Mark E. Tourtellotte

Mycoplasma membrane lipids: variations in fatty acid composition.

The fatty acid composition of the membrane polar lipids of Mycoplasma laidlawii B can be dramatically altered. These variations reslut in characteristic morphological chlanges, and in most cases the cells remain viable. This organism should provide a useful system for clarifying the role of fatty acyl chains in biological membranes.

Freeze-Fractured Acholeplasma laidlawii Membranes: Nature of Particles Observed

Freeze-fracture of Acholeplasma laidlawii membranes from cells incubated in the presence of puromycin or omission of amino acids reveals a decrease in the number of particles between 50 and 100 angstroms in the hydrophobic fracture plane, which strongly suggests that these particles are protein. Additional evidence indicates that they may be involved in substrate transport.

Mycoplasma gallisepticum--control by immunization.

The earliest report in the literature indicating an immune response for Mycoplasma gallisepticum was made by Nelson in 1935, when he reported that a recovery from coryza was followed by an altered susceptibility. Delaplane and Stuart (1943), in some of the early work with chronic respiratory disease. observed that “recovered birds reinoculated with this virus failed to show any symptoms, indicating that prior infection had stimulated development of a significant degree of immunity.” The agent responsible for this condition was later shown to be a pleuropneumonia-like organism (Markham and Wong, 1952) and named M . gallisepticum (Edward and Kanarek, 1960). Numerous laboratory t r ia ls (Fahey and Crawley, 1954; Domermuth, 1957, 1962; Adler, 1960; Adler et al., 1960; McMartin and Adler, 1961; Olson et al., 1962, 1964; Fabricant and Levine, 1963) and field observations (Fahey and Crawley, 1956; Van Roekel and Olesiuk, 1952, 1953; Olesiuk et al., 1953; Olesiuk and Van Roekel, 1960a, b; Adler et al., 1960) suggested the development of an immune response after an infection with M . gallisepticum. During a review of these articles, it became evident that the strain of organisms used, the route of inoculation, and the number of organisms inoculated were important factors relating to the degree of immunity induced. This paper documents the experience of the authors in laboratory and field trials on the use of a live culture in an immunization procedure for the control of M . gallisepticum infection.

The relationship between fatty acid structure and the positional distribution of esterified fatty acids in phosphatidyl glycerol from Mycoplasma laidlawii B

Abstract 1. 1. The distribution of a number of fatty acids between the 1- and 2-positions of phosphatidyl glycerol from Mycoplasma laidlawii B has been investigated. The fatty acid composition of the phosphatidyl glycerol was systematically altered so that the positional distribution values determined here are accurate reflections of the relative positional affinities of the individual fatty acids examined. 2. 2. The positional distribution of any given fatty acid constituent was independent of the amount of that acid in the phosphatidyl glycerol, provided that the fatty acid in question was not present in sufficient quantity to saturate the preferred position. 3. 3. Within any single class of fatty acids, the positional specificity was markedly dependent on the chain length of the fatty acid. The smaller the total number of carbon atoms, the lesser was the affinity for the 1-position. 4. 4. Positional distribution was also dependent on the structural characteristics of the fatty acids. The following is the order of the fatty acid classes arranged according to a decreasing affinity for the 1-position: straight-chain saturated > branched-chain saturated > trans -monounsaturated > cis -monounsaturated > cyclopropane > cis -diunsaturated.

Localization of an immunodominant 64kDa lipoprotein (LP 64) in the membrane of Mycoplasma gallisepticum and its role in cytadherence

A64kDa lipoprotein (LP 64) haemagglutinin (pI 4.9–5.0) was isolated from the membrane of Mycoplasma gallisepticum. Triton X‐114 phase partitioning has demonstrated that the hydrophobic nature of this haemagglutinin is due to a lipid portion of the molecule. Autoradiography of [3H]‐palmitate‐labelled M. gallisepticum revealed the presence of several additional lipoproteins. Immunoelectron microscopy demonstrated the localization of LP 64 to the base of the terminal structure. Densitometric scans of stained polyacrylamide gels of M. gallisepticum showed that LP 64 constitutes 1.7% of the total protein. Scans of immunoblots of M. gallisepticum indicate that LP 64 is highly immunogenic in chickens, accounting for 7.4% of the total serum IgG response at four weeks post‐infection. A quantitative value for the IgG response to LP 64, relative to the percentage of total protein (the Relative Immunogenicity Index) was 4.4. LP 64 is conserved among several strains of M. gallisepticum, but its presence could not be detected in Mycoplasma synoviae. Antiserum raised to electroeluted LP 64 reacted specifically with this lipoprotein when assessed on either one‐or two‐dimensional immunoblots of M. gallisepticum. This antiserum, as well as Fab fragments, inhibited haemagglutination of chicken erythrocytes and inhibited the attachment of 14Clabelled M. gallisepticum to chicken tracheal epithelium in vitro by 62%.

Physiological and serologic comparisons of PPLO from various sources.

The relationship between pleuropneumonialike organisms (PPLO) and L forms of bacteria has been the subject of numerous reviews (Dienes and Weinberger, 1951; Edward, 1954; and Klieneberger-Nobel, 1954). Wittler et a,?. (1956) and Smith et al. (1957) have demonstrated physiological and serologic similarities between PPLO and Corynebacterium spp. That PPLO are L forms of bacteria, however, is not yet an accepted fact (Edward and Freundt, 1956). Many of these organisms have been studied morphologically, pathologically, and serologically. With the exception of a few strains, little is known of their physiology and metabolism. Dujardin-Beaumetz (1900) identified acetic acid as the only volatile acid produced by Mycoplasma mycoides from carbohydrate. Holmes and Pirie (1932) demonstrated lactic dehydrogenase in this organism. Rodwell and Rodwell (1954) showed that cell suspensions of M . mycoides oxidized glucose and pyruvate aerobically to acetate and COz . Anaerobically, glucose was not utilized, and pyruvate was dismutated to lactate, acetate, and COZ . Small amounts of acetylmethylcarbinol were also produced in the latter case. Tricarboxylic acid cycle intermediates were not utilized. Metabolic similarities between M . mycoides and Streptococcus faecalis were noted. Lecce and Morton (1954) showed that certain human strains do not utilize carbohydrates or tricarboxylic acid cycle intermediates. Active alcohol dehydrogenases were observed in these organisms. Smith (1955, 1957a, 19573, 1957c), studying the amino acid metabolism of the same organisms, showed the phosphorolytic deamidation of glutamine to be the only reaction of amino acids capable of producing energy that could be utilized for growth. In order to understand this group of organisms further, a physiological and serologic study of various strains was made. With the exception of four strains, all organisms produced acid from carbohydrate. Metabolic studies were confined to the fermentative group. These are compared serologically to the n onfermen tative organisms. With present techniques, numerous PPLO have been isolated.

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.

PROTEIN SYNTHESIS IN MYCOPLASMA

Although the mycoplasma replicate in nonliving bacteriological media, their extreme small size and complex nutritional requirements have raised questions concerning their biosynthetic abilities. The ability to synthesize protein and the mechanisms thereof are obviously of paramount importance. In the past several years, the mechanism of protein biosynthesis in plant, animal and bacterial systems has been extensively investigated and at the present time is relatively well understood. The basic mechanisms in all these free-living forms appear to be essentially the same.1,2 Recent reports, however, have shown some differences in effects of antibiotics on protein synthesis in bacteria, on one hand, and the fungi, protozoa, plants and animals on the ~ t h e r . ~ . ~ ~ ~ In a brief report,” Tourtellotte and Morowitz described cell-free protein synthesis in Mycoplasma laidlawii B in which similarities with other organisms were noted. Other than this, virtually nothing is known of protein synthesis in the Mycoplasmatales. In this report, previous work together with some new observations will be summarized. Data suggest (1) that protein synthesis in several strains of mycoplasma is essentially similar to that in other systems, and (2) that, on the basis of antibiotic sensitivity of protein synthesis, the mycoplasma are more closely related to bacteria than to higher forms.