el exterior’ granted by Colciencias and the government of Norte de Santander, Colombia. LA was financed with a fellowship (ED481A-2019/081) from Xunta de Galicia (Spain), co-financed by ESF (European Social Fund)Xunta de Galicia; GRC2018/018Xunta de Galicia; 2021-CP112Xunta de Galicia; GRC2018/039Xunta de Galicia; ED481A-2019/08

Selective Detection of Aeromonas Spp. By a Fluorescent Probe Based on the Siderophore Amonabactin

[Abstract] Amonabactins, the siderophores produced by some pathogenic bacteria belonging to Aeromonas genus, can be used for the preparation of conjugates to be imported into the cell using their specific transport machinery. Herein, we report the design and synthesis of a new amonabactin-based fluorescent probe by conjugation of the appropriate amonabactin analogue to sulforhodamine B (AMB-SRB) using a thiol-maleimide click reaction. Growth promotion assays and fluorescence microscopy studies demonstrated that the AMB-SRB fluorescent probe was able to label the fish pathogenic bacterium A. salmonicida subsp. salmonicida through its outer membrane transport (OMT) protein FstC. The labelling of other Aeromonas species, such as the human pathogen A. hydrophila, indicates that this probe can be a very useful molecular tool for studying the amonabactin-dependent iron uptake mechanism. Furthermore, the selective labelling of A. salmonicida and other Aeromonas species in presence of other fish pathogenic bacteria, suggest the potential application of this probe for detection of Aeromonas in water and other fish farming samples through fluorescence assays.This work was supported by grants RTI2018-093634-B-C21/C22 from the State Agency for Research (AEI) of Spain, cofunded by the FEDER Programme from the European Union (MCIN/AEI/10.13039/501100011033/FEDER). M.B. was supported by grant PID2019-103891RJ-100 from MCIN/AEI (Spain). Work in University of Santiago de Compostela and University of A Coruña was also supported by grants GRC2018/018 and GRC2018/039, respectively, from Xunta de Galicia and BLUEBIOLAB (0474_BLUEBIOLAB_1_E), Programme INTERREG V A of Spain-Portugal (POCTEP). D.R-V. thanks Xunta de Galicia (Spain) for a predoctoral fellowship. J.C.-S. thanks to the FPU National Program (FPU16/02060) of the Spanish Ministry of Science, Innovation and Universities for a predoctoral fellowshipXunta de Galicia; GRC2018/018Xunta de Galicia; GRC2018/03

The Outer Membrane Protein FstC of Aeromonas salmonicida subsp. salmonicida Acts as Receptor for Amonabactin Siderophores and Displays a Wide Ligand Plasticity. Structure–Activity Relationships of Synthetic Amonabactin Analogues

[Abstract] Amonabactins are a group of four related catecholate siderophores produced by several species of the genus Aeromonas, including A. hydrophila and the fish pathogen A. salmonicida subsp. salmonicida. Although the gene cluster encoding amonabactin biosynthesis also contains a gene that could encode the ferri-siderophore receptor (fstC), to date there is no experimental evidence to explain its role. In this work, we report the identification of the amonabactins’ outer membrane receptor and the determination of the minimal structural parts of these siderophores involved in the molecular recognition by their cognate receptor. The four natural amonabactin forms (P750, T789, P693, and T732) and some mono and biscatecholate amonabactin analogues were chemically synthesized, and their siderophore activity on A. salmonicida FstC(+) and FstC(−) strains was evaluated. The results showed that each amonabactin form has quite different growth promotion activity, with P750 and T789 the most active. The outer membrane receptor FstC recognizes more efficiently biscatecholate siderophores in which the length of the linker between the two iron-binding catecholamide units is 15 atoms (P750 and T789) instead of 12 atoms (P693 and T732). Analysis of the siderophore activity of synthetic analogues indicated that the presence of Phe or Trp residues is not required for siderophore recognition. The results together point toward evidence that the amonabactin receptor FstC admits a high degree of ligand plasticity. We also showed that FstC is present in most Aeromonas species, including relevant human and animal pathogens as A. hydrophila. From the results obtained, we concluded that the ferri-amonabactin uptake pathway involving the outer membrane transporter FstC possesses a considerable functional plasticity that could be exploited for delivery of antimicrobial compounds into the cell. This would allow the use of the siderophore-based iron uptake mechanisms to combat infections caused by species of the genus Aeromonas.This work was supported by grants AGL2015-63740-C2-1/2-R and RTI2018-093634–B-C21/C22 (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, both cofunded by the FEDER Programme from the European Union. Work in University of Santiago de Compostela and University of A Coruña was also supported by grants GRC2018/018 and GRC2018/039, respectively, from Xunta de Galicia. D.R.V. thanks Xunta de Galicia (Spain) for a predoctoral fellowship. J.C.-S. thanks to the FPU National Program (FPU16/02060) of the Spanish Ministry of Science, Innovation and Universities for a predoctoral fellowshipXunta de Galicia; GRC2018/018Xunta de Galicia; GRC2018/03

The Marine Bivalve Molluscs Pathogen Vibrio neptunius Produces the Siderophore Amphibactin, Which Is Widespread in Molluscs Microbiota

This is the peer reviewed version of the following article:Galvis, F., Ageitos, L., Martínez‐Matamoros, D., Barja, J.L., Rodríguez, J., Lemos, M.L., et al. (2020) The marine bivalve molluscs pathogen Vibrio neptunius produces the siderophore amphibactin, which is widespread in molluscs microbiota. Environmental Microbiology 22: 5467–5482, which has been published in final form https://doi.org/10.1111/1462-2920.15312. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.[Abstract] Amphiphilic siderophores, including amphibactins, are the most abundant siderophores in oceans. Genes putatively encoding the amphibactin system were proposed in some bacteria and homologues of these genes are particularly abundant in multiple bacterial lineages inhabitant of low-iron seawater. However, since no defective mutant strains in any of these genes were studied to date, their role in amphibactin synthesis or uptake was not demonstrated. In this work, an in silico analysis of the genome of the mollusc pathogen Vibrio neptunius leads us to identify a gene cluster (denoted absABDEF) that is predicted to encode an amphibactin-like siderophore and several mutant strains unable to synthesize or use siderophores were constructed. The results showed that genes absABDEF are required for amphibactin synthesis. A comparative chemical analysis of V. neptunius wild type and biosynthesis mutants allowed us to identify a mixture of nine amphibactin forms produced by this bacterium. In addition, the gene abtA is predicted to encode the ferri-amphibactin outer membrane transporter. The prevalence of the amphibactin system in bivalve hemolymph microbiota was also studied. We found that the amphibactin system is widespread in hemolymph microbiota including both commensal and pathogenic bacterial species. Thus, its contribution to bacterial fitness must be more related to environmental persistence than to pathogenicity.This work was supported by grants AGL2017-86183-R (AEI/FEDER, EU), RTI2018-093634-B-C21/C22 (AEI/FEDER, EU) and PID2019-103891RJ-100 (AEI) from the State Agency for Research (AEI) of Spain. AGL2017-86183-R and RTI2018-093634-B-C21/C22 were co-funded by the FEDER Programme from the European Union. Work in University of Santiago de Compostela and University of A Coruña was also supported by grants GRC2018/018 and GRC2018/039, respectively, from Xunta de Galicia (Spain). Fabian Galvis was financed with a fellowship ‘Programa de formación de recurso humano de alto nivel doctorado en el exterior’ granted by Colciencias and the government of Norte de Santander, Colombia. Lucía Ageitos was financed with a fellowship (ED481A-2019/081) from Xunta de Galicia (Spain) co-financed by ESF (European Social Fund)Xunta de Galicia; GRC2018/018Xunta de Galicia; GRC2018/039Xunta de Galicia; ED481A-2019/08

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

Author accepted manuscript[Abstract] In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described. Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the Stöber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.The authors gratefully acknowledge Professor Klaus Hantke (University of Tübingen, Germany) for kindly supply the Yersinia enterocolitica strains used in this work. This work was supported by grants AGL2015-63740-C2-1/2-R and RTI2018-093634-B-C21/C22 (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, co-funded by the FEDER Programme from the European Union. Work in University of Santiago de Compostela and University of A Coruña was also supported by grants GRC2018/018, GRC2018/039, and ED431E 2018/03 (CICA-INIBIC strategic group) from Xunta de Galicia. Finally, we want to thank to Nuria Calvo for her great collaboration doing the voice-off this video protocolXunta de Galicia; GRC2018/018Xunta de Galicia; GRC2018/039Xunta de Galicia; ED431E 2018/0

O-versus S-Metal Coordination of the Thiocarboxylate Group: An NMR Study of the Two Tautomeric Forms of the Ga(III)-Photoxenobactin E Complex

[Abstract] Photoxenobactin E (1) is a natural product with an unusual thiocarboxylic acid terminus recently isolated from an entomopathogenic bacterium. The biosynthetic gene cluster associated with photoxenobactin E, and other reported derivatives, is very similar to that of piscibactin, the siderophore responsible for the iron uptake among bacteria of the Vibrionaceae family, including potential human pathogens. Here, the reisolation of 1 from the fish pathogen Vibrio anguillarum RV22 cultured under iron deprivation, its ability to chelate Ga(III), and the full NMR spectroscopic characterization of the Ga(III)-photoxenobactin E complex are presented. Our results show that Ga(III)-photoxenobactin E in solution exists in a thiol–thione tautomeric equilibrium, where Ga(III) is coordinated through the sulfur (thiol form) or oxygen (thione form) atoms of the thiocarboxylate group. This report represents the first NMR study of the chemical exchange between the thiol and thione forms associated with thiocarboxylate-Ga(III) coordination, including the kinetics of the interconversion process associated with this tautomeric exchange. These findings show significant implications for ligand design as they illustrate the potential of the thiocarboxylate group as a versatile donor for hard metal ions such as Ga(III).The work was supported by grants PID2021-122732OB-C22/C21 from MCIN/AEI/10.13039/501100011033/FEDER “A way to make Europe” (AEI, Spanish State Agency for Research and FEDER Programme from the European Union). M.B. was supported by grant PID2019-103891RJ-100 from MCIN/AEI/10.13039/501100011033 (Spain). Work at the University of Santiago de Compostela and University of A Coruña was also supported by grants GRC2018/018 and ED431C 2022/39, respectively, from Xunta de Galicia. L.B. thanks Horizon Europe Marie Skłodowska-Curie Actions Postdoctoral Fellowship funded by the European Union (ID: 101066127). L.A. thanks Xunta de Galicia (Spain) for a predoctoral fellowshipXunta de Galicia; GRC2018/018Xunta de Galicia; ED431C 2022/3