Amplification of synthesis and secretion of haemolysin using a run-away plasmid in Escherichia coli

Molin and co-workers have described the construction of a ‘run-away’ plasmid, pOU71 which could be useful for the amplification of cloned genes at high temperature when the plasmid replicates to high copy number.In this paper we describe the kinetics of synthesis of a plasmid-coded gene product, β-lactamase, concomitant with pOU71 amplification at 42°C. Maximum amplification was obtained by shifting a culture growing at 30–42°C for 60 min resulting in a 70- to 80-fold amplification for the β-lactamase gene product when the culture was returned to 30°C.The haemolytic determinant LE2001 from an Escherichia coli strain of human origin was cloned into plasmid pOU71 giving rise to plasmid pLG570. Using an identical amplification procedure a 20-fold amplification of the synthesis and secretion of haemolysin was achieved.     

Study on the relationship of protease production and luminescence in Vibrio harveyi

AIMS: To demonstrate that Vibrio harveyi produces various types of toxins and how the production of those toxins is related with luminescence. METHODS AND RESULTS: Luminescence and toxicity of eight V. harveyi were evaluated. We demonstrated that all V. harveyi emitting luminescence were isolated from marine organisms and also showed that they were highly pathogenic when compared with culture collection V. harveyi based on cytotoxic assay test. On the contrary, V. harveyi isolated from shrimp farm showed no luminescence but showed high pathogenicity based on toxicity test. The effect of protease inhibitors on pathogenicity and luminescence was also investigated. We demonstrated that light emission of pathogenic V. harveyi remarkably decreased after addition of protease inhibitor. Furthermore, extracellular proteins from cell-free culture supernatant of luminescent and nonluminescent V. harveyi were compared using SDS-PAGE analysis. Results showed that there were differences in molecular weight and amount of proteins. CONCLUSIONS: Vibrio harveyi parasiting marine organisms have both luminescence and pathogenicity. Based on this study, luminescence and protease toxin activity in V. harveyi are related. Moreover, this paper clarified that V. harveyi produces various types of toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study demonstrated that V. harveyi produces two kinds of toxins, haemolysin and protease toxin. It may be clear roots of V. harveyi toxin.     

Topological and functional studies on HlyB of Escherichia coli

Summary The topology of HlyB, a protein located in the inner membrane of Escherichia coli and involved in the secretion of -haemolysin (HlyA), was determined by the generation of HlyB-PhoA and HlyB-LacZ fusion proteins. The data obtained by this biochemical method together with computer predictions suggest that HlyB is inserted in the cytoplasmic membrane by six stable hydrophobic, -helical transmembrane segments. These segments extend from amino acid positions 158 to 432 of HlyB. The cytoplasmic loops between these transmembrane segments are relatively large and carry an excess of positively charged amino acids, while the periplasmic loops are rather small. In addition to these six transmembrane segments, two additional regions in the 78 N-terminal amino acids of HlyB appear to be also inserted in the cytoplasmic membrane. However, the association of these two segments with the cytoplasmic membrane seems to be less tight, since active PhoA and LacZ fusions were obtained by insertion into the same positions of these segments. A LacZ-HlyAs fusion protein carrying, at the C-terminus of LacZ, the 60-amino acid signal sequence of HlyA was not secreted in the presence of HlyB/HlyD. However, transport of this fusion protein into the cytoplasmic membrane appeared to be initiated, as suggested by the tight association of this protein with the inner membrane. A similar close association of LacZ-HlyA_s with the inner membrane was also observed in the presence of HlyB alone but not in its absence. These data suggest that HlyB recognizes the HlyA signal sequence and initiates the transport of HlyA into the membrane.     

Glutaraldehyde Treated Gelatin as a Cation Exchanger

Glutaraldehyde tanned gelatin proved to be a cation of high capacity and may be used advantageously in the separation and purification of serum components. Being proteinaceous it is attacked by microorganisms. This may be prevented by storage in the presence of 0.1% chloroform.     

Purification of Uricase by Biospecific Adsorption-Desorption

A simple procedure for the purification of uricase from bovine kidney is described. The procedure involves the following steps: 1) processing of kidney mince by borate/butanol, 2) ammonium sulphate precipitation, and 3) biospecific adsorption-desorption. The adsorbents were prepared by chemical attachment of urate or xanthine to agarose gel beads. The desorption was performed by a xanthine solution. The adsorption-desorption procedure resulted in an 11 000–12 000-fold purification. The specific activity of the purified uricase was 19.8 U/mg using either “urate” or “xanthine” adsorbent. The recovery was about 70%.The adsorbents were also used for the purification of commercial uricase preparations from hog liver. In this case the purified uricase also possessed a specific activity of 19.8 U/mg. The products were homogenous as judged by gradipore electrophoresis and gel filtration.     

Effect of Fusobacterium necrophorum haemolysin on peripheral leukocytes in vitro

Abstract Fusobacterium necrophorum haemolysin was cytotoxic for animal leukocytes. The most sensitive cells to the haemolysin were rabbit leukocytes, of which disruption of the cells and protoplastic extrusion were induced. The response was dose- and time-dependent, and was neutralised by antiserum. Scanning electron microscopy revealed that the haemolysin induced destruction of rabbit leukocytes, leaving membrane fragments.     

Multifactorial determination of systemic invasivity in Escherichia coli

Abstract Studies of aerobactin excretion, production of antimicrobial agents, haemolysin production, serum resistance and presence of plasmids were made in 157 clinical isolates of Escherichia coli , 96 from blood and 61 from faeces. Only serum resistance and the presence of a small number of plasmids (< 4 per cell), appeared more frequently in strains from blood than in faecal isolates. It was noteworthy that combination of aerobactin, antimicrobial agents and haemolysin production was more frequent in blood isolates than in strains from faeces. A substantial number of strains from blood presented all 5 virulence factors (9.4%) while none of the faecal strains exhibited this phenotype. Definition of the systemic invasivity of E. coli as a multi-factorial phenomenon is suggested.