Identification of porins in the outer membrane of Pseudomonas aeruginosa that form small diffusion pores

The purified outer membrane proteins of Pseudomonas aeruginosa were reconstituted with phosphatidylcholine and dicetylphosphate into membrane vesicles, and these were tested by the liposome swelling method for the diffusion of saccharides with different Mr. Proteins C (Mr, 70,000), D (Mr, 46,000), and E (Mr, 43,000) were found to confer the monosaccharide-permeable pores in the reconstituted liposome membranes. The membrane vesicles containing proteins F (Mr, 34,000), G (Mr, 25,000), or H (Mr, 19,000) showed no detectable pore-forming activity. The pores formed by proteins C, D, or E appeared to be smaller than that formed by the Escherichia coli porins. The size of the solutes that permeated through the newly identified porins is similar to that through the intact and purified outer membrane of P. aeruginosa (Yoneyama, H., and Nakae, T. (1986) Eur. J. Biochem. 157, 33-38; Yoshihara, E., Gotoh, N., and Nakae, T. (1988) Biochem. Biophys. Res. Commun. 156, 470-476).     

Stimulatory effect of glycine betaine on l-lysine fermentation

Summary The growth rate, sugar consumption rate, and production rate of an l-lysine producing Brevibacterium lactofermentum mutant were stimulated by addition of exogenous glycine betaine. Glycine betaine stimulated the growth rate especially in media of inhibitory osmotic stress, and the stimulation was independent of any specific solute. Therefore growth stimulation by glycine betaine was considered to be an osmoprotective effect. A strong enhancement of the sugar consumption rate and the l-lysine production rate was observed even with resting cells under osmotic stress as well as in a fermentation with growing cells. These data indicated that the osmoprotective effects of glycine betaine on l-lysine production can be independent of protein synthesis. Offprint requests to: Yoshio Kawahara     

Serological Characterization of Trichosporon cutaneum and Related Species

Recent molecular biological, chemical, physiological and morphological studies indicate that Trichosporon cutaneum and related species should be reclassified. In this study, antigenic characteristics of the species were determined. The results of adsorption experiments revealed that there were at least three serological types: I, II and III. Specific factor sera I, II and III were prepared on the basis of adsorption experiments and isolates were serotyped by cell slide agglutination (CSA). Since the CSA test was difficult to read in some strains, the results of the CSA test were compared with the findings from an enzyme-linked immunosorbent assay (ELISA). For the ELISA, crude polysaccharide antigens prepared from the culture supernatant were used as the antigen. The types determined by ELISA correlated well with those determined by the CSA test. These data suggest that T. cutaneum and related species have at least three serological types, and that the typing can be done by either CSA or ELISA.     

An improved method for the measurement of total lipid-bound sialic acids after cleavage of α2,8 sialic acid linkage withVibrio cholerae sialidase in the presence of cholic acid,SDS and Ca2+

In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD₃ and GD₂) are not involved because their 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of 2,8 linkages byV. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca²⁺ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.Presented at the Second International Glycobiology Symposium which was held in San Francisco, CA, USA (14 February 1994).     

3-Aminobenzamide,a potent inhibitor of poly (ADP-ribose) polymerase,causes a rapid death of HL-60 cells cultured in serum-free medium

HL-60 cells transferred from serum-supplemented to serum-free culture medium initially bound to culture plate tightly and then released from the plate on increasing the culture time and resumed exponential growth after about 8 h lag. At the initial stage of the culture, the cells became extremely sensitive to 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, and, at 1 mM, 80 to 90% of the cells were lysed within 20 h, whereas the inhibitor was totally ineffective on the cell growth in serum-supplemented medium at the concentration. Non-inhibitory analogs of the inhibitor were ineffective. Assay of poly(ADP-ribose) polymerase activity in permeable cells indicated that a transient activation of the enzyme occurred during the culture in serum-free medium (the maximum activation was observed at 8 h of the culture). The cells conditioned in serum-free medium for 24 h acquired significant resistancy to the inhibitor. A low concentration of fibronectin (5 to 10/ml) and a relatively high concentration of bovine serum albumin (0.5 to 1 mg/ml) effectively blocked the cell attachment to plate and also the 3-aminobenzamide-induced cell lysis. These results suggest that poly(ADP-ribose) polymerase is involved in a process essential for HL-60 cells to adapt to a serumdeprived growth condition.     

Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene.

Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.     

Staining method of poly(3-hydroxyalkanoic acids) producing bacteria by nile blue

Summary Using an ethanol solution of nile blue, we have developed an efficient method to detect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on the agar plate. When the bacterial colonies with PHA granules were stained with nile blue, the stained colonies fluoresced bright orange on the irradiation of UV light. In the fluoresce emission spectra, fluorescence intensity increased with an increase in the PHA content of bacterial cells.Alcaligenes eutrophus andA.latus colonies with poly(3-hydroxybutyric acid) (PHB) homopolymer exhibited an emission maximum at 580nm on the excitation at 490nm. On the other hand,Pseudomonas oleovorans andP.putida with medium-chain-length (mcl-) PHA copolymers of C6, C8 and C10 units exhibited an emission maximum at 570nm.