Membrane Association of the Deoxyribonucleic Acid Growing-Point Region in Mycoplasma gallisepticum

Newly replicated deoxyribonucleic acid (DNA) in Mycoplasma gallisepticum A5969 is membrane associated. Cells pulse-labeled 1 to 3 min with (3)H-thymidine are lysed by a freeze-thaw procedure. After brief sonic treatment to shear the DNA, differential centrifugation gives a cell fraction (P2) that is enriched sevenfold for pulse-labeled DNA. P2 contains 80% of the total adenosine triphosphatase activity, 65% of the total cholesterol, and morphologically intact terminal bleb structures. Three to four minutes are needed to fully label the DNA growing-point region, whereas 7 to 8 min are required to "chase" 50% of the (3)H-labeled DNA. This pulse-chase removes 80 to 85% of the nascent DNA from the P2 fraction.     

Transposon mutagenesis of Mycoplasma gallisepticum

There are few systems available for studying the genetics of the important avian respiratory pathogen, Mycoplasma gallisepticum. These techniques are needed to develop a mechanism to study the molecular pathogenesis of M. gallisepticum. Tn916 has the ability to transpose into the M. gallisepticum genome by both transformation and conjugation. In this study, PEG-mediated transformation was employed for the transfer of Tn916 into M. gallisepticum and create a transposon mutant library. Transformants were obtained at a frequency of approximately 5 x 10(-8) per recipient CFU. A total of 424 MG/Tn916 mutants were constructed and sequence data from the transposon junctions of 71 mutants was obtained and used to identify transposon insertion sites. Insertions were found throughout the genome in nearly all of the major gene categories, making this the first extensive characterization of a transposon mutant library of M. gallisepticum. Transposon stability was also examined, and it was determined that for two mutants the element was stably maintained in vivo in the absence of selective pressure.     

Sugar transport in Mycoplasma gallisepticum

Mycoplasma gallisepticum cells were found to contain two different sugar transport systems, one for d-glucose and alpha-methyl-d-glucoside (alpha-MG) and the other for d-mannose and d-fructose. Both systems were noninducible, stereospecific, dependent on temperature and pH, and sensitive to sulfhydryl-blocking reagents. The rate of sugar uptake depended on its external concentration, obeying Michaelis-Menten kinetics. The sugar accumulated in the cells against a concentration gradient, and an energy requirement for accumulation was demonstrated with alpha-MG. Both transport systems thus meet the criteria of active transport. The exit of alpha-MG from the cells, like its entry, depended on temperature and was accelerated by energy supplied by the oxidizable d-mannose. d-Glucose accelerated alpha-MG exit, apparently by an exchange reaction. A method for measuring the intercellular space and intracellular free-water volume of Mycoplasma was devised, and several of its applications are described.     

Physical map of Mycoplasma gallisepticum.

Physical chromosomal maps of two Mycoplasma gallisepticum strains, R and ATCC 19610, were constructed by using field inversion gel electrophoresis. To assist in the ordering of chromosomal fragments and the construction of the chromosomal maps, the gram-positive transposon Tn4001 was modified to serve as a mobile restriction site. The total sizes of the M. gallisepticum R and ATCC 19610 genomes were estimated to be 1,037 and 998 kb, respectively. The restriction enzyme locations for EagI and SmaI were determined along with several transposon insertion sites. The two strain maps were similar except for three small deletions and one additional EagI site in strain ATCC 19610.     

Mycoplasma gallisepticum 16S rRNA genes

Abstract The genome of Mycoplasma gallisepticum A5969 contains a truncated pseudogene for 16S rRNA in addition to a single unsplit rRNA-operon and a second discontinuous set of rRNA genes. Other M. gallisepticum strains tested do not posses the truncated gene. This gene is almost identical to full-size isolated 16S rRNA gene starting from at least 500 nucleotides upstream of the coding sequence and ending at the 977th nucleotide within the structural part of 16S rRNA.     

Histochemical Localization of Phosphatases in Mycoplasma gallisepticum

Histochemical studies of adenosine triphosphatase and acid phosphatase activity were performed on Mycoplasma gallisepticum. The adenosine triphosphatase activity appears to be localized in the bleb and infrableb regions exclusively and is associated with the cell membrane; acid phosphatase activity is localized in the infrableb region and does not appear to be membrane-associated. These findings are consistent with data from biochemical studies of Mycoplasma cell fractions but, unlike them, reveal that adenosine triphosphatase activity is restricted to a particular part of the cell membrane.     

Adherence of Mycoplasma gallisepticum to glass.

Attachment of washed Mycoplasma gallisepticum cells to glass was quantified with organisms in which membrane lipids were labelled with 3H. Siliconization of the test tubes decreased attachment, while centrifugation increased it. Attachment increased with temperature, decreased with increasing pH and ionic strength of the attachment mixture, but was unaffected by Ca2+, Mg2+ and EDTA. This suggests that ionic bonds, but not salt bridges, participate in the attachment process. Glycophorin, the major receptor responsible for M. gallisepticum attachment to erythrocytes, partially inhibited the attachment of the organisms to glass. However, bovine serum albumin also decreased attachment. Extensive pretreatment of the organisms with trypsin decreased their ability to attach to glass by about 35 to 40%. Trypsin and pronase failed to detach the organisms already bound to glass, suggesting that external mycoplasma cell components, other than membrane proteins, also participate in attachment of the organisms to glass.     

Ultrastructure and Ribosomes of Mycoplasma gallisepticum.

Maniloff, Jack (Yale University, New Haven, Conn.), Harold J. Morowitz, and Russell J. Barrnett. Ultrastructure and ribosomes of Mycoplasma gallisepticum. J. Bacteriol. 90:193-204. 1965.-The ultrastructure of Mycoplasma gallisepticum strain A5969 has been studied by electron microscopy (thin-section and negative staining), ultracentrifugation, and chemical analysis. The list of ultrastructure is: membrane, nuclear material, ribosomes, ribosomal structures, infra-bleb region, and blebs. The nuclear material, containing the cell's deoxyribonucleic acid, appears as an unbounded region containing 30-A fibrils. The ribosomes have a diameter of about 140 A, a ribonucleic acid-protein ratio of 0.68, and an uncorrected sedimentation coefficient of 70.2S. The 70.2S particle can be broken into 49.3S and 32.4S particles. Ribosomal arrays were found filling the intracytoplasmic space between the nuclear material and the membrane. Under certain conditions, these arrays formed cylindrical arrangements of ribosomes. The infra-bleb region is composed of a granular material, although little internal structure could be found. The bleb was highly structured.     

Analysis of the life cycle of Mycoplasma gallisepticum

Morowitz, Harold J. (Yale University, New Haven, Conn.), and Jack Maniloff. Analysis of the life cycle of Mycoplasma gallisepticum. J. Bacteriol. 91:1638-1644. 1966.-A series of electron microscope observations on Mycoplasma gallisepticum strain A5969 have been made by use of thin-section techniques and negative staining. The methods presented a consistent picture of a postdivision cell, which contains a fibrillar nuclear region, surrounding ribosomal region, highly organized bleb at one end of the cell, granular infrableb region, and bounding unit membrane. Cell division commenced with the appearance of a second infrableb area at the end of the cell opposite the original bleb. A new bleb grew in this area, and the cell then elongated. The nuclear material segregated into two parts separated by a band of ribosomes. A constriction appeared, in this central ribosome-packed area, leading to the formation of two daughter cells. The following was noted: the cells were very small (volume, 5 x 10(-14) cm(3)); each cell was highly structured and strongly ordered; and the replication appeared to be a very precisely programmed series of events.     

Intraspecies genotypic heterogeneity among Mycoplasma gallisepticum strains

The DNA cleavage patterns and protein profiles of six Mycoplasma gallisepticum strains from various parts of the world were compared. Obvious differences among the strains were obtained by DNA restriction analysis. Reflection of genotypic variations in the polypeptide patterns was less pronounced; slight differences in the protein profiles of the strains were found. The data presented here indicate that some intraspecies polymorphism exists among M. gallisepticum strains.     

Cell volume regulation in Mycoplasma gallisepticum.

Mycoplasma gallisepticum cells incubated in 250 mM NaCl solutions in the absence of glucose showed a progressive fall in intracellular ATP concentration over a period of 2 to 3 h. When the ATP level fell below 40 microM the cell began to swell and become progressively permeable to [14C]inulin and leak intracellular protein and nucleotides. The addition of nondiffusable substances such as MgSO4 or disaccharides prevented swelling, suggesting that NaCl (and water) entry was due to Gibbs-Donnan forces. The addition of glucose after the initiation of cell swelling increased intracellular ATP, induced cell shrinkage, and prevented the release of intracellular components. The ATPase inhibitor dicyclohexylcarbodiimide, which collapsed the chemical and electrical components of the proton motive force, caused rapid cell swelling in the presence of glucose (and high intracellular ATP levels). Extracellular impermeable solutes such as MgSO4 and disaccharides prevented swelling of dicyclohexylcarbodiimide-treated cells incubated in NaCl. It was postulated that Na+ that diffused into the cell was extruded by an electrogenic Na+-H+ exchange (antiport) energized by the proton motive force established by the dicyclohexylcarbodiimide-sensitive H+-ATPase.     

Experimental infection of house finches with Mycoplasma gallisepticum

Mycoplasma gallisepticum (MG) has caused an endemic upper respiratory and ocular infection in the eastern house finch (Carpodacus mexicanus) after the epidemic first described in 1994. The disease has been studied by a number of investigators at a population level and reports describe experimental infection in group-housed MG-free house finches. Because detailed observation and evaluation of individual birds in group-housed passerines is problematic, we studied individually housed house finches that were experimentally inoculated with the finch strain of MG in a controlled environment. To accomplish this, a study was conducted spanning the period of November 2001-April 2002 with 20 MG-free (confirmed by the rapid plate agglutination assay and polymerase chain reaction [PCR] assay) eastern house finches captured in the Cayuga Basin area of central New York (USA) in the summer of 2001. After a period of acclimatization and observation (12 wk), 20 finches were inoculated with a 0.05-ml aliquot of MG (3.24 x 10(5) colony-forming units/ml) via bilateral conjunctival sac instillations. Two additional finches acted as controls and were inoculated in the same manner with preservative-free sterile saline solution. After inoculation, all finches except the controls exhibited clinical signs of conjunctivitis within 2-6 days. The progression of the disease was evaluated by several methods, including PCR, behavioral observations, and physical examination including eye scoring, body weight, and body condition index. Over a period of 21 wk, MG-infected finches developed signs of disease and recovered (80%), developed signs of disease and progressed to become chronically infected (15%), or died (5%). We hypothesize that the high survival rate and recovery of these finches after infection was associated with the use of controlled environmental conditions, acclimatization, a high plane of nutrition, and low stocking (housing) density, all of which are factors documented to be important in the outcome of MG infections in domestic poultry and other species.     

Intraspecies genotypic heterogeneity among Mycoplasma gallisepticum strains.

The DNA cleavage patterns and protein profiles of six Mycoplasma gallisepticum strains from various parts of the world were compared. Obvious differences among the strains were obtained by DNA restriction analysis. Reflection of genotypic variations in the polypeptide patterns was less pronounced; slight differences in the protein profiles of the strains were found. The data presented here indicate that some intraspecies polymorphism exists among M. gallisepticum strains.     

Deoxyribonucleic Acid Synthesis in Synchronously Growing Mycoplasma gallisepticum

Early log-phase cells of Mycoplasma gallisepticum A5969 were synchronized by holding in Eagle minimal essential medium (MEM) for 2 h. When transferred out of MEM into tryptose medium, the cells exhibited synchronous growth. Deoxyribonucleic acid (DNA) synthesis proceeded continuously during this growth but stopped during the period of cell division. One round of DNA replication was observed per cell doubling, and a unique region of DNA was found to be permanently bound to the membrane.