The growth and long term survival of Acholeplasma laidlawii in media products used in biopharmaceutical manufacturing

Acholeplasma laidlawii is a potential contaminant of bovine serum and has also been found as a contaminant in serum free cell culture media products. Anecdotal evidence of A. laidlawii contamination of tryptone soya broth circulated for a number of years before it was acknowledged that the organism could contaminate microbiological broth powders. The occasional occurrence of A. laidlawii in broth powders and possibly in powdered components of cell culture media as part of the normal bioburden poses a serious threat to routine pharmaceutical and biopharmaceutical operations where filtration is the sterilisation method of choice. Absence of visual evidence of contamination cannot be relied upon as there is variation with both organism strain and media product in the ability to produce turbidity. Strains of A. laidlawii which have been isolated from broth powders are not significantly different in temperature or media preferences from other strains. A. laidlawii is capable of growing to high titre at refrigeration and ambient temperatures in unsupplemented bacteriological sterility media or serum free cell culture media and can survive for prolonged periods in these products.     

Lipid and protein membrane components associated with cholesterol uptake by mycoplasmas

Membranes of Mycoplasma species take up 2–4 times more exogenous cholesterol than membranes of Acholeplasma species. To test whether the lower cholesterol uptake capacity of Acholeplasma is due to the high glycolipid content of their membranes, the phospholipids of Acholeplasma laidlawii and Mycoplasma capricolum membranes were hydrolyzed by phospholipase A². Digestion removed about 30% of the polar lipids of A. laidlawii, leaving the glycolipids and phospholglycolipids intact, and about 70% of the polar lipids of M. capricolum, the residue consisting mostly of sphingomyelin. Cholesterol uptake by the treated membranes from phosphatidylcholine/cholesterol vesicles decreased in rough proportion to the amount of polar lipid removed, indicating that the glycolipids in A. laidlawii membranes can participate in cholesterol uptake.Trypsin digestion of growing cells and isolated membranes of M. capricolum decreased cholesterol uptake by about one-half. Similar treatment of A. laidlawii cells and membranes had no effect on cholesterol uptake. These findings suggest the existence of protease-sensitive receptors on the cell surface of M. capricolum responsible for tighter contact with the cholesterol/phosphatidylcholine vesicles. It is proposed that the ability of Mycoplasma species to take up large quantities of exogenous cholesterol and phospholipids depends on the presence of protein receptors for cholesterol donors, receptors which are absent in Acholeplasma species.     

The nature and location of Acholeplasma laidlawii membrane proteins investigated by two-dimensional gel electrophoresis

The high resolution, two-dimensional electrophoresis system for the separation of proteins described by O'Farrell, (O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007–4021) has been modified for the separation of Acholeplasma laidlawii proteins.Reproducible protein patterns have been obtained from A. laidlawii cell, membrane and soluble protein preparations. The isoelectric focusing of membrane proteins was greatly improved by removing the bulk of the membrane lipid before solubilizing the protein.A. laidlawii peripheral membrane proteins were removed from the membrane by low ionic strength washing and by treatment with EDTA. The effect of an exhaustive EDTA treatment and a rapid, warm EDTA treatment were compared. By comparing the protein patterns obtained in these ways it was possible to distinguish two separate groups of peripheral membrane proteins and one integral membrane protein group. The peripheral membrane proteins which were removed from the membrane at low ionic strength (group I) were also insoluble in Triton X-100, whereas additional peripheral membrane proteins extractable by subsequent EDTA treatment (group II) were soluble in Triton X-100.Exterior-facing membrane proteins were distinguished from the interiorfacing ones by lactoperoxidase-catalyzed iodination of intact cells and membranes. Group I peripheral membrane proteins faced the cell interior whereas group II proteins faced the cell exterior. We counted approximately 320 individual whole cell proteins. Of these, about 140 were membrane associated and a maximum of 40 proteins were iodinated after iodinationg intact cells.A. laidlawii was also grown in the presence of NaH₂³²PO₄ and whole cell proteins were separated by two-dimensional gel electrophoresis. One membrane protein and two soluble proteins were labelled.     

Cholesterol-phosphatidylcholine dispersions as donors of cholesterol to Mycoplasma membranes

Growing cells of sterol-requiring Mycoplasma hominis and sterol non-requiring Acholeplasma laidlawii were used to test the ability of cholesterol-dipalmitoyl phosphatidylcholine dispersions to serve as cholesterol donors to these organisms. Dispersions with high cholesterol to phosphatidylcholine ratios were more effective than dispersions with low cholesterol to phosphatidylcholine ratios in donating cholesterol to the membranes of both mycoplasmas and in promoting growth of the sterol-requiring species. M. hominis took up almost three times as much cholesterol as did A. laidlawii. In addition, significant quantities of the phosphatidylcholine component of the dispersions were found to be associated with M. hominis membranes as against none in the A. laidlawii membrane preparations. In all cases, the percentage of cholesterol taken up by M. hominis from the dispersions exceeded that of phosphatidylcholine by a factor of 3–5. These results were interpreted to suggest that all the cholesterol taken up by A. laidlawii is transferred from the dispersion to the membranes by a process which involves only a transient contact between the organisms and the lipid dispersions, whereas a certain amount of the cholesterol taken up by M. hominis may also be derived from lipid dispersions adhering to or fusing with the cell membranes.     

Comparative proteomic characteristic of mycoplasmas (Mollicutes)

Using one- and two-dimensional gel electrophoresis, various types of mass-spectrometry, and combinations of these two methods, saturating identification of mycoplasm Acholeplasma laidlawii proteome and study of proteins carrying posttranslational modifications were performed. We compared our data with earlier identified proteome of Mycoplasma gallisepticum. It was shown that M. gallisepticum and A. laidlawii expressed 61 and 58% of the proteins of the annotated open reading frames, respectively. All subunits of DNA-polymerase III were identified for A. laidlawii during our study, which indicates that our techniques enable detection of single copies of mollicutes proteins per cell. Metabolic pathways of respective mycoplasmas is compared in this research.     

Effect of acriflavine on ultraviolet inactivation of Acholeplasma laidlawii

An increased sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawiiAn increase sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawii cells were plated on medium containing either acriflavine or chloramphenicol. Chloramphenicol reduced liquid holding recovery (dark repair) to about 10 percent of that in untreated irradiated cells. In acriflavine treated cells no dark repair could be observed and there was a progressive degradation of cell DNA during holding. While the primary effect of acriflavine may be to inhibit excision repair, since ultraviolet-irradiated Mycoplasma gallisepticum (cells which lack an excision repair mechanism) show a slight increase in inactivation when plated on medium containing acriflavine, the dye must also have some other effects on ultraviolet repair processes. Acriflavine treatment of A. laidlawii cells before ultraviolet irradiation has a protective effect, as seen by an increased cell survival.     

Role of cholesterol in the action of cereolysin on membranes.

The following evidence supports the concept that cholesterol in membranes is the receptor and target site for the cytolytic action of cereolysin. (i) Of the various phospholipids, gangliosides, and steroids tested, only cholesterol and closely related sterols (sitosterol and dihydrocholesterol) significantly inhibited the hemolytic activity of cereolysin. (ii) Acholeplasma laidlawii cells grown in the presence of cholesterol inhibited the hemolytic activity of cereolysin, but A. laidlawii grown in the absence of cholesterol did not. (iii) Incubation of A. laidlawii cells, grown in the absence of cholesterol, with a cholesterol-Tween 80 mixture reestablished the ability of the cells to bind cereolysin. (iv) Treatment of erythrocyte membranes and A. laidlawii cells containing cholesterol with cholesterol oxidase (EC 1.1.3.6, Brevibacterium species) abolished the ability of these membranes to bind cereolysin and inhibit the hemolytic activity of the toxin, (v) Cereolysin could bind to and alter the permeability of both right-side-out ghosts and inside-out vesicles prepared from human erythrocytes, in agreement with other data that cholesterol is present on both sides of the erythrocyte membrane, (vi) Cereolysin caused the release of [¹⁴C]glucose from liposomes containing cholesterol, and this release was dependent on the amount of cholesterol in the liposomes.     

Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment

Mycoplasmas (class Mollicutes) are wall-less prokaryotes phylogenetically related to gram-positive bacteria. This study describes the construction of recA mutants of the mycoplasma Acholeplasma laidlawii. An internal fragment of the recA gene from A. laidlawii was cloned into a plasmid that does not replicate in this organism. When this plasmid construct was used to transform A. laidlawii, it inserted into the chromosome, disrupting the recA gene. The pheno-type of the resulting recA mutant was compared to that of wild-type cells and to that of a strain that has a naturally occurring ochre mutation in its recA gene. As found in other bacterial systems, loss of RecA activity resulted in cells deficient in DNA repair.     

Electrical Properties and Ultrastructure of Mycoplasma Membranes

Mycoplasma, in particular species laidlawii and gallisepticum, are found to have a very small, low frequency conductivity as would be predicted by the dielectric model for bacteria and their apparent lack of cell wall structure. Membrane capacitance values for the two organisms are both about 0.9 μF/cm², although electron micrographs show that the membrane of M. gallisepticum is 20-40 A thicker than that of M. laidlawii.     

Freeze-etch electron microscopy of erythrocytes,Acholeplasma laidlawii cells and liposomal membranes after the action of filipin and amphotericin B

Freeze-etch electron microscopy demonstrated that filipin induces the formation of aggregates 150–250Åin diameter, in the membranes of rat erythrocytes, in cholesterol-containing membranes ofAcholeplasma laidlawii cells and in egg lecithin-cholesterol liposomes. No change in fracture faces was observed when cholesterol was absent in the membranes ofA. laidlawii, and lecithin liposomes.Amphotericin B does not visibly affect the freeze-etch morphology of erythrocytes, cholesterol-containingA. laidlawii cells and lecithin-cholesterol liposomes.     

Identification of lipoglycan antigens from the Acholeplasma laidlawii cell membrane in crossed immunoelectrophoresis

Membranes from the wall-less prokaryote Acholeplasma laidlawii contain a component termed lipoglycan or lipopolysaccharide (LPS). The lipoglycan has extraction properties, which are similar to those of LPS of gram-negative bacteria, but it is chemically distinct from bacterial LPS. The membrane-bound lipoglycan of A. laidlawii did not seem to be particularly immunogenic and antibodies against it could not always be detected by rocket immunoelectrophoresis (RIE) or crossed immunoelectrophoresis (CIE) in hyperimmune sera raised against membranes. The immunoprecipitate corresponding to the lipoglycan, obtained by CIE of Tween 20-solubilized A. laidlawii membranes, has been identified and shown to be both a cathodically and anodically migrating component at pH 8.6. The shape of the immunoprecipitate in both RIE and CIE showed that the lipoglycan antigen is composed of at least two components, which are immunologically related.     

The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii

Small heat shock proteins (sHSPs) control the proteins stability in the cell preventing their irreversible denaturation. While many mycoplasmas possess the sHSP gene in the genome, Acholeplasma laidlawii is the only mycoplasma capable of surviving in the environment. Here we report that the sHSP IbpA directly interacts with the key division protein FtsZ in A. laidlawii, representing the first example of such interaction in prokaryotes. FtsZ co-immunoprecipitates with IbpA from A. laidlawii crude extract and in vitro binds IbpA with K_D ~ 1 μM. Proteins co-localize in the soluble fraction of the cell at 30–37 °C and in the non-soluble fraction after 1 h exposition to cold stress (4 °C). Under heat shock conditions (42 °C) the amount of FtsZ decreases and the protein remains in both soluble and non-soluble fractions. Furthermore, in vitro, FtsZ co-elutes with IbpA_His6 from A. laidlawii crude extract at any temperatures from 4 to 42 °C, with highest yield at 42 °C. Moreover, in vitro FtsZ retains its GTPase activity in presence of IbpA, and the filaments and bundles formation seems to be even improved by sHSP at 30–37 °C. At extreme temperatures, either 4 or 42 °C, IbpA facilitates FtsZ polymerization, although filaments under 4 °C appears shorter and with lower density, while at 42 °C IbpA sticks around the bundles, preventing their destruction by heat. Taken together, these data suggest that sHSP IbpA in A. laidlawii contributes to the FtsZ stability control and may be assisting appropriate cell division under unfavorable conditions.     

Requirements ofAcholeplasma laidlawii A,strain LA 1, for nucleic acid precursors

The requirements for nucleic acid precursors byAcholeplasma laidlawii type A, strain LA 1, were studied. The absence of a requirement for ribose or deoxyribose and the interconvertibility of uridine and cytidine contrasts with previously reported results with strain pG 8. Growth of the LA 1 strain was only moderately inhibited by a ten-fold excess of RNA over DNA. The only required nucleic acid precursors were guanine, thymine and either cytosine or uracil.     

Survival of Frozen Mycoplasmas

Cooling to -70 C killed a higher percentage of Acholeplasma laidlawii and Mycoplasma mycoides var. capri cells than cooling to -20 C. However, to preserve cell viability for prolonged periods storage at -70 C was much more preferable. The percentage of cells surviving freezing could be increased by increasing the initial cell concentration or by the addition of dimethyl sulfoxide or glycerol as cryoprotective agents. In the presence of 1.5 M of any one of these agents survival rates of up to 100% could be obtained. The optimal cooling rates for maximal survival of A. laidlawii under the experimental conditions tested were 11 C/min for cooling to -20 C and about 15 C/min for cooling to -70 C. Increasing the warming rate during thawing from 0.6 to 67 C/min increased survival by 3 log. Oleic acid enrichment of A. laidlawii membrane lipids, or reduction in the cholesterol content of M. mycoides var. capri membranes, increased the percentage of organisms surviving freezing. Hence, the composition of membrane lipids appears to have a marked influence on the susceptibility of mycoplasmas to freezing injury.     

Lipid distribution in Acholeplasma laidlawii membrane. A study using the lactoperoxidase-mediated iodination

The lactoperoxidase-mediated radioiodination has been applied to study the transbilayer distribution of phospho- and glycolipids in Acholeplasma laidlawii membranes. After radioiodination, about 5% of the ¹²⁵I-iodine was found in membrane lipids. A comparison of the labeling intensities of the various lipid species between iodinated intact cells and isolated membranes revealed that the glycolipids monoglucosyldiglyceride and diglucosyldiglyceride are located almost exclusively in the outer half of the bilayer, whereas the phospholipids phosphatidylglycerol and diphosphatidylglycerol as well as the phosphoglycolipids glycerophosphoryl-diglucosyldiglyceride and glycerophosphoryl-monoglucosyldiglyceride are almost equally distributed in the outer and inner halves of A. laidlawii membranes.     

Nutritional effects of culture media on mycoplasma cell size and removal by filtration

Careful media filtration prior to use is an important part of a mycoplasma contamination prevention program. This study was conducted to increase our knowledge of factors that influence efficient filtration of mycoplasma. The cell size of Acholeplasma laidlawii was measured after culture in various nutritional conditions using scanning electron microscopy. The maximum cell size changed, but the minimum cell size remained virtually unchanged and all tested nutritional conditions resulted in a population of cells smaller than 0.2 μm. Culture in Tryptic Soy Broth (TSB) resulted in an apparent increase in the percentage of very small cells which was not reflected in increased penetration of non-retentive 0.2 μm rated filters. A. laidlawii cultured in selected media formulations was used to challenge 0.2 μm rated filters using mycoplasma broth base as the carrier fluid. We used 0.2 μm rated filters as an analytical tool because A. laidlawii is known to penetrate 0.2 μm filters and the degrees of penetration can be compared. Culture of A. laidlawii in TSB resulted in cells that did not penetrate 0.2 μm rated filters to the same degree as cells cultured in other media such as mycoplasma broth or in TSB supplemented with 10% horse serum.     

Cholesterol uptake is dependent on membrane fluidity in mycoplasmas

The transfer of elaidate-enriched Acholeplasma laidlawii cells in culture from 37°C to 4°C virtually arrested exogenous cholesterol incorporation into the cell membrane. Cholesterol uptake continued, though at a slower rate, in oleate-enriched A. laidlawii cells undergoing similar temperature shift-down. It is concluded that the incorporation of exogenous cholesterol into the cell membrane of living mycoplasmas is rapid when the membrane lipid bilayer is in the liquid-crystalline state and very slow when the lipid bilayer is in the gel state.     

The influence of the fatty acid composition of Acholeplasma laidlawii membranes on the growth inhibitory activity of narasin,a polyether ionophorous antibiotic

Narasin, a polyether ionophorous antibiotic capable of acting as a transmembrane carrier of cations, has a growth inhibitory effect on Acholeplasma laidlawii, permitting only 20% survival when present at 0.1 μg/ml in an undefined growth nutrient or fatty acid-deficient nutrient supplemented only with palmitic acid. When A. laidlawii is propagated in fatty acid-deficient nutrient supplemented with linoleic acid, however, the organisms become 40 times more sensitive to the growth inhibitory effect of this antibiotic. The actual fatty acid compositions of the membranes would indicate that a higher degree of unsaturation enhances ionophore activity.     

On the possible role of respiratory activity of Acholeplasma laidlawii cells in sugar transport

1. Out of 20 exogeneous substrates only ethanol and, to a much lesser extent, lactate and pyruvate were shown to be capable of stimulating the respiration of Acholeplasma laidlawii cells. However, none of these substrates changed the initial rate of active transport of 3-O-methyl-d-glucose (3-O-MG).
2. From inhibitory analyses and spectroscopic data, it is apparent that the respiratory chain of A. laidlawii has no cytochromes and is probably not responsible for oxidative phosphorylation.
3. Valinomycin and nigericin stimulated cell respiration only in the presence of K⁺-ions, while monensin stimulated it in the presence of Na⁺-ions.
4. 3-O-MG transport was shown to be sensitive to uncouplers, ATPase inhibitors and arsenate are resistant to a majority of respiratory inhibitors tested. This suggested that there was no relationship between respiration and carbohydrate transport in the A. laidlawii cells. Further evidence was provided by the absence of respiratory stimulation during the transport of non-metabolizing carbohydrates.