Marion Bonnet

Jean Christophe Lagier

Prof Didier Raoult
email: didier.raoult@gmail.com

Saber Khelaifia

Cocci

Cin Agar

Sps Agar

Tsn Agar

Oxford Agar

Campylosel Agar

Thayer-Martin Selective Agar

Broth with Gentamicin Todd Hewitt

Nalidixic Acid

Azide Blood

Agar Base

Azide Dextrose Broth

Esculin Agar

Eva Broth

* Mkttn

Broth Delarras

Fraser Broth Base

Mannitol Salt Agar

* Bile

V J Agar

Baird-Parker Agar Base

Methylen Blue

Macconkey Agar

Mitis Salivarius Agar

Drigalski Medium

Bacterial culture through selective and non-selective conditions: the evolution of culture media in clinical microbiology

Keywords: Culture media, liquid and solid media, enriched media, selective media, gelling agents

Microbiology has been largely developed thanks to the discovery and optimization of culture media. The first liquid artificial culture medium was created by Louis Pasteur in 1860.

Previously, bacterial growth on daily materials such as some foods had been observed. These observations thus highlighted the importance of the bacteria's natural environment and their nutritional needs in the development of culture media for their isolation. A culture medium is essentially composed of basic elements (water, nutrients), to which must be added different growth factors that will be specific to each bacterium and necessary for their growth.

The evolution of bacterial culture through the media used for their culture began with the development of the first solid culture medium by Koch, allowing not only the production of bacterial colonies, but also the possibility of purifying a bacterial clone. The main gelling agent used in solid culture media is agar. However, some limits have been observed in the use of agar because of some extremely oxygen sensitive bacteria that do not grow on agar media and other alternatives were proposed and tested. Then, the discovery of antimicrobial agents and their specific target induced the emergence of selective media. These inhibiting agents thus make it possible to eliminate undesirable bacteria from microbiota and select the bacteria desired. Thanks to a better knowledge of the bacterial environment, it will be possible to develop new culture media and new culture conditions, better adapted to certain fastidious bacteria, difficult to isolate.

Cefalotin (1st generation)

Entrobacteria

Introduction

The discovery of culture media allowed the development of microbiology in the 19 th century (1). Bacterial culture was the first method developed to study the human microbiota (2) using an artificial medium that allows growth and isolation of bacteria. The first to have cultured a bacterium in a reproducible way was Louis Pasteur in 1860 thanks to the development of the first so-called artificial culture medium (3). Recently, after the emergence of molecular techniques in the 1970s, such as PCR, sequencing and more particularly metagenomics, microbiologists have favoured these innovative techniques to the detriment of culture.

Nevertheless, metagenomics presents certain disadvantages and in particular a depth bias, due to the lack of sensitivity of the primers used, since it does not detect bacteria present at concentrations below 10 5 bacteria per gram of stool [START_REF] Lagier | The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota[END_REF]. Moreover, these techniques only detect DNA: it is impossible with these techniques to differentiate DNA belonging to living bacteria, from that of the transient bacteria of the microbiota studied or from that of dead bacteria.

A few years ago, culturomics, a new culture technique, which uses a very large number of culture media and culture conditions to extend the repertoire of bacteria, was developed in our laboratory [START_REF] Lagier | The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota[END_REF]. This technique demonstrates the complementarity between metagenomics and culturomics. Therefore, the metagenomic identification of bacterial species existing in a given microbiota can be exploited by culturomics through the optimization of new specific culture media for the isolation of these species. This complementarity allows culturomics to become a targeted technique.

Nowadays, new culture media mimic the natural environment of bacteria by adding different elements in culture medium to cultivate bacteria previously uncultivated.

We propose here a bibliographical review of culture media and techniques evolution through the development of microbiology over time.

I.

Empirical approach of microbiology I.1. Observational microbiology

Microbiology is defined by the organisms it studies, but also by the tools used to study them.

The first observation of a bacterium was made around 1673 by the German microscopist Anton van Leeuwenhoek thanks to the microscopes he had developed. Those enlarged from 50 to 300 times what he observed (1,3).

In the course of his research, he highlighted small structures that he called "animalcules" (4), because he thought he was observing small animals [START_REF] Weeks | Alcamo's Microbes and Society[END_REF]. Throughout his observations, he described and designed yeast cells, filiform fungi, microscopic algae and protozoa [START_REF] Pommerville | Fundamentals of microbiology[END_REF].

Leeuwenhoek was also the first to observe a parasite, Giardia lamblia (3). However, microscopy alone cannot address all the questions about the microorganisms studied. For about 200 years, microbiology has been stagnating until the development of microbial isolation techniques in pure cultures. This is another important milestone in the history of microbiology.

I.2. Cultural microbiology

I.2.1. The birth of culture broth

In the 13 th century AD, 400 years before Leeuwenhoek, a blood-like substance appeared on the communion bread and, into beliefs, this red substance was assimilated to the blood of the Christ. Bartholomeo Bizio, an Italian pharmacist, solved this mystery in 1817 thanks to advances in microbiology and showed that it was not blood, but a microorganism that he named Serratia marcescens. This bacterium appeared as red colonies on bread when stored in a warm and humid atmosphere [START_REF] Yu | Serratia marcescens: Historical perspective and clinical review[END_REF]. It is one of the first natural cultures of a bacterium. The origins of culture media date back to the 19 th century. Many bacteriologists have tried, with varying degrees of success, to grow bacteria on the food or material on which the microorganism was developed in the first place.

The evolution of microbiology has been made possible by challenging the theory of spontaneous generation, according to which living organisms could develop from dead or decaying matter and thus spontaneously appear in cultures. So it was inconceivable to obtain pure cultures. In 1861, Louis Pasteur, a French microbiologist (3), solved this problem through an experiment. He placed nutrient solutions in flasks, heated their necks with flame and stretched them in various ways, keeping the end open to the air. Pasteur boiled the solutions for a few minutes and cooled them. No growth appeared even if the contents of the flasks had been exposed to air. Pasteur noticed that there was no growth because dust and germs had been trapped on the walls of the curved tubes. If the tubes were broken, growth began immediately. Pasteur thus demonstrated the spontaneous generation nonsense and also found ways to keep sterile solutions (3).

The first to have cultured bacteria reproducibly in a liquid culture medium was Louis Pasteur.

In 1860, he developed a culture medium containing "yeast soup", ashes, sugar and ammonium salts [START_REF] Murray | Medical microbiology[END_REF][START_REF] Sandle | History and development of microbiological culture media[END_REF]. His objective was to create a fermentation medium to demonstrate that each fermentation (alcoholic, acetic, lactic ...) was associated with the development of a particular microorganism [START_REF] Vallery-Radot | Les oeuvres de Pasteur[END_REF]. The presence of these different elements in the medium allowed him to observe that some of these components could promote or inhibit the growth of certain bacteria and that they could also allow the emergence of certain bacteria compared to others [START_REF] Sandle | History and development of microbiological culture media[END_REF].

Indeed, in the course of the study of fermentation of beer, he was confronted with a problem: when the beer was healthy, the microscope only showed brewer's yeast, but when the beer was acid, Pasteur noticed that the tanks contained "tiny rod-shaped objects" producing lactic acid. This fermentation medium therefore made it possible to highlight the multiplication of this bacterium (1,3).

In 1881, Robert Koch demonstrated optimal growth of bacteria when they were incubated in a broth composed of fresh beef serum or meat extract. However, the use of a liquid culture medium did not produce pure bacterial cultures. Koch therefore sought a way to solidify the media (8) (figure 1).

I.2.2. Emergence of pure cultures in solid media

First, he tested coagulated egg albumin, starch paste or an aseptically cut slice of potato.

However, not all these techniques allowed him to isolate colonies. Koch then added gelatin to his broth and poured it all over a flat glass plate. However, gelatin had disadvantages since it liquefied at temperatures above 25°C and could be consumed by gelatinase, an enzyme produced by certain bacteria. Thanks to the wife of one of his assistants, Fannie Hesse, who used agar to solidify his jams, he replaced gelatin with agar, which allowed him to obtain firm agars and isolate bacteria. In 1887, Julius Richard Petri replaced the glass plate with a circular culture box, the Petri box has just been created, and is still used today [START_REF] Sandle | History and development of microbiological culture media[END_REF]. This allowed him to obtain and observe isolated colonies and to limit contamination.

II.

How to design an enriched culture medium?

An enriched culture medium is a medium to which have been added elements essential for the growth of bacteria.

The nutrients

In order to grow, bacteria need a minimum of nutrients: water, a carbon source, a nitrogen source and some mineral salts [START_REF] Kumar | Textbook of microbiology[END_REF].

Water

Water plays a fundamental role in solubilizing nutrients, transporting them and ensuring hydrolysis reactions. Some bacteria need free water for their growth. If evaporation occurs during the incubation of the agar, there may be a loss of this water, resulting in a decrease in colony size and inhibition of bacterial growth [START_REF] Kumar | Textbook of microbiology[END_REF][START_REF] Power | Difco™ & BBL™ manual[END_REF].

Carbon sources

Carbon is the most abundant constituent element in bacteria. It is essential for bacteria to produce carbon molecules, such as fats, carbohydrates, proteins and nucleic acids. Bacteria can use inorganic carbon sources, such as carbon dioxide, or organic sources such as sugars and alcohols [START_REF] Kumar | Textbook of microbiology[END_REF][START_REF] Atlas | Handbook of microbiological media[END_REF].

Nitrogen sources

As for nitrogen sources, they are numerous and can be found in a large number of compounds used in the composition of a culture medium. It is found in the organic form, corresponding to protein hydrolysates, particularly in case of hydrolysate, proteose-peptone or tryptone [START_REF] Atlas | Handbook of microbiological media[END_REF], but also in an inorganic form, nitrates [START_REF] Latge | Croissance et sporulation de 6 espèces d'Entomophthorales II. Influence de diverses sources d'azote[END_REF]. Nitrogen allows bacteria to synthesize their proteins.

Finally, among the common mineral salts, phosphate, sulphate, magnesium or calcium [START_REF] Power | Difco™ & BBL™ manual[END_REF] are regularly found.

Energy sources

There are two types of bacteria, phototrophic bacteria, such as Thiocapsa roseopersicina [START_REF] Yurkov | Aerobic anoxygenic phototrophic bacteria[END_REF] which uses light as an energy source by transforming it into an electrochemical gradient of protons ( 14) and chemotrophic bacteria which uses the energy of oxidation of mineral or organic compounds as energy sources [START_REF] Thauer | Energy conservation in chemotrophic anaerobic bacteria[END_REF][START_REF] Vallery-Radot | Les oeuvres de Pasteur[END_REF]. Among these bacteria, we can find Listeria monocytogenes [START_REF] Van Der Horst | Photosensing in chemotrophic, nonphototrophic bacteria: let there be light sensing too[END_REF].

Growth factors

The use of a minimal medium does not allow the growth of certain bacteria that need specific elements to grow. It is sometimes necessary to add growth factors to culture media in order to boost the multiplication of bacteria. Growth factors are elements that bacteria are unable to synthesize from the nutrients available in the environment [START_REF] Kumar | Textbook of microbiology[END_REF][START_REF] Werkman | Bacterial physiology[END_REF]. Growth factors are required in small quantities in the culture medium and their need is justified by the absence or blocking of a metabolic pathway in the bacterium.

Purine and pyrimidine bases

There are different categories of growth factors among which we find purine and pyrimidine bases. They are necessary for synthesis of nucleic acids. Indeed, some lactic acid bacteria need adenine, guanine, thymine or uracil for growth [START_REF] Werkman | Bacterial physiology[END_REF]. This is the case in particular for the bacterium Leuconostoc mesenteroides, for which guanine is essential for its growth [START_REF] Snell | Purine and pyrimidine as growth substances for lactic acid bacteria[END_REF].

Amino acids

Amino acids are also growth factors and are used for protein synthesis. Dunn et al [START_REF] Dunn | The amino acid requirements of twenty-three lactic acid bacteria[END_REF] showed in 1946 that only 2 amino acids were essential for Leuconostoc mesenteroides, glutamic acid and valine, while for Lactobacillus brevis, no less than 15 amino acids were necessary for its growth. This high requirement for amino acids can be explained by the fact that the base medium used at the time was not rich in common nutrients. Indeed, nowadays, Lactobacillus brevis grows on a COS agar (Columbia Blood Agar) (Biomérieux, France), not supplemented with amino acids [START_REF] Magdoub | Probiotic properties of some lactic acid bacteria isolated from Egyptian dairy products[END_REF], but composed of casein hydrolysate and peptone proteose, themselves sources of amino acids [START_REF] Power | Difco™ & BBL™ manual[END_REF].

Vitamins

Vitamins are also part of growth factors. They are coenzymes or precursors of coenzymes. A vitamin is an organic substance, necessary in small quantities for the metabolism of a living organism, which cannot be synthesized in sufficient quantity by that organism.

Faecalibacterium prausnitzii, which is a tedious bacterium, requires a large number of vitamins to grow, such as biotin, folic acid, riboflavin or vitamin B12 [START_REF] Duncan | Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb[END_REF]. This bacterium also requires the presence of other growth factors such as volatile fatty acids (acetic acid, propionic acid or valeric acid) [START_REF] Duncan | Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb[END_REF].

Blood use and its derivatives

Blood and its derivatives will also boost the growth of certain bacteria. In culture media, it is common to use sheep's blood or horse's blood. The role of blood is to act as a protective agent against toxic oxygen radicals ( 22), but also as a nutritional supplement. For instance, cooked sheep's blood provides the factor X (heme) necessary for the growth of many pathogenic species, including Haemophilus influenzae [START_REF] Vastine | Comparison of media for the isolation of Haemophilus species from cases of seasonal conjunctivitis associated with severe endemic trachoma[END_REF]. Most often, 5% blood is added to the culture media [START_REF] Collins | Microbiological methods[END_REF]. Serum, such as fetal calf or lamb serum, can also be used as a growth factor by providing a large number of elements such as lipids, vitamins, triglycerides, minerals and others [START_REF] Cézard | Biotechnologies en 27 fiches[END_REF]. This blood has been cleared of cells, platelets and clotting factors. Fetal calf serum, which is most commonly used in microbiology [START_REF] Albertson | Growth and survival of Helicobacter pylori in defined medium and susceptibility to Brij 78[END_REF][START_REF] Northoff | Fetal Calf Serum[END_REF], must be inactivated by heat before use (56°C for 30 min) in order to inactivate all components of the complement system present in this serum [START_REF] Meenakshi | Cell Culture Media: A Review[END_REF][START_REF] Rahman | Fetal calf serum heat inactivation and lipopolysaccharide contamination influence the human T lymphoblast proteome and phosphoproteome[END_REF]. It must be stored at -20°C and heated to room temperature rather than in a water bath to avoid deterioration of the proteins it contains.

Rumen fluid

Finally, it is also possible to use the rumen fluid (29, 2) to promote the growth of certain bacterial species by mimicking their natural environment. The rumen fluid used corresponds to the rumen of the sheep. To prepare it, it is first necessary to recover the juice from the fermented plants in the stomach by filtering it through a fine cloth. This juice is then centrifuged (10,000 rpm for 90 min) and the supernatant collected. Then, it undergoes 3 successive filtrations at 0.8µm, 0.45µm and 0.22µm [START_REF] Lagier | The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota[END_REF][START_REF] Goodman | Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice[END_REF]. 5% rumen juice is usually added to the culture media. The rumen fluid can promote the growth of certain Treponema, such as

Treponema hyodysenteriae and Treponema innocens [START_REF] Szynkiewicz | Evaluation of selective media for primary isolation of Treponema hyodysenteriae and Treponema innocens[END_REF].

Antioxidants

Some anaerobic bacteria are fastidious to grow and new culture strategies have been developed to isolate them. Indeed, anaerobic bacteria are most abundant in the human intestinal microbiota, accounting for up to 99.9% of total bacteria [START_REF] Bennett | Bennett's Principles and Practice of Infectious Diseases[END_REF] and are extremely sensitive to oxygen, which is toxic to them. Antioxidants have therefore been added to culture media to allow the culture of strict anaerobic bacteria under aerobic conditions [START_REF] Lagier | Current and past strategies for bacterial culture in clinical microbiology[END_REF][START_REF] Scola | Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report[END_REF]. A number of antioxidants have been tested and have shown satisfactory results. This is the case for ascorbic acid and glutathione [START_REF] Scola | Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report[END_REF] or uric acid [START_REF] Khelaifia | A quasi-universal medium to break the aerobic/anaerobic bacterial culture dichotomy in clinical microbiology[END_REF]. The addition of these antioxidants to the culture medium and its incubation under aerobic conditions allowed the growth of 135 strict anaerobic bacteria, 12 microaerophilic bacteria and 22 strict aerobic bacteria [START_REF] Khelaifia | A quasi-universal medium to break the aerobic/anaerobic bacterial culture dichotomy in clinical microbiology[END_REF].

III. How can we have a selective culture medium?

A selective culture medium is used to isolate a particular bacterial species or genus. After the addition of a number of inhibitors to the culture medium, the objective of this type of media is to eliminate unwanted microbial flora. The selective medium is composed of a basic medium to which antibiotics, chemicals, dyes, antiseptics, sodium salts or phages can be added [START_REF] Harrigan | Laboratory methods in food microbiology[END_REF].

Antibiotics

Antibiotics are the most commonly used selective agents. Their spectrum of action being well known, it is easier to anticipate their action on bacteria. There are a large number of antibiotics that can be used in culture media, some of which are called antibacterial because they target bacteria and others antifungal because they eliminate fungi and yeasts [START_REF] Entis | Food Microbiology: The Laboratory[END_REF]. Some molecules target Gram positives, such as penicillin G, bacitracin or vancomycin, while others target Gram negatives such as colistin or polymixin B. Amphotericin B, cycloheximide or nystatin have an action against fungi and yeasts (Table 1) [START_REF] Delarras | Pratique en microbiologie de laboratoire? Recherche de bactéries et de levures-moisissures[END_REF][START_REF] Kumar | Textbook of microbiology[END_REF]. Several antibiotics can be combined to obtain a more selective medium [START_REF] Entis | Food Microbiology: The Laboratory[END_REF].

Antiseptics

Antiseptics are more rarely used in culture media. However, cetrimide [START_REF] Brown | Use of an improved cetrimide agar medium and other culture methods for Pseudomonas aeruginosa[END_REF][START_REF] Oa | Antimicrobial activities of chlorhexidine gluconate and cetrimide against pathogenic microorganisms isolated from slaughter houses in rivers state, Nigeria[END_REF] or acriflavin [START_REF] Kumar | Textbook of microbiology[END_REF] can be found in some culture media. Chlorhexidine can be used to select Mycobacterium tuberculosis [START_REF] Asmar | Chlorhexidine decontamination of sputum for culturing Mycobacterium tuberculosis[END_REF][START_REF] Asmar | A chlorhexidine-agar plate culture medium protocol to complement standard broth culture of Mycobacterium tuberculosis[END_REF] (Table 2). Ethanol can also select bacterial species, including sporulated bacteria [START_REF] Browne | Culturing of 'unculturable' human microbiota reveals novel taxa and extensive sporulation[END_REF][START_REF] Koransky | Use of ethanol for selective isolation of sporeforming microorganisms[END_REF] such as Clostridiodes difficile [START_REF] Browne | Culturing of 'unculturable' human microbiota reveals novel taxa and extensive sporulation[END_REF].

Cetrimide Agar Base is a culture medium used to selectively isolate and identify Pseudomonas aeruginosa. Cetrimide is a quaternary ammonium that inhibits a large number of bacteria, including those of the genus Pseudomonas, other than P. aeruginosa [START_REF] Kumar | Textbook of microbiology[END_REF].

Sodium salts

Sodium salts are known for their inhibitory properties. The best known is sodium chloride, used to select halophilic bacteria that resist very high amounts of salts [START_REF] Seck | Halophilic & halotolerant prokaryotes in humans[END_REF]. In addition, sodium deoxycholate has a strong solvent action on bacteria (54) (Table 3).

The Marine Agar 2216E culture medium is used to enumerate marine heterotrophic bacteria.

It is composed of a high concentration of salt, which eliminates a large number of bacteria and preserves marine bacteria of interest [START_REF] Patrick | The use of membrane filtration and marine agar 2216E to enumerate marine heterotrophic bacteria[END_REF].

Chemical substances

Chemical substances can be added to culture media to inhibit certain bacteria. These inhibiting substances include potassium tellurite and bile salts, which inhibit Gram-positive bacteria [START_REF] Hoyle | A tellurite Blood-Agar Medium for the rapid diagnosis of diphtheria[END_REF][START_REF] Kumar | Textbook of microbiology[END_REF][START_REF] Delarras | Pratique en microbiologie de laboratoire? Recherche de bactéries et de levures-moisissures[END_REF] or lithium chloride [START_REF] Kumar | Textbook of microbiology[END_REF][START_REF] Delarras | Pratique en microbiologie de laboratoire? Recherche de bactéries et de levures-moisissures[END_REF], which eliminates Gram-negative bacteria (Table 4).

Brayton et al [START_REF] Brayton | New selective plating medium for isolation of Vibrio vulnificus biogroup 1[END_REF] have created a selective culture medium for Vibrio vulnificus, which is a pathogenic halophilic bacterium. This medium, VV agar, consists, among other things, of potassium tellurite as selective agent for inhibiting Enterobacteriaceae.

Dyes

Dyes can be used as a color indicator in a culture medium or as a selective agent against certain bacteria. Cristal violet is one of the most commonly used dyes to inhibit bacteria [START_REF] Entis | Food Microbiology: The Laboratory[END_REF][START_REF] Nichols | A new selective medium for Streptococcus pneumoniae[END_REF]. Malachite green and methylene blue are also used to inhibit Gram positive and negative bacteria and Gram positive bacteria respectively (10, 50) (Table 5).

A selective medium of Streptococcus pneumoniae has been developed, containing crystal violet. This dye is used to select Streptococci and inhibit Staphylococci as well as other Grampositive bacteria [START_REF] Nichols | A new selective medium for Streptococcus pneumoniae[END_REF].

Phages

Bacteriophages are specific viruses of bacteria that can infect and even destroy bacteria, in the case of lytic phages. In order to isolate Mycobacterium tuberculosis, the use of phage lysin decontaminates the sputum of other bacteria present in the pulmonary microbiota [START_REF] Subramanyam | Phage lysin as a substitute for antibiotics to detect Mycobacterium tuberculosis from sputum samples with the BACTEC MGIT 960 system[END_REF].

Sillankorva et al [START_REF] Sillankorva | Efficacy of a broad host range lytic bacteriophage against E. coli adhered to urothelium[END_REF] worked on permanent urinary tract infections due to Escherichia coli. In order to treat these infections, they tested different phages (T1, T4, and phiX174 like phages) against E. coli. After 2 hours of treatment, phage T1 reduced the E. coli population by 45%, thus demonstrating the efficacy of this selective agent.

IV. Disadvantages of gelling agents use

IV.1 Liquid culture media versus solid culture media

There are two main types of culture media, liquid and solid culture media.

In liquid culture media, also called culture broths, nutrients are dissolved in water. The growth of bacteria in this type of medium can be demonstrated by the appearance of a turbidity in the medium, although this is not always the case. It is difficult to isolate a bacterium specifically in this type of medium. Indeed, the bacteria obtained from a sample inoculated into the culture broth are all mixed with each other. In addition, this type of culture medium does not allow the morphological characteristics of bacterial species to be identified [START_REF] Vallery-Radot | Les oeuvres de Pasteur[END_REF].

However, liquid culture media facilitate access to nutrients for bacteria. These nutrients are all the more accessible as the culture media are incubated under agitation, allowing a renewal of nutrients for bacteria.

Solid culture media are obtained by adding a gelling agent, such as agar, to the culture broth.

They make it possible to obtain isolated colonies of different bacterial species, which can be identified. The different morphological characteristics of the bacterium can be described from these cultures [START_REF] Vallery-Radot | Les oeuvres de Pasteur[END_REF][START_REF] Denyer | Russell's pharmaceutical microbiology[END_REF]. However, in solid culture media, access to nutrients for bacteria may be limited. Media with high gel content, such as agar, will form smaller colonies than low gel content media because nutrient flow and toxin removal are reduced [START_REF] Murray | Medical microbiology[END_REF].

In addition, it has been shown that agar, in excessive quantities, can inhibit the growth of certain bacteria, highlighting the need to find other gelling agents [START_REF] Vallery-Radot | Les oeuvres de Pasteur[END_REF].

IV.2 Different gelling agents

One of the first gelling agents used in culture media was gelatin. The problem with this gelling agent is that it melts at 37°C, which is the incubation temperature of most bacteria.

Moreover, the presence of an enzyme in certain bacteria, gelatinase, causes the digestion of gelatin and therefore its degradation. Agar was then used in culture media. However, overconsumption of agar has led to a reduction in its source, red algae [START_REF] Mclachlan | Macroalgae (seaweeds): industrial resources and their utilization[END_REF], thus increasing costs.

In addition, agarase, present in some bacteria, destroys agar, thus preventing the isolation of these bacteria [START_REF] Das | Progress in the development of gelling agents for improved culturability of microorganisms[END_REF]. Moreover, agar can inhibit the growth of some anaerobic bacteria because inhibitory growth compounds can be produced from autoclaving phosphate with agar [START_REF] Tanaka | A hidden pitfall in the preparation of agar media undermines microorganism cultivability[END_REF].

Finally, agar can cause inhibition of PCR of fungal DNA when extracted directly from the solid culture medium [START_REF] Schmidt | Faster genetic identification of medically important aspergilli by using gellan gum as gelling agent in mycological media[END_REF]. For all these reasons, new gelling agents have been sought. These gelling agents include κ-carrageenan, ι-carrageenan, sodium alginate, HM (high-methoxyl)

and LM (low-methoxyl) pectins, gellan gum [START_REF] Das | Progress in the development of gelling agents for improved culturability of microorganisms[END_REF][START_REF] Doublier | Les systèmes alimentaires gélifiés: structure et propriétés fonctionnelles. 9ème Colloque de l'Alliance 7 / CEDUS : Le sucre et la conservation des produits à base de fruits[END_REF]. All these gelling agents have different properties and particular needs to gel (Table 6).

Carrageenan gums

κ-carrageenan and ι-carrageenan are part of the carrageenan gums.

The first will form a firm gel with a rapid mass build-up when combined with potassium ions.

It allows the growth of some bacteria. This gelling agent resists very alkaline pH values above 12.5, thus isolate highly very alkalineophilic bacteria [START_REF] Datta | Novel application of κ-carrageenan: as a gelling agent in microbiological media to study biodiversity of extreme alkaliphiles[END_REF]. It can be used to replace agar because many bacteria grow on κ-carrageenan-based media [START_REF] Abbott | Evaluation of kappa carrageenan as a substitute for agar in microbiological media[END_REF].

ι-carrageenan is rarely used because it gives elastic gels that make bacterial culture difficult [START_REF] Abbott | Evaluation of kappa carrageenan as a substitute for agar in microbiological media[END_REF].

Sodium alginate

This gelling agent is produced from brown seaweed extract and form a flexible gel in the presence of calcium ions. However, this gelling agent does not provide a gel firm enough to grow bacteria [START_REF] Doublier | Les systèmes alimentaires gélifiés: structure et propriétés fonctionnelles. 9ème Colloque de l'Alliance 7 / CEDUS : Le sucre et la conservation des produits à base de fruits[END_REF].

HM and LM pectins

HM pectins require sugar and high acidity to gel. Gel setting is slow and results in the formation of a "spreadable" gel.

LM pectins form brittle gels in the presence of Ca2+ [START_REF] Schmidt | Faster genetic identification of medically important aspergilli by using gellan gum as gelling agent in mycological media[END_REF].

Gellan gum

Gellan gum is a polysaccharide produced by a bacterial genus, Sphingomonas spp. According to Tamaki et al [START_REF] Tamaki | Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment[END_REF], 108 bacteria tested on media with gellan gum as gelling agent showed growth.

The use of these different gelling agents could allow the culture of new bacteria, which do not grow on the agar, because of the presence of an agarase for example or because the agar forms a network too dense to allow motility and optimal growth of certain bacteria [START_REF] Vallery-Radot | Les oeuvres de Pasteur[END_REF].

IV.3 Colony size

The amount of nutrients available in a culture medium will determine the size of bacterial colonies [START_REF] Denyer | Russell's pharmaceutical microbiology[END_REF]. An overly firm culture medium, due to a high concentration of gelling, causes a decrease in the flow of nutrients and thus a decrease in the access of these nutrients to bacteria [START_REF] Murray | Medical microbiology[END_REF][START_REF] Vallery-Radot | Les oeuvres de Pasteur[END_REF]. On the other hand, in some culture media, the amount of nutrients available is too high and can be toxic for certain bacteria that necessitate a poor culture medium to grow [START_REF] Ferrari | Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria[END_REF].

Microcolonies are colonies that are barely visible to the naked eye (between 100 and 300 µm in diameter). To obtain larger colonies, it is sometimes necessary to mimic the bacterium's natural environment by providing it with specific elements. This is the case, for example, for Phascolarctobacterium faecium and Phascolarctobacterium succinatutens, which form microcolonies. However, when the medium is supplemented with succinate, the colonies have a diameter ranging from 0.8 to 1.2 mm (69, 70).

Conclusion

After a stagnation in the development of new culture techniques, due to the rapid evolution of new microbiological methods such as metagenomics, bacterial culture is experiencing a new boom. In recent years, culturomics, with the use of new culture media and new culture conditions, has enabled the enrichment of the bacterial repertoire through the isolation of new bacterial species. This shows that, despite the abandonment of culture by a large number of microbiologists, culture media remain a fundamental tool for bacteriologists for the isolation of commensal but also pathogenic bacteria.

Studying the natural environment of bacteria that remain uncultivated to date would be interesting because it would provide the essential elements for the bacteria to grow. Indeed, although there are many enriched culture media, each bacterium is unique and requires specific needs. The use of new gelling agents could also allow the isolation of new species for which agar was not suitable for their growth. Although many gelling agents have been tested, few are still used in commercial culture media and therefore in laboratories. Many developments in bacterial culture are therefore still to come, making it possible to enrich the bacterial repertoire and gain a better understanding of certain diseases.

In addition, intracellular bacteria such as Coxiella burnetii or Tropheryma whipplei require a host cell to survive and multiply. Some of these bacteria cause severe diseases and pose a diagnostic problem because of their fastidious growth or lack of growth on conventional media [START_REF] Lamoth | Approches diagnostiques des bactéries intracellulaires et des germes fastidieux[END_REF]. It would therefore be interesting to develop culture media that allow faster and easier detection of these bacteria. In addition, as the microbiota plays an increasingly important role in human health [START_REF] Sekirov | Gut microbiota in health and disease[END_REF][START_REF] Guinane | Role of the gut microbiota in health and chronic gastrointestinal disease: understanding a hidden metabolic organ[END_REF], the development of probiotics is on the rise [START_REF] Kerry | Benefaction of probiotics for human health: A review[END_REF]. The use of targeted culture media to select certain bacteria with an important medical role therefore remains a priority.

Table 1 :

 1 Antibiotics agents used in bacterial culture: Action against bacteria or fungiNo action against bacteria or fungi

	Microorganisms
	*A7 Agar modified	ECN Pilly 2018 (38) Power et al, 2009 (11)
	*PC agar	Power et al, 2009 (11)
	*Bolton broth * Preston Agar and Broth	Delarras, 2014 (39)
	*Differentiation test	Delarras, 2014 (39)

Dyes


Stéphane Dupas

Fabrice Neiers

Emma Granon

Erwan Rougeux

Sébastien Dupont

Laurent Beney

Haq François Bousquet

Abdul Shaik

Loïc Briand

Hubert Wojtasek
email: hubert.wojtasek@uni.opole.pl.

Haq Abdul Shaik

Loic Briand

Jean-Philippe Charles
email: philippe.charles@u-bourgogne.fr

Collisional mechanism of ligand release by Bombyx mori JHBP, a member of the TULIP / Takeout family of lipid transporters

Keywords:

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Juvenile hormones (JHs) regulate important processes in insects, such as postembryonic development and reproduction [START_REF] Goodman | The juvenile hormones[END_REF][START_REF] Jindra | Molecular basis of juvenile hormone signaling[END_REF]. In the hemolymph of Lepidoptera, JHs are transported from their site of synthesis (the corpora allata) to target tissues by high affinity carriers, the juvenile hormone binding proteins (JHBPs). It is believed that these proteins protect the hormones from degradation and non-specific adsorption to lipophilic depots [START_REF] Goodman | Biosynthesis, Titer Regulation and Transport of Juvenile Hormones[END_REF]; [START_REF] Koeppe | Juvenile Hormone-Binding Proteins[END_REF].

For a long time, the details of JHBP-JH interactions have remained elusive. The X-ray structure of apoJHBP was first determined for the protein from Galleria mellonella [START_REF] Kolodziejczyk | Insect juvenile hormone binding protein shows ancestral fold present in human lipid-binding proteins[END_REF].

The structure consists of a long α-helix surrounded by a twisted, anti-parallel ß-sheet with two cavities forming potential binding sites at the opposite ends of the molecule (Figure S1). It closely resembles the structure of several other lipid binding proteins, such as the Takeout 1 protein of the moth 3 Epiphyas postvittana [START_REF] Hamiaux | Ligand promiscuity within the internal cavity of Epiphyas postvittana Takeout 1 protein[END_REF][START_REF] Hamiaux | Crystal structure of Epiphyas postvittana Takeout 1 with bound ubiquinone supports a role as ligand carriers for Takeout proteins in insects[END_REF], as well as the mammalian bactericidal/permeability increasing protein (BPI) [START_REF] Beamer | Crystal structure of human BPI and two bound phospholipids at 2.4 angstrom resolution[END_REF] and the cholesteryl ester transfer protein (CETP) [START_REF] Qiu | Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules[END_REF]. Phylogenetic analyses have shown that these proteins and many others, including plant proteins, share one or two copies of an ancestral TULIP lipid binding motif (Tubular Lipid binding), which allows lipid transport, as well as interaction with phospholipids at the surface of membranes or lipoproteins [START_REF] Alva | The TULIP superfamily of eukaryotic lipid-binding proteins as a mediator of lipid sensing and transport[END_REF][START_REF] Wong | Tubular lipid binding proteins (TULIPs) growing everywhere[END_REF].

Although very ancient and therefore likely playing fundamental functions in lipid trafficking for most eukaryotes and prokaryotes, the functions of the members of this emerging family are mostly known from studies in mammals. In invertebrates, the TULIP domain proteins Takeout and Takeout-related proteins have been involved in various processes (courtship behavior, feeding, aging, JH transport, perception [START_REF] Fujikawa | A novel Takeout-like protein expressed in the taste and olfactory organs of the blowfly, Phormia regina[END_REF][START_REF] Galikova | Dietary restriction and other lifespan extending pathways converge at the activation of the downstream effector takeout[END_REF][START_REF] Lazareva | A role for the adult fat body in Drosophila male courtship behavior[END_REF][START_REF] Meunier | Regulation of feeding behaviour and locomotor activity by Takeout in Drosophila[END_REF][START_REF] Sarov-Blat | The Drosophila Takeout gene is a novel molecular link between circadian rhythms and feeding behavior[END_REF][START_REF] Yoshizawa | Ligand carrier protein genes expressed in larval chemosensory organs of Bombyx mori[END_REF]). Lepidopteran JHBPs appear to constitute a subclass of the Takeout proteins, characterized by the presence of a conserved disulfide bridge dividing the large internal hydrophobic cavity into two smaller ligand binding pockets [START_REF] Hamiaux | Ligand promiscuity within the internal cavity of Epiphyas postvittana Takeout 1 protein[END_REF][START_REF] Hamiaux | Crystal structure of Epiphyas postvittana Takeout 1 with bound ubiquinone supports a role as ligand carriers for Takeout proteins in insects[END_REF][START_REF] Kolodziejczyk | Insect juvenile hormone binding protein shows ancestral fold present in human lipid-binding proteins[END_REF][START_REF] Kolodziejczyk | Two disulphide bridges are present in juvenile hormone binding protein from Galleria mellonella[END_REF]. To this day, JHBPs are the only members with well characterized endogenous ligands.

The structure of JHBP from Bombyx mori with a bound hormone was solved by both X-ray diffraction and NMR spectroscopy [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]. This study revealed that ligand uptake and release are accompanied by substantial conformational changes of the protein, in agreement with earlier findings on G. mellonella JHBP [START_REF] Krzyzanowska | UV-difference and CD spectroscopy studies on juvenile hormone binding to its carrier protein[END_REF][START_REF] Wieczorek | Conformational change of the haemolymph juvenile-hormonebinding protein from Galleria mellonella (L)[END_REF].

It was shown that the N-terminal helix functions as a gate, which closes upon ligand binding. In the apo form of the protein in the crystalline state, the gate is open, allowing access of the ligand to the binding site. In solution, the gate of the apoJHBP is highly mobile and the protein exists in multiple conformations, including the gate-open conformation, seen in the crystalline state, and the gate-closed conformation similar to the ligand-bound form. This flexibility of the gate in the apo-JHBP disappears upon JH-binding. Based on these structural changes a mechanism of JH delivery to target cells has been proposed ( [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF], Figure S1).

Release of a hydrophobic ligand from a complex with a binding protein has been previously demonstrated for a number of proteins. B. mori pheromone-binding protein (PBP) [START_REF] Horst | NMR structure reveals intramolecular regulation mechanism for pheromone binding and release[END_REF] binds its ligand only at neutral and basic pH, whereas low pH induces the formation of an additional α-helix (helix 7), which fills the binding pocket and prevents ligand binding. Analysis by CD and fluorescence spectroscopy has demonstrated that structural changes, very similar to those evoked by low pH, were also induced by phospholipid vesicles [START_REF] Wojtasek | Conformational change in the pheromone-binding protein from Bombyx mori induced by pH and by interaction with membranes[END_REF], and it was therefore hypothesized that pheromone release could be triggered by phospholipid membranes on the surface of olfactory neurons, thus providing a mechanism of ligand delivery to target cells.

Evidence for pH-induced ligand release has also been shown for several other hydrophobic ligand binding proteins, including serum retinol binding protein [START_REF] Ptitsyn | Mechanism of pH-induced release of retinol from retinol-binding protein[END_REF] and tear lipocalin [START_REF] Gasymov | Interstrand loops CD and EF act as pH-dependent gates to regulate fatty acid ligand binding in tear lipocalin[END_REF]. More generally, acidic pH and interaction with phospholipidic membranes are two common triggers for membrane-active proteins, such as pore-forming bacterial toxins or viral fusion proteins [START_REF] Butko | Membrane interactions and surface hydrophobicity of Bacillus thuringiensis delta-endotoxin CryIC[END_REF][START_REF] Harrison | Viral membrane fusion[END_REF][START_REF] Jiang | Involvement of denaturation-like changes in Pseudomonas exotoxin a hydrophobicity and membrane penetration determined by characterization of pH and thermal transitions. Roles of two distinct conformationally altered states[END_REF][START_REF] London | Diphtheria toxin: membrane interaction and membrane translocation[END_REF][START_REF] Menestrina | Interaction of tetanus toxin with lipid vesicles. Effects of pH, surface charge, and transmembrane potential on the kinetics of channel formation[END_REF].

We have investigated the effects of pH and phospholipid membranes on ligand binding by B. mori and Manduca sexta JHBPs by several methods. We show that acidic pH substantially reduces JHBP affinity for JH III, apparently increasing its koff, which suggests that low pH favors ligand release, presumably by keeping the protein in the open conformation. We also report that JH III binding is diminished in the presence of anionic phospholipid liposomes, and that B. mori JHBP significantly interacts with these membranes. Taken together, the results support a collisional model of ligand release mechanism triggered by specific electrostatic and hydrophobic interactions on the surface of the plasma membrane of target cells.

Material and methods

2.1.

Purification of native JHBP [START_REF] Kurata | Purification and characterization of a juvenile hormone binding protein from hemolymph of the silkworm, Bombyx mori[END_REF]. The hemolymph was collected from fifth instar day 4 larvae into Falcon tubes on ice, frozen in liquid nitrogen and lyophilized. The lyophilizate was dissolved in 0.5 mM phenylthiourea in water before purification, centrifuged for 10 min at 10000 g and dialyzed against 20 mM Tris-HCl, pH 7.5 overnight at 4 °C. The dialyzed sample was applied to a DEAE-Sepharose (DFF-100, Sigma-Aldrich) column and the proteins were eluted with a stepwise gradient of 50-300 mM NaCl in 20 mM Tris-HCl, pH 7.5. Fractions containing JHBP were pooled, mixed with an equal volume of 2 M ammonium sulfate and applied to a Phenyl-Sepharose (Fast Flow, high substitution, GE HealthCare) column. Proteins were eluted with a stepwise gradient of 0.75-0.1 M ammonium sulfate in 20 mM Tris-HCl, pH 7.5 and then water. Fractions containing JHBP were pooled again and applied to a hydroxyapatite (Ceramic Hydroxyapatite Type I, BioRad) column. Proteins were eluted with a stepwise gradient of 10-300 mM sodium phosphate buffer, pH 6.8. Fractions from each purification step were analyzed by SDS-PAGE and DCC binding assay (Figure S2). This protein preparation of native B. mori JHBP (nBmJHBP) was used for initial binding studies (Figure 1), testing the effect of ionic strength (Figure S6), and estimation of koff (Figures 5,S8). Other experiments were performed with recombinant B. mori (rBmJHBP, see below).

Expression of recombinant JHBPs

B. mori larvae were reared on mulberry leaves under natural daylight conditions. Fat body was isolated from fifth instar day 5 larvae, frozen in liquid nitrogen and stored at -70 °C. Total RNA was isolated with the single phenol-chloroform-isothiocyanate extraction method [START_REF] Chomczynski | Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction[END_REF], and mRNA was isolated with the PolyATtract mRNA Isolation system III (Promega).

The cDNA was synthesized with AMV reverse transcriptase and a primer corresponding to the mRNA fragment encoding the C-terminus of JHBP. The reverse transcription reaction mixture was used directly for PCR reactions with Pfu DNA polymerase and primers designed based on the published B.

mori JHBP cDNA sequence [START_REF] Vermunt | The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis[END_REF]: JHBPBmF (5'-GCAGAATTCCCATGGGAGATGGAGATGCTCTT) and JHBP-BMX-rev2 (5'-CGGTGAATTCTTAATTAAGATTTTCG), and the product coding the mature form of the protein (starting with Asp19 immediately after the signal peptide excision site [START_REF] Vermunt | The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis[END_REF]) was ligated at the EcoRI site of pGEM™-T Easy Vector (Promega). An insert in the right orientation was excised with EcoRI and NcoI, and ligated at the EcoRI and NcoI sites of Pet-22b vector (Novagen, Merck) in frame with the pelB leader, allowing periplasmic localization. We found 28 nucleotides differing from the original published sequence [START_REF] Vermunt | The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis[END_REF], among which 10 yielded amino acid substitutions. These differences likely stem from the natural allelic diversity amongst B. mori strains (Figure S3). A cDNA kindly provided by W.G. Goodman (University of Wisconsin-Madison), was used as a template to amplify the sequence encoding M. sexta JHBP with primers JHBP-Manduca-fw (5'-ATATCCATGGGAGATCAAGGGGCACT) and JHBP-Manduca-rev (5'-CGCTGAATTCAAAATTAATTAACATCC). The product coding the mature form of the protein (starting with Asp19 immediately after the signal peptide excision site [START_REF] Touhara | Ligand binding by a recombinant insect juvenile hormone binding protein[END_REF] was fused in frame with the pelB leader in Pet-22b. As we failed to obtain soluble protein with the pelB leader system, the sequence coding for pelB was removed by digesting pET-22b (+) with NdeI and MlsI, the ends were polished with Klenow, and ligated to yield pET-22b-(minus)-pelB plasmids, encoding the mature forms with two extra residues (Met1 and Gly2) at the amino-terminus. Shuffle bacteria (New England Biolabs) were transformed with either B. mori or M. sexta Pet-22b-(minus)-pelB JHBP plasmid, grown in the presence of 0.5% glucose and protein expression was induced by IPTG. Bacterial pellets were lysed for 15 min at 30 °C in a lysis buffer (25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 100 µg/mL lysozyme, and proteases inhibitors), sonicated on ice, and centrifuged for 10 min at 12000 g. Recombinant JHBPs from M. sexta (rMsJHBP) and B. mori (rBmJHBP) were purified from bacterial lysates essentially as described above for the native protein from B. mori (Figure S2).

Ligand Binding Assays

The DCC (Dextran Coated Charcoal) assay was performed essentially as described previously [START_REF] Touhara | Ligand binding by a recombinant insect juvenile hormone binding protein[END_REF]. Unless indicated otherwise, the continuous buffer system of McIlvaine composed of citrate and phosphate was used to explore the binding for various pH values [START_REF] Mcilvaine | A buffer solution for colorimetric comparison[END_REF]. For typical DCC assays, JHBP (50-300 nM) was diluted in a final volume of 100 µL of buffer containing a JH esterase inhibitor (OTFP: 3-octylthio-1,1,1-trifluoro-2-propanone, a kind gift of B.D.

Hammock (University of California) at a final concentration of 1 µM, and about 10000 dpm [ 3 H]-JH III (~ 20 Ci/mmol, Perkin-Elmer). After incubation at room temperature (ca. 20 °C) for one hour in a 6

x 50 mm borosilicate glass tube (Kimble-Chase, # 73500-650) precoated with a 10 % polyethylene glycol solution ( [START_REF] Goodman | The Juvenile Hormones[END_REF], Sigma # 95172), 20 µL of a DCC slurry (5% (w/v) activated charcoal (Norit A, ACROS Organics # 134342500), 1 % (w/v) Dextran (Sigma # D4751), 1.5 mM EDTA) was added and the mixture was gently mixed by vortexing for about 2 s. After a 30 s incubation, the tubes were centrifuged (ca. 10000 g, 2 min), and the radioactivity of a 50 µL aliquot of the supernatant was measured in a scintillation counter to estimate the amount of bound [ 3 H]-JH III. In these conditions, 10-50 % of [ 3 H]-JH III was bound by JHBP. For dialysis assays, 70 µL of a buffer containing about 70000 dpm of [ 3 H]-JH III was loaded into each chamber of a disposable dialysis device with a 10 kDa MW cut-off (Harvard Apparatus, Holliston, MA, U.S.A.). Proteins (1-5 µg of purified JHBP or bovine serum albumin (BSA) used as a control), were added to one chamber of the device. After overnight incubation at 4 °C, an aliquot of each chamber was counted in a scintillation counter, and the percentage of bound ligand was determined. For experiments involving lipid membranes, varying amounts (5-600 µg) of liposomes were added to the chamber of a dialysis device with or without JHBP.

Isothermal titration calorimetry

Isothermal titration calorimetry (ITC) experiments were carried out at 293 K and 298 K using a VP-ITC microcalorimeter (GE Healthcare). rBmJHBP was dialyzed against 100 mM sodium phosphate buffer, pH 7.0, or 100 mM sodium acetate buffer, pH 4.5. Solutions of JH III were prepared in the same buffer as used for dialysis. The titration sequence included a single 3 µL injection followed by 25 injections of 10 µL of ligand solution, with 210 s intervals between injections.

Measurements were performed for 5 µM protein with 70 µM ligand, or 2.5 µM protein with 35 µM ligand. The OriginLab software (GE Healthcare) was used to fit the raw data using a single bindingsite model. For the analysis the data points obtained after the first 3 µL injection were discarded. The experiments were performed 3 times for each ligand and protein concentrations indicated, and the dissociation constants (Kd) were calculated.

Circular dichroism

Near UV CD spectra were recorded between 250 and 320 nm in a JASCO J-815 spectropolarimeter at 20 °C, using a 1 cm path length quartz cell (Hellma). rBmJHBP (25 µM) was dissolved in McIlvaine buffer at pH 3.5, 4.5, 5.5, 6.5 or 7.5. Data were averaged over 10 accumulated scans recorded at 1 nm intervals with a 50 nm/min scan speed. The spectra were corrected for buffer contributions and converted to molar ellipticity in deg•cm²•dmol -1 .

Tryptophan fluorescence spectroscopy

The intrinsic fluorescence of rBmJHBP was measured with a Cary Eclipse spectrofluorometer (Varian Instruments) at 20 °C, in a 2 mL quartz cuvette with a 1 cm path length equipped with a magnetic stirrer. The effect of ligand addition was examined using rBmJHBP at 10 µM final concentration before and after 15 min equilibration with JH III at various concentrations (1.18, 2.32, 3.42, 6.5, 11.87 and 20.25 µM) in 100 mM sodium phosphate buffer, pH 7.0. The proteins were excited at 293 nm and emission spectra were recorded from 320 to 450 nm with a 10 nm bandwidth for both emission and excitation. The results were corrected for the dilution due to the addition of different volumes of JH III solution. For each ligand concentration, two or three spectra were recorded and averaged.

To study pH-dependent changes in fluorescence, a small volume of the concentrated rBmJHBP protein (28 µL), was added to a large volume of McIlvaine buffer (972 µL) at different pH values (4.5, 5.5, 6.5, 7.0, 7.5), thus ensuring a minor pH change of the final 10 µM protein solution.

Fluorescence was recorded as described above. Three independent measurements were performed for each pH value and the spectra were averaged.

Light scattering

The size and homogeneity of rBmJHBP at neutral and acidic pH in McIlvaine buffers were examined by light scattering using a Zetasizer Nano S apparatus (Malvern, U.K.).

Liposome preparation

Solutions of lipids (DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, Sigma #43096), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine, Sigma #P-2663), or a 1:1 mixture of both) at a 10 mg/mL concentration in chloroform were evaporated, first by heating at 45 °C under a nitrogen stream, then for further 2-6 h with a centrifugal evaporator (Speedvac). The lipids were suspended in water (10 mg/ml), aliquoted and stored at -20 °C. Unilamellar liposomes were obtained by extrusion of the phospholipid suspension through a 100 nm pore size polycarbonate filter (Liposofast, Avestin), at 37 °C. The final amount of phospholipid was verified by gas chromatography, and the quality (size and distribution) of the unilamellar liposomes was verified by light scattering. respectively. G = Ihv/Ihh is a correction factor for the monochromator's transmission efficiency for vertically (v) and horizontally (h) polarized light.

Determination of fluidity of DMPG liposomes

Statistical analyzes

All analyses (Student's test, curve fittings, determination of pH50, determination of the temperature of phase transition) were performed with Graphpad Prism version 5.0d for Mac OS X (GraphPad Software, La Jolla, California USA).

Results

Low pH reduces affinity of native and recombinant lepidopteran JHBPs for JH III

A sharp decrease of binding affinity of Lepidopteran JHBP in the low pH range was first discovered for the G. mellonella protein [START_REF] Ozyhar | Juvenile-hormone-binding protein from the hemolymph of Galleria mellonella (L). Isolation and characterization[END_REF], and a similar correlation was later reported for the recombinant B. mori JHBP [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]. We have examined the affinity of the native JHBP from B. mori, and recombinant proteins from B. mori and M. sexta with the Dextran Coated Charcoal (DCC) assay, and also observed a steep decrease of affinity at low pH in all three cases, with a 50% reduction of binding in the pH range between 4 and 5.5 (Figure 1A). The native proteins were more sensitive to hydrogen ion concentration (pH50 values for nBmJHBP and nGmJHBP (inferred from data in [START_REF] Ozyhar | Juvenile-hormone-binding protein from the hemolymph of Galleria mellonella (L). Isolation and characterization[END_REF])) were 5.08 and 5.45, respectively), than the recombinant proteins from B. mori and M. sexta (pH50 values were 4.18 and 4.67, respectively). The cause of the lower sensitivity of the recombinant proteins is unclear. The effect of low pH on nBmJHBP, rBmJHBP and rMsJHBP was also examined using a dialysis assay (Figure 1B).

The results showed a significant (two-to four-fold, p<0.001, Student's t test, n=3) reduction of binding at pH 4, when compared to pH 7. In addition, the relative sensitivity of the three proteins was the same as found with the DCC assay (Figure 1B): nBmJHBP was the most sensitive, whereas rBmJHBP showed only a ca. twofold reduction in affinity at pH 4 in this assay.

The dissociation constant (Kd) of the recombinant B. mori JHBP for JH III was then determined at pH 4.5 and pH 7.0 by isothermal titration calorimetry (Figure 1C,D). Consistently with the above data, the Kd is increased at acidic pH. Interestingly, increasing the temperature to 25 °C had opposite effects at pH 4.5 and pH 7, as the Kd was increased at pH 4.5, but lowered at pH 7. This difference of dependence of the Kd on temperature may reflect the existence of distinct conformations of rBmJHBP at acidic and neutral pH [START_REF] Vega | On the link between conformational changes, ligand binding and heat capacity[END_REF]. The Kd measured at 25 °C and pH 7 (37 nM) is similar to the Kd for JH II reported for a baculovirus-produced M. sexta JHBP (42 nM, [START_REF] Touhara | Ligand binding by a recombinant insect juvenile hormone binding protein[END_REF]), but much lower than the Kd for JH III reported earlier for a B. mori JHBP expressed in E.

coli (450 nM, [START_REF] Vermunt | The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis[END_REF]). The Kds reported in this study are, however, higher than those reported for enantiomerically-pure JH III and native M. sexta JHBP which were determined by equilibrium dialysis (1.86 nM, [START_REF] Park | Affinity purification and binding analysis of the hemolymph juvenile hormone binding protein from Manduca sexta[END_REF]). These differences likely result from the varying conditions used in the different studies, notably the procedures for protein production and/or purification, the nature and purity of the ligands, and the methods used for binding assays [START_REF] Park | Affinity purification and binding analysis of the hemolymph juvenile hormone binding protein from Manduca sexta[END_REF]). The effect of low pH cannot be accounted for by pH-dependent solubility of the ligand or ligand breakdown (such as through hydrolysis of the epoxide ring by nucleophilic attack at low pH (Figure S4)). Moreover, light scattering analysis showed that the apparent size and aggregation of recombinant rBmJHBP were essentially the same at acidic and neutral pH (Figure S5).

Since the ionic strength of McIlvaine buffers changes with pH (0.157 M at pH 4, and 0.427 M at pH 7, [START_REF] Elving | Preparation of buffer systems of constant ionic strength[END_REF])), we were also concerned that this factor may contribute to changes in binding. However, binding experiments in the presence of 0-0.6 M NaCl showed that the binding affinity decreases with increasing ionic strength (Figure S6). Therefore, reduced binding at low pH can likely be attributed to increased proton concentration, rather than reduced ionic strength.

Reduced JH III binding at low pH is reversible

We next asked whether this effect of low pH on JHBP affinity could be reversed. When nBmJHBP was first allowed to bind JH at neutral pH and reach equilibrium, adjusting the pH to a value of 4 was sufficient to reduce dramatically the amount of bound JH. Conversely, after one hour incubation at pH 4, full binding activity could be recovered by adjusting the pH back to neutrality (Figure 2).

Effect of pH on JHBP structure

We next tested the hypothesis that reduced binding at acidic pH could be evoked by a switch from the closed to the open conformation, as proposed by Suzuki and colleagues (Figure S1) [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]. Far and near UV circular dichroism (CD) analyses were performed to examine if the structure of JHBP is different at acidic and neutral pH. For these and further experiments rBmJHBP was used, except as indicated. Little change was observed in far UV CD spectra (Figure S7), suggesting that the secondary structure of rBmJHBP is essentially unchanged at these two pH values,

consistently with the open and closed forms determined by NMR [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]. These data show in addition that rBmJHBP is well structured at both pHs.

In contrast, however, near UV CD spectra taken at several pH values showed significant differences, indicating changes in the tertiary structure of the protein (Figure 3). We also obtained fluorescence spectra, taking advantage of the fact that B. mori JHBP contains a single tryptophan residue (Trp38) [START_REF] Vermunt | The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis[END_REF], in the loop forming the gatepost for the gate-forming α1 helix near the entrance of the ligand binding pocket (Figure 4, Figure S1, [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]). At neutral pH, ligand binding induces a change in the environment of Trp38, as evidenced by a significant drop of fluorescence emission at 293 nm with JH III concentrations ranging from about 1 to 20 µM (Figure 4A). This result shows that the environment of Trp38 changes, and is consistent with the fact that the nearby α1 helix, which forms the "gate" of the ligand binding pocket, undergoes a major change of position upon ligand binding (Figure S1, [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]). Importantly, lowering the pH also strongly affected fluorescence intensity (Figure 4B). The fact that a drop in fluorescence emission is observed in both experiments is not paradoxical, because the fluorescence of tryptophan is known to be exquisitely sensitive to changes in the local environment [START_REF] Hellmann | Hands on: using tryptophan fluorescence spectroscopy to study protein structure[END_REF][START_REF] Royer | Probing protein folding and conformational transitions with fluorescence[END_REF], and the mere presence (Figure 4A) or absence (Figure 4B) of ligand, may have strong and hardly predictable effects.

Although we cannot exclude that the change in fluorescence seen with decreasing pH is partly due to the protonation of His206, which is bound to Trp38 by π-π stacking interactions [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF], this observation, together with UV CD spectra, suggests that at acidic pH, rBmJHBP undergoes a significant change in tertiary structure that may involve the switch of the α1 helix into the " open " conformation, and hence trigger ligand release.

Binding kinetics

Considering the hypothesis that the reduced binding observed at low pH (Figure 1), or at high ionic strength (Figure S6), results from the opening of the gate-forming α1 helix, we expected that in both conditions the dissociation rate constant (koff) of the nBmJHBP-JH III complex would be increased. To address this point, we attempted to estimate koff using a modified DCC assay. As shown in Figure 5A, the estimated koff of the complex was unchanged at high ionic strength, suggesting that low JH binding in this condition is caused by reduced access of JH III to the binding pocket, rather than by increased ligand release. In contrast, at acidic pH, the koff appeared roughly three times greater than at neutral pH (Figure 5B). This result suggests that a high proton concentration may contribute to ligand release by nBmJHBP. The absolute values for koff measured here should be considered as preliminary and rough estimates (since protein loss was evident for longer incubation times in this assay (Figure S8)). The koff values determined at pH 7 in this study (ca. 0.033 s -1 , Figure 5) are similar to those measured for the native M. sexta JHBP-JH III complex by two different approaches (0.0525 s - 1 and 0.0357 s -1 , [START_REF] Park | Affinity purification and binding analysis of the hemolymph juvenile hormone binding protein from Manduca sexta[END_REF]), but must be confirmed in future experiments with a dedicated and accurate method (such as stop-flow spectroscopy). Together with the observation that low pH provokes a change in fluorescence intensity (Figure 4B), however, these results are consistent with the hypothesis that low pH contributes to a switch from the "closed" to the "open" conformation of the α1 helix.

Interaction of JHBP with lipid vesicles

It was previously shown that if the dielectric constant of a binding mixture is increased by adjusting the solution to a final concentration of 30 % ethanol, thereby mimicking the hydrophobic environment of the cell membranes, JH III was released from JHBP [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]. We have therefore decided to examine if ligand binding by rBmJHBP is modified in the presence of phospholipid membranes. Unilamellar phospholipid vesicles composed of either pure anionic 1,2dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), or a 1:1 mixture of DMPG and 1,2-dimyristoylsn-glycero-3-phosphocholine (DMPC, a zwitterionic phospholipid) were prepared, and their influence on JH binding was assessed by a dialysis assay (Figure 6). JHs are lipophilic hormones, and were shown earlier to partition into phospholipid membranes [START_REF] Barber | Perturbation of phospholipid membranes by juvenile hormone[END_REF]. Therefore, as expected, DMPG liposomes loaded in one chamber of a dialysis device bound [ 3 H]-JH III in a dose-dependent manner from 5 µg to 200 µg per assay, with a plateau at about 70% bound ligand (Figure 6, white columns). In this dialysis assay, rBmJHBP alone showed robust binding to [ 3 H]-JH III at neutral pH (Figure 6, black column). However, when JHBP was incubated with the ligand in the presence of DMPG vesicles at an amount equal to or greater than 40 µg, the percentage of bound [ 3 H]-JH III was significantly lower than when rBmJHBP was incubated alone (Figure 6, striped columns). This result therefore suggests that interaction with DMPG liposomes prevents binding of JH III by rBmJHBP. The anionic nature of the phospholipid was apparently important for this effect, since even a high (600 µg) amount of liposomes composed of a 1:1 mixture of the anionic DMPG and zwitterionic DMPC, was barely sufficient to significantly reduce JH III binding by rBmJHBP (Figure 6, grey columns).

To examine the effect of anionic phospholipids more closely, the interaction of rBmJHBP with DMPG liposomes was analyzed by a steady-state fluorescence polarization method ( [START_REF] Borenstain | Characterization of liposomes and other lipid assemblies by multiprobe fluorescence polarization[END_REF], Figure 7). The fluorescence anisotropy of the TMA-DPH probe embedded in lipid membranes is a measure of the motional order within the headgroup region of the phospholipids, as far down as C8-C10 [START_REF] Prendergast | 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene: synthesis, fluorescence properties, and use as a fluorescence probe of lipid bilayers[END_REF]. DMPG liposomes were incubated first with the hydrophobic fluorescent probe TMA-DPH alone. The decreasing slope of TMA-DPH fluorescence anisotropy with temperature was relatively shallow, consistently with earlier observations [START_REF] Lamy-Freund | The peculiar thermo-structural behavior of the anionic lipid DMPG[END_REF]; Figure 7: liposomes alone). The shape of the temperature-dependent anisotropy curve was significantly different in the presence of rBmJHBP (Figure 7: rBmJHBP). Below 20 °C, the motion of acyl chains was increased, while membrane phospholipids were stabilized between 20 °C and 27 °C.

Importantly, neither BSA nor heat-denatured rBmJHBP had an effect on TMA-DPH fluorescence.

These results indicate that rBmJHBP strongly interacts with DMPG membranes at neutral pH, and that this interaction requires a proper folding of the protein.

Discussion

More than three decades ago, in their study of G. mellonella JHBP, Ozyhar and Kochman reported a steep decrease in its JH binding activity below pH 7, and proposed that this peculiar activity profile may be shared by JHBPs from other species ( [START_REF] Ozyhar | Juvenile-hormone-binding protein from the hemolymph of Galleria mellonella (L). Isolation and characterization[END_REF], Figure 1). A similar pH-sensitivity was later observed for a recombinant B. mori JHBP [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]. We report here that the native JHBP purified from larval B. mori hemolymph, as well as recombinant JHBPs from this species and M. sexta, share indeed with their G. mellonella homologue a similar sensitivity profile at low pH (Figure 1).

We think that this conserved feature may hint to the existence of a biologically relevant ligand release mechanism involving the switch of the gate-forming α1 helix, as proposed by Suzuki and coworkers (Figure S1, [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]). This hypothesis is supported by UV fluorescence and circular dichroism spectrometry, as well as light scattering data, showing that the secondary structure of B. mori JHBP is very similar at acidic and neutral pH, whereas in contrast, significant changes in tertiary structure are observed, consistently with a conformational switch of the α1 helix (Figures 3, S5, S7).

Our fluorescence spectrometry data indicate that low pH also strongly influences the fluorescence intensity of rBmJHBP (Figure 4B), which likely reflects a significant change occurring in the environment of Trp 38, located in the "gatepost" of B. mori JHBP (Figure S1). This result would be expected if the α1 helix switches to the closed conformation upon ligand binding, and is consistent with the apparent increase of koff of the nBmJHBP-JH III complex (Figure 5). In their NMR studies, Suzuki and colleagues observed that there were only small differences in chemical shifts for most residues when JHBP was incubated at pH 4 or pH 6, and that the intermolecular NOEs between JH III and JHBP were conserved in both conditions, indicating that JH III was bound at acidic pH. Although these results may appear in contradiction with ours, this discrepancy may be explained by the high (0.6-1 mM) concentrations of JHBP and ligand applied in NMR experiments [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF].

Considering the Kd values determined in this study (Figure 1D), and the generally accepted maximal concentration range for juvenile hormones in aqueous solutions (30 to 50 µM [START_REF] Kramer | The juvenile hormone binding protein in the hemolymph of Manduca sexta Johannson (Lepidoptera: Sphingidae)[END_REF]), the law of mass action predicts a high fractional JHBP occupancy (> 90%) at both neutral and acidic pH at concentrations applied in NMR experiments.

The TULIP superfamily of LTPs, to which JHBP belongs, has recently been recognized as very ancient, since it is present in a wide range of animal, fungal, algal, and plant cells, as well as in prokaryotic cells [START_REF] Alva | The TULIP superfamily of eukaryotic lipid-binding proteins as a mediator of lipid sensing and transport[END_REF][START_REF] Wong | Tubular lipid binding proteins (TULIPs) growing everywhere[END_REF]. TULIP domain proteins are involved in a variety of processes related to lipid shuttling, either between membrane compartments or lipoprotein particles, such as HDL and LDL. In vertebrates, the plasma phospholipid transport protein (PLTP) and the cholesteryl-ester transfer protein (CETP), both harboring two TULIP domains, have well known functions in neutral lipid and phospholipid transfer between lipoprotein particles [START_REF] Masson | The role of plasma lipid transfer proteins in lipoprotein metabolism and atherogenesis[END_REF][START_REF] Tzotzas | Plasma phospholipid transfer protein (PLTP): review of an emerging cardiometabolic risk factor[END_REF]. Although the details of the molecular mechanisms of docking and 10 lipid transfer of CETP and PLTP to lipoproteins are still debated, it is now well established that CETP interacts strongly with the outer phospholipid layer of lipoproteins [START_REF] Lauer | Cholesteryl ester transfer between lipoproteins does not require a ternary tunnel complex with CETP[END_REF][START_REF] Qiu | Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules[END_REF][START_REF] Zhang | Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein[END_REF]. Its N-terminal end penetrates ca. 50 Å into HDL particles, thus fully crossing the 18-27 Å phospholipid layer. Interestingly, shallow penetration into liposomes has also been observed [START_REF] Charles | New molecular insights into CETP structure and function: a review[END_REF], and CETP can efficiently load lipids from liposomes and deliver them to lipoproteins [START_REF] Morton | The surface cholesteryl ester content of donor and acceptor particles regulates CETP: a liposome-based approach to assess the substrate properties of lipoproteins[END_REF][START_REF] Swenson | Plasma cholesteryl ester transfer protein has binding sites for neutral lipids and phospholipids[END_REF], or transfer lipids from lipoproteins to liposomes [START_REF] Morton | Cholesteryl ester transfer proteins from different species do not have equivalent activities[END_REF]. We report here that JH binding by JHBP is reduced in the presence of DMPG liposomes (Figure 6), and that JHBP strongly interacts with these phospholipid membranes, as judged by its effect on TMA-DPH fluorescence anisotropy in DMPG liposomes (Figure 7). It is therefore tempting to speculate that lepidopteran JHBPs, with their single TULIP domain, could deliver their lipidic hormonal cargo upon docking to plasma membranes, through a mechanism analogous to lipid delivery at the phospholipid surface of lipoproteins by their vertebrate relatives CETP and PLTP. The fact that this effect seems modulated by anionic charge density (since liposomes composed of a mixture of DMPG and DMPC, a zwitterrionic phospholipid, had a much smaller effect on JH binding), suggests a primary involvement of electrostatic interactions. This is reminiscent of lipid transfer by CETP and PLTP, which is initiated by the electrostatic interaction of positively charged residues of CETP with negative charges (probably carried by phospholipids) at the lipoprotein surface [START_REF] Desrumaux | Influence of the electrostatic charge of lipoprotein particles on the activity of the human plasma phospholipid transfer protein[END_REF][START_REF] Lagrost | Regulation of cholesteryl ester transfer protein (CETP) activity: review of in vitro and in vivo studies[END_REF].

The data reported here are also similar to the findings on fatty acid binding proteins (FABPs), a family of small LTPs characterized by an antiparallel ß-barrel fold. Fatty-acid binding by Liver FABP (L-FABP), for example, shows a steep decline towards the acidic region, and is strongly inhibited with increased ionic strength, similarly to BmJHBP [START_REF] Stewart | Fatty acid binding to rat liver fatty acid-binding protein is modulated by early glycolytic intermediates[END_REF]. Moreover, L-FABP binds to anionic phospholipid vesicles, and the mode of delivery of fatty acids to target membranes is thought to be collisional, rather than to depend on simple diffusion. It is believed to depend first on electrostatic interactions with anionic phospholipids at the membrane surface, leading to a conformational change inducing ligand release, but involves a subsequent membrane insertion or equivalent hydrophobic interactions [START_REF] Corsico | The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes[END_REF][START_REF] Davies | Liver fatty acid binding protein (FABP) binds to anionic phospholipid vesicles with release of ligand[END_REF][START_REF] Davies | Binding of recombinant rat liver fatty acid-binding protein to small anionic phospholipid vesicles results in ligand release: a model for interfacial binding and fatty acid targeting[END_REF]. Since we found evidence for both electrostatic and hydrophobic interactions with phospholipids (Figures 6 and7), our data also suggest a collisional model of ligand release for BmJHBP. It is then conceivable that key interactions stabilizing the α1 helix in the closed conformation, such as the hydrogen bonds between the α1 helix and the C terminus described by Suzuki and colleagues (Figure S1, [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF], may be destabilized as part of the release mechanism.

Further experiments will be required to examine if ligand release actually depends on a locally lower pH, or alternatively, if low pH in vitro only mimics a combination of electrostatic forces prevailing in the vicinity of target membranes in vivo. For example, ligand release on target membranes enriched in acidic phospholipids could be explained by the creation of a negative surface potential, leading to a local increase in proton concentration.

Collectively, the data presented here support the model of ligand release first envisioned for G.

mellonella JHBP and E. postvittana Takeout 1 [START_REF] Hamiaux | Crystal structure of Epiphyas postvittana Takeout 1 with bound ubiquinone supports a role as ligand carriers for Takeout proteins in insects[END_REF][START_REF] Kolodziejczyk | Insect juvenile hormone binding protein shows ancestral fold present in human lipid-binding proteins[END_REF]1, and described in detail for B. mori JHBP by Suzuki and colleagues [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF], whereby JH is released by a movement of the α1 helix to the open conformation, and suggest that ligand release could be triggered by both electrostatic and hydrophobic interactions with the target membranes.

These findings may be of general relevance for other JHBPs and Takeout proteins, since the structure of G. mellonella JHBP and E. postvittana Takeout 1 proteins are strikingly similar, presenting also an N terminal α1 helix followed by a loop possibly playing the role of a gatepost [START_REF] Hamiaux | Crystal structure of Epiphyas postvittana Takeout 1 with bound ubiquinone supports a role as ligand carriers for Takeout proteins in insects[END_REF][START_REF] Kolodziejczyk | Insect juvenile hormone binding protein shows ancestral fold present in human lipid-binding proteins[END_REF][START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF], and because molecular simulations have suggested that ligand entry and release by E. postvittana Takeout 1 may occur in this region of the protein [START_REF] Zhang | Investigations of Takeout proteins' ligand binding and release mechanism using molecular dynamics simulation[END_REF]. Ligand release from the other side of the protein may coexist with this mechanism, however, in the case of large ligands fitting in the continuous binding pocket of Takeout proteins [START_REF] Hamiaux | Crystal structure of Epiphyas postvittana Takeout 1 with bound ubiquinone supports a role as ligand carriers for Takeout proteins in insects[END_REF].

  Membrane fluidity of DMPG liposomes was assessed by the steady-state fluorescence polarization of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) (Sigma Aldrich). The concentration of liposomal preparations was adjusted to 10 mg/mL in McIlvaine buffer, pH 7.0. Protein solutions (rBmJHBP, heat-denatured rBmJHBP or BSA) were prepared in 1 mM Tris-HCl, pH 7.0, at a concentration of ca. 40 nM. TMA-DPH was prepared as a 1 mM stock solution in tetrahydrofuran (THF). The liposomal preparations (30 µL) were transferred to a cuvette containing 2970 µL of buffer (1 mM Tris-HCl, pH 7.5) and labeled with TMA-DPH by addition of 4 µL of the stock solution. The suspension was stirred for 15 min at 35 °C to allow the probe to insert in the lipid bilayer. For the study of the effect of the proteins on liposome fluidity, 50 µL of protein solutions were added to the cuvette before labeling with TMA-DPH. Anisotropy measurements were performed in a range of temperatures between 35 °C and 11 °C, with a Fluorolog-3 spectrometer (Jobin-Yvon, Horiba Group, USA), using T-format fluorescence polarizers, equipped with a thermoelectric Peltier junction. The excitation and emission wavelengths were 360 nm (5 nm bandwidth) and 431 nm (5 nm bandwidth), respectively. Steady-state fluorescence anisotropy (r) was calculated as follows: r = (Ivv-GIvh)/(Ivv + 2 GIvh), where I stands for the fluorescence intensity and the first and second subscripts refer to the setting of the excitation and emission polarizers,

Fig. 1 .

 1 Fig. 1. Ligand binding by lepidopteran JHBPs is reduced at acidic pH.

Fig. 3 .

 3 Fig. 3. Near UV CD spectra of rBmJHBP at different pH values.Near UV CD spectra of rBmJHBP were recorded between 320 and 250 nm. The spectra were recorded for a 10 µM rBmJHBP solution in McIlvaine buffer with varying pH.

Fig. 4 .

 4 Fig. 4. Fluorescence emission spectra of rBmJHBP. (A) The spectra of a 10 µM protein solution were recorded after excitation at 295 nm before (solid line) and after 15 min equilibration with JH III at various concentrations. The measured intrinsic fluorescence is given in arbitrary units (A.U.) In (B), the spectra were recorded for a 10 µM protein solution without ligand, in McIlvaine buffer at varying pH.

Fig. 5 .

 5 Fig. 5. Dissociation kinetics of JH III from JHBP. To estimate the half-life (t1/2) of the nBmJHBP-JH III complex and the rate constant of dissociation (koff), nBmJHBP was first incubated in McIlvaine buffer at pH 7, 0 M NaCl with labelled hormone ([ 3 H]-JH III) to achieve equilibrium. At t0, an equal volume of DCC slurry was added, allowing buffer change (0.6 M NaCl (A), or pH 4 (B)), as well as quick and complete removal of unbound hormone (Fig. S8 (A)). DCC was next eliminated by centrifugation, after incubation for various periods of time. Since the amount of JHBP left in solution decreased only slowly with time, and was similar in the different conditions (Fig. S8 (B)), the radioactivity in the supernatant presumably reflects the release of [ 3 H]-JH III by JHBP. (A) Effect of high ionic strength. (B) Effect of pH. n = 3-6 for each time point. *** indicates P < 0.0001.

Fig. 6 .

 6 Fig. 6. Inhibition of binding of JH III to JHBP by model membranes. Binding of [ 3 H]-JH III by rBmJHBP was assayed with a dialysis assay (see Material and Methods for details). The black column represents [ 3 H]-JH III bound to rBmJHBP at equilibrium. Note that DMPG liposomes alone were also able to bind a significant, but smaller amount of [ 3 H]-JH III (white columns). DMPG/DMPC liposomes bind similar amounts of [ 3 H]-JH III (data not shown). When rBmJHBP was incubated together with 40 µg or higher amounts of anionic liposomes, the total amount of bound [ 3 H]-JH III was significantly reduced (DMPG, striped columns) compared to the JHBP-only control, and was comparable to the amount bound by DMPG vesicles alone. In contrast, 600 µg of liposomes composed of a 1:1 ratio of DMPC and DMPG only slightly inhibited binding (DMPG/DMPC liposomes, grey columns). ***: p<0.0001, N=3-9, Student's t test, two-tailed.

Fig. 7 .

 7 Fig. 7. Steady-state fluorescence anisotropy of TMA-DPH in DMPG liposomes.

  Binding of [ 3 H]-JH III by native B. mori JHBP (nBmJHBP), recombinant B. mori JHBP (rBmJHBP), and recombinant M. sexta JHBP (rMsJHBP) was assayed by the Dextran Coated Charcoal (DCC) method (A) and dialysis assay (B). To allow comparison, the highest percentage of bound [ 3 H]-JH III observed is set to 100% for each protein. The pH values inducing a 50% reduction in binding (pH50) were determined from the curves in (A) and are respectively equal to 4.18, 4.67, 5.08 and 5.45 for rBmJHBP, rMsJHBP, nBmJHBP and native G. mellonella JHBP (nGmJHBP, data inferred and

	redrawn with permission from figure 7 in (Ozyhar and Kochman, 1987)). (C, D) The Kd values of
	rBmJHBP for JH III at two different temperatures were determined by isothermal titration calorimetry
	(ITC). (C) Illustration of results of a typical ITC experiment, with 5µM rBmJHBP and 70 µM JH III,
	at pH 7 and 20 °C: raw data (top), and integrated data (bottom). The arrow indicates the slope of the
	curve used to determinate Kd. (D) Summary of Kd determinations by ITC. *: p <0.05, Student's t test,
	unpaired, one-tailed, n = 3.
	Fig. 2. Reversibility of the effect of low pH. Recombinant B. mori JHBP (ca. 1.2•10 -7 M) was incubated with 2•10 4 dpm [ 3 H]-JH III for 2 h at pH 7
	(black column), for 1 h at pH 7, then for 1 h at pH 4 after adjusting the pH (hatched column), or first
	for 1 h at pH 4, then for 1 h at pH 7 after adjusting the pH (white column). Initial incubations were
	done in 100 µL diluted McIlvaine buffer (1/10), and pH adjustment was obtained by addition of an
	equal volume of undiluted buffer at the desired pH. The percentage of bound hormone was determined
	with the DCC assay. ***: p < 0.001 Student's t test, unpaired, two-tailed, n = 3 (black and hatched
	columns) or n = 5 (white column).

Acknowledgments

This work was supported by INRA, CNRS, Université de Bourgogne Franche-Comté, and in its early stage by a grant from Région de Bourgogne to JPC.

liposomes and rBmJHBP, BSA or heat-denatured rBmJHBP. Each curve represents the average of three replicates. Protein concentration was ~ 4 nM, and DMPG concentration was ~ 145 µM, thus the lipid:protein molar ratio was ~ 1:35000 (lipid:protein mass ratio ~ 1/1000).

Legends of supplementary figures

Supplementary figure S1. Crystal structure of B. mori holo-(A) and apo-JHBP (B).

(A) Juvenile hormone II (JH II, shown as balls and sticks) bound to JHBP (PDB id: 3AOS). The gateforming amino-terminal α helix (α1) is shown in red, and the "gatepost" is highlighted in green. W38 is the only tryptophan residue of B. mori JHBP, and the main contributor to the JHBP fluorescence (Fig. 4). The residues in yellow are involved in direct hydrogen bonds that participate in keeping the α1 helix in its closed position. They are shown here only as examples of candidate bonds that may be disturbed at low pH. The closed conformation is further stabilized by polar interactions involving water molecules, as well as polar and hydrophobic interactions with JH II [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]. (B) Apo-JHBP (PDB id: 3AOT). The arrow depicts the ca. 70 ° swing of the α1 helix allowing JH II release in the open conformation [START_REF] Suzuki | Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori[END_REF]. The images were done with PyMOL 2.3.1.

Supplementary figure S2. Purification of native and recombinant JHBPs.

(A) Flow-chart for purification. The Coomassie stained gel at the bottom shows purified proteins used in this study. Purity is estimated to be greater than 95%. Note that both native and recombinant proteins migrate with apparent molecular weights around 30 kDa instead of the predicted molecular weight (~ 25 kDa). This electrophoretic behavior has been already noted in previous studies (see for example [START_REF] Vermunt | The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis[END_REF]). The first codon (GAT) of the mature protein (after the excision of the putative signal peptide predicted in [START_REF] Vermunt | The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis[END_REF] starts at position 7, and the 6 first nucleotides encode 2 residues added in this study for expression purposes (Met1, Gly2). We found 28 nucleotide differences in our strain (top sequence), when compared to the sequences published in [START_REF] Vermunt | The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis[END_REF] (bases in red). Among these base changes, 13 were synonymous, and the remaining 15 lead to a total of 10 amino acid substitutions. Three of these amino acid changes (highlighted in yellow) were also found by Vermunt and colleagues in an independent cDNA referred to as JHBP2. These variants presumably reflect the allelic variability of the different strains of silkworms used in the two studies. The apparent radius of rBmJHBP was not affected by pH (r = 2.56 ± 0.77 nm at pH 6.9; r = 2.49 ± 0.48 nm at pH 4.5). Some aggregation was evidenced at pH 4.5 (bottom left), but it represented a negligible fraction of the total protein. The % volume (right) reflects more accurately the relative 13 abundance in each size category, because intensity depends on light scattering, which is much greater for a large particle than for a small particle (the intensity of scattering is proportional to the sixth power of its diameter).

Supplementary figure S4. Stability of JH III at various pHs.

Supplementary figure S6

. Effect of ionic strength on JH III binding by nBmJHBP. JH III binding was measured using the DCC assay, in 10 mM Tris-HCl, pH 7, with NaCl concentrations as indicated.

Supplementary figure S7. Far-UV CD spectral analysis of rBmJHBP at acidic and neutral pH.

Supplementary figure S8.

Control experiments for analysis of ligand release by the DCC method.

(A) Unbound labeled hormone ([ 3 H]-JH III) is efficiently removed by Dextran Coated Charcoal (DCC). [ 3 H]-JH III (about 100000 dpm) was incubated for 30 min at 20 °C, in the same buffers as those used for analysis of ligand release (Figure 5) and exposed for 20 s at 20 °C to DCC. After centrifugation, (30 s, ca.10000 g), the percentage of labelled hormone left in solution was measured by scintillation counting. Note that this short DCC treatment is sufficient to virtually eliminate all free [ 3 H]-JH III in the three different conditions. (B) Assessment of nBmJHBP removal by DCC. To confirm that the apparent different half-lives of the complexes shown in Fig. 5 are not merely reflecting the differential binding of nBmJHBP to charcoal in different buffers, nBmJHBP was incubated in the same conditions as those used in Figure . 5. After 20 s (top) or 2 min (bottom) exposure to DCC, followed by a centrifugation to pellet charcoal (30 s, ca. 10000 g), the amount of protein remaining in solution was examined by SDS-PAGE and silver staining (Pierce). Note that there is no or little protein loss after 20 s exposure to DCC. Protein loss occurs after 2 min exposure, but appears similar in all three conditions. The asterisk indicates a 31 kDa MW marker.