Université de Pau et des Pays de l’Adour offers a PhD position “Molecular mechanisms of the acquisition of essential metals in heterotrophic bacteria degrading particulate organic matter (POM) in marine environments”.

In oceans, the greatest part of the heterotrophic activity that remineralizes the organic carbon into CO₂, resides in biofilms colonizing the particulate fraction of organic matter (POM). POM consists of aggregated compounds (mostly proteins, polysaccharides and lipids). Several experimental and theoretical hints suggest that metal availability, particularly iron, have a strong impact on organic carbon remineralization.

This project will focus on the acquisition of iron and other metals by bacteria that degrade the particulate organic matter. It aims at understanding the biological significance of siderophores and others metallophores produced by POM degrading bacteria. The general intended strategy is to link the metal pool to the genes involved in ligands biosynthesis and to the regulation of their expression in relation to the environmental conditions. The methodology used will involve molecular genetics of bacteria in close association with analytical chemistry. We are looking for a motivated and dynamic student with a solid background mainly in Microbiology and Molecular Biology. The student must have good writing and public presentation skills. She/He will have to develop his autonomy and to show initiative. It is essential that the student appreciates teamwork

HOST LABORATORY PROFILE:

This PhD subject is part of the project MesMic (Metals in Environmental Systems Microbiology) which mobilizes over 20 permanent researchers of the research cluster: “Environmental Chemistry and Microbiology” of IPREM for a period of 5 years, gathering competences in analytical chemistry, microbiology, molecular biology, biochemistry and Ecology.

MISSION – PRINCIPAL ACTIVITIES:

I. Scientific Context

In oceans, remineralization of the organic carbon into CO₂ occurs mostly through the respiration of heterotrophic bacteria that degrade the organic matter released by lysed or decaying phytoplankton cells (1). The greatest part of the heterotrophic activity resides in the particulate fraction of the organic matter (POM) consisting of aggregated compounds (mostly proteins, polysaccharides and lipids) that is colonized by biofilm-forming bacteria (2). Metal availability, particularly iron, is expected to have a strong impact on organic carbon remineralization since heterotrophic bacteria have higher iron content than eukaryotic phototroph and respiration is a highly iron demanding process, the respiratory chain alone containing approximately 94% of the cellular iron (3) (4).

In response to the challenge of metal acquisition, marine heterotrophic bacteria have evolved very efficient pathways designed to extract trace amount of iron and most likely other metals from their surrounding environment. The best documented acquisition pathway is the siderophore-mediated iron uptake. Siderophores bind iron with a very high affinity and hence are able to scavenge iron at low concentration or to displace iron from other ligands having a lower affinity. Siderophore-Fe(III) complexes are then recognized and transported across the cell membranes through an energy dependent process involving outer membrane receptors, periplasmic binding proteins and inner membrane transporters. Relatively few siderophore structures from marine bacteria have been elucidated in comparison to those of terrestrial or pathogenic bacteria (5). The majority of marine siderophores identified to date are amphiphilic or/and photoreactive (6). Some bacterial species produce several siderophores or suite of siderophores with various hydrophobic tails. For instance, in Marinobacter sp. DS40M6, the amphiphilic character of the marinobactin siderophore appears to be controlled in a growth-phase dependent manner by hydrolysis of the acyl tail (7) (8). The eco-physiological role of the different siderophores produced by one strain as well as the function of the amphipathy and photoreactivity are not understood.

II. Objectives

We will focus on the acquisition of iron and other metals by bacteria that degrade the particulate organic matter (POM). The challenge is to understand the biological significance of siderophores and others metallophores produced by POM degrading bacteria. The general intended strategy is to link the metal pool and their ligands produced by a bacterium to the genes and the regulation of their expression in relation to environmental conditions. The more specific objectives include:

(i) identification of the type of metallophore produced in POM degrading biofilm and determine if they are specifically produced in this growth mode

(ii) exploration of isotopic fractionation of iron during its assimilation by POM degrading bacteria.

(iii) gaining insight into the significance of the amphiphilic and photoreactive characters of marine siderophore in regards to biofilm development on POM.

(iv) identification of the genes involved in the metallophores biosynthesis and investigation of the regulation of their expression in relation to POM degrading biofilms formation.

III. Work plan

The experiments will be performed on model strains belonging to the class Gamaproteobacteria (Alteromonadaceae, Vibrionaceae and Alcanivoracaceae) as they are often associated to POM particles degradation and have been less investigated than bacteria from the class Alphaproteobacteria with respect to organic matter degradation and iron acquisition. All the strains have their genome sequenced, are genetically amenable and grow on insoluble/polymeric substrates (lipids, alkanes, chitin and proteins) that will be used as models for POM.

Task 1. Determination of the metallophore profiles of POM degrading bacteria.

Determination of the metallophore profile will consist in identifying the whole set of metallophores produced by POM degrading model bacteria including siderophores that only bind iron, siderophores that bind others metals, and metallophores that bind non-iron metals. The metallophores and their metal-complexes will be determined by HPLC – high resolution high mass accuracy MS in collaboration with chemists of the institute. The metallophore profiles of POM degrading strains will be established in different growth conditions. Strains will be grown as biofilms on their insoluble/polymeric substrates which are representative of particulate organic matter (proteins, polysaccharides, lipids or hydrocarbons). As amphipathy is the hallmark of marine siderophores, the partition of siderophore between biofilms and culture supernatants will be determined. Effect of light on siderophore profiles will also be examined as many marine siderophores are photoreactive. This will allow to determine if bacteria adapt the metallophores produced to their growth status and to the environmental conditions encountered. In parallel, in order to assess the impact of metals availability on POM degradation, the architecture and kinetic of development on POM and the biodegradation rate of POM will be established in metal limiting and iron depleted conditions. Along with the determination of metallophore profiles of the model strains, isotopic fractionation of iron will be investigated.

Task 2. Genes involved in the biosynthesis of metallophores and regulation of their expression.

The strain exhibiting the most interesting metallophone profile determined in Task 1 will be chosen for a deeper investigation of the physiological role of siderophores in biofilms degrading POM. The main criteria to select the strain will be the production of a metallophore profile characteristic of the biofilm growth mode and/or light dependency. In a global approach, we will link the genome with the metallophore profile. A miniTn5 transposon insertion mutant library will be generated and screened for alteration of the metallophore profile. This approach will enable the identification of genes involved in metallophore biosynthesis, modification and regulation. In parallel, the genes putatively involved in Fe acquisition, including siderophore biosynthesis pathway and transport, will be identified by genome mining. In order to demonstrate the role of these genes in iron acquisition, the corresponding deletion mutants will be constructed. Mutants phenotypes regarding production of siderophores and growth on POM in iron depleted condition will be examined. The biosynthetic pathway of newly identified siderophore will be elucidated by combining genetic analysis with analytical chemistry. Regulation of siderophores or suite of siderophores production during biofilm development on POM will be determined by (i) measuring directly the presence of siderophore by HPLC-MS and (ii) measuring gene expression levels by RT-qPCR.

The combination of results obtained in Tasks 1 and 2 will give a picture of different siderophores or siderophores suites during degradation of POM and pose the first hint about the relation between structure and physiological function of siderophore in biofilm degrading POM.

IV Teaching

The student will have to teach biology in French, 96 hours at the undergraduate level.

Eligibility criteria

• the candidate’s motivation, scientific maturity and curiosity
• candidate’s marks and rankings in Master
• French and English proficiency

Skills/Qualifications

• good knowledge in physiology, molecular biology and biochemistry of bacteria are required. Basic skills in bioinformatics (protein sequence and genome analysis) are also desired.
• taste and aptitude for multidisciplinary work, the candidate will have to be interested in microbiology and analytical chemistry.
• scientific rigor
• good level of scientific English
• good ability to communicate and write in French and English