Evolution of a minimal cell.

Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life1,2. Here we report on how an engineered minimal cell3,4 contends with the forces of evolution compared with the Mycoplasma mycoides non-minimal cell from which it was synthetically derived. Mutation rates were the highest among all reported bacteria, but were not affected by genome minimization. Genome streamlining was costly, leading to a decrease in fitness of greater than 50%, but this deficit was regained during 2,000 generations of evolution. Despite selection acting on distinct genetic targets, increases in the maximum growth rate of the synthetic cells were comparable. Moreover, when performance was assessed by relative fitness, the minimal cell evolved 39% faster than the non-minimal cell. The only apparent constraint involved the evolution of cell size. The size of the non-minimal cell increased by 80%, whereas the minimal cell remained the same. This pattern reflected epistatic effects of mutations in ftsZ, which encodes a tubulin-homologue protein that regulates cell division and morphology5,6. Our findings demonstrate that natural selection can rapidly increase the fitness of one of the simplest autonomously growing organisms. Understanding how species with small genomes overcome evolutionary challenges provides critical insights into the persistence of host-associated endosymbionts, the stability of streamlined chassis for biotechnology and the targeted refinement of synthetically engineered cells2,7-9.

Mycoplasma mycoides ssp. mycoides biotype small colony-secreted components induce apoptotic cell death in bovine leucocytes.

Contagious bovine pleuropneumonia, caused by Mycoplasma mycoides ssp. mycoides biotype small colony (MmmSC), is one of the most serious cattle diseases in Africa. Several observations suggested that MmmSC had evolved an efficient way to escape the bovine immune responses by triggering host-cell cytotoxicity. This study was implemented to determine whether the cytotoxic effect was due to apoptotic cell death. To that end, bovine blood cells were cultured for up to 3 days in the presence of viable or heat-killed MmmSC compared to unstimulated cultures. The findings provided evidence for a viable MmmSC-induced, time-dependent apoptosis in bovine blood leucocytes, whereas heat-killed MmmSC had no effect. Morphological and physiological changes (evidenced by TUNEL and annexin V staining) typical of apoptosis were observed in response to viable MmmSC. All the lymphocyte subsets as well as the monocyte/granulocyte subset exhibited extensive apoptosis after exposure to viable MmmSC. Our results demonstrated a potential role for MmmSC-secreted components as pathogenic factors able to induce programmed cell death in bovine blood leucocytes.

Effect of HEPES buffer systems upon the pH, growth and survival of Mycoplasma mycoides subsp. mycoides small colony (MmmSC) vaccine cultures.

The use of a buffer system based on N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), in conjunction with standard Gourlay's culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T(1)44. When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose. Compared to growth in standard unbuffered Gourlay's medium, the final culture titre was found to be one log(10) higher, at 10(11) colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37 degrees C. The titre remained above 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of 1 month. After 4 month's storage at 37 degrees C the titre had fallen to 5x10(4) CCU ml(-1). In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4. No significant difference in growth rate between the two media was observed. Adoption of a HEPES-NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).

Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. Na+-dependent K+ accumulation.

We have studied some features of K+ accumulation by glycolysing Mycoplasma mycoides var. Capri cells. We report that when Na+ is absent from the external medium, K+ accumulates up to the level predicted by the amplitude of the transmembrane electrical potential, delta psi m, measured by Rb+ and methyltriphenylphosphonium cation (TPMP+) distribution. Therefore, under these experimental conditions, the coupling mechanism of K+ uptake consists of a delta psi m-driven uniport. More important, when Na+ is present in the external medium, the level of K+ accumulation by glycolysing Mycoplasma cells is far too steep to be equilibrium with delta psi m (-120 mV for delta muK+ compared with -90mV for delta muRb+ or delta muTPMP+). Our results clearly indicate the presence in Mycoplasma of an active K+-transport system specifically stimulated by Na+. Furthermore, by controlling the amplitude of the energy-dependent delta muH+, we obtain strong evidence that this specific Na+-stimulated K+ transport is modulated by the transmembrane electrical potential. Finally, we show that ATP is consumed when such a transport system is in activity.

Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. The nature of the link between K+ and Na+ transport.

We have studied the links between the mechanisms of Na(+), K(+) and H(+) movements in glycolysing Mycoplasma mycoides var. Capri cells. In the light of the results reported in the preceding paper [Benyoucef, Rigaud & Leblanc (1982) Biochem. J.208, 529-538], we investigated certain properties of the membrane-bound ATPase of Mycoplasma cells, with special reference to its ionic requirements and sensitivity to specific inhibitors. Our findings show, first, that, although Na(+) stimulated ATPase activity, K(+) did not affect it, and, secondly, that NN'-dicyclocarboidi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were potent inhibitors of the basal ATPase activity, which was unaffected by vanadate and ouabain. We also investigated the movements of Na(+) and H(+) under the experimental conditions applied to the study of the K(+) uptake reported in the preceding paper, and found that when ;Na(+)-loaded cells' previously equilibrated with (22)Na(+) were diluted in a sodium-free medium, addition of glucose induced a rapid efflux of (22)Na(+). This energy-dependent efflux was independent of the presence of KCl in the medium. Studies of the changes in internal pH by 9-aminoacridine fluorescence or [(14)C]methylamine distribution indicated that the movement of Na(+) was coupled to that of protons moving in the opposite direction, a finding that supports the presence of an Na(+)/H(+) antiport. When Na(+)-loaded cells are diluted in an Na(+)-rich medium the Na(+)/H(+) antiport is still active, but cannot decrease the intracellular Na(+) concentration. Under such conditions, net (22)Na(+) extrusion is specifically dependent on the presence of K(+) in the medium. The present results and those derived from the study of K(+) accumulation (the preceding paper) can be rationalized by assuming that Mycoplasma mycoides var. Capri cells contain two transport systems for Na(+) extrusion: an Na(+)/H(+) antiport and an ATP-consuming Na(+)/K(+)-exchange system.

Immunoelectrophoretic analysis of Mycoplasma mycoides var. mycoides.

Acrylamide gel electrophoresis was used to show the similarities and differences in the membrane proteins of two vaccine and two virulent strains of Mycoplasma mycoides var. mycoides. Immunoelectrophoretic (IEP) analysis was also used to partially characterize the associated antigens. Antibody spectra to the antigens of M. mycoides differ in rabbit, pig, and cattle sera. Rabbits produce better precipitating antibody against the anodic migrating protein mycoplasma antigens than cattle and pigs as seen in IEP. However, rabbit anti-M. mycoides serum did not show precipitating antibody against the heat-stable carbohydrate antigen. As judged by IEP, the major carbohydrate antigen extracted from the media, or boiled whole organism, is similar to that present in the sera-infected cattle and knee joints of calves. This carbohydrate antigen has a cathodic migration in IEP at pH 8.6. Periodate oxidation, classically used to destroy carbohydrate, also destroys most of the protein antigens. Heating the antigens to 56 C for 10 min destroys many of the noncarbohydrate antigens and 100 C eliminates all but the carbohydrate antigen. Extraction of M. mycoides with chloroform-methanol, phenol, ethanol, or ethanol-acetone reduced or eliminated most of the protein antigens. Some of the isolated antigenic fractions of M. mycoides were tested to determine their activity in the diagnostic complement fixation test for contagious bovine pleuropneumonia and their inhibitory effect in this test by using bovine anti-M. mycoides antisera having precipitating antibody and circulating antigen. The complement fixation antigen is not the galactan, cannot be extracted by chloroform-methanol, but is stable to boiling at 100 C and may be extracted by phenol and partially precipitated by ethanol-acetone.

Binucleate classical mycoplasmas pathogenic for goats.

The growth of three pathogenic goat mycoplasmas, strains Y, KH1 and Mycoplasma mycoides var. capri (PG3), was studied. They formed classical colonies on agar containing 1/500 thallium acetate. They were inactivated during storage at 2 to 4 C and by freezing and thawing but not by shaking. Only KH1 was killed by sonic treatment. Ultraviolet inactivation curves showed that their colony-forming units were single binucleate cells. Details of their growth curves are given. Filtration through 0.45- or 0.3-mum membrane filters removed up to 97% of the cells. Less than 0.003% passed 0.22-mum membranes. In electron micrographs, the cells were seen replicating by budding and most were 0.6 to 0.9 mum in diameter; but cells between 0.1 and 0.2 mum reproduced. They usually multiplied by producing one bud, a form of binary fission. However, two buds were produced by some synchronized cells, indicating that both nuclei had divided simultaneously to form progeny, an alternate method of multiplication.