The structure of cloned hemolysin DNA from plasmid pHly185

Stark, James M.; Shuster, C. W.

doi:10.1016/0147-619x(83)90056-2

Cited by 16 publications

(7 citation statements)

References 19 publications

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“…This is effected by a specific envelope transport system formed by two proteins, HlyB and HlyD, which are encoded by genes of the contiguous four-gene hemolysin determinant (12,21,28,30). Examination of the highly conserved hly determinant sequence in E. coli has revealed surprisingly that the four genes have a G+C content of 39% rather than the expected 50% of the genome (6,14).…”

Section: Discussionmentioning

confidence: 99%

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The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli

Koronakis¹,

Cross²,

Senior³

et al. 1987

J Bacteriol

220

134

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Secreted hemolysins were extremely common among clinical isolates of Proteus mirabilis, Proteus vulgaris, and Morganella morganii, and hemolytic activity was either cell associated or cell free. Southern hybridization of total DNA from hemolytic isolates to cloned regions of the Escherichia coli alpha-hemolysin (hly) determinant showed clear but incomplete homology between genes encoding production of hemolysins in the four species.One of the two E. coli secretion genes, hlyD, hybridized only with DNA from P. vulgaris and M. morganii, which produced cell-free hemolysis, but not with that from P. mirabilis, which showed only cell-associated activity. Molecular cloning of the genetic determinants of cell-free hemolytic activity from P. vulgaris and M. morganii chromosomal DNA allowed their functional analysis via inactivation with the transposons TnlOOO and TnS. Both hemolysin determinants were about 7.5 kilobase pairs and comprised contiguous regions directing regulation, synthesis, and specific secretion out of the cell. Transposon mutations which eliminated secretion of the Proteus and Morganella hemolysins could be complemented specifically by the E. coli hemolysin secretion genes hlyB or hlyD. Alignment of the physically and functionally defined hly determinants from P. vulgaris and M. morganii with that of the E. coli alpha-hemolysin confirmed a close genetic relationship but also indicated extensive evolutionary divergence.

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Section: Discussionmentioning

confidence: 99%

“…Both the organization (4,18,20,21,24,28,30) and nucleotide sequence (6,14) of the four contiguous hemolysin genes (hlyC, hlyA, hlyB, and hlyD) are rigidly conserved on both plasmids and chromosomes throughout E. coli strains.…”

mentioning

confidence: 99%

The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli

Koronakis¹,

Cross²,

Senior³

et al. 1987

J Bacteriol

220

134

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“…Plasmid hemolysin determinants and their function. Noegel and associates (75,76) and Stark and Shuster (98,99) investigated the location and functions of the genes from pHlylS2 and pHlyl85, respectively. Strains harboring these plasmids produce a cell-free extracellular pool of hemolysin (Hlyex) as well as an intracellular pool (Hlyin).…”

Section: Genetic Aspectsmentioning

confidence: 99%

Escherichia coli alpha-hemolysin: characteristics and probable role in pathogenicity.

Cavalieri¹,

Bohach²,

Snyder³

1984

Microbiological Reviews

170

106

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HISTORICAL PERSPECTIVE Kayser in 1903 reported that some E. (oli cultures lysed erythrocytes (RBC) (55). He noted that the culture supernatants retained hemolytic activity after being filtered through a Chamberland filter. Dudgeon and Pulvertaft in 1927 also reported hemolytic activity in E. coli cultures (19) but could not demonstrate hemolysin in culture filtrates. They assumed that all activity was associated with the E. coli cell. Several other reports confirmed the existence of cell-associated hemolytic activity (2, 19, 20, 51, 81, 86). Kayser's report of a filterable hemolysin was confirmed in 1960 when Lovell and Rees (64) obtained a bacteria-free hemolysin preparation by filtration of cultures grown in alkaline meat infusion broth. Smith in 1963 was the first to clearly differentiate cellbound and cell-free hemolysin in cultures of E. (oli grown in alkaline meat extract broth (88). He showed that under the same growth conditions some hemolytic strains of E. coli produce cell-free and cell-bound hemolysin simultaneously. Cell-bound hemolysin was not neutralized by antiserum prepared against the cell-free hemolysin, indicating that the two hemolysins might be different. Smith designated the cell-bound hemolytic factor as P-hemolysin and the cell-free factor as oa-hemolysin (AH). This was perhaps an unfortunate designation and has caused some confusion since both the ot-and ,-hemolysins cause 3-hemolysis (clear zone of lysis) around colonies on blood agar plates. Walton and Smith (113) found a third hemolysin (yhemolysin) produced by mutants resistant to nalidixic acid. Hemolysin production by these mutants is enhanced by growth in nalidixic acid. Unlike the cxand 3-hemolysins, the 326

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“…Investigation of the haemolysin secretion system (Goebel and Hedgepeth. 1982;Stark and Schuster. 1983;Welch et ai, 1983;Mackman and Holland.…”

Section: Introductionmentioning

confidence: 99%

Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or β‐galactosidase fused to the Hly C‐terminal signal domain

Kenny

Haigh

Holland

1991

Molecular Microbiology

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Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA, when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the beta-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain.

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The structure of cloned hemolysin DNA from plasmid pHly185

Cited by 16 publications

References 19 publications

The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli

The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli

Escherichia coli alpha-hemolysin: characteristics and probable role in pathogenicity.

Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or β‐galactosidase fused to the Hly C‐terminal signal domain

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