Coordinate synthesis and turnover of heat shock proteins in Borrelia burgdorferi: degradation of DnaK during recovery from heat shock.

The synthesis and turnover of heat shock proteins (Hsps) by Borrelia burgdorferi, the Lyme disease spirochete, was investigated by radiolabeling of whole spirochetes and spheroplasts, comparison of one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and use of immunochemistry. The approximately 72-kDa DnaK homolog and three additional Hsps of 39, 27, and 21 kDa increased in amount by 3- to 15-fold between 2 and 6 h following temperature upshift from 28 to 39 degrees C. Temperature downshift experiments following the transfer of spirochetes from 40 to 28 degrees C showed that within 15 to 30 min, synthesis of most of the major Hsps returned to levels seen in spirochetes statically maintained at the lower temperature. Spheroplasts of B. burgdorferi produced by treatment with EDTA and lysozyme were radiolabeled, and specific Hsps were localized to either the cytoplasm or membrane fraction. Further analysis by two-dimensional electrophoresis demonstrated three constitutively expressed DnaK isoforms with pIs near 5.5. A pattern suggestive of DnaK degradation was observed following recovery from heat shock but not in spirochetes maintained entirely at a low temperature. Some of these putative degradation products were recognized by monoclonal antibodies directed against the B. burgdorferi DnaK protein. These data suggest that following a period of peak synthesis, DnaK is actively degraded as the spirochete reestablishes its metabolic thermometer. These findings provide a new interpretation of previous work suggesting that 10 to 15 B. burgdorferi polypeptides, including DnaK have a common epitope.

Thermoregulation of protein synthesis in Borrelia burgdorferi.

Borrelia burgdorferi, the etiological agent of Lyme disease, infects humans via the bite of a tick. The microbe survives in at least two vastly different environments: an arthropod vector and a warm-blooded host. We examined protein synthesis in B. burgdorferi B31 in response to sudden heat stress, which is similar to that which occurs during the transmission from vector to host. Proteins synthesized after shifts from 28 degrees C to higher temperatures and in pulse-chase experiments were labeled with 3H-labeled amino acids for 4 h and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The synthesis of four proteins we designated as heat stress proteins (HSPs) was increased by shifts to higher temperatures (HSP-1, 75 kilodaltons [kDa]; HSP-2, 42 kDa; HSP-3, 39 kDa; and HSP-4, 27 kDa); and the amount of one protein we designated as heat-labile protein 1 (29.5 kDa) was decreased at higher temperatures. At 37 to 40 degrees C, the major heat stress protein, HSP-1, represented 14 to 18% of the total cell protein compared with 1 to 2% of the total cell protein at 28 degrees C. HSP-1 was stable during a 4-h chase at either 40 or 28 degrees C. Demonstration of similar HSPs in low-passage, pathogenic strains of B. burgdorferi suggests that the heat stress response may be common among B. burgdorferi strains and may play a role in Lyme disease.

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp.

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.

Simple staining procedure permits rapid counting of mycoplasma colonies.

A staining procedure employing oil red O and Coomassie R-250 was developed to increase visualization of mycoplasma colonies. This procedure permits CFU determination of mycoplasmas without additional microscopy.

Liposomes replace serum for cultivation of fermenting mycoplasmas.

Cholesterol and albumin are limiting factors in the growth of Mycoplasma species. These nutrients are usually supplied in the culture medium by the addition of serum. The growth of M. pneumoniae in a serum-free medium containing an ethanolic cholesterol suspension and albumin was about one-half the level attained in serum-containing medium. M. gallisepticum and M. fermentans were not cultivable in the cholesterol suspension medium even after supplements were included. In another culture medium containing phosphatidylcholine-cholesterol liposomes and albumin as serum replacements, the growth of M. pneumoniae was approximately equal to that in serum-containing medium, and the growth of M. gallisepticum and M. fermentans was significantly greater than that in medium containing serum. M. fermentans produced even higher yields in liposome medium supplemented with arginine. These fermenting mycoplasmas readily adapted to the liposome medium and by the fifth or sixth serial passage produced thick confluent growth on the lower surface of culture bottles. To obtain maximum growth, we serially transferred the mycoplasmas at least 10 times in serum-free medium before quantitations of growth were made. This is the first report of a serum-free mycoplasma medium of high growth-promoting ability.