Analysis of the Life Cycle of Mycoplasma Pneumoniae by Synchronized Division and by Ultraviolet and X Irradiations

Furness, G.; Pipes, Florence J.; McMurtrey, Marion J.

doi:10.1093/infdis/118.1.7

Cited by 35 publications

(15 citation statements)

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“…The results of such observations on M. pneumoniae as described here show binary fission (or division) to be the nor mal mode of multiplication. Furthermore they indicate a replica tion time of about 3 h, a time much shorter than the previously calculated It) h [12] or 6.5 h [6]. Media other than those used here might result in still shorter periods.…”

Section: Discussionmentioning

confidence: 38%

“…Media other than those used here might result in still shorter periods. The differences between these microscopic results and those obtained by colony counting could be due to the formation of 'spherules' [9], 'clusters' [6] or 'colonies' ]2] in the broth. These structures are preventing an exact evalua tion by colony counting of the true number of single organisms in broth.…”

Section: Discussionmentioning

confidence: 99%

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Motility and Multiplication of Mycoplasma pneumoniae

Bredt¹

1968

Pathobiology

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Section: Discussionmentioning

confidence: 38%

Section: Discussionmentioning

confidence: 99%

Motility and Multiplication of Mycoplasma pneumoniae

Bredt¹

1968

Pathobiology

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“…Eubacteria have been synchronized by a variety of methods, including amino acid starvation (8), gradient sedimentation (2, 10), and membrane (Millipore) adsorption and elution (5). It has been reported that some Mycoplasma species, including M. gallisepticum A5969, can be synchronized by starving them of serum components (1,4,6); however, this procedure has not yielded positive results in this laboratory.Therefore, we investigated another method of synchronizing M. gallisepticum A5969 based on a previous observation that, when held in Eagle minimal essential medium (MEM), M. gallisepticum cells are unable to replicate and retain their viability for 6 h, while DNA synthesis continues for 60 min and then stops (12). This suggests that in MEM each cell may be arrested at the same point in its life cycle and hence, if transferred back into fresh mycoplasma tryptose medium, the culture may grow synchronously.…”

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confidence: 81%

Deoxyribonucleic Acid Synthesis in Synchronously Growing Mycoplasma gallisepticum

Quinlan

Maniloff

1973

J Bacteriol

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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.The deoxyribonucleic acid (DNA) replication complex in Mycoplasma gallisepticum A5969 has been shown to be membrane associated (11). In order to continue these studies and to examine DNA synthesis during the cell life cycle, it was necessary to obtain synchronously growing cultures. Eubacteria have been synchronized by a variety of methods, including amino acid starvation (8), gradient sedimentation (2, 10), and membrane (Millipore) adsorption and elution (5). It has been reported that some Mycoplasma species, including M. gallisepticum A5969, can be synchronized by starving them of serum components (1,4,6); however, this procedure has not yielded positive results in this laboratory.Therefore, we investigated another method of synchronizing M. gallisepticum A5969 based on a previous observation that, when held in Eagle minimal essential medium (MEM), M. gallisepticum cells are unable to replicate and retain their viability for 6 h, while DNA synthesis continues for 60 min and then stops (12). This suggests that in MEM each cell may be arrested at the same point in its life cycle and hence, if transferred back into fresh mycoplasma tryptose medium, the culture may grow synchronously. This paper reports studies of this method of synchronization and its utilization to examine DNA synthesis and the attachment of DNA to the membrane-associated replication complex during the cell life cycle. I Visting Scientist, John Innes Institute, Colney Lane, Norwich, England. MATERIALS AND METHODSOrganism and medium. M. gallisepticum A5969 was grown in tryptose broth (containing 1% glucose and 1% PPLO serum fraction) as previously described (9). Tryptose broth, with 1% agar (Difco) added was used for agar plates. MEM was made as described by Quinlan et al. (12).Protocol for synchronizing cells. A sample of early log-phase cells (12 to 13 h) was diluted 100-fold with tryptose medium and then 100-fold with MEM. The cells were held in MEM at 37 C for 90 to 120 min. The cells were then diluted 10-fold into fresh tryptose medium and incubated at 37 C. At 20-to 30-min intervals, samples (0.05 ml) were plated (in triplicate) on tryptose-agar. Control cells were handled in the same way except that tryptose medium was substituted for MEM in the synchronization protocol. Plating, colony staining, and counting of colony-forming units (CFU) have been described previously (12).Radioactive label and scintillation counting. [methyl-3H]thymidine (18 Ci/mM) was obtained from New England Nuclear Corp. (Boston, Mass.). 'H-DNA was assayed as described by Quinlan et al. (12).Harvesting and washing of cells. Log-phase cells were harvested by centrifugation and washed, as described previously (11).DNA sy...

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“…elementary units) of Mycoplasmas. It has been reported that these particles are 100-150 in// for M. gallisepticum [1] and about 150 m/< for M. pneumoniae [2], Grace et al [3] have reported the interesting observation that, after filtration through 100 m// millipore membranes, M. mergenhagen (i.e. M. pulmonis) suspensions were no longer able to grow in acellular media hut only in cellular cultures ('a stage of obliged intracellular parasitism?…”

Section: Introductionmentioning

confidence: 99%