Dr Fraser Bergersen, plant scientist

Dr Fraser Bergersen

Plant scientist

Dr Fraser Bergersen is a distinguished plant scientist whose research in the field of microbiology, particularly through the study of symbiotic nitrogen fixation in legumes, has led to improved crop performance in Australia and Asia. After completing an MSc (Hons) from the University of Otago he joined the CSIRO Division of Plant Industry in Canberra. Initially appointed as a Research Scientist, he remained with the organisation for the rest of his working life, retiring from his position of Chief Research Scientist in 1994. He received a DSc from the University of New Zealand in 1962. Elected a Fellow of the Australian Academy of Science in 1985, he served the Academy as a member of Council (1987-1993) and Foreign Secretary (1989-1993). Dr Bergersen is also a Fellow of the Royal Society.

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Interviewed by David Salt in 2004.

Contents


Born to tinker with things

Fraser, you were born in the large country town of Hamilton, New Zealand, in 1929. What are your memories of childhood there?

They are very clear. My paternal grandparents, for most of our time in Hamilton, lived only two or three houses away, so I had close contact with them. And the primary school was only one block away, within walking distance - I used to walk home for lunch. It was a congenial location.

I believe your father, grandfather and great-grandfather were all inventors, tinkerers. Do you think you inherited some of this from them?

I probably did. I had access to my grandfather's and my father's workshops and I was always making something, such as a boat for sailing on the lake or (less successfully) a radio. My grandfather had a small engineering business in Hamilton and had been in engineering all his life - his father had had a number of engineering enterprises in Palmerston North, where he was located. My father would have liked to be an engineer but was prevented by illness, so when he retired at the age of 65 he went to work in a foundry [laughs]. And I always like tinkering with things and making small apparatus for use in my science.

I believe your grandfather came up with potentially a quite important invention.

He invented a wheel which was patented. At that time most vehicles were either steel shod or shod with solid rubber, but pneumatic tyres were just coming onto the market. He invented a wheel made of steel with solid rubber outside and a pneumatic inner tube, which worked quite well and would have been very useful for mobile vehicles at war, I think. But the patent was bought up by a company and trashed, so the wheel was never built.

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School years: inspirations and challenges

In primary school you were awarded a book prize for being Dux, and you chose Chemistry Today. You later commented that this was your introduction into science.

Yes. That book, with its shiny cover, stimulated my interest in science and chemistry, and when I went on to high school I used it as a source of material for my school work for a little while. But it was soon outdated. I don't even remember the author and the book no longer exists, so I can't tell you any more about it!

You attended Hamilton High School and once again you did very well. I gather that one of your science teachers, Fred Mason, had a most interesting style of teaching.

Fred was a very particular sort of a fellow. He laced his teaching with endless anecdotes and stories, many of which I remember. He had a way that caught my imagination and I found him interesting, although some of the students thought he was dreary. He was innovative: within a year and a half of the official release of information about atomic fission and the atomic bomb, he had copies of the documents and we studied them in the Lower Sixth. I found that a very challenging thing to be doing.

Fred had a practical bent, being from an engineering family and related to the Masons of Mason and Porters, the manufacturing firm in Auckland. He made us take an interest in things like internal combustion engines and learn how they worked, simply by taking an engine to pieces and studying it. Later I used what I had learnt from him about the internal combustion engine for servicing my car and motorbike.

Are science teachers important to the careers of budding scientists?

Oh yes, absolutely. Another of our teachers was Horrie Sayers, a biologist and a zoologist who taught those subjects. He had a friend who was the pathologist at the local hospital, and very early he would confront us with a bucket containing a human brain preserved in formalin. He was able to bring reality to things - this was certainly not just a skeleton of a cat on the sideboard.

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From would-be medical student to committed microbiologist

You studied for a Bachelor of Science at the University of Otago, in Dunedin. Were you interested in microbiology from the beginning?

Well, I originally intended to be a medical student. But this was immediately after the war and access to the medical school was taken up 95 per cent by returned servicemen, so to enhance my chances of getting into medical school I decided to do full degree subjects. I did chemistry, physics and botany in the first year, but in the second year I began to specialise in chemistry, biochemistry and microbiology - things I thought would be useful when I went into medical school, which in fact I never did. I went into microbiology as a professional pretty well from that time.

You were also getting work experience in microbiology, weren't you?

Yes, at the University's Department of Bacteriology. This was the main diagnostic laboratory for the province of Otago, dealing with medical specimens from all over. (There was a pathology department as well, on the next floor.) The laboratory was staffed mainly with trainees and graduates of the program of medical technology, and it was sensible for me to learn my microbiology from the roots up in that way, so I spent my time doing sterilising and washing up and making media, just the same as all the other trainees. That became very useful when I advanced in microbiology.

Soon afterwards your first scientific paper was published in the prestigious journal Nature. Can you tell us a little bit about that?

A senior lecturer, Solomon Faine - who later became the Professor of Microbiology at Monash University and is now retired - saw the work I had been doing and said, 'This is quite important. Why don't you write a letter to Nature?' So I did and with the department's approval I sent it off, and it was published. The letter was about cross-resistance to antibiotics in Staphylococcus aureus, and it was one of the first reports of cross-reactions: that some strains resistant to penicillin were also resistant to aureomycin.

I see, this would be the bacterium that we commonly call golden staph. To get a letter in Nature when you have just finished your Bachelor of Science is fairly good going.

Well, it is nowadays, but then it was less exotic. [Laughs] It was a pretty unimportant thing but our department then became quite active in trying to prevent the overuse of antibiotics by clinicians just to remove symptoms. It is so easy to specify an antibiotic to relieve the symptoms of a sore throat, for example, but that impacts on the future usefulness of antibiotics. In those days we were using only four antibiotics - penicillin, chloromycetin, aureomycin and streptomycin - and they were important against infectious diseases. We could see that cross-resistance was going to be important, and I remember addressing seminars to clinicians in the hospital, trying to persuade them not to prescribe any antibiotic until it was known that the particular causative organism of any disease was sensitive to that one.

So we were warned 50 years ago about the massive, growing problems we now have?

Yes. Many people were involved in the story, but the clinicians were not persuaded by it. It seems more important to remove or relieve a patient's symptoms than to think of the long-term aspects.

For your Masters you did a microscopic study of bacterial structures. That may sound fairly straightforward, but when you did it, it was quite novel.

This was when almost all work in microbiology was still being done with the light microscope. Bacteriologists in clinical work just stained the bacteria and looked at their shape and colour - what was inside was thought to be below the resolution of the light microscope, and often it was. We were able to use some techniques such as staining in certain ways for nuclear material, for example, for nucleic acids and for sites of metabolic activity, but these were later discounted because of the artefacts introduced by the chemical reactions. Very soon after I did that study, the work with thin sections of bacteria for electron microscopy was first published and we moved on to that. In fact, I made use of it later when I came to Australia.

During your Masters you found out about a microbiology job in Canberra with CSIRO Division of Plant Industry. Did you think you had much chance of getting it?

Well, I just made an application and put it in the mail, but within a very short time I had an invitation from the Division to come over for an interview. So I was flown from New Zealand to Australia, which in those days was quite unusual. The aircraft, a DC4, was unpressurised and flew at about 4000 feet over the Tasman. It was a pretty rough journey and we arrived late in Melbourne. Then I had to transfer to a DC3 for Canberra. The plane was held for me and a meal was put on board because I hadn't had one on the trans-Tasman flight. In the DC3 I was seated next to a rather large gentleman, with another rather large gentleman behind: beside me was the Treasurer of the Commonwealth, Sir Arthur Fadden, and in front was the Prime Minister, Mr Menzies. And I had a dinner and they didn't!

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A new vision: developments in CSIRO legume nutrition work

In Canberra, the Chief of Plant Industry was Dr - later Sir - Otto Frankel. Can you tell us a little bit about him?

I'm not sure now about my first impressions, except that I thought he was a powerful little man. Perhaps I was terrified. But he soon passed me over to people who were a little more friendly and approachable.

Otto was a great man. He had a long view of science. He, like Rivett - the first Chairman of CSIR, which became CSIRO - had the philosophy that the job of the administrator of science was to give the scientists the things they needed and let them get on with it. And that came through.

He had taken over the Division two or three years before I came. Seeing that it was a heavily practical, not science oriented, division he took it as his first task to strengthen the scientific base for all of the agricultural work that was being done. He found that one of the strengths of the Division was a group of people looking at the mineral nutrition of plants - agricultural plants, pastoral plants - and that certain parts of their work had had a great impact. For example, Alf Anderson and his group had discovered, first in Adelaide and later in Canberra, the effects of deficiencies of molybdenum in soils, and had made the connection that this was expressed through the nitrogen status of the legumes in the pastures.

So Otto said, 'Well, we want to know more about that process. What's the link between molybdenum and pasture nitrogen status?' During an early trip to England soon after his appointment, he met Dr Phillip Nutman, who was working at Rothamsted Experimental Station, at Harpenden, in Hertfordshire. Otto Frankel was a geneticist; Phillip Nutman was working on the genetics of nodulation of legumes - of red clover, as a matter of fact. Otto made the connection that there was bound to be a genetic solution to the molybdenum-nitrogen link and he recruited Philip Nutman to come to Canberra for three years as a senior fellow of the Division.

Actually, there were two such positions in the Microbiology Section of the Division. The other one was held by Professor K O Müller, a graduate of Berlin who had worked in Cambridge during and just after the war. He was working on the mechanisms involved in disease resistance in plants. I was offered a position with either one or the other; I chose to work with Philip Nutman, and so I came to work on legume nodules.

Can you provide us with a bit of background on what nitrogen-fixing nodules are, why they are important and why you chose to study them?

After some initial work I moved on to soybean as a model system, growing the soybean plants in a glasshouse. The nodules on soybean roots are typically round bumps about 2 or 3 millimetres in diameter, filled with special tissue in which there are symbiotic bacteria - the agents of nitrogen fixation. The nitrogen which is fixed is moved out of the bacteria into the plant and translocated from the roots to the shoots, where it joins up with the photosynthetic system of the plant.

Nodules are very complex organs. If you cut through a root nodule on a soybean plant, you can see that the central part is pale pink and surrounded with a white or greenish white cortex. It is these pinkish cells in the centre which are the site of activity.

Isn't the nitrogen fixing role of nodules important in providing nutrients for agricultural plants?

Yes, and also for pasture, which I worked on first. At that time, pasture systems in much of Australia relied on subterranean clover - or, elsewhere in the country, other clovers and just a few other legumes - for the production of good balanced food for cattle and sheep, and to produce a vigorous growth of the plants. (Later, soybeans, lupins and other crops which were nodulated became important too.) In addition, the nitrogen which was fixed in the nodules became available, through the processes in the soil, for the grasses and other plants which grew in association with the legumes. This system was very important to Australia. Indeed, the world depends on nitrogen fixation by this system almost as much as on photosynthesis for its cycles of nutrition.

So it was known already that nodules, and the bacteria living within them, were important. But not much was known about how these bacteria functioned, what products they formed or how you could maximise their growth or interaction in a pasture system, and the Division of Plant Industry decided that it needed to get a greater understanding of the science behind it all. Is that right?

That's pretty right. Despite the lack of basic knowledge, there was quite an existing program in Australia on the nitrogen-fixing bacteria of pasture legumes. For instance, Professor Jim Vincent, in Sydney University, and Professor Lex Parker, in Western Australia, had active programs in selecting better bacteria for subterranean clover and other legumes. They knew that you could select better bacteria, but they didn't know what the functions of these bacteria exactly were. It was at about that time that all this knowledge began to come together, and I was a part of the scheme - but not the only one, by any means!

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The Rhizobium Research Group gets under way

You were brought in, then, as a member - and, later, leader - of the group which Dr Frankel set up, the Rhizobium Research Group. What is rhizobium?

'Rhizobium' is a general term for the bacteria in the root nodule system. They live in the soil, from where they infect the roots of the leguminous plants and build the nodules in which they function. We now know that Rhizobium is only one of about six genera of soil bacteria involved, but loosely they are all called rhizobia.

And the initial work of the group was to examine the structures of the nodules?

Well, in a way. The first project I was given was to look at the red clovers that Phillip Nutman had selected and bred for nodulation differences. With him, I began to cut sections and look at the inside of the nodules of his plants - the defective ones and the better ones. But to do that first work, I had to learn how to grow sterile plants and to grow them with bacteriological controls; you had to be able to see that you were really getting the effects you thought you were working with.

When I came, there was already a small group. John Brockwell and Frank Hely had begun a good deal of work with nitrogen-fixing legumes. Frank was in fact involved in designing the modification needed so that you could grow these plants in a glasshouse in the Canberra summer, when it was very hot, without cooking them. He designed a louvre system which went over the roof of the glasshouse and could be adjusted with levers. It was oriented in the correct way so that with these louvres the plants growing in test tubes in the glasshouse experienced bands of sunlight and shade, sunlight and shade; it controlled the temperature in the tubes; and also - funnily enough, because that wasn't the intention - it enhanced the ventilation of the tubes, because as they heated and cooled they were able to pull air and expel it from the tubes, keeping them ventilated. Since that work was already under way, my first weekend job was to go and adjust the louvres of the greenhouse.

So that's where we started. But gaining an understanding didn't happen all at once. We were first involved with some field studies in the Armidale area of New England, where there was a nodulation problem. I participated in the discovery of some microbial factors in the soil which were preventing the successful inoculation of the plants in that area with good bacteria. The work was quite exciting at the time but later it was totally superseded, because Australians were learning how to make better inoculants - that is, better products which would produce populations of Rhizobium in the soil for the nodulation of the plants.

I think an important part of the next phase of the research was to understand the effects that oxygen had on the nitrogen fixation rates.

Yes. In about 1957 I decided that we should go to a model system which was tractable for laboratory work. Work with pasture legumes was very difficult because the nodules were tiny and you couldn't get enough material to do any biochemical work with any great ease. At much the same time, a lab in Wisconsin, America, had done some work with soybeans that showed what the early products of nitrogen fixation might have been. They were using the stable isotope 15N as a tracer to help with the study of the system, so I persuaded Otto Frankel to allow me to visit Wisconsin to learn how to use this technique and what its fundamentals were, because there was then no capacity in Australia to do 15N work. At about the same time other people developed it as well, but this was the first time in Canberra that we were able to get 15N to work. That was a key development, and for some years afterwards all of our mechanistic work was based on the incorporation of 15N from nitrogen gas into the plants and tracing the products and the mechanisms.

What did you find were the first products being made by the bacteria?

Among our early experiments were some very short-term experiments: you exposed root nodules in a little bottle to a gas containing some oxygen and some nitrogen gas containing 15N, for a short period - one or two minutes - and then stopped the reaction reasonably quickly and extracted the products from the nodules. The Americans had already found that the first product appeared in the soluble fraction, so I concentrated on looking at the soluble fraction. We found that ammonia was the first free product of nitrogen fixation which could be detected in the soluble fraction. And that was soon incorporated into amino acids and translocated to the plant. It was not until perhaps 20 years later, though, that the products after ammonia could be elucidated.

Didn't your visit to Wisconsin involve something that looked like a promising lead but actually went nowhere?

Yes. I have to backtrack to 1958, when the structural work which we were talking about began to develop into electron microscope work. An electron microscope was to have been bought by CSIRO in 1955, but we didn't get it because the budget was cut. Then Sir Ian Clunies Ross, the Chairman of CSIRO, was able to pull strings so that CSIRO helped to pay for an electron microscope which was installed in the John Curtin School of Medical Research, in the Australian National University, and in return was promised use of the machine. And we used it.

Prior to that we had had a few thin sections cut for us at the Division of Chemical Physics, in Melbourne, which had an electron microscope, and from using those thin sections we had the idea that the bacteria weren't just sitting in the nodules but were in a structure. That put us on the way, and with Margaret Briggs - who was very helpful in those early years - I took it further. Being able now to cut some thin sections for ourselves, we found that the bacteria were in fact done up in little packets, with a plant membrane between the bacteria and the plant cytoplasm.

Then, when I went to Wisconsin, I developed a hypothesis that this packet was the important thing. And we did 15N experiments which seemed to suggest that 15N was incorporated in the membrane around the bacteria. Wrong. It wasn't that way. So I developed a hypothesis about how the nodules functioned, based on the idea that the membrane was important. (Subsequent work has shown that it is very important, but not for those reasons!)

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Serendipity and the 'prepared mind': a microaerobic system steps forward

A number of research institutions around the world were trying to unravel the nitrogen fixing topic. Was there a collegiate approach among these different groups, all wanting to contribute? Or were each of you trying to beat the others to the punch?

It was both, really. There were not so many people looking at the legume system. While I was in Wisconsin there was a major breakthrough: a group at the Du Pont chemical company in Delaware succeeded in producing a nitrogen-fixing extract from the non-symbiotic bacterium Clostridium pasteurianum. So the basic biochemistry of the nitrogen fixing process, the nitrogenase action, began then. It was some time, though, before we were able to apply that to the nodule system, because it was harder to work with. You couldn't grow it in a big fermenter and get a pound of bacteria very easily.

Research on the basic mechanism of nitrogenase action went on elsewhere for quite a while. We were more concerned with how the bacteria were able to do this magic reaction, this 'black box' of nitrogenase. Some other people were working on that, but we concentrated on how it might work in the nodule system. Although it was suspected that the bacteria were the source of the nitrogenase, we didn't really know, so one of the first things to do was to make a preparation from active nodules, with which we could work separate from the nodules themselves. In the mid-'60s we succeeded in making our extracts of nodules which contained active bacteria, and the active bacteria were proved - for the first time - to be the source of the nitrogenase.

It was an accident that we found out how to do it. We knew from the work in America that the nitrogenase was very sensitive to oxygen, which inactivated it. So we tried to make anaerobic preparations. We found out how to make them and they were certainly anaerobic, but they didn't fix any nitrogen. Then one day, in one experiment, one flask - out of about 10 - had activity. Why? The clue came from the way in which we did our experiments, monitoring the gas phase of these flasks: we found that the one which was active had had a little air leak in it. We knew then that although the only way to make the preparation active was to make it anaerobically, the secret was to drive it with a smidgin of oxygen.

So that was the beginning of understanding that this whole nodule system is a microaerobic system. To make it go, you had to give the nodules oxygen, and we worked out what the oxygen responses were and what was needed. But obviously the air concentration of oxygen around the outside of the nodule was very much less than on the inside of the nodule.

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The contribution of partially oxygenated leghaemoglobin

About the same time, Cyril Appleby, who had joined the biochemistry group, had been extracting the haemoglobin, the pink material inside the cells of the central tissue, to find out some of its physical properties. He found that it was half-saturated with oxygen at a very low oxygen concentration. This was a key point. Human haemoglobin, which is vaguely similar to the nodule haemoglobin, and myoglobin (in muscles), which is very similar to it, are half-saturated with oxygen at quite high concentrations - several micromolar concentrations - but the pigment in these cells was half-saturated with oxygen at a thousand times lower concentration.

Cyril and I began to do a few experiments together. We shook bacterial suspensions, made from the nodules, in various concentrations of oxygen and with leghaemoglobin, the red pigment, added to them. We found that there was a specific effect: the respiration was stimulated a little bit but nitrogen fixation was stimulated very much more when partially oxygen-saturated haemoglobin was present. That was the beginning of quite a long series of experiments with Cyril and with visiting scientists, finding out what the mechanism was.

Also involved at that time was Nick Stokes, a physicist who worked in a different division of CSIRO and had certain ideas as to what was happening in these shaken assays. From our first experiments, however, it looked as if a specific interaction with partially oxygenated leghaemoglobin was driving the bacteria to fix nitrogen. But this idea vastly offended an American worker in New York. He came out and visited CSIRO and worked with Cyril, and together we did a lot of experiments from which we found that it was a mechanism which was delivering oxygen at a low concentration to the bacteria. It wasn't a specific reaction - other haemoproteins and oxygen-carrying proteins would work - but leghaemoglobin was the best, because it had the lowest-saturated oxygen characteristics. We have since worked for a long time with the interaction between the bacteroids (the symbiotic form of the bacteria in the nodule) and this partially oxygenated leghaemoglobin.

Do bacteroids normally exist in the soil?

No. Free living bacteria exist in the soil. Once they enter the tissue of the nodule, they are changed into a different form. Sometimes they are grossly enlarged; sometimes they are just the same size but their biochemistry changes. The bacteria in the soybean nodule are about the same size as those living in the soil but they have quite different biochemistry. And the changes in the biochemistry have been studied, both in our lab and elsewhere.

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Not a program but a long-lived, productive research group

How long did the Rhizobium Research Group operate for? Was it ever given the status of a program?

The group operated before we had programs. Its name was actually an informal one: it was in the microbiology group, and the Microbiology Section was where the work was done, using central CSIRO funds. (Most of its projects did not require external funds.) We have had various heads of the Microbiology Section - I was the head for some time - but the rhizobium group remained constant from 1955 until about 1979, when the management of all CSIRO's science changed and these efforts had to be located within programs, in a formal structure which was much more rigid than originally. Nevertheless, we continued to follow many of the leads which came from the earlier work. For instance, for a while a program called Nitrogen in Agriculture embraced the work of the rhizobium group.

What had been some of the other major breakthroughs of the group?

The rhizobium group became quite large, with at one stage 12 people working in it, including Dr Alan Gibson, a graduate of Sydney University who was there from fairly early on, and Dr Bill Dudman, who had come to us from the UK via Jamaica and the CSIRO laboratories at Merbein. He worked on the use of serology - immunology - as a tool to look at the surfaces of bacteria involved in this process. I have already mentioned John Brockwell, who was in the group from a very early stage and still works on the subject as a research fellow at CSIRO Plant Industry. And Bob Gault was involved with the field work.

They worked together to develop several things. There were methods of inoculation, which they worked on very successfully: they were able to develop new techniques for adding the bacteria in an active form which survived the rigours of isolation in the soil from other things. And they developed very good techniques which involved pelleting of seeds and the way in which the inoculum was applied to the seeds, sometimes - in later developments - in the soil directly as a suspension of bacteria. Those sorts of things were done for practical reasons.

It was a numbers game, however. There were always bacteria present in the soil so you were trying to introduce a new member of the population in competition with a lot of things that were already there. And many of these bacteria were rhizobia from other plants, other systems, which were not useful on the plant of interest, so it was necessary to develop methods which would get a large, viable population present when the seed germinated, the roots grew out and the infection was able to proceed. Many of the other people in the microbiology group were involved in processes like that, with a lot of publications about counting the numbers of bacteria in various locations and how they interacted with the plants, and fertiliser systems and so on, just optimising the way in which they could be used. Many of those techniques are now a part of agricultural practice in Australia.

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Secrets of success: far-sightedness, stubbornness, collaboration

Can you tell us the secrets of the rhizobium group's success?

I find that very hard to answer. There were a lot of reasons, including the legacy of Otto Frankel's original far-sightedness, and the stubbornness of the people involved: they didn't want to change it unnecessarily when their work was being productive. There was a mix of fundamental and practical work, and as leader of the group I encouraged that from the start. We needed to keep our feet on the ground, so to speak - or in the soil, because this was a soil problem. The work developed very strongly in the direction of practical agriculture. For example, the new methods of inoculation had to be tested in the field before they could be applied properly in agriculture.

So there was demonstration work, there was work in collaboration with Departments of Agriculture right across the country but particularly in New South Wales, where many of the field experiments were done with members of the agriculture department. Collaborative work was an important feature - our people were good collaborators with people on the ground, and some of our work was done on farm, with the collaboration of interested farmers - and was one reason, I think, why the work was able to continue. And it supported my work in the lab on this highfalutin biochemical stuff, because I was able to draw the connection between that and the practical stuff. Irrigated soybeans, in particular, are quite an important agricultural crop now, and I was able to combine my fundamental work with an interest and expression in the field work with soybeans.

Similar work was done, very successfully, in Western Australia with lupins, which were an important agricultural crop there. That sort of pattern was able to be used for some time, and it wasn't until the 1980s that we needed to seek support from other sources of funding.

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International activities with mixed consequences

Did the group undertake any successful international collaboration?

Yes. We had joint projects in South-East Asia - in Malaysia and Thailand - as well as in Papua New Guinea and Indonesia, sponsored by funds from ACIAR, the Australian Centre for International Agricultural Research. This was truly collaborative work: we had partners in those countries and we were transferring the skills which we had in Australia to be used in those countries. That was a major objective of the work, but it also carried quite a bit of the fundamental work.

For example, we were able to put together a program of collaborative experiments on the translocation of fixed nitrogen from nodules into the rest of the plant - as I mentioned before, we didn't know much about the products that were translocated out of nodules - and that involved specific biochemistry which was studied both in Canberra and in other Australian laboratories. And an alternative method to 15N came up, which we were able to test and calibrate against 15N methods (which, by the way, had become quite different in the interim). This was the ureide method of measuring nitrogen fixation. Some plants translocate ureides from the roots to the shoots, and by analysing the content of the bleeding sap that is being translocated from the roots to the sap - you could cut the plant and sample what was coming out - the chemical nature of the products could be identified and the ratio of certain products in those could be correlated with the amount of nitrogen fixation which was going on. It was good to find an alternative method, because 15N methods were quite expensive to conduct and not many countries were able to use them.

You yourself have been involved in many international experiences. I believe there is even a Chinese version of a textbook you did on nitrogen-fixing nodules.

That's right! In about 1979 a visit to Australia by Chinese nitrogen fixing interests was sponsored by the agreement between the Academy of Sciences in China and the Australian Academy of Science. Then in 1983 a reciprocal visit to China by a party of Australian nitrogen-fixers was sponsored by this Academy. (I led this group.) Very early in our extensive travels in China we visited the headquarters of the Chinese Academy of Sciences in Beijing and were graciously received by the President, in sumptuous surroundings. I was able to present to him, as a memento of our visit, two books which I had authored or edited, and he graciously thanked us.

In later years, however, a Chinese researcher working in Sydney University visited China and found a copy of one of the books - in Chinese - in the library. It had been made without our knowledge and was available to Chinese students for a few American dollars, whereas the English original cost about $150. So those students were able to make use of the book. I'm sure the publisher was very unhappy about it!

Workshop support and collaboration

Otto Frankel supported the work in CSIRO by bringing in good people, allowing excellent teams to be assembled and then making sure that you had well-equipped workshops and research infrastructure. I imagine that you needed several decades of understanding the field to come up with something like the apparatus you have just shown us, but you would also have needed access to workshop facilities and the ability to build laboratory equipment from scratch.

That was very important. These days it is much more difficult to maintain workshop facilities, because they are quite expensive. They have to be re-equipped with up-to-date equipment, just as a scientific lab has, and it can be very hard to identify an outcome in dollar terms. Otto saw the need for workshops, and that was generally appreciated. The Division of Entomology, next door, was another of the many divisions which saw it as essential to have their own workshops.

Within a year of my coming and beginning to work with the respiration of bacteria, the workshop helped me to make a useful microrespirometer. And then through the years they helped me to make an anaerobic press, which enabled me to make nodule extracts. They modified French Presses with Holden motorcar parts so that cheap disruptive presses for bacteria could be made, this flow chamber (pictured) which I used in my own work was another product, and there were many others. They helped me to make a gadget to measure the diffusion coefficients of leghaemoglobin in solution, and that produced a paper. It was absolutely crucial for our modelling work that we should know these things.

In all of these instances I took a concept to somebody in the workshop - the chief engineer, perhaps, or somebody working at the bench - and discussed what I wanted to do. They made or drew something, then they made what they thought would do the job, and I might say, 'Well, this isn't quite right, you need such-and-such.'

There was a close interaction with the staff who were making these gadgets. Along with the capacity for things to be made, that collaboration was really important. It applied also to the electronic side. The little controller of the speed at which this apparatus worked was just made very quickly by somebody in the electronic workshop, and we used it for years.

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Bacteria in, ammonia out: an apparatus to focus on the nitty-gritty

You have brought along today an experimental rig to demonstrate how you conducted some of your studies. Could you talk to us about that?

I mentioned that we were studying the role of leghaemoglobin. Out of that work evolved a system by which we were able to monitor the activities of bacteria obtained from nodules. Similar experiments could be done with any nitrogen-fixing bacteria, and in fact some have been done and some work has been published. But we were interested in getting to the nitty-gritty of what the interactions between bacteria were, as they functioned in the nodule, in relation to experimental systems.

We had already found that the most activity occurred in the bacteroids when leghaemoglobin bathing their surface was partially oxygenated, but we didn't have any good quantitative relationships. So I built this system, which evolved over several years. We had a flow chamber and a stirred chamber, with a little electric motor (M) driving the stirrer, and we had a conical chamber in which one could put a suspension of bacteria in a solution containing leghaemoglobin. We were able to inject bacteria into that solution through an injection port so that we knew how much was in there and exactly what its properties were.

By changing the flow rate of medium through another port, we could perfuse the contents of the chamber with a solution containing leghaemoglobin, whose state of oxygenation was known and whose concentration was known analytically. The solution passed through the stirred chamber (A) in which the bacteria were respiring and fixing nitrogen - just the solution, the bacteria were retained in the chamber (A) behind the membrane filter - and out through a flow cuvette (F), mounted in the sample beam of a spectrophotometer (S). So, we could scan the properties of the leghaemoglobin before and after it had gone through the chamber.

In that way we were able to make changes to substrates, to the degree of oxygenation of the leghaemoglobin. We could relate the concentration of the product - which was ammonia in the solution, with no transformation into anything else so you just monitored the amount of ammonia - and that was initially calibrated with 15N so we knew we were in the right direction.

In doing that we were able to study these very low concentrations of free oxygen in the solution, maintained by the action of leghaemoglobin. That is quite a complex matter. The leghaemoglobin binds oxygen very strongly and releases it slowly, and the physics of that process are well calibrated and known. So from the degree of oxygenation of the leghaemoglobin in the solution which came out, compared with the degree of oxygenation as it went in, we could calculate how much oxygen had been used and how much nitrogen had been fixed, by analysing for ammonia in the effluent solution. And we could do all sorts of experiments like that.

We were able to find that there was quite precise control of the conditions in the chamber - by the bacteria! We could influence those conditions by changing the rates of flow and so on, but within a certain period of a few minutes it would re-establish a new metabolic activity to cope with the increased level of oxygenation, or oxygen supply. We found that there is a cyclical control: you can produce a sine wave control just like a control system in an engineering set-up, simply by the changes in the activities of the bacteria. And those degrees of change of respiratory activity were correlated with changes in nitrogen fixation activity.

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The plant component: mitochondrial bodies versus leghaemoglobin

It seems that the bacterial response to different circumstances, different environments, is amazingly complex and intricate. Have we got a long way now towards understanding these nodules, or is most of it still to be figured out?

Well, we have been talking about the bacterial component, which I have studied exhaustively. But the plant component is very important as well. We had found in our structural work that plant mitochondria and plastids are also involved in the structure of these infected cells where the bacteria are sitting, and that it is a very specific structure. When I left CSIRO and became a Research Fellow at the ANU, the collaborative work on this developed further. We were able to go back and look at sections which had been cut in CSIRO in 1973 for electron microscopy, and we cut new sections off these same blocks and began using a different orientation to study the way in which mitochondria and plastids were arranged in the infected cells.

It is fascinating. They have quite enormous numbers of mitochondria, which are also involved in the oxygen demand of the central tissue, accounting for perhaps a half of the respiratory activity of the nodule. These mitochondrial bodies are lined up along the structures of the infected cell where there are intercellular spaces, so we have had to explore the three-dimensional structure of that cell and try to understand it rather better than we did. And we were able to collaborate with some graduate students working in the ANU to actually study the mitochondria which were isolated from nodules, looking at them in relation to the deoxygenation of leghaemoglobin. Because these mitochondria are lining the surfaces of the air spaces which ventilate the nodule tissue, they get first bite of the cherry, the first cut of the oxygen, and leghaemoglobin is competing with that sink at the surface of the cells.

One of the graduate students was able to measure the oxygen affinity of the respiratory activity of these mitochondria, and we have used that in a model system to explore how the plant cells are modifying the respiration and the distribution of oxygen within the affected cell. That work has been published as a simulation model of how the cell is working, and for me it has been a quite good exercise, combining the structural work which began 50 years ago and the recent, fascinating work on the activity of these mitochondria.

We seem to be good at pulling a system apart, isolating individual components and modelling them individually. Do we need also to be able to put the pieces of the jigsaw puzzle together to understand how the system works and where we should make a modification or whatever to achieve certain desirable outcomes?

Yes. It is very hard to know what modifications to seek, unless you understand the system better than we do at the moment.

In the new age of biotechnology we are asked to believe that understanding the genetic code of a cell may be enough to know that cell. Yet we are just beginning to realise how complex and intricate the system is in the way it behaves.

That's right. That applies to the nodule system, certainly, and to many systems. Gene expression is one thing, and the regulation of gene expression is very important; having the genes there is a primary requirement for any activity. But you can't derive information from a metabolic map to tell you about the regulation of activity and the way in which systems interact with each other.

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Guiding a science topic to maturity

You have been a part of CSIRO for about 43 years. What is it like to have been in charge of a research unit overseeing an entire area of research from its infancy to a mature science?

Well, such development is rare. Mostly present-day science is divided up into fundable-sized bits, and it is very hard to have a continuing topic studied. For reasons which are both practical and obvious, some science becomes stale. It takes a long time to come to conclusions, to have a mature piece of science, and few projects can be sustained for that long. Some people have been quite successful in being able to carry on a continuous series of experiments for perhaps 10 or 15 years, but that is rare and these days it is more difficult, because no funding agency wants to fund science that is going to make a continuing demand on its resources. This was easier in my day. I did most of my work in areas funded entirely through CSIRO's central structure, but now it is almost impossible to do that. I don't know of any current projects which are not funded, at least to a very large extent, by external funds from various sources.

Our funds from CSIRO were seldom enough but they were adequate to enable us to pursue our work pretty well free of interference, as long as we were responsible about it - always, of course, you had to have terms of reference and you had to do responsible work. But such freedom is now largely gone and it took some modifications of our approach as scientists in order to cope with those changes. Some of us didn't do it at all well. I was privileged not to have to do it very much, but others have found it very difficult and I believe the whole stream of scientific advance has been grossly affected by it.

Did you think the investment in you and your work was a great one?

From a scientific point of view I think it was. And it was a product of its time. We have to learn how to use the different funding structures better than we do at the moment. Long-term research is very difficult to fund, but every branch of science agrees that such research is necessary. It has been a little easier in the university system to maintain that, but it is increasingly difficult there and almost impossible in CSIRO.

I think there is too much emphasis on outcomes. Of course outcomes are important, but if you knew what the outcomes were going to be before you did the work, you wouldn't have to do it! It is important that flexibility remains, no matter how hard it is to maintain it in the current managerial atmosphere.

This is really the drift throughout the world, isn't it?

Oh yes. It has been so in America for many, many years. When I worked in America in 1958 and '59 I would not have been supported but for a continuing NSF [National Science Foundation] grant to the professor of the lab, who was able to work me into his program. In England and this country it has now followed the same pattern. It is just different and we have to learn to do our best with it. And we still need to interact with our masters, telling them constantly about the importance of long-term public-interest work which doesn't have an easily identified outcome but without which we will run out of steam very quickly.

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Always more questions to be asked and answered

Are there still any major gaps in our understanding of nitrogen-fixing nodules?

There must be, because you never finish science. There is always something new to find out. We know pretty well, having done a lot of work with soybeans, how the soybean system works. We know almost nothing about many other systems, some of which will become increasingly important as they are used in broadacre agriculture or even in farming systems in small countries. For instance, peanuts are grown in many countries, yet we know only a little bit about the systems of the peanut. And you could say that over and over again.

Also, we know very little about non-leguminous nitrogen-fixing systems which are important in an ecological sense in natural systems. What we do know has been growing steadily over the years, but we don't know enough to use those systems wisely. We don't even have a proper inventory in Australia of the nitrogen fixing capacity of the components of natural systems. I remember discussing this in Western Australia with people in the forestry systems. They knew that they had acacias and that these fix nitrogen, but they didn't know how much, or the acacia life expectancy in terms of the overall cycles of the systems - or anything about the enormous number of lichens, for example, and which are important for fixation in situ and on trees, rocks and so on.

I am reminded of some work by my former colleague Don Norris, who worked in Queensland. He went to Brazil before I did, and he said he was talking there about the use of legumes in the field. The people said, 'But we have no legumes that are capable of doing that,' when they were standing knee-deep in legumes which they knew nothing about. And that can be so at all stages of agricultural development.

Is there just less chance these days of setting up a long-scale research program that exhaustively studies such problems?

In this country, certainly, it is difficult. The Brazilians set up quite an advanced laboratory from 1970. I visited it in 1997 and found it astonishing: they had a mass spectrometer and very sophisticated work going on, even if it had taken 20 years or 30 years to come to fruition. That too was an international effort.

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A congenial home for science and the scientist

You moved to Canberra in 1954, when its population was much less than the present 350,000. What was it like as a place to live in?

It had only 23,000 people then, and because it was smaller you had more access to people. We used to see Bob Menzies and Dame Pattie shopping in J B Young's at Civic when we first came, and I remember once being nearly knocked over by the British premier as he ran down the stairs from the UK High Commission, in the Melbourne Building. You just saw these people all the time, and so in that sense it is quite different.

It is culturally different too. There was very little theatre. There were two (later three) movie theatres, and you used to have to front up to the frigid Albert Hall for any concert or play that was on. But it was a friendly place. I think everybody who comes to Canberra takes a couple of years to become really accustomed to it, but that's probably true wherever you go.

Has Canberra been a good place to do science?

Oh yes. I don't think anywhere would match it or exceed its usefulness in that direction. We've always had good relationships between the CSIRO and the ANU, and I have utilised those. We have done a great many joint things which over the years have been important. That's now expanded to include the ADF [Australian Defence Force] Academy and the University of Canberra, which is increasingly collaborating with CSIRO and other laboratories. So there is a great draw in bringing people to Canberra. When people have left Canberra for other places they have realised what a good place it was. Fortunately for me, I didn't have to leave.

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© 2017 Australian Academy of Science

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