A cation/proton antiport activity in Acholeplasma laidlawii

Sealed membrane vesicles of Acholeplasma laidlawii were obtained by controlled lysis of carotenoid-rich intact cells. An imposed delta pH was created by loading membrane vesicles or intact Acholeplasma laidlawii cells with 0.25 M NH4Cl and diluting them into 0.25 M choline chloride. The passive efflux of NH3 from the membrane vesicles or cells resulted in the creation of a delta pH (inside acid) that could be visualized by the quenching of the fluorescence of the weak base acridine orange. Whereas with isolated membrane vesicles, the fluorescence was dequenched by the addition of Na+, with intact cells, K+ in addition to Na+ was required. These results strongly suggest a Na+/H+ exchange activity that in intact Acholeplasma laidlawii cells is K+-dependent. The possible role of the Na+/H+ exchange activity in pH homeostasis at the more alkaline pH range, as well as in the extrusion of excess Na+ from the cells is discussed.     

Complete genome and proteome of Acholeplasma laidlawii

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.     

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 interior-facing 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 iodinating 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.     

Binding of lectins to membranes of mycoplasmas from aging cultures

The binding of iodinated concanavalin A (Con A) and Ricinus communis agglutinin (RCA) to intact cells and isolated membranes of Acholeplasma laidlawii, Mycoplasma hominis and Mycoplasma capricolum decreased with the progression of the culture from the mid- to the late-logarithmic phase of growth. The binding of the lectins to Acholeplasma laidlawii membranes had no significant effect on membrane fluidity, as assessed by electron-paramagnetic resonance spectroscopy of spin-labelled fatty acids, and had no effect on several membrane-associated enzymic activities. Temperature affected the binding of Con A and RCA in an opposite manner: the binding of Con A increased, whereas that of RCA decreased, on raising the temperature from 4 degrees C to 37 degrees C. No significant difference in lectin binding was found between oleate- and elaidate-enriched membranes at low temperatures where the former was in the liquid-crystalline state and the latter in the gel state, suggesting that membranes fluidity does not influence the binding of Con A and RCA to Acholeplasma laidlawii membranes.     

Oligomeric forms, functions and cellular localization of alpha-crystallin type protein from Acholeplasma laidlawii

Alpha-Crystallin type heat shock protein (alpha-HSP) IbpA from Acholeplasma laidlawii was expressed in Escherichia coil and isolated from cell extract on Ni-sepharose column. Recombinant IbpA, like other alpha-HSPs, spontaneously formed oligomeres in vitro. High resolution electron microscopy revealed regular structures with 15 nm in diameter. Evaluation of molecular mass of IbpA oligomers was performed by gel filtration. Most of oligomers consist of 24 subunits. Recombinant IbpA prevents heat denaturation of soluble proteins in cell extract of E. coli and displays a mild positive effect on thermotolerance of E. coli cells during severe heat shock. We investigated a localization of IbpA in A. laidlawii cell by immunocytochemistry. We suppose that IbpA may protect various intracellular structures from damage during heat shock.     

Crossed immunoelectrophoresis, in the presence of tween 20 or sodium deoxycholate, of purified membrane proteins from Acholeplasma laidlawii.

Five membrane proteins from Acholeplasma laidlawii have been previously purified on a large scale. These proteins have been used to establish the relationship between the precipitation lines obtained by crossed immunoelectrophoresis of solubilized cell membrane proteins from A. laidlawii in the presence of the neutral detergent Tween 20 or those obtained in the presence of the anionic detergent sodium deoxycholate. This relationship, which was unambiguously established for four of the five proteins, was determined by tandem or "parallel" crossed immunoelectrophoresis of the sodium deoxycholate-solubilized membrane together with the purified proteins. Membranes from strain A of A. laidlawii were composed of proteins, which were immunologically related to and probably identical to membrane proteins from strain B of this organism.     

Interaction of mycoplasma virus type 2 with cellular components of Acholeplasma laidlawii strain JA1.

Mycoplasma virus type 2 was shown to adsorb specifically to intact cells, membranes, and lipoglycan of Acholeplasma laidlawii strain JA1 but not to these components of Acholeplasma oculi. The oligosaccharide chain of the lipoglycan defined the specificity of the receptor site since deacylation not only did not reduce adsorption but increased it threefold. Actual adsorption of virus to lipoglycan was demonstrated by sucrose density gradient separation of the virus-lipoglycan complex. A strain of A. laidlawii, JA1r, resistant to infection with mycoplasma virus type 2, was incapable of adsorbing the virus and was devoid of lipoglycan.     

Is the vertical disposition of Mycoplasma membrane proteins affected by membrane fluidity?

The influence of the physical state of the membrane lipid matrix on the vertical disposition of membrane proteins was studied with Acholeplasma laidlawii. Changes in membrane fluidity were brought about by altering the fatty acid composition of membrane lipids, by changing the growth temperature, by aging of cultures and by inducing changes in the membrane lipid-to-protein ratio through treatment with chloramphenicol. The lactoperoxidase-mediated iodination technique was used to label membrane proteins exposed to the aqueous surroundings. The degree of exposure of the iodine-binding sites of membrane proteins on the external surface of intact cells was found to undergo significant changes on varying growth conditions, but the changes could not be consistently correlated with changes in membrane fluidity, nor were they discernible on iodination of isolated membranes.     

Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.: Fr. in strawberry (Fragariaxananassa Duch.) using PCR

Part of the gyrase A gene (gyrA) of Acholeplasma laidlawii was cloned and incorporated directly downstream from a 6 x His tag segment of the pQE expression vector. The 23-kDa fusion protein was expressed as a 6 x His-tagged protein in Escherichia coli. The fusion protein was purified and used as an antigen for rabbit immunization. Western immunoblot analysis revealed that the antiserum raised against the gyrase A fragment had a specific affinity for a 108-kDa protein of A. laidlawii and cross-reacted with a 107.5-kDa protein of Acholeplasma axanthum, a 107-kDa protein of Acholeplasma granularum, and 95-97-kDa proteins of several phytoplasma-infected plants. The antiserum could also detect phytoplasmas in infected plant sap. These results demonstrate that the gyrase A protein (GyrA) of A. laidlawii shares antigenicity with the GyrA of other Acholeplasma species and also with those of phytoplasmas including some from a few groups with unrelated 16S rRNAs.     

Ultrastructural Study of Salmonella typhimurium Treated with Membrane-Active Agents: Specific Reaction of Dansylchloride with Cell Envelope Components

Amino groups of cell envelope proteins, lipids, and lipopolysaccharides cannot be labeled in intact cells of Salmonella typhimurium G 30 by using 5-dimethylaminonaphthalene-1-sulfonylchloride incorporated in lecithin-cholesterol vesicles. However, application of membrane-interacting agents like tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, ethylenediaminetetraacetate (Na salt) (EDTA), divalent cations, and sublethal doses of the cationic antibacterial agents polymyxin B and chlorhexidine induced specific fluorescent labeling of envelope proteins and lipids but not of cytoplasmic compounds, with the exception of a soluble protein with a molecular weight of 46,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with Tris-hydrochloride buffer produced labeling of the heat-modifiable protein B/B(+) and of proteins with molecular weights of 26,000, 22,000, and below 17,000. A combination of Tris-hydrochloride and EDTA induced additional dansylation of the major protein A and of proteins of molecular weights 80,000, 60,000, and 44,000. Polymyxin B and chlorhexidine caused similar labeling patterns. In every case, except with divalent cation treatment, protein B/B(+) was the most prominently labeled species. Phosphatidylethanolamine was dansylated up to 30%. Lipopolysaccharide was not reactive under any condition or treatment. In addition, the peptidoglycan-bound lipoprotein did not react with dansylchloride in either intact or Tris-hydrochloride-treated cells. The results are discussed with regard to a possible localization of labeled and unlabeled compounds of the cell envelope on the basis of a model placing cell envelope amino groups into ion-ion interactions with anionic components of other envelope compounds like phosphate and carboxyl groups.     

Disposition of exposed antigens on the faces of isolated Mycoplasma gallisepticum membranes.

The transverse disposition of exposed protein antigens on the two faces of isolated Mycoplasma gallisepticum membranes have been investigated by using indirect immunoferritin labeling to accomplish visualization of the antigens at the ultrastructural level. Comparison between the labeling patterns obtained with unabsorbed specific mycoplasma antiserum and antiserum from which antibodies directed against outer side determinants had been removed revealed that the majority of protein antigens were the same on the opposed membrance faces or at least displayed extensive interside cross-reactivity. The relatively scarce tagging of isolated Acholeplasma laidlawii membranes, contrary to membranes on intact organisms observed in this investigation, precluded conclusions regarding the disposition of membrane antigens of this species. The advantages and limitations of the employed method in disposition studies and the factors influencing the transverse distribution of membrane proteins in mycoplasmas are discussed.     

Animal viruses are able to fuse with prokaryotic cells. Fusion between Sendai or influenza virions and Mycoplasma

Sendai and influenza virions are able to fuse with mycoplasmata. Virus-Mycoplasma fusion was demonstrated by the use of fluorescently labeled intact virions and fluorescence dequenching, as well as by electron microscopy. A high degree of fusion was observed upon incubation of both virions with Mycoplasma gallisepticum or Mycoplasma capricolum. Significantly less virus-cell fusion was observed with Acholeplasma laidlawii, whose membrane contains relatively low amounts of cholesterol. The requirement of cholesterol for allowing virus-Mycoplasma fusion was also demonstrated by showing that a low degree of fusion was obtained with M. capricolum, whose cholesterol content was decreased by modifying its growth medium. Fluorescence dequenching was not observed by incubating unfusogenic virions with mycoplasmata. Sendai virions were rendered nonfusogenic by treatment with trypsin, phenylmethylsulfonyl fluoride, or dithiothreitol, whereas influenza virions were made nonfusogenic by treatment with glutaraldehyde, ammonium hydroxide, high temperatures, or incubation at low pH. Practically no fusion was observed using influenza virions bearing uncleaved hemagglutinin. Trypsinization of influenza virions bearing uncleaved hemagglutinin greatly stimulated their ability to fuse with Mycoplasma cells. Similarly to intact virus particles, also reconstituted virus envelopes, bearing the two viral glycoproteins, fused with M. capricolum. However, membrane vesicles, bearing only the viral binding (HN) or fusion (F) glycoproteins, failed to fuse with mycoplasmata. Fusion between animal enveloped virions and prokaryotic cells was thus demonstrated.     

New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy.

The structure of the flagellar base in Salmonella typhimurium has been studied by rapid-freeze techniques. Freeze-substituted thin sections and freeze-etched replicas of cell envelope preparations have provided complementary information about the flagellar base. The flagellar base has a bell-shaped extension reaching as far as 50 nm into the bacterial cytoplasm. This structure can be recognized in intact bacteria but was studied in detail in cell envelopes, where some flagella lacking parts of the bell were helpful in understanding its substructure. Structural relationships may be inferred between this cytoplasmic component of the flagellum and the recently described flagellar intramembrane particle rings as well as the structures associated with the basal body in isolated, chemically fixed flagella.     

Heat shock response in mycoplasmas, genome-limited organisms.

We have measured the effect of heat shock on three mycoplasmas (Acholeplasma laidlawii K2 and JA1 and Mycoplasma capricolum Kid) and demonstrated the induction of mycoplasma heat shock proteins under these conditions. Increased synthesis of at least 5 heat shock proteins in A. laidlawii K2, 11 heat shock proteins in A. laidlawii JA1, and 7 heat shock proteins in M. capricolum was observed by electrophoretic analysis of proteins from heat-shocked cells in sodium dodecyl sulfate-polyacrylamide gels. In all three strains, major heat shock proteins (66 to 68 and 26 to 29 kilodaltons [kDa]) were found. The 66- to 68-kDa protein cross-reacted with antibody to Escherichia coli DnaK protein, suggesting that this heat shock protein has been conserved in spite of major reductions in genetic complexity during mycoplasma evolution. A. laidlawii also contained a 60-kDa protein that cross-reacted with eubacterial GroEL protein and a 40-kDa protein that cross-reacted with E. coli RecA protein. Unlike with coliphages, the mycoplasma virus L2 progeny yield was not increased when virus was plated on heat-shocked A. laidlawii host cells. However, UV-irradiated L2 virus could be host cell reactivated by both A. laidlawii SOS repair and heat shock systems.     

Peptidoglycan hydrolases in an autolytic mutant of Salmonella typhimurium

Two peptidoglycan hydrolases were isolated from the autolytic mutant Salmonella typhimurium DA361 (envD). One of them, resistant to penicillin, was found free in the supernatant of partially purified envelopes sedimented by ultracentrifugation, and the other bound to the envelopes proved to be sensitive to the antibiotic. Both were able to hydrolyse in vitro high molecular weight non-specific peptidoglycan isolated from E. coli W7 labelled with [14C]diaminopimelic acid. Similar enzymatic activities were separated also from S. typhimurium DA362 (envD+) a non-lytic isogenic pair of the above and from the wild type strain LT-2. All of the hydrolytic activities reported here were strongly inhibited when DNA was added to the assay systems. The peptidoglycan hydrolases isolated from the autolytic mutant suffered a competitive inhibition while those from the non-lytic strains were apparently inhibited in uncompetitive modal relationship. It is postulated that the inhibitory effect may bear affinity with the preservation of DNA sites of attachment to cell membranes sustaining peptidoglycan structure and functions.     

Isolation of Acholeplasmatales from rabbit faeces

Acholeplasma laidlawii was isolated from the faeces of 23.5% and 24% of groups of 51 conventional and 45 specified-pathogen-free (SPF) rabbits respectively. Isolation of the organism from individual animals could often be repeated, suggesting that infection was not merely transient. Two further acholeplasmas were isolated from two SPF rabbits. One was serologically related to Acholeplasma modicum. The other could not be identified and may be a new species.     

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.     

Alternative to fluorescence assays to monitor fusion between Acholeplasma laidlawii cells and liposomes

AIMS: To develop a new technique as an alternative to the fluorescence assays and electron microscopy for the purpose of monitoring the cell-liposome fusion. METHODS AND RESULTS: Acholeplasma laidlawii whole cells did not oxidize Glucose-6-phosphate (G6P) or Fructose-1,6 diphosphate (F1,6DP) as free (unentrapped) substrates, at concentrations 47 and >270 mM, respectively. Lysed A. laidlawii cells oxidized G6P and F1,6DP at lower concentration of 0.8 and 15 mM, respectively. When these substrates were entrapped inside liposomes, at a final concentration of 1.5 mM, and interacted with A. laidlawii whole cells, in an oxygen electrode chamber, an increase in oxygen uptake was evident. This interaction does not have any effect on cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental system described here is advantageous over classical fluorescence assays in determining the fate of liposome-entrapped material and raises the possibility of studying the kinetics of metabolic substrates, which are normally excluded from the cell by the cell membrane.     

Comparative studies to determine the efficiency of 6 methylpurine deoxyriboside to detect cell culture mycoplasmas

Summary Studies were performed to compare three methods to detect mycoplasmal infection of cell cultures. The methods included microbiological assay by inoculation into broth and onto agar with anaerobic incubation, fluorescent DNA staining by Hoechst 33258, and mycoplasmal mediated cytotoxicity by 6 methylpurine deoxyriboside (6MPDR). Fluorescent DNA staining and 6MPDR assays were performed in an indicator cell culture system. A total of 2589 cell cultures were assayed. Mycoplasmas were detected in 174, an incidence of 6.7%. Species isolated were:Acholeplasma laidlawii, Mycoplasma orale, M. arginini, M. hyorhinis, M. fermentans, M. pirum, and M. pneumoniae. In separate studies, 6MPDR also detected infection withSpiroplasma mirum when this organisms was deliberately inoculated into cell cultures. The efficiencies of microbiological testing, fluorescent DNA assays, and 6MPDR were 43.1, 98. 8, and 97.1%, respectively. The work was supported by grant AI-15748 from the National Institutes of Health, Bethesda, MD     

Infrared spectroscopic studies of Acholeplasma laidlawaii B membranes. Comparison of the gel to liquid-crystal phase transition in intact cells and isolated membranes

The physical state of the membrane lipids in the plasma membranes of intact, live Acholeplasma laidlawii B cells was probed by Fourier-transform infrared spectroscopy and compared with that in isolated membranes. Infrared spectra of live A. laidlawii B cells, enriched biosynthetically in the presence of avidin, with saturated deuterated and unsaturated non-deuterated fatty acids have been recorded at a variety of temperatures. The results indicate that within the temperature range of the gel to liquid-crystal phase transition, the live cells are able to keep the 'fluidity' of their plasma membranes at a considerably higher value compared to that in the isolated membranes at the same temperature. While this is a generally valid observation, the degree by which live and isolated membranes differ in their liquid-crystal-phase content at a given temperature depends on the nature of the exogenous fatty acid and the temperature of growth.