When PPLO Became Mycoplasma
The smallest cell has had a long career in the spotlight
PPLO Gets a Conference
In January 1959 a conference on Biology of the Pleuropneumonialike Organisms was held by the New York Academy of Sciences. There were about 200 attendees with speakers from nine countries. The meeting was chaired by D. W. Edward of the Wellcome Research Laboratory in England. The speakers included most of the pioneers, largely from veterinary and agricultural institutions, who were struggling to understand organisms that did not fit the paradigm of bacteria or viruses. Another feature appeared at this juncture. Tissue-culture technology had come into widespread use and the media were often sterilized by filtration. Pleuropneumonia-like organisms were starting to show up frequently as tissue-culture contaminants. When we began culturing PPLOs at Yale, I recall frequent phone calls from laboratories around the university that were growing tissue cultures. Some were requests for information on how to avoid PPLO contamination and some were accusatory, as if our sloppy ways were spreading these organisms around New Haven.
We plowed ahead trying to measure the amount of DNA and the number of colony-forming units in cultures under varying conditions. We continued using the strain of avian pathogen known as A5969. Jack Maniloff began detailed studies of this strain using electron microscopy. Word got around that PPLOs were among the smallest of genomes and this proved of interest to a number of investigators. I recall meeting Leo Szilard, the famous nuclear physicist turned molecular biologist, at a meeting of the American Physical Society in Washington. On being introduced and hearing my name he asked, “What is the size of the PPLO genome?” Although we had values, the uncertainties about morphology, cellularity and mode of replication left the meaning of these values uncertain.
Our discussions suggested a method of confirming cellularity by means of predictions about closed vesicles made of membranes of known high impedance. This allowed us to use a technique discovered 100 years earlier. James Clerk Maxwell, in the early days of electromagnetic theory, worked on the problem of the impedance of a suspension of conducting spheres surrounded by nonconducting shells and embedded in a conducting medium. In the 1920s this theory was tested for biological materials by placing a suspension of red blood cells between two platinum plates and measuring the impedance as a function of frequency of the applied field. From the values obtained, one is able to calculate the inductance of the nonconducting shell. With these results and a knowledge of the dielectric constant of the shell, arrived at by assuming it was made of lipids, a value was obtained for the thickness of red blood cell membranes. This was the first experimental determination of this important biological parameter.
We reasoned that if PPLOs were vesicular in nature, we should be able to demonstrate it to be so. A center for work on dielectric dispersion was the Moore School of Engineering at the University of Pennsylvania under the direction of Herman Schwan. I knew Schwan from various meetings of biophysicists and made contact to carry out the necessary experiments. And so with a Styrofoam cooler packed with ice and a number of pellets of PPLO A5969, I made my way from New Haven to Philadelphia for several days of intensive experiments with my engineering mentor. The result was a paper, “Electrical properties of the membranes of pleuropneumonia-like organism A5969,” and a conviction that the cells were indeed vesicles surrounded by the usual type of cell membrane.