Characterization of group H streptococcal temperate bacteriophage phi 227

Abstract

¢227, a temperate phage from a group H streptococcus (Streptococcus sanguis), was propagated vegetatively in group H strain Wicky 4-EryR, and its characteristics were determined. A procedure dependent on multiplicity of infection, incubation time, and treatment of crude lysates with diatomaceous earth was found to optimize phage yield, resulting in titers of 1 x 1010 to 2 x 1010 PFU/ ml. Without prior treatment with diatomaceous earth, subsequent purification procedures (methanol, ammonium sulfate, polyethylene glycol) gave recoveries of less than 1% of crude lysate titers. Adsorption of k227 to host cells was relatively unaffected by the medium, but calcium (not substituted by magnesium) was required for formation of infectious centers. The phage receptor was present on purified cell walls, resisted trypsin and heat, and was removed by hydrochloric acid, trichloroacetic acid, and hot formamide: however, formamideextracted material failed to inactivate phage, and the nature of the receptor is unknown. Single-step growth experiments showed a latent period of 39 min and a burst size of 100 PFU/infectious center; results were unaffected by omission of supplemental Ca2+, by supplementation with Mg2, addition of glucose, or changes of pH between 6.35 and 8.0; but increased temperature (40 to 430C) shortened the latent period and decreased the burst size. The latent period was prolonged in genetically competent host cells and in chemically defined medium; and in the latter, the burst size was smaller. Phage replication was sensitive to those metabolic inhibitors which inhibited the host streptococcus: these included rifampin, fluorodeoxyuridine, hydroxyurea, dihydrostreptomycin, and 6-P-hydroxyphenylazouracil. The data suggest that 0227 does not code for a rifampinresistant RNA polymerase. However, in a rifampin-resistant host strain, phage replication and lysogen formation were both decreased, suggesting that altered host core polymerase had less affinity for (some) promoters on the k227 template. In transfection, a Ca2+-dependent stabilization step that was inhibited by Mg2+ was demonstrated; transformation was not affected by either Ca2+ or Mg2+, and the site and nature of the stabilization are unknown. More than one molecule of DNA was required for plaque formation. Biophysical characterization showed a type B phage of buoyant density (CsCl) 1.50, containing five proteins and 54.8% DNA. The duplex linear DNA had a molecular weight (calculated from contour length) of 23.2 x 106 and a guanine plus cytosine content (calculated from melting point) of 42.3 mol%. Similar characterizations of streptococcal phages, including biophysical data, have not been previously available. Streptococci designated as "group H" (17) in-(69), as well as naturally occurring lysogeny clude strains subject to DNA-mediated trans-(64), inhibit transformationa phenomenon formation (18, 20, 63, 65), and it is probable that also reported in Bacillus subtilis (70, 92, 93). this mode of genetic exchange can contribute to This inhibition in strepto...

Purification and immunochemical characterization of Streptococcus sanguis serotype I carbohydrate antigen

“…The bacterial surface structures, especially cell wall components, have been shown to confer various biological properties (6,21) and to mediate bacterial attachment to tooth and mucosal surfaces of the oral cavity (12). The serotype I antigen may participate in the attachment of S. sanguis to tooth surfaces; however, the mechanism of cell attachment remains to be elucidated.…”

Protease-sensitive transfection of Streptococcus pneumoniae with bacteriophage Cp-1 DNA

“…The importance of calcium ions for transfection of intact competent cells of Streptococcus species has been documented (23,30). Calcium ions are also required for uptake of prebound transforming DNA in S. pneumoniae (38).…”

“…knowledge on the transfection of several species of Streptococcus has been achieved recently (10,23). To our knowledge, the existence of a transfection system in procaryotic cells that is sensitive to proteolytic enzymes and uses intact competent cells as receptors has not been described so far, apart from the case of Bacillus species.…”

DNA binding and entry during transfection in Streptococcus pneumoniae

“…This finding is in contrast to the absence of a divalent cation effect in the binding of bacterial DNA to competent pneumococci [11 ]. Nugent and Cole [12] reported an analogous observation in transfection of Streptococcus sanguts.…”