Nucleic acid homology studies between Peptococcus saccharolyticus and various anaerobic and facultative anaerobic Gram-positive cocci

Abstract

The taxonomic position of Peptococcus saccharo-l~epticus is'uncertain [1 ]. The exclusion from the genus tococcus, due to its capacity to ferment sugars [1 ], was justified by results of Schleifer and Nimmermann [2] who showed that the cell wall composition of P. saceharolyticus was characteristic of staphylococci [3,4] ; a finding that may indicate a certain relationship between these two groups of bacteria. The aim of the present study was to determine the genetic relationship ofP. saccharolyticus to other anaerobic and facultative anaerobic Gram.positive cocci usi/ag DNA-rRNA and DNA-DNA hybridization techniques. It could be dearly demonstrated that P. saccharolyticus is genetically related to staphylococci. Materials and methods Organisms and growth conditionsThe strains used are listed in Table 1. Apart from anaerobic and facultative anaerobic cocci, Lactobacillus acidophilus was included as an unrelated reference strain with a similar DNA base composition. Staphylococci were cultivated aerobically in peptone-yeast extract-glucose-NaC1 bouillon [5]. All the other strains, except for L. acidophilus and Pediococcus pentosaceus, which were grown in MRS medium [6], were cultivated anaerobically in a simplified PYG medium [7]. Glucose in this medium was replaced by glycine or glutamine for cultivation ofP. variabilis and P. aerogenes. The incubation temperature was 37°C. Ceils were harvested in the late logarithmic growth phase. In order to label DNA or rRNA, the bacteria were grown in broth, in which yeast-extract was replaced by 2/aCi/ml of [6-3H] thymidine and [5,6-3H]uracil, respectively. Specific activities obtained by this method were in the range of 1 • 104-7 • l0 s for [3H]DNA and of 1 • 104-1 • l0 s for [3H]-rRNA. DNA isolation and re-associationExtraction of labelled and unlabelled DNA was carried out using Marmur's method [8], with modifications described by Meyer and Sehleifer [9,10]. Fixation of unlabelled DNA on nitrocellulose filters was performed according to the method of Gillespie and Spiegelmann [11 ]. DNA-DNA hybridization experi. ments were accomplished following the procedure described by McConaughy et al. [12] in 3 X SSC (Standard Saline Citrate: 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0) containing 10% formamide at 58°C for 30 h. At an average G + C-content of 34 mol%, these conditions correspond to a temperature of 25°C below the thermal melting point of the DNA [13]. The ratio of filter-bound DNA to labelled, frag. mented, DNA in solution was approx. 75 : 1.The percentage of radio-activity bound in the heterologous reaction was normalized to the percentage of radio-activity bound in the homologous reaction and expressed as an homology value. rRNA isolationUptake of [5,6-all]uracil into cells was followed by measuring the decrease of radio-activity in the

A Phylogenetic Analysis of Staphylococci, Peptococcus saccharolyticus and Micrococcus mucilaginosus

“…Later, Kilpper & Schleifer (1978) and Crosa et al (1979) found a significant degree of relationship of P. saccharolyticus to coagulase-negative staphylococci, based on DNA-DNA homology studies. More extensive nucleic acid reassociation studies using DNA-DNA and DNA-23s rRNA hybridization techniques established the close genetic relatedness of P. saccharolyticus to the staphylococci (Kilpper et al, 1980). This finding is confirmed by our results.…”

“…To determine the genealogical relationship of the genus Staphylococcus on a broader basis, we have extended these RNA sequencing studies, using an in vitro labelling procedure (Stackebrandt et al, 1981), to several species shown by DNA-DNA reassociation studies to be only rather distantly related (Schleifer et al., 1979 a). Peptococcus saccharolyticus was included because this organism was recently described as an anaerobic member of Staphylococcus (Kilpper et al, 1980). Micrococcus mucilaginosus (Staphylococcus salivarius) CCM 24 17 was investigated because its DNA G + C content of 59 mol % (Kocur & Bohacek, 1974) does not fall in the usual range for either Staphylococcus or Micrococcus.…”

“…Peptococcus saccharolyticus was grown anaerobically in a medium described by Kilpper et al (1980). All other strains were grown aerobically in 200 ml of a peptone/yeast extract/glucose broth (0.5 :0.25 :0.5; %, w/v).…”

Molecular Relationships and Classification of Some Viridans Streptococci as Streptococcus oralis and Emended Description of Streptococcus oralis (Bridge and Sneath 1982)

“…Cells were harvested at the end of the exponential growth phase for the isolation of deoxyribonucleic acid (DNA) and at the stationary growth phase for the preparation of cell walls. Isolation of DNA and ribosomal ribonucleic acid, radioactive labeling, and nucleic acid hybridization experiments were carried out by the filter method as described previously (19,20,22). DNA base composition was determined by thermal denaturation with a Gilford model 2600 spectrophotometer (16).…”

Staphylococcus muscae, a New Species Isolated from Flies

“…DNA from Escherichia coli K-12 was used as a standard (13). Radioactive labeling of DNA and the DNA-DNA hybridization experiments were carried out by using the filter method as described previously (12). 16s rRNA genes (16s rDNA) were amplified in vitro by using the polymerase chain reaction as described previously by Ludwig et al (16).…”