Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli

Noegel, A.; Rdest, Ursula; Goebel, Werner

doi:10.1128/jb.145.1.233-247.1981

Cited by 89 publications

(80 citation statements)

References 21 publications

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“…ShlA was obtained from the culture supernatant of IPTGinduced E. coli BL21(pRO3 shlA shlB ) by precipitation with 50% ammonium sulphate. ShlA was purified as described ᮊ 1997 Blackwell Science Ltd, Molecular Microbiology, 26, 853-865 Noegel et al (1981) above for ShlA* on a MonoS HR5/5 column (single chromatographic step). ShlA-255 was obtained by precipitation with 50% ammonium sulphate (final concentration) from the culture supernatant of IPTG-induced E. coli BL21 (pRO2 shlA shlB ).…”

Section: Haemolysis After Complementation With Pementioning

confidence: 99%

Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity

Hertle

Brutsche

Groeger

et al. 1997

Molecular Microbiology

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The cytolytic and haemolytic activity of Serratia marcescens is determined by the ShlA protein, which is secreted across the outer membrane with the aid of the ShlB protein. In the absence of ShlB, inactive ShlA* remains in the periplasm of Escherichia coli transformed with an shlA‐encoding plasmid, which indicates that ShlB converts ShlA* to active ShlA. ShlA* in a periplasmic extract and partially purified ShlA* were activated in vitro by partially purified ShlB. When both proteins were highly purified, ShlA* was only activated by ShlB when phosphatidylethanolamine (PE) or phosphatidylserine was added to the assay, while phosphatidylglycerol contributed little to ShlA* activation. Lyso‐PE, cardiolipin, phosphatidylcholine, phosphatidic acid, lipopolysaccharide and various detergents could not substitute for PE. Although radioactively labelled PE was so tightly associated with ShlA that it remained bound to ShlA after heating and SDS–PAGE, it was not covalently linked to ShlA as PE could be removed by thin‐layer chromatography with organic solvents. The number of PE molecules associated per molecule of ShlA was 3.9 ± 2.2. Active ShlA was inactivated by treatment with phospholipase A2, which indicated that PE is also required for ShlA activity. ShlA‐255 (containing the 255 N‐terminal amino acids of ShlA) reversibly complemented ShlA* to active ShlA and was inactivated by phospholipase A2, which demonstrated that PE binds to the N‐terminal portion of ShlA; this region has previously been found to be involved in ShlA secretion and activation. Electrospray mass spectroscopy of ShlA‐255 determined a molar mass that corresponded to that of unmodified ShlA‐255. An E. coli mutant that synthesized only minute amounts of PE did not secrete ShlA but contained residual cell‐bound haemolytic activity. Since PE binds strongly to ShlA* in the absence of ShlB without converting ShlA* to haemolytic ShlA, ShlB presumably imposes a conformation on ShlA that brings PE into a position to mediate interaction of the hydrophilic haemolysin with the lipid bilayer of the eukaryotic membrane.

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Section: Haemolysis After Complementation With Pementioning

confidence: 99%

Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity

Hertle

Brutsche

Groeger

et al. 1997

Molecular Microbiology

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“…The hemolytic activity of E. coli strains was analyzed by cultivation of the strains on human blood agar plates as described previously [16]. S-¢mbrial adhesin expression was analyzed by a hemagglutination assay with plate-grown bacteria.…”

Section: Characterization Of Strainsmentioning

confidence: 99%

The leuX-encoded tRNA5Leu but not the pathogenicity islands I and II influence the survival of the uropathogenic Escherichia coli strain 536 in CD-1 mouse bladder mucus in the stationary phase

Dobrindt

1998

FEMS Microbiology Letters

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The uropathogenic Escherichia coli strain 536 carries two pathogenicity islands, each of which is associated with either of the tRNA genes selC or leuX, respectively. Growth competition in CD-1 mouse mucus between the wild-type strain E. coli 536, its leuX mutant 536v102 and its mutant 536R3, lacking both pathogenicity islands but expressing a functional tRNA S veu , revealed a major impact of leuX on E. coli survival in bladder mucus. The impaired survival in CD-1 mouse mucus observed upon deletion of the leuX gene was abolished after complementation with the leuX gene. The survival of bacteria in bladder mucus was not influenced by the presence of pathogenicity islands I and II. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

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“…Bacteria were grown at 30°C in L-broth medium or on blood-agar plates containing the appropriate antibiotics at 50 ~g/ml. Plasmids pAC50 and pAC2001 are pACYC184 bearing the E. coli hly determinant from the wild-type plasmid pHly152 [11] and the chromosomal determinant LE2001 [10], respectively.…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

“…1 E. coli 5K and mutant strain Hsb. 1 were transformed with plasmids pAC50 and pAC2001 to determine production of hemolysin when carrying the complete plasmid [11] or chromosomalderived [10] hly determinants. Extracellular hemolytic activity was measured in culture supernatants and showed that the extracellular hemolytic activity of strain Hsb.…”

Section: Reduction-of Hemolysin Synthesis and Secretionmentioning

confidence: 99%

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Loss of secreted hemolysin activity in the mutant strain Hsb. 1 is due to a lesion in a plasmid copy number locus

Dı́az

Juárez

1991

FEMS Microbiology Letters

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Further studies have been carried out on mutation hsb which was previously suggested to block hemolysin secretion (Muñoa et al., 1988, FEMS Microbiol. Lett. 56: 167–172). We show that the reported reduction in the extracellular hemolytic activity of mutant Hsb. 1 is due to lower hemolysin synthesis and that this is itself a consequence of a decrease in plasmid copy number. We suggest that the hsb is identical to the pcnB lesion located at minute 3.6 of the chromosome.

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Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli

Cited by 89 publications

References 21 publications

Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity

Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity

The leuX-encoded tRNA5Leu but not the pathogenicity islands I and II influence the survival of the uropathogenic Escherichia coli strain 536 in CD-1 mouse bladder mucus in the stationary phase

Loss of secreted hemolysin activity in the mutant strain Hsb. 1 is due to a lesion in a plasmid copy number locus

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