Laidlawii Strain Research Articles

Influence of lipid composition on the orientational order in Acholeplasma laidlawii strain B membranes: a deuterium NMR study.

2H NMR techniques have recently been developed to determine the complete orientational order profile of lipid bilayers employing lipids containing perdeuteriated palmitic acid [Lafleur, M., Fine, B., Sternin, E., Cullis, P.R., & Bloom, M. (1989) Biophys. J. 56, 1037-1041]. In this work, these techniques have been applied to study order profiles in intact membranes derived from Acholeplasma laidlawii strain B. It is shown that complete orientational order profiles can be readily obtained from the intact membranes of A. laidlawii B grown on equimolar amounts of perdeuteriated palmitic acid and a nondeuteriated fatty acid of varying length and unsaturation. By variation of the fatty acid composition employing mixtures of perdeuteriated palmitic acid with myristic, elaidic, oleic, or linoleic acid, a range of hydrocarbon order compatible with high rates and extents of cell growth has been obtained where the average order parameter, mean value of S, varies over the range 0.140-0.176. This same variation in order is seen for liposomes derived from total lipids extracted from these intact membranes. 2H NMR studies on liposomes composed of individual species of the extracted lipids indicate that modulation of the membrane lipid headgroup composition has the potential to play an important role in maintaining the membrane order within this range.

Cloning and DNA sequence of a mycoplasmal recA gene

Mycoplasmas are wall-less prokaryotes phylogenetically related to gram-positive bacteria. In order to investigate DNA recombination in these organisms, we have cloned the recA gene from the mycoplasma Acholeplasma laidlawii. DNA sequence data indicate extensive homology between the A. laidlawii recA gene and recA genes from other bacteria, particularly Bacillus subtilis. The recA sequences from three A. laidlawii strains (strains JA1, K2, and 8195) were compared, and surprisingly, the gene from A. laidlawii 8195 was found to contain a nonsense mutation that results in truncation of 36 amino acids from the carboxyl terminus of the RecA protein. By using sensitivity to UV irradiation as a measure of DNA repair, strain 8195 had an apparent RecA- phenotype. When carried on a multicopy plasmid, the wild-type A. laidlawii recA gene was detrimental to growth of Escherichia coli, perhaps because of improper regulation of the RecA protein.

The effect of variations in growth temperature, fatty acid composition and cholesterol content on the lipid polar head-group composition of Acholeplasma laidlawii B membranes

We have systematically investigated the effect of variations in growth temperature, fatty acid composition and cholesterol content on the membrane lipid polar headgroup composition of Acholeplasma laidlawii B. Two important lipid compositional parameters have been determined from such an analysis. The first parameter studied was the ratio of the two major neutral glycolipids of this organism, monoglucosyldiacylglycerol (MGDG) and diglucosyldiacylglycerol (DGDG). As the former lipid prefers to exist in a reversed hexagonal phase at higher temperatures, with unsaturated fatty acyl chains or in the presence of cholesterol, the ratio of these two lipids reflects the phase state preference of the total A. laidlawii membrane lipids. Although we find that the MGDG/DGDG ratio is reduced in response to an increase in fatty acid unsaturation, increases in growth temperature or cholesterol content reduce this ratio only in cells enriched in a saturated but not an unsaturated fatty acid. The second parameter studied was the ratio of these neutral glycolipids to the only phosphatide in the A. laidlawii membrane, phosphatidylglycerol (PG); this parameter reflects the relative balance of uncharged and charged lipids in the membrane of this organism. We find that the MGDG + DGDG/PG ratio is lowest in cells enriched in the saturated fatty acid even though these cells already have the highest lipid bilayer surface charge density. Moreover, this ratio is not consistently related to growth temperature or changes in cholesterol levels, as expected. We therefore conclude that A. laidlawii strain B, apparently unlike strain A, does not possess coherent regulatory mechanisms for maintaining either the phase preference or the surface charge density of its membrane lipid constant in response to variations in growth temperature fatty acid composition or cholesterol content.

Cholesterol uptake capacity of Acholeplasma laidlawii is affected by the composition and content of membrane glycolipids

The composition of the cell membrane of 20 Acholeplasma laidlawii strains grown under identical conditions was studied and correlated with the capacity of these strains to incorporate cholesterol. Membranes of these strains had similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns and contained the same lipid species, but the relative amounts of the major polar lipids varied. Statistical analyses revealed that the glycolipids, monoglucosyldiglyceride, and an unidentified glycolipid (glycolipid-X) succeeded in explaining 90% ( R 2 = 0.90) of the cholesterol uptake variations. The regression coefficients for both glycolipids were negative ( P < 0.001), indicating that the capacity of A. laidlawii strains for cholesterol incorporation is inversely proportional to the relative amounts of these glycolipids. Accordingly, an increased capacity for cholesterol incorporation was detected upon aging of A. laidlawii cells. The aged cells contained significantly smaller amounts of both monoglucosyldiglyceride and glycolipid-X, and a higher amount of diglucosuldiglyceride. The change in cholesterol incorporation as a response to glycolipid composition and content can be explained by the low solubility of cholesterol in glycolipids as well as by the induction by the sterol molecule of a nonlamellar phase state that will destabilize a membrane structure containing monoglucosyldiglyceride and glycolipid-X.

Acholeplasma laidlawii retains sensitivity to exogenous virus while releasing endogenous, mitomycin C induced, virus. Brief report.

Three of five Acholeplasma laidlawii strains were found to carry Mitomycin C inducible acholeplasmaviruses. These virus hosts were capable of propagating (1) virus homologous to the one it carried, and (2) exogenous virus while releasing induced endogenous virus.

Difference in packing properties between iso and anteiso methyl-branched fatty acids as revealed by incorporation into the membrane lipids of Acholeplasma laidlawii strain A

Acholeplasma laidlawii strain A was grown on the long-chain, branched, saturated fatty acids 12-methyltetradecanoic acid (anteiso-C 15), 14-methylhexadecanoic acid (anteiso-C 17), 13-methyltetradecanoic acid (iso-C 15) and 15-methylhexadecanoic acid (iso-C 17), and on the branched-chain fatty acid precursors 2-methylbutanoic acid (anteiso-C 5) and 3-methylbutanoic acid (iso-C 5). The membrane lipid composition as a function of the fatty acid supplement and the growth temperature was determined: (1) The ratio between the two dominating lipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol, is decreased when anteiso acyl chains replace iso acyl chains in the lipids, and when the acyl chains are elongated from 15 to 17 carbon atoms; (2) the ratio iso / anteiso acyl chains is reduced when the growth temperature is decreased; (3) the average length of the de novo synthesized branched fatty acids is decreased when the growth temperature is lowered, and when anteiso-C 5 is exchanged for iso-C 5 in the growth medium. The lipid regulation mechanisms are interpreted in terms of lipid molecular geometry and self-assembly of lipid molecules. It is concluded that iso and anteiso fatty acids have distinctively different packing properties in a biological membrane, and they mimic straight-chain saturated and cis-monounsaturated fatty acids, respectively, as seen by the way they affect the physicochemical properties of membrane lipids and by the way they are used in lipid regulation mechanisms.

Transfection of REP- mycoplasmas with viral single-stranded DNA.

Double-stranded DNA from mycoplasma virus L2 can transfect Acholeplasma laidlawii cells in the presence of polyethylene glycol (T. L. Sladek and J. Maniloff, J. Bacteriol. 155:734-741, 1983). We report here that both single-stranded DNA and double-stranded replicative form DNA, from the single-stranded DNA mycoplasma virus L51, are also infectious in this system. For both DNAs transfection frequencies were in the range of 10(-8) transfectants per DNA molecule and 10(-3) transfectants per CFU. An unexpected finding was that both DNAs could transfect A. laidlawii strain REP-, a variant which is a nonpermissive host for single-stranded DNA mycoplasma viruses due to a block in viral DNA replication (Nowak et al., J. Bacteriol. 127:832-836, 1976). The number of viruses produced by transfected REP- cells was comparable to the number produced by both transfected and infected wild-type cells. Therefore, transfected L51 DNAs are able to bypass the replication block in REP- cells that occurs when these cells are infected by L51 virions.

Characterization of the enveloped plasmavirus MVL2 after propagation on threeAcholeplasma laidlawii hosts

Plasmavirus MVL2 was propagated on three Acholeplasma laidlawii strains, JA1, S2, or BC1-13. Previously reported host-controlled modification (HCM) of MVL2, as reflected by changes in plating efficiency, was observed. Adsorption rates and one-step growth curves varied according to host used. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the three MVL2 populations revealed differences in polypeptide profiles.

Cytosine methylation of the sequence GATC in a mycoplasma.

Mycoplasma virus L2 is subject to host-specific restriction and modification in Acholeplasma laidlawii strains JA1 and K2. We have examined the DNAs from both host cells and viruses propagated on these strains with respect to susceptibility to cleavage by restriction endonucleases and for DNA base modifications. We show that, in strain K2 and L2 virus grown on K2 cells, cytosine in the sequence GATC is methylated to 5-methylcytosine and, although strain K2 and L2 viruses grown on K2 contain N6-methyladenine in their DNA, adenine in the sequence GATC is not methylated. In contrast to K2, strain JA1 and L2 virus grown on JA1 cells contain no detectable methylated bases. It is not known which of the methylated bases in K2 is the basis for the K2 restriction-modification system operative on L2 virus.

Molecular organization and selective solubilization of lipids and proteins in the envelope of mycoplasmal virus L2.

The composition and molecular organization of mycoplasmal virus L2 (MVL2) were studied and compared with those of the cell membrane of the host, Acholeplasma laidlawii strain JA1. The virus contained 0.2-0.25 mumol of polar lipids per mg of viral protein. The lipid species of the MVL2 were the same as those of the host cells. Nevertheless, the proportions between the various polar lipids were different, with a much lower content of phosphatidylglycerol in the viral lipids. Despite these quantitative differences, the fatty acid composition of MVL2 was similar to that found in the host cells, a similarly allowing alteration of the fatty acid composition and study of its effect on viral absorption, penetration, and release. Pulse-chase experiments revealed that the lipids incorporated into the virus were synthesized before and after infection. Electron paramagnetic resonance spectrometry suggested that the viral lipid domain had the properties of a lipid bilayer. Nevertheless, the hydrocarbon chains in the MVL2 envelope were less mobile than those in membranes of the host cells, a difference apparently due to the different content, composition, and disposition of proteins in the MVL2 envelope. The electrophoretic pattern of MVL2 polypeptides was dominated by four major and five minor bands distinct from the polypeptides present in A. laidlawii membranes. None of the polypeptides gave a positive periodic acid-Schiff reaction, a result suggesting the absence of glycoproteins. Selective solubilization experiments excluded the possibility that one or more of the major polypeptides was associated with a capsid structure. Lactoperoxidase-mediated iodination of intact viral particles revealed that at least two of the major polypeptides were localized on the external surface of the viral envelope. The susceptibility of these polypeptides to brief proteolytic treatment and the finding that the infectivity of the virus was dramatically affected by such treatment suggested that these polypeptides were playing a role in recognition and/or attachment of the virus to the host cells.

Two chemically defined media for the growth of Acholeplasma laidlawii strain A

A total of 11 tissue culture media and one synthetic medium were screened to determine whether they could support the growth of two strains of Acholeplasma. Two similar chemically defined media were found in which Acholeplasma laidlawii Strain A (Buchanan & Gibbons 1974) could grow. This is the first report of a fully defined medium for the growth of this organism (Greenaway 1981).

Isolation and characterization of Mycoplasma and Acholeplasma from apparently healthy and diseased (infectious sinusitis) turkeys.

Investigation of 136 turkeys (24 manifesting infra-orbital sinusitis, 112 apparently healthy) resulted in isolation of 79 strains of Mycoplasma and 4 of Acholeplasma. By the disc growth inhibition test with 16 reference antisera of avian serogroups, 55 strains were identified serologically and 28 remained unidentified. Thirteen strains of Mycoplasma gallisepticum, 1 of M. meleagridis, and 2 of Acholeplasma laidlawii were isolated from turkey sinusitis whereas serogroups C (2), D (19), F (8), M. meleagridis (4), M. anatis (4), A. laidlawii (2), and 28 unidentified strains were isolated from apparently healthy turkeys. Three patterns were recognized on the basis of glucose, maltose, and sucrose, fermentation. The most frequent, pattern I, included 13 M. gallisepticum strains whereas 5 M. meleagridis strains belonged to fermentation pattern III. Isolates were also studied for reduction of tetrazolium, methylene blue, potassium tellurite, resistance to methylene blue and sodium taurocholate, and production of arginine deiminase and "film and sports." Inoculation of selected isolates into developing chick embryos revealed that 2 A. laidlawii strains were nonpathogenic and 13 M. gallisepticum, 1 serogroup D and 2 serogroup F strains were pathogenic, causing 50--100% mortality. In vitro antibiotic disc sensitivity tests indicated that rovamycin (solubilized spiramycin) may be recommended for turkey mycoplasmosis. Isolation of 2 A. laidlawii strains from turkey sinusitis and 4 M. anatis strains from apparently healthy turkeys appears interesting.

Lipid Composition of Two Mycoplasmaviruses, MV-Lg-L172 and MVL2

SUMMARY The enveloped mycoplasmaviruses, MV-Lg-L172 and MVL2, were purified from the culture filtrates of infected Acholeplasma laidlawii strains by precipitation with polyethylene glycol followed by centrifugation in metrizamide gradients. Purified viruses were found to contain lipids by thin-layer chromatography. The total lipid content of the two viruses was estimated to be 10 to 12% of the dry wt. of purified virus. Isotopic labelling with inorganic 32P and 1-14C-oleic acid revealed the presence of phospholipids, glycolipids and phosphoglycolipids, similar to those found in the host cells. The fatty acid composition of the two viruses and their hosts were investigated by gas-liquid chromatography and found to be nearly identical. Virus-infected cells exhibited a slight increase in ratio of diglucosyl diglyceride to monoglucosyl diglyceride relative to uninfected cells. Viral glycolipid ratios more closely approximated those found in uninfected cells.

Colony morphology, ultrastructure and morphogenesis in Mycoplasma hominis, Acholeplasma laidlawii and Ureaplasma urealyticum.

Colonies of Mycoplasma hominis, Acholeplasma laidlawii (three strains) and Ureaplasma urealyticum were examined by light and electron microscopy and their characteristic morphology, ultrastructure and morphogenesis are described. Mycoplasma hominis and A. laidlawii, PG8 and oral strains, developed typical 'fried-egg' colonies which were remarkably heterogeneous in size. The colonies of A. laidlawii strain NCTC 10116 were more homogeneous and grew mainly on the surface of the agar showing a fine granular appearance. Ureaplasma urealyticum produced smaller, granular colonies which grew deeply embedded in the agar and generally without much surface growth. The cellular ultrastructure in these colonies was also examined. The results indicate that several aspects of colony morphogenesis and ultrastructure varied for each of the three species examined.

Presence of viruses in a strain of mycoplasma pulmonis.

Filtrates prepared from heavily grown agar cultures of M. pulmonis strain Negroni-52 formed plaques on lawns of A. laidlawii strain JAl but not on those of M. pulmonis strains Ash or Negroni-52. The plaque-forming agent proved to be rod-shaped particles morphologically identical with mycoplasmavirus group 1. Evidence supporting the conclusion that the virus originated from Negroni-52 has been obtained. Electron microscopy revelaed that Negroni-52 is also a carrier of long-tailed phage-like particles.

Demonstration by electron microscopy of intracellular virus in Acholeplasma laidlawii infected with either MV-L3 or a similar but serologically distinct virus (BN1 virus).

Cultures of Acholeplasma laidlawii strain M1305/68 were inoculated with Mycoplasmatales virus-laidlawii 3 (MV-L3) and examined by electron microscopy. Particles resembling MV-L3 were observed both intra- and extracellularly in thin sections prepared from MV-L3 infected cultures, but not from uninfected cultures. Similar particles were occasionally observed in uninoculated cultures of A. laidlawii strain BN1 cells, from which a virus (BN1 virus) was subsequently isolated. This virus was morphologically similar but not identical to MV-L3. It also differed serologically from, and in its resistance to, MV-L3 and the other mycoplasma viruses.

Growth of an enveloped mycoplasmavirus and establishment of a carrier state

The growth of an enveloped DNA-containing mycoplasmavirus (MVL2 obtained from R.N. Gourlay) has studied, by using the indicator host Acholeplasma laidlawii strain JA1. From virus one-step growth curves, artificial lysis experiments, and infected cell growth curves, it was found that virus infection is nonlytic. Newly infected cells grow slower and are osmotically more stable than uninfected cells. However, 4 to 6 h after infection, the cells reach a carrier state in which cell growth rate and osmotic fragility are indistinguishable from uninfected cells. Carrier cultures contain free virus. Every carrier culture cell gives rise to either a clone of carrier cells or a clone of MVL2-resistant cells.

Mycoplasma infection of geese II. Studies on pathogenicity of mycoplasmas in goslings and goose and chicken embryos

Of various mycoplasma strains of goose original, axanthum strains (609 and 612) caused the death both of goose and chicken embryos, A. laid-lawii strain (606) killed only goose embryos, whereas M. gallinarum (598) failed to kill either. Infection of 3-day-old goslings with these mycoplasmas resulted in no mortality but lesions were produced with A. axanthum in 9 of 10 birds. Less severe lesions were seen in fewer birds infected with other strains. Dual infection of 3-day-old goslings, with maternal antibody to goose parvovirus, with M. gallinarum (598) or A. axanthum (612) and a virulent parvovirus resulted in some death and all birds showed lesions.

Direct Visualization of the Hydrophobic Portion of Acholeoplasma Laidlawii Membranes

Alteration and extraction of lipids occur during the preparation of biological tissues for electron microscopy. This problem has rendered the interpretation of membrane fine structure in molecular terms particularly difficult. By using a water-miscible, glutaraldehyde-carbohydrazide (GACH) resin polymerized at neutral pH, we have previously demonstrated lipid retention in biological tissues during embedding. Since A. laidlawii membranes have been characterized with regard to the composition and structural organization of the lipid component, they provided an ideal model system for direct visualization of the retained lipids after GACH embedding.High resolution micrographs of the membranes were taken on a Hitachi HU-11B electron microscope. At the same time, the instrument was calibrated according to established guidelines and found to give a standard deviation of 2.9% in magnification over a period of approximately one year. A. laidlawii strain B cells were generously provided by Dr. James Gilliam. The presence of an intact, hydrophobic core in sections of GACH-embedded membranes was confirmed by vanatomolybdate staining of the thin sections.

Mycoplasma infection of Geese I. Incidence of Mycoplasmas and Acholeplasmas in Geese

This paper presents data about the isolation of members of the order Mycoplasmatales from material of goose origin. Acholeplasma laidlawii strains were isolated from 2 to 8 day old goslings with heavy fibrinous airsacculitis, peritonitis and perihepatitis. Losses reached 30% of the flock by the end of the 8th week of age. Acholeplasma axanthum strains were detected in goose-embryos that died on the 13th day of incubation. A significant loss (up to 60%) of embryos was observed in the flock and some layers died showing fibrinous peritonitis, salpingitis and abdominal airsacculitis. Mycoplasma gallinarum also was isolated from goose-embryo fibroblast tissue cultures. All strains except A. laidlawii caused cytoplasmic vacuolization and intracytoplasmic inclusion bodies in goose-embryo fibroblast tissue cultures. The alteration observed in chicken-embryo fibroblast cell cultures were similar; in addition, the A. laidlawii caused a marked pycnosis of the cells.