Protein L. A novel bacterial cell wall protein with affinity for Ig L chains.

A novel Ig-binding protein has been isolated from the surface of bacteria belonging to the anaerobic species Peptococcus magnus. To solubilize the protein, peptococci were treated with different proteolytic enzymes (papain, pepsin, and trypsin) or with mutanolysin, a bacteriolytic agent known to digest the cell walls of streptococci. Papain, trypsin, and mutanolysin all solubilized peptides showing affinity for radiolabeled human IgG in Western blot analysis. Compared with papain and trypsin, mutanolysin liberated a more homogeneous material, which also had a higher m.w. This mutanolysin-solubilized protein (Mr 95 kDa) was obtained highly purified by a single isolation step on IgG-Sepharose, and the molecule was found to exhibit unique Ig-binding properties. Thus, in dot blots and in Western blots, human IgG, F(ab')2 and Fab fragments of IgG, and human kappa and lambda L chains all showed affinity for the protein. Moreover, the molecule also bound human IgM and IgA, whereas no binding was recorded for IgG-Fc fragments or IgG H chains. Finally, the protein bound to human polyclonal Ig L chains immobilized on polyacrylamide beads. These different data demonstrate that the isolated peptococcal protein binds Ig through L chain interaction. The name protein L is therefore suggested for this novel Ig-binding bacterial cell wall protein.

A phylogenetic analysis of staphylococci, Peptococcus saccharolyticus and Micrococcus mucilaginosus.

The intra- and intergeneric relationships of the genus Staphylococcus, and the phylogenetic position of Peptococcus saccharolyticus and Micrococcus (Staphylococcus salivarius), were investigated by comparative oligonucleotide cataloguing of 16S rRNA. All the staphylococci investigated form a phylogenetically coherent group at the genus level that, in addition, contains the anaerobic species Peptococcus saccharolyticus. The genus Staphylococcus belongs to the broad Bacillus-Lactobacillus-Streptococcus cluster that is defined by Gram-positive bacteria with a low DNA G+C content. Micrococcus mucilaginosus is not a genuine member of the genus Micrococcus. The binary matching coefficients between the 16S rRNA of Micrococcus mucilaginosus and those representatives of the Arthrobacter/Micrococcus group and related genera indicate that Micrococcus mucilaginosus should be regarded as a member of a new genus.

Differentiation of Peptococcus and Peptostreptococcus by gas-liquid chromatography of cellular fatty acids and metabolic products.

Gas-liquid chromatographic (GLC) profiles of cellular fatty acids and metabolic products were useful in identifying strains of Peptococcus saccharolyticus, Peptococcus asaccharolyticus, Peptostreptococcus anaerobius, Peptostreptococcus micros, and Streptococcus intermedius. The GLC results supported the recent taxonomic decision to transfer aerotolerant Peptostreptococcus species to the genus Streptococcus. Because inconsistencies in the results prevented our differentiating Peptococcus prevotii. Peptococcus magnus, and Peptococcus variabilis by GLC, additional strains will have to been examined. These GLC techniques are amenable to routine use; however, for interlaboratory results to be meaningful, the classification and nomenclature of the anaerobic gram-positive cocci should be standardized.

Determination of cooperative interaction between clusters in Clostridium pasteurianum 2 (4Fe-4S) ferredoxin.

The effect of reducing one 4Fe-4S cluster in Clostridium pasteurianum 2 (4Fe-4S) ferredoxin on the reduction potential of the unreduced cluster has been investigated. While such an effect is suggested by both the x-ray structure of Peptococcus aerogenes 2 (4F-4S) ferredoxin and the polypeptide conformational change on reduction present in clostridial-type 2 (4Fe-4S) ferredoxins, present studies indicate that cluster-cluster cooperative interaction is not strong enough to be of functional importance in these proteins.

Assignment of the cysteinyl 13C nuclear magnetic resonances and comparison of other aliphatic amino acid resonances of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins.

13C NMR spectra of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins show that some 13C resonances of the aliphatic amino acid residues are shifted significantly from their corresponding resonance positions in the spectra of model polypeptides or apoferredoxin. Thirteen 13C resonances are shifted into the 80- to 120-ppm (from CS2) region, and have been assigned to the cysteinyl alpha and beta carbon atoms. The remaining shifted resonances in the 120- to 190-ppm region are tentatively assigned to amino acid residues that may be close to [4Fe-4S] clusters of the oxidized and reduced ferredoxins. The similarity in the shift pattern of the corresponding 13C resonances of the cysteinyl alpha and beta carbon atoms in the three ferredoxins studied suggests that the three-dimensional amino acid environments of the corresponding [4Fe-4S] clusters in each protein are similar.

Cellular fatty acids of Peptococcus variabilis and Peptostreptococcus anaerobius.

Cellular fatty acids of Peptococcus variables and Peptostreptococcus anaerobius were identified by gas chromatography, mass spectrometry, and associated analytical techniques. Iso- and anteiso-branched-chain acids were major components in both species.

Long-chain fatty acids of peptococci and peptostreptococci.

The long-chain fatty acids extracted from the whole cells of 12 clinically significant species of peptococci and peptostreptococci were characterized by gas-liquid chromatography. The resulting methylated fatty acid profiles (and some unidentified compounds) of 82 strains allowed the 12 species to be separated into four groups. Fifteen strains of Peptostreptococcus anaerobius were placed in group I because they had a unique, prominent compound that occurred in the area where a C8 to C10 fatty acid would be expected. Group II, consisting of Peptostreptococcus intermedius, Peptostreptococcus micros, Peptostreptococcus parvulus, Peptococcus morbillorum, and Peptococcus constellatus, produced C14, C16:1, C18:1, and C18 fatty acids. Peptococcus prevotii, Peptococcus variabilus, Peptococcus magnus, Peptococcus asaccharolyticus, and Peptostreptococcus productus were placed in group III because they contained three to six additional, unidentified compounds that strikingly differentiated them from group II. Peptococcus saccharolyticus was the single species assigned to group IV because it yielded C14, C16, C18:1, C18, and C20 fatty acids and a prominent unidentified peak that occurred between C14 and C16 fatty acids. This study indicated that cellular long-chain fatty acids may be an important tool in clarifying the taxonomy of the peptococci and peptostreptococci.

Structure of Peptococcus aerogenes ferredoxin. Refinement at 2 A resolution.

The model for ferredoxin from Peptococcus aerogenes, derived from a 2.8-A resolution map, has been refined with a 2-A resolution data set. The conventional index, R, decreased from 0.449 for the initial model to 0.188 for the unconstrained one and 0.206 for the constrained one. The standard deviations of the iron and sulfur atoms are 0.04 to 0.05 A and for the oxygen, nitrogen, and carbon atoms they range from 0.14 to 0.26 A. One hundred forty-six water oxygen atoms were included in the solvent part of the model and were checked by a highly selective criterion, suggesting that most of them represented water molecules.

NH---S hydrogen bonds in Peptococcus aerogenes ferredoxin, Clostridium pasteurianum rubredoxin, and Chromatium high potential iron protein.

Results from refinement of the crystal structures of P. aerogenes ferredoxin and C. pasteurianum rubredoxin determined by x-ray diffraction show that there are 15-18 NH---S bonds in the former and six in the latter with lengths in the range 3.1-3.9 A. Earlier tritium exchange experiments are consistent with the presence of these hydrogen bonds in the ferredoxin structure and show that more peptide hydrogen atoms are available for exchange in apoferredoxin than in intact ferredoxin. Four types of NH---S bonds are observed and two of these are geometrically similar to the two types of 3(10) NH---O bonds. The existence of more NH---S bonds in ferredoxin than in high potential iron protein suggests why the -2 form of the Fe4S4 cluster is preferred in ferredoxin over the -1 form found in high potential iron protein. From comparison of Cys-X-Y-Cys sequences in rubredoxin, ferredoxin, and high potential iron protein we suggest that two Cys-X-Y-Cys-Z sequences, where Z may have conformation angles similar to glycine, are required to make a one-iron cluster, no more than one Cys-X-Y-Cys-Z-Gly sequence is required to form a Fe2S2 ferredoxin, and a Cys-X-Y-Cys-Gly sequence where Y has a conformation such that the cysteines bond to different iron atoms is necessary to form the tetrameric cluster.

Evaluation of a test kit for identification of anaerobic bacteria.

A test kit for identification of anaerobic bacteria--API--has been compared for accuracy in individual tests and for identification on the genus or species level with pre-reduced anaerobically sterilized media methods on 241 anaerobic strains. The microsystem was found to be reliable and permits identification of the clinically most significance anaerobic bacteria if it is supplemented with other tests such as gaschromatographic analysis, morphology, lecithinase, lipase and antibiotic sensitivity pattern.