Jack Maniloff

Phylogeny and Evolution

Beginning in the late 1970s, results from a variety of studies enabled an increasingly detailed Mollicutes phylogenetic tree to be reconstructed4. The overall tree is based on 16S rRNA sequence analyses and provides a framework for results on other relationships between Mollicutes strains and between Mollicutes and Gram-positive bacteria; e.g., other macromolecule sequences, lipid composition, metabolic enzymes and pathways, gene organization, and antibiotic sensitivity.

Viruses of mycoplasmas and spiroplasmas.

Publisher Summary The term mycoplasma refers to a group of microorganisms (Class Mollicutes, Order Mycoplasmatales), previously known as pleuropneumonia-like organisms or PPLO. The many different isolates have been cataloged into the genera mycoplasma, acholeplasma, ureaplasma or T-strains, spiroplasma, thermoplasmn, and anaeroplasma. Mycoplasmas are clinically important microorganisms, and include the etiologic agents of a variety of plant and animal diseases. In human studies, mycoplasma pneumoniae has been found to be a pathogen and other mycoplasmas (of unknown etiology) have been isolated in clinical situations. Mycoplasmas are also frequently found as contaminants in tissue cultures. The biology of the mycoplasmas has been reviewed recently and some of their properties are summarized in this chapter. These prokaryotes do not have cell walls and are bounded by a single lipoprotein cell membrane. Mycoplasma, ureaplasma, spiroplasma, and anaeroplasma require sterol for growth. Some mycoplasma isolates are the smallest cells that have been found, and the genome sizes of the mycoplasma and ureaplasma are among the smallest reported.

Isolation of Mycoplasmatales viruses and characterization of MVL1, MVL52, and MVG51.

Eleven Mycoplasmatales viruses are now known. Burst size, burst time, sensitivity to ultraviolet and the host range of three viruses that were studied are different. However, all three are naked, rod-shaped particles of similar size. Plaque morphology and the isolation of immune cells suggest that both virulent and nonvirulent infections are possible.

Infection of Acholeplasma laidlawii by MVL51 virus

The infection of Acholeplasma laidlawii cells by MVL51 virus has been studied, MVL51 is a bullet-shaped virus, about 14 nm wide and 71 nm long. At multiplicities of infection less than 10, virus infected cells continue to grow during virus production, but the cell doubling time is 160 min, rather than the normal 110 min. Progeny virus assembly and maturation occur at the cell membrane as the viral particles are extruded. This model is suggested by the data of one-step growth curves, premature lysis experiments, single burst size measurements, and electron microscopy of infected cells.

Sequence analysis of a unique temperate phage: mycoplasma virus L2

Mycoplasma virus L2 is a quasi-spherical enveloped virion containing circular double-stranded DNA. L2 infection of Acholeplasma laidlawii host cells leads to a noncytocidal productive infection cycle followed by establishment of lysogeny in all (or most) infected cells, with viral DNA integrated into the host cell genome. The L2 genome has been sequenced and analyzed. L2 DNA is 11,965-bp long and contains 15 open reading frames (ORFs). One of these, ORF13*, has its start codon within and in the same reading frame as ORF13. The ORFs are clustered in four groups separated by noncoding intergenic regions, suggesting that gene expression involves transcription of genes in a cluster into polycistronic mRNA and translation of these genes via translational coupling or reinitiation. Fifteen L2 start codon sites have been defined and resemble those of eubacteria. The N-terminal sequences of two ORFs appear to be signal peptides, and the gene product of one of these may be an L2 virion integral membrane protein. The ORF 5 product has been tentatively identified as an integrase, based on its sequence similarity to site-specific recombinases. The putative attP integration site has been mapped to an intergenic region, 280-bp downstream from ORF 5. Two putative DNA replication ori sites have been mapped. Each is in an intergenic region and contains a DnaA-box bounded by A + T-rich 6-mer repeats.

Characterization of Mycoplasmatales virus DNA

Abstract The DNA of the group L1 Mycoplasmatales virus, MVL51, was analyzed using alkaline sucrose velocity sedimentation, neutral and alkaline CsCl isopycnic sedimentation, and treatment of the DNA with nucleases. These treatments show that the viral chromosome is a covalently linked single-stranded DNA circle of molecular weight 2×106 daltons.

Optical analysis of electron micrographs of cytochrome oxidase membranes

Abstract Optical analysis of electron micrographs of membranous cytochrome oxidase shows that the membrane lattice has the symmetry of the ppg two-dimensional space group. The unit cell size is 87A by 115 A. After optical filtering, some details of the particle structure can be seen.

THE MOLECULAR BIOLOGY OF MYCOPLASMA VIRUSES

Since the isolation of the first mycoplasma virus by Gourlay,’ three years ago, more than fifty virus isolates have been reported.2-4 These form two serologically and morphologically distinct groups: ( 1 ) L1-type, consisting of unenveloped rods, and (2) L2-type, consisting of enveloped viruses of unknown capsid morphology. Both types of virus contain DNA. Little has been reported about L2-type viruses. In this paper, we consider the biology and infective process of an L1-type Mycoplasmatales virus (MV), MVLS1. The isolation of this virus has been previously d e s ~ r i b e d . ~ , ~

Replication of mycoplasmavirus MVL51. I. Replicative intermediates

The intracellular replication of the single stranded DNA of the non-lytic bullet-shaped Group L1 mycoplasmavirus, MVL51, has been shown to involve three virus specific DNAs: RFI, RFII and SS. The relative sedimentation rates and ethidium bromide CsCl gradient analysis show that RFI is covalently closed circular double stranded DNA and RFII is a nicked form of RFI. SS is circular single stranded progeny viral DNA. RFI and RFII serve as precursors for the synthesis of progeny SS.

BIOSYNTHESIS AND SUBCELLULAR ORGANIZATION OF NUCLEIC ACIDS IN MYCOPLASMA GALLISEPTICUM A5969

The “replicon” model of Jacob and associatesg hypothesized that in eubacteria, deoxyribonucleic acid (DNA) was attached to the cell membrane. Membrane synthesis, together with replication of the chromosome, would allow for segregation of the DNA during cell division. This segregation would ensure that both daughter cells received a full complement of the genetic material. Subsequently, Goldstein and Brown,6 using Escherichia coli, presented evidence that DNA synthesis itself was a membrane-associated phenomenon, and other data has accumulated indicating that membrane-bound DNA synthesis seems to be a general occurrence, being found in procaryotes,1° for bacteriophage replication,lo and in eucaryotes.15 The correlation between cell division and DNA synthesis in eubacteria has been most extensively studied by Helmstetter and colleagues7 and by Clark.’ Using E . coli, these investigators found that synchronized cells (with generation times exceeding 60 minutes) exhibited a period of active DNA synthesis (the C-period), which commences as the cell is separating from its daughter cell; this was followed by a “gap” where there was no net DNA synthesis (D-period) prior to the cell-separation phase of the next division cycle. DNA synthesis resumed as the cells began separating again. This is analogous to the cell cycle in mammalian system^,^^^^^ where S and G2 correspond to the Cand D-periods, respectively, in E. coli. Relative to studying the membrane-bound DNA growing-point complex in eubacteria, technical problems arise because of the necessity of removing the cell wall prior to cell lysis and fractionation. Another problem involves the use of detergents to lyse cells; this can affect the interpretation of any subsequent chemical analyses on a membrane fraction. Mycoplasma gallisepticum A5969 provides a system for studying the replication of DNA and its relationship to other cellular processes without the above technical problems. There is no cell wall to remove and no intracellular membrane system. In addition, there is a polar bleb ~ t r u c t u r e ~ ~ J 4 that can serve as a morphological marker. Munkres and Wachtel16 have shown that adenosine triphosphatase ( ATPase) activity resides along the inner surface of the bleb-infrableb membrane; rhus, the bleb can serve, morphologically and biochemically, to distinguish one area of the membrane from another. The life cycle of M . gallisepticum A5969 has been shownl4 to begin with a pear-shaped daughter cell with a terminal bleb-infrableb structure. These structures are replicated and form opposite poles of the cell at division, which takes