by Jean-Marie Frère
Jean-Marie Ghuysen was one of the greatest Belgian contributors to the field of bacteriology. He was a major (if not the major) actor in the deciphering of the structure of peptidoglycan, the central component of the bacterial cell wall. He died as the result of septicaemia on August 31st, 2004 at the age of 79.
He was born in Blégny-Trembleur, a village not far from Liège, on January 26th 1925, and grew up in the local pharmacy owned by his father. As a boy, he went to primary school in Blégny and later to the Saint-Hadelin secondary school in Visé, a small town north of Liège.
Before he was eighteen, the young Jean-Marie had virtually no contact with scientific matters except through his father’s activities. The only influence he could remember was when his father decided around 1933-34 to install a water supply in the village (that he was the mayor of) to replace the central well. Together they explored the countryside and collected samples from springs to analyse them in a small laboratory next to the pharmacy. As a youth, Jean‑Marie lived in a mostly literary world (old Greek, Latin, philosophy), but he also dreamt of becoming a sailor when he left high school. His adventurous nature led him to head for Antwerp in the hope of boarding a ship. This plan was foiled when he was arrested by the police (in war time) and brought back home.
He then decided to follow his father’s steps and to become a pharmacist. This is how he discovered the sciences at the University of Liège and became fascinated by chemistry and physics. His passion for chemistry was so great that he decided to enrol in chemistry school in parallel with his curriculum. He obtained his degree in pharmacy in 1947 and graduated in chemistry in 1948, after presenting a final term essay on the “Isolation and purification of RNA”.
While Belgium was under German occupation, Jean-Marie was not registered as a regular university student in order to avoid being sent to Germany as a “voluntary worker”. Instead, he joined the resistance movement where he helped as a messenger and a bomb maker. His degrees were authenticated after the war was over.
From 1948 to 1951, he obtained a research fellowship to pursue his work on RNA with Professor Victor Desreux in the department of chemistry. He published four papers that formed the core of a PhD thesis on “The study of the heterogeneity of RNA” that he presented in October 1951 and for which he was awarded the Stas-Spring Prize, the first of many distinctions.
He was then approached by the Labaz Laboratories, a company that was planning to create a biochemistry and microbiology facility. He was offered the post of research director and while the laboratory was being set up, he decided to improve his knowledge in microbiology. With the support of the IRSIA and the Labaz Laboratories, Jean-Marie worked with Professor Maurice Welsch, director of the general and medical microbiology unit of the medical school at the University of Liège. Louis Dierickx, a young chemical engineer was hired at the same time as his deputy. The team studied bacteriolytic enzymes that were known to be part of the actinomycetin secreted by some bacteria of the Streptomyces family. Quite rapidly they identified and separated several peptidolytic activities as well as different bacteriolytic enzymes. Two enzymatic fractions were submitted to detailed studies and shown to have various peptidase activities with bacterial cell walls as substrates. Results collected over a six year period were published in twelve papers and constituted the basis of a thesis for the Agregation Degree (at that time, in Belgium, the highest possible degree) in Pharmaceutical Sciences which was awarded in 1957. In the meantime, in 1955, Jean-Marie had received the Louis Empain prize.
When the unit at the Labaz Laboratories was ready, Jean‑Marie initiated research on brain glutamate decarboxylase. However, he resigned from the company after a few months and returned to university research in January 1958. After negotiations with the University of Liège, it was agreed that he would not be involved in practical courses for the students but that he would be free to develop his research as he pleased. With the help of Maurice Welsch and the Rector of the University, Professor Marcel Dubuisson, he was rapidly promoted and became associate professor in April 1966.
From 1958 to 1969 his research focussed on the determination of the chemical structure of the bacterial cell wall peptidoglycan with the help of the various bacteriolytic enzymes he had purified and characterized previously. During this period he collaborated extensively with a number of microbiologists in related fields: Milton Salton (structure), Jack Strominger (biosynthesis) and Gerald Shockman (lytic enzymes). These collaborations were particularly fruitful and these investigations represented an essential contribution to the understanding of the chemical structure of the bacterial cell wall. In 1966, at a roundtable organized during a Symposium of the American Chemical Society in Detroit, Jean-Marie proposed, with colleagues including Gerald Shockman, the word “peptidoglycan” to define the macromolecular structure which forms the extracellular skeleton of the cell wall and constitutes the target of autolysins and other lytic enzymes such as lysozyme. These results were the basis of an important review entitled ‘Use of Bacteriolytic Enzymes in Determination of Wall Structure and their Role in Cell Metabolism’ (Bacteriological Reviews 32, 425-464, 1968). The work was developed in 1972 by Professors Karl-Heinz Schleifer and Otto Kandler who used the different peptidoglycan structures as taxonomic criteria for bacterial classification.
Jean-Marie was appointed full Professor in Liège in 1969 and formed a nucleus of local research workers that included Melina Leyh-Bouille, Jacques Coyette, Martine Distèche, Jean Dusart and Jean-Marie Frère. The stimulation provided by Jean Marie and his laboratory attracted a large number of international scientists (microbiologists, biochemists, chemists). The atmosphere was so invigorating that it increased the enthusiasm of all participants and paved the way for future successes. At the same time, the general orientation of the research also shifted from the elucidation of the structure of the bacterial peptidoglycan (a problem Jean‑Marie considered as solved in 1971) to that of the mode of action of penicillin, which was known to interfere with peptidoglycan biosynthesis. The enzymes produced by various strains of actinomycetes that Jean-Marie had used as tools for the study of peptidoglycan structure became model proteins for the analysis of the interactions between penicillin and its targets. With the help of Harold Perkins (at Mill Hill and later at the University of Liverpool) who supplied a large number of synthetic peptides as model substrates. Assay systems were developed to enable the study of the complex peptidoglycan transpeptidation reaction using purified enzymes so that the kinetics of the interactions with substrates and inactivators could be investigated. The studies were later extended to Penicillin-Binding Proteins from E. coli and enterococci. In 1976, the team contributed a seminal advance in the field by demonstrating that penicillin acylated a serine residue in one of the Streptomyces penicillin-sensitive D-alanyl-D-alanine carboxy-transpeptidases (Frère J.M., Duez C., Ghuysen J.M. and Vandekerckhove J., Occurrence of a serine residue in the penicillin-binding site of the exocellular DD-carboxy-transpeptidase of Streptomyces R61., FEBS Letters, 70, 257-260, 1976), a reaction which was later found to account for the inactivation of all PBPs by b-lactam antibiotics. Surprisingly, this important paper is seldom cited, it seems that the result is now considered as self-evident! Not surprisingly, the “sensitivity” of a PBP to a particular penicillin was found to be dependent mainly on the rate of this acylation reaction.
Further progress required the utilisation of new methods and collaborations were initiated with crystallographers in Connecticut (Jim Knox and Judith Kelly) and Liège (Otto Dideberg). The first complete structure of a D-alanyl-D-alanine carboxypeptidase was solved in Liège in 1978 (Dideberg O., Charlier P., Dupont L., Vermeire M., Frère J.M. and Ghuysen J.M., The 4.5 Å resolution structure of the exocellular DD-carboxypeptidase of Streptomyces albus G., FEBS Letters, 117, 212-214, 1980). It was a Zn++ metallo enzyme that was in consequence not sensitive to penicillin. It was the first 3D protein structure to be solved in Belgium. Later, the collaboration with the Connecticut group resulted in the determination of the first 3D structure of a penicillin sensitive enzyme (the Streptomyces DD carboxy-transpeptidase again) and in the demonstration of the structural relationship between Penicillin-Binding Proteins and beta-lactamases (Kelly J.A., Dideberg O., Charlier P., Wéry J.P., Libert M., Moews P.C., Knox, J.R., Duez, C., Fraipont, C., Joris, B., Dusart, J., Frère, J.M. and Ghuysen, J.M., On the origin of bacterial resistance to penicillin. Comparison an active-site serine beta-lactamase active-site serine D-alanyl-D-alanine-cleaving peptidase, Science, 1986, 231, 1429-1431, 1986). At this time, Jean-Marie also felt the need for theoretical approaches and Georges Dive and Josette Lamotte joined the team to start a group specialising in molecular modelling and quantum chemistry.
The group had now become multidisciplinary, covering such diverse areas as microbiology, enzymology, protein crystallography and theoretical chemistry. New staff members, namely Paulette Charlier, Bernard Joris and Colette Duez obtained permanent positions. The need for large quantities of proteins became an important factor and, since the focus was still mainly on Streptomyces, Jean Dusart visited David Hopwood’s laboratory in Norwich to acquire expertise in the cloning of Streptomycetes genes and in expression systems for the production of large amounts of the corresponding proteins. Several genes encoding D‑alanyl-D-alanyl-peptidases and b-lactamases were cloned to satisfy the requirements of the protein chemists and crystallographers (Dehottay P.H., Dusart J., Duez C., Lenzini M.V., Martial J.A., Frère J.M., Ghuysen, J.M. and Kieser, T., Cloning and amplified expression in Streptomyces lividans of a gene encoding the extracellular beta-lactamase from Streptomyces albus G, Gene, 42, 31-36, 1986). The structural and functional characteristics of several enzymes were thus determined and attempts were made to establish correlations between both types of properties, an old dream of Jean-Marie’s. In 1990, when Jean-Marie reached the age of mandatory retirement, the Centre for Protein Engineering was created to avoid dispersion of the equipment and of the various experts who formed the team. The Rector of the University organized the appointment of Jean-Marie as the first director of the Centre until, at 70, he had to completely retire but he remained very active as a scientific advisor. Meanwhile, new collaborations had been initiated, mainly with Josef Van Beeumen, a protein chemist at the University of Ghent and with Léon Ghosez, an organic chemist at the University of Louvain. Jean-Marie had an encyclopaedic knowledge of microbiology and biochemistry. He was generous with his time and keen to discuss their data or more general scientific problems with his younger collaborators: this often led to the birth of new ideas, many of which turned out to be seminal. In his most recent publications (out of a total of more than 350), he discussed the molecular basis of the lack of efficiency of penicillins against mycobacteria (the “Mycobacterial Paradox”) and the evolutionary relationship between penicillin-binding enzymes (Ghuysen J.M. and Goffin C., Lack of cell wall peptidoglycan versus penicillin sensitivity: new insights into the chlamydial anomaly, Antimicrob. Agents Chemother, 43, 2339-2344, 1999; Goffin C. and Ghuysen J.M., Biochemistry and Comparative genomics of SxxK superfamily acyltransferases offer a clue to the mycobacterial paradox: presence of penicillin-susceptible target proteins versus lack of efficiency of penicillin as therapeutic agent, Microbiol. Mol. Biol. Rev., 66, 702-738, 2002).
Over the years, he was the recipient of a large number of prestigious awards: the Prix Joseph Maisin of the National Research Foundation of Belgium (FNRS), the Prix de l’Innovation Technologique of the Walloon Region (shared with four of his o-workers: Colette Duez, Melina Leyh-Bouille, Jean Dusart and Jean-Marie Frère), the Gairdner Foundation International Award in Medical Science, the UNESCO Carlos J. Finlay Award in Microbiology, the Albert Einstein World Award of Science and the Bristol-Myers-Squibb Award in Microbiology. He was chairman of numerous symposia at international conferences and a much sought after lecturer at several Belgian and foreign universities and at a large number of meetings. He received “Honoris Causa” doctorates from the Universities of Nancy, Debrecen and Montréal. He was a member of the Biochemical Society from 1984 and an Editorial Advisor of the Biochemical Journal form 1987 to 1999.
The last years were saddened by his wife’s poor health. To the end, in August 2003, he devoted a lot of time, attention and love to her.
Jean-Marie leaves behind him a thriving Centre, composed of more than 70 scientists and technicians headed successively by Jean-Marie Frère (not unexpectedly called “Jean-Marie the second”), Bernard Joris, Moreno Galleni and presently, Paulette Charlier. Over the years, investigations in the Centre have evolved to cover both a deeper understanding of bacterial physiology (including phenomena such as secondary metabolitic production and resistance to beta-lactam antibiotics) and different fields related to protein structure and function. In the first orientation, the following studies are worth mentioning: beta-lactamases (Moreno Galleni, André Matagne, Frédéric Kerff, Jean Dusart, Jean-Marie Frère), Penicillin-Binding Proteins and other components of the cell wall metabolism machinery (Martine Nguyen-Distèche, Mohammed Terrak, Frédéric Kerff, Bernard Joris), Streptomyces genetics (Sébastien Rigali) and the control of beta-lactamase biosynthesis (Christine Jacobs, Moreno Galleni, jean-Marie Frère , Bernard Joris). The field of cyanobacteria is explored by Annick Wilmotte’s group. In the second orientation, studies in protein folding were developed (André Matagne (who often makes uses of beta-lactamases as model enzymes) extending to the study of the mechanism of formation of amyloid fibres (Mireille Dumoulin).
In conclusion, Jean-Marie Ghuysen has made a lasting mark on Belgian and international science and his numerous contributions will long be remembered not only by scientists all over the world, but also by his three children and eight grandchildren, one of whom is … a pharmacist!
J. Coyette, J.M. Frère, P.E. Reynolds.
This biography is an updated version of a text that was first published in The Biochemist (Coyette, Frère and Reynolds, The Biochemist, April 2005, pp 46-48). A slightly modified obituary was later published in Molecular Microbiology (Coyette, Frère and Reynolds, Mol. Microbiol., 57, 871-873, 2005). It is reproduced here with the permission of the Biochemical Society (for The Biochemist) and of Wiley, the present commercial editor of Molecular Microbiology. © 2005 Biochemical Society, Molecular Microbiology, 57, 871-873.