One of the useful things they learnt about disease-causing, or ‘pathogenic’, bacteria was that the behaviour of the disease, and thus of the bug itself in relation to its infected host, could be altered by various deliberate means: for example, through repeated cultures in the laboratory, or by repeatedly passing generations of the bug through a series of experimental animals. Through such manipulations it was possible to make the disease worse or less severe by making the bug either ‘more virulent’ or ‘attenuated’. Bacteriologists looked for ways to extrapolate this to medicine. In France, for example, the eminent Louis Pasteur used this principle of attenuation to develop the first vaccine to be used successfully against the otherwise universally fatal virus infection of rabies.
One fascinating observation that came out of these studies was the fact that, once a bug had been attenuated or been driven to greater virulence, the change in behaviour could be ‘passed on’ to future generations. Could it be that some factor of the bug’s own heredity had been altered to explain the change in behaviour?
Bacteriologists talked about ‘adaptation’, using the same term that was coming into vogue with evolutionary biologists when referring to evolutionary change in living organisms as they adapted to their ecology over time. While it was too early to be sure if bacterial heredity depended on genes, these scientists linked it to the physical appearance of bugs and colonies, or to the bugs’ internal chemistry, and even to their behaviour in relation to their hosts. These were measurable properties, the bacterial equivalents of what evolutionary biologists were calling the ‘phenotype’ – the physical make-up of an organism as opposed to what was determined by the hereditary make-up, or ‘genotype’.
Bacteriologists also came to recognise that the same bacterium could exist in different subtypes, which could often be distinguished from one another using antibodies. These subtypes were called ‘serotypes’. In 1921 a British bacteriologist, J. A. Arkwright, noticed that the colonies of a virulent type of dysentery bug, called Shigella, growing on the jelly-coated surfaces of culture plates, were dome-shaped with a smooth surface, whereas colonies of an attenuated, non-virulent, type of dysentery bug were irregular, rough-looking and much flatter. He introduced the terms ‘Smooth’ and ‘Rough’ (abbreviated to S and R) to describe these colonial characteristics. Arkwright recognised that the ‘R’ forms cropped up in cultures grown under artificial conditions, but not in circumstances where bacteria were taken from infected human tissues. He concluded that what he was observing was a form of Darwinian evolution at work.
In his words: ‘The human body infected with dysentery may be considered a selective environment which keeps such pathogenic bacteria in the forms in which they are usually encountered.’
Soon researchers in different countries confirmed that loss of virulence in a number of pathogenic bacteria was accompanied by the same change in colony appearance from Smooth to Rough. In 1923, Frederick Griffith, an epidemiologist working for the Ministry of Health in London, reported that pneumococci – the bugs that caused epidemic pneumonia and meningitis which were of particular interest to Oswald Avery at the Rockefeller Laboratory – formed similar patterns of S and R forms on culture plates. Griffith was known to be a diligent scientist and Avery was naturally intrigued.
Griffith’s experiments also produced an additional finding, one that really shook and puzzled Avery.
When Griffith injected non-virulent R-type pneumococci from the strain known as type I into experimental mice, he included an additional ingredient in the injections, a so-called ‘adjuvant’, which usually pepped up the immune response to the R pneumococci. A common adjuvant for these purposes was mucus taken from the lining of the experimental animal stomach. But for some obscure reason Griffith switched adjuvant to a suspension of S pneumococci, derived from type II, that had been deliberately killed off by heat. The experimental mice died from overwhelming infection. In the blood of these dead mice Griffith expected to find large numbers of multiplying R-type I bacteria – the type that he had injected at the start of the experiment. Why then had he actually found S-type II? How on earth could adding dead bacteria to his inoculum have changed the actual serotype of the bacterium from non-virulent R-type I to highly virulent S-type II?
Researchers, including Avery himself, had previously shown that S and R types were determined by differences in the polysaccharide capsules coating the cell bodies of the bugs. Griffith’s findings suggested that the test bacteria, initially R-type pneumococci, had changed their polysaccharide coats inside the infected bodies of the mice to that of the virulent strain. But they could not have achieved this by just flinging off the old coat and putting on the new one. The coat was determined by the bacteria’s heredity – it was an inherited characteristic. Further cultures of the recovered bacteria confirmed that the S type bred true. There appeared to be only one possible explanation: adding the dead S bacteria to the living R bacteria had induced a mutation in the heredity of the living R-type bacteria, so they literally transformed into S-type II.
In the words of Dubos: ‘[At the time] Griffith took it for granted that the changes remained within the limits of the species. He probably had not envisaged that one pneumococcus type could be transformed into another, as this was then regarded as the equivalent of transforming one species into another – a phenomenon never previously observed.’
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It is little wonder that Avery was astonished by Griffith’s findings. Like Robert Koch before him, Avery subscribed to the view that bacterial strains were immutable in terms of their heredity. The very concept of a mutation – that heredity was capable of an experimentally induced change – was a highly controversial issue within biology and medicine at this time. To understand why, we need to grasp the concept of what a mutation means.
By the late nineteenth century Darwinian theory had entered a crisis. Darwin himself had been well aware that natural selection relied on some additional mechanism, or mechanisms, capable of changing heredity, so that natural selection would have a range of ‘hereditable variation’ to choose between. Generations later, in the opening chapters of his innovative book Evolution: The Modern Synthesis, Julian Huxley put his finger on the nub of the problem. ‘The really important criticisms have fallen upon Natural Selection as an evolutionary principle and centred round the nature of inheritable variation.’ In 1900, a Dutch biologist, Hugo de Vries, put forward a novel mechanism that would be capable of providing the necessary variation: the concept of a random change in a unit of inheritance. Opportunity for change exists when genes are copied during reproduction, when a random change in the coding of a gene might arise from an error in copying the hereditary information. De Vries called this source of hereditary change a ‘mutation’. It was only with what Julian Huxley termed ‘the synthesis’ of Mendelian genetics – the potential for change in the inherited genes through mutation – and Darwinian natural selection operating on the hereditary choices presented within a species, that Darwinian theory became credible again to the great majority of scientists.
In time Griffith’s finding would be confirmed to be what Avery was now wondering about: it was a mutation. Geneticists would show that the change from the R to the S strain of pneumococcus involved the transfer of a gene from the dead S-type II bacteria to the living R-type I bacteria, which was incorporated into subsequent bacterial reproductive cycles, transforming the cells of the R-type I bacterium into the cells of the S-type II bacterium. It was indeed the bacterial equivalent of a change of species. And Griffith was proven right in inferring that Darwinian