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In order to investigate the availability of laser-induced mass spectrometry for the determination of bacteria, ten strains of the genera Escherichia and Streptococcus were chosen. Gram-negative as well as Gram -positive lysogenic and non-lysogenic. pathogenic and non-pathogenic strains were examined. Giant pulses of a Nd: YAG laser (frequency quadrupled, wave-length 265 nm) were used to vaporize and partly ionize the bacterial material. The time-of-flight mass spectra obtained could be discriminated by using the stepwise discriminant analysis provided by BM DP software package. Two different methods relating centrifugation and washing procedures were used. With the first method a correct classification between 50.0 and 90.9 per cent (S. sanguis) by means of mass spectra of positive ions and a correct classification between 27.1 and 80.4 per cent (S. lactis-25) by means of mass spectra of negative ions was observed. Using the second method a correct classification between 40.7 and 93.3 per cent (5. lactis-25) by means of mass spectra of positive ions and a correct classification between 56.7 and 95.0 per cent by means of mass spectra of negative ions was obtained. Here 5. sanguis with 93.3 and S. lactis-25 with 81.7 per cent were significantly differentiated. In this context it must be em phasized that a correct classification of 27 per cent must be considered statistically significant, if 10 different strains are used.

Mycoplasmas are potent macrophage stimulators. We describe the isolation of macrophage-stimulatory lipopeptides S-[2, 3-bisacyl(C16:0/C18:0)oxypropyl]cysteinyl-GQTDNNSSQSQQPGS GTTNT and S-[2,3-bisacyl(C16:0/C18:0)oxypropyl]cysteinyl-GQTN derived from the Mycoplasma hyorhinis variable lipoproteins VlpA and VlpC, respectively. These lipopeptides were characterized by amino acid sequence and composition analysis and by mass spectrometry. The lipopeptides S-[2,3-bis(palmitoyloxy)propyl]cysteinyl-GQTNT and S-[2, 3-bis(palmitoyloxy)propyl]cysteinyl-SKKKK and the N-palmitoylated derivative of the latter were synthesized, and their macrophage-stimulatory activities were compared in a nitric oxide release assay with peritoneal macrophages from C3H/HeJ mice. The lipopeptides with the free amino terminus showed half-maximal activity at 3 pM regardless of their amino acid sequence; i.e., they were as active as the previously isolated M. fermentans-derived lipopeptide MALP-2. The macrophage-stimulatory activity of the additionally N-palmitoylated lipopeptide or of the murein lipoprotein from Escherichia coli, however, was lower by orders of magnitude. It is concluded that the lack of N-acyl groups in mycoplasmal lipoproteins explains their exceptionally high in vitro macrophage-stimulatory capacity. Certain features that lipopolysaccharide endotoxin and mycoplasmal lipopeptides have in common are discussed. Lipoproteins and lipopeptides are likely to be the main causative agents of inflammatory reactions to mycoplasmas. This may be relevant in the context of mycoplasmas as arthritogenic pathogens and their association with AIDS.

The polysaccharide intercellular adhesin (PIA) is an important factor in the colonization of medical devices by Staphylococcus epidermidis. The genes encoding PIA production are organized in the icaADBC (intercellular adhesion) operon. To study the function of the individual genes, we have established an in vitro assay with UDP-N-acetylglucosamine, the substrate for PIA biosynthesis, and analyzed the products by thin-layer chromatography and mass spectrometry. IcaA alone exhibited a low N-acetylglucosaminyltransferase activity and represents the catalytic enzyme. Coexpression of icaA with icaD led to a significant increase in activity. The newly identified icaD gene is located between icaA and icaB and overlaps both genes. N-Acetylglucosamine oligomers produced by IcaAD reached a maximal length of 20 residues. Only when icaA and icaD were expressed together with icaC were oligomer chains that react with PIA-specific antiserum synthesized. IcaA and IcaD are located in the cytoplasmic membrane, and IcaC also has all the structural features of an integral membrane protein. These results indicate a close interaction between IcaA, IcaD, and IcaC. Tunicamycin and bacitracin did not affect the in vitro synthesis of PIA intermediates or the complete PIA biosynthesis in vivo, suggesting that a undecaprenyl phosphate carrier is not involved. IcaAD represents a novel protein combination among beta-glycosyltransferases.

The effect of NNMG on the template activities of different polynucleotides (polyuridylic acid, polycytidylic acid, polyadenylic acid and copolymer of adenylic and guanylic acid 5,5:1) and t-RNS was studied. The maximum inhibition of the messenger activity was found for poly-C, followed by poly-Α and poly-U. The acceptor activity of t-RNA was found to be inhibited by NNMG: maximum for proline, followed by serine, leucine, phenylalanine and lysine. The mechanism of these inhibitions was studied using NNMG radioactively labelled on the methyl group. Different amounts of radioactivity were found in the various polynucleotides and t-RNS.

1-Nitroso-3-nitro-1-methyl-guanidine (NNMG) (at pH 5,5 resp. 6,0) methylates and deaminates the bases of nucleic acids. We treated nucleic bases, nucleosides, nucleoside phosphates and homopolyribonucleic acids with NNMG and we found the following methyl derivates of adenine: 1-methyladenine, 3-methyladenine and N6-methyladenine : of guanine: 7-methylguanine and of cytosine: 1-methylcytosine and 3-methylcytosine. Moreover bases of the nucleic acids were deaminated : adenine to hypoxanthine and guanine to xanthine.