Mycoplasma melaleucae sp. nov., a sterol-requiring mollicute from flowers of several tropical plants

Three sterol-requiring mollicutes from floral surfaces of two tropical plant species (Melaleuca quinquenervia and Melaleuca decora) and a single isolate from a flower of the silk oak (Grevillea robusta) were serologically indistinguishable. Strain M1T (T = type strain), isolated from Melaleuca quinquenervia, was chosen for characterization. Light and electron microscopic observations of strain M1T revealed nonhelical, nonmotile, pleomorphic coccoid cells surrounded by a single cytoplasmic membrane. No evidence of a cell wall was observed. The organism grew well in SP-4 medium, but no sustained growth occurred in conventional mycoplasma media containing horse serum. The optimum temperature for growth was 23 degrees C, but multiplication occurred over a temperature range of 10 to 30 degrees C. Growth was not observed at temperatures above 30 degrees C. Strain M1T and related strains (strains M5, M10, and SO1) catabolized glucose but hydrolyzed neither arginine nor urea. The size of the strain M1T genome was about 561 megadaltons, while the guanine-plus-cytosine content of the DNA was about 27.0 mol%. The organism was serologically unrelated to the type strains of the 80 previously recognized Mycoplasma species or to 18 other unclassified sterol-requiring strains cultivated from animal, plant, or insect sources. Recent sequencing studies of 16S rRNA demonstrated that strain M1T is a member of a clade that contains the type species of the genus Mycoplasma. Strain M1 (= ATCC 49191) is the type strain of Mycoplasma melaleucae sp. nov.     

The spiroplasma deformation test,a new serological method

The deformation test is a simple and highly sensitive technique capable of demonstrating significant antigenic differences among helical, wall-less prokaryotes (spiroplasmas). Specific identified. Quantitative relationships among various antisera are determined by examining, under dark-field microscopy, samples containing serum dilutions and a measured number of organisms which is held constant in each test. Antisera dilutions of 1:2,000 to 1;16,000 deformed spiroplasmas in homologous tests involvingSpiroplasma citri and the corn stunt and suckling mouse cataract spiroplasmas. With the exception of some heterologous cross-reactions in the deformation test betweenS. citri and corn stunt spiroplasmas, antisera and preimmunization sera failed to deform heterologous spiroplasmas at dilutions higher than 1:16.     

Cultivation,serology, ultrastructure,and virus-like particles of spiroplasma 277F

Spiroplasma 277F, a helical, motile mycoplasma from rabbit ticks in Montana, was cloned and cultivated in liquid and solidified spiroplasma or mycoplasma media. Serum was required, glucose was fermented, and digitonin inhibited growth. Colonies of spiroplasma 277F possessed granular centers and were surrounded by smaller, subsurface “satellite” colonies. Cloned agent 277F was antigenically distinct from the suckling mouse cataract agent, the corn stunt organism, andSpiroplasma citri by growth inhibition and deformation tests, but exhibited weak cross-reactivity withS. citri in the precipitin ring tests. Although current passage levels did not cause cataracts in neonatal rats, kill embryonated hen's eggs, or cause bovine mastitis, definitive tests must await the availability of fresh isolates. Ultrastructurally, 277F closely resembledS. citri but displayed a system of 3-nm threadlike filaments in the exterior layer of its membranous covering. Two phagelike entities, similar to viruses associated withS. citri, were present.     

Revised serological classification of spiroplasmas,new provisional groups,and recommendations for serotyping of isolates

Five new spiroplasma strains were analyzed in reciprocal growth inhibition, metabolism inhibition (MI), and deformation (DF) serological tests. New provisional groups from the waspMonobia quadridens (VII) and the syrphid flyEristalis arbustorum (VIII) were added to the existing classification. Three serovars—represented by the LB-12 green leaf bug spiroplassignificantly with subgroup I-4 and with each other. These new groups and serovars bring the number of recognized spiroplasma serovars to 16. Serological distinctiveness of a sixth group (VI) fromIxodes ticks was confirmed. Simple DF and/or MI procedures are described for typing new isolates.     

Spiralin: Major membrane protein specific for subgroup I-1 Spiroplasmas

Preparations of spiralin from membranes ofSpiroplasma citri, strain C189, purified by sequential solubilization with detergents followed by agarose-suspension electrophoresis induced rabbit antibodies that were largely specific forSpiroplasma citri Group I-1 spiroplasmas, as demonstrated by metabolic inhibition (MI), growth inhibition (GI), and deformation (DF) tests. By contrast, antibodies againstS. citri whole-membrane protein preparations reacted broadly with representative type cultures of seven subgroups of theS. citri complex. Neither antimembrane nor antispiralin sera reacted withS. floricola, S. mirum, or Group IV, (VI), (VII), or (VIII) spiroplasmas. Minor cross-reactions in MI and DF tests between antispiralin serum and Subgroup I-2 and I-3 antigens may have represented shared epitopes in a set of homologous membrane proteins of the three spiroplasmas, or antibodies against highly antigenic traces of other common membrane proteins in the purified spiralin preparations. The unique antigenic properties of spiralin, the most abundant protein in theS. citri membrane, explain in part the unique profiles shown by this spiroplasma species in comparative taxonomic serological tests.