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Cloning, and expression in Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa. 总被引:15,自引:7,他引:8
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A hemolysin determinant was cloned from Pseudomonas aeruginosa PA103 by inserting Sau3a-generated DNA fragments between the BamHI sites of the lambda replacement vector WL47.1. A 9.5-kilobase HindIII fragment encoding the hemolysin was subcloned from this phage and inserted into the plasmid vector pHC79 to generate the recombinant plasmid pKC95. Escherichia coli K-12 strains harboring pKC95 exhibited zones of hemolysis after several days of growth on blood agar plates. Hemolysis was shown to be due to phospholipase C activity by using the chromogenic substrate p-nitrophenylphosphorylcholine. Deletion mutants of pKC95 were isolated, and polypeptides expressed from these plasmids were examined by using the E. coli minicell system. A polypeptide of 78,000 daltons was associated with phospholipase C activity. The hemolytic activity was cell associated when expressed in E. coli. 相似文献
3.
Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli. 总被引:53,自引:18,他引:35
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We cloned the DNA containing the Escherichia coli hemolysin determinant on a small, high-copy plasmid. We generated plasmids containing fragments of this DNA and used them either alone or in two-plasmid complementation systems to define the limits of the structural genes. This system also allowed us to partially characterize the function of each of the gene products in the production and transport of hemolysin. Taken with previously published data, the present experiments indicate the following. (i) At least three cistrons, hlyC, hlyA, and hlyB (these were previously designated cisC, etc. [Noegel et al., Mol. Gen. Genet. 175:343-350, 1979]), contain the specific genetic information for the hemolytic phenotype, (ii) hlyA encodes a 107,000-kilodalton protein, which seems to be an inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity require only the products of hlyA and hlyC. This activity was found in the periplasm; very little hemolysin activity was found in the cytoplasm, suggesting that the hlyC product is required for transport or activation of the hlyA product or both. (iv) Active hemolysin remains in the periplasm in the absence of hlyB function, hence the hlyB product seems to be necessary for the transport of hemolysin to the exterior of the cell. We further show that overproduction of the hlyA product is lethal, probably causing lysis of the cell. 相似文献
4.
Identification of a new hemolysin from diarrheal isolate SSU of Aeromonas hydrophila 总被引:2,自引:0,他引:2
Erova TE Sha J Horneman AJ Borchardt MA Khajanchi BK Fadl AA Chopra AK 《FEMS microbiology letters》2007,275(2):301-311
A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene (hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine beta synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence. 相似文献
5.
Active and inactive forms of hemolysin (HlyA) from Escherichia coli 总被引:11,自引:0,他引:11
The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W. & Hedgpeth, J. (1982) J. Bacteriol. 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle. However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase. External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication. The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished. On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA. Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes. Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein. An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product. This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase. 相似文献
6.
Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins. 总被引:10,自引:5,他引:5
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R L Oropeza-Wekerle E Müller P Kern R Meyermann W Goebel 《Journal of bacteriology》1989,171(5):2783-2788
Extra- and intracellular Escherichia coli hemolysin expressed by two cloned hly determinants, both under the control of the activator element hlyR, were analyzed. One determinant carried all four hly genes (hlyC, hlyA, hlyB, and hlyD), whereas the other carried only the two genes (hlyC and hlyA) required for synthesis of active hemolysin but not those essential for its secretion. It was shown that the total amounts of HlyA protein and of hemolytic activity are similar in both cases in logarithmically growing cultures. The E. coli strain carrying the complete hly determinant released most hemolysin into the media and accumulated very little HlyA intracellularly. The active extracellular hemolysin (HlyA*) was inactivated in the stationary phase without degradation of the HlyA protein. In contrast, the hemolysin which accumulated intracellularly in the E. coli strain carrying hlyA and hlyC only was proteolytically degraded at the end of the logarithmic growth phase. Immunogold labeling indicates that active intracellular HlyA bound preferentially to the inner membrane, whereas that part of the extracellular HlyA which remained cell-bound was located exclusively at the cell surface. It was shown by fluorescence-activated cell sorter analysis that active extra- and intracellular HlyA* bound with similar efficiency to erythrocytes, whereas hemolytically inactive HlyA protein did not bind to these target cells. 相似文献
7.
Relationship between plasmid and chromosomal hemolysin determinants of Escherichia coli 总被引:19,自引:6,他引:13
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Plasmid hemolysin (hly) determinants have been shown previously to comprise three cistrons (hlyA, hlyB, hlyC), coding for the synthesis and transport of hemolysin. Using recombinant plasmids as specific probes for these cistrons, we were able to analyze the chromosomal hly determinants of nine Escherichia coli strains which belonged to serotypes O4, O6, O18, and O75 and were isolated from urinary tract infections and fecal flora. The chromosomal hly genes shared extensive sequence homology with the cloned plasmid hly determinant. Nevertheless, small differences were observed, and these were found to lie mainly within cistron A (hlyA), which has been shown to determine the hemolysin protein itself. These fine variations were not specific for the O-serotype. 相似文献
8.
Transport of hemolysin by Escherichia coli 总被引:25,自引:0,他引:25
Michael Hrtlein Sigrid Schießl Wilma Wagner Ursula Rdest Jürgen Kreft Werner Goebel 《Journal of cellular biochemistry》1983,22(2):87-97
The hemolytic phenotype in Escherichia coli is determined by four genes. Two (hlyC and hlyA) determine the synthesis of a hemolytically active protein which is transported across the cytoplasmic membrane. The other two genes (hlyBa and hlyBb) encode two proteins which are located in the outer membrane and seem to form a specific transport system for hemolysin across the outer membrane. The primary product of gene hlyA is a protein (protein A) of 106,000 daltons which is nonhemolytic and which is not transported. No signal peptide can be recognized at its N-terminus. In the presence of the hlyC gene product (protein C), the 106,000-dalton protein is processed to the major proteolytic product of 58,000 daltons, which is hemolytically active and is transported across the cytoplasmic membrane. Several other proteolytic fragments of the 106,000-dalton protein are also generated. During the transport of the 58,000-dalton fragment (and possible other proteolytic fragments of hlyA gene product), the C protein remains in the cytoplasm. In the absence of hlyBa and hlyBb the entire hemolytic activity (mainly associated with the 58,000-dalton protein) is located in the periplasm: Studies on the location of hemolysin in hlyBa and hlyBb mutants suggest that the gene product of hlyBa (protein Ba) binds hemolysin and leads it through the outer membrane whereas the gene product of hlyBb (protein Bb) releases hemolysin from the outer membrane. This transport system is specific for E coli hemolysin. Other periplasmic enzymes of E coli and heterologous hemolysin (cereolysin) are not transported. 相似文献
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10.
Molecular characterization of the hemolysin determinant of Serratia marcescens. 总被引:24,自引:9,他引:15
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The nucleotide sequence of a 7.3-kilobase-pair fragment of DNA encoding a hemolytic activity from Serratia marcescens was determined. Two large open reading frames were identified, designated shlA (Serratia hemolysin) and shlB, capable of encoding polypeptides of 165, 056 and 61,897 molecular weight, respectively. Both reading frames were expressed in vivo. The shlB gene product was localized to the outer membrane of Escherichia coli cells harboring the S. marcescens hemolysin determinant. Consistent with this location, a signallike sequence was identified at the N terminus of the polypeptide predicted from the nucleotide sequence of the shlB gene. Hyperexpression of the shlB locus permitted the identification of two shlB-encoded polypeptides of 65,000 and 62,000 molecular weight, respectively. Determination of the N-terminal amino acid sequence of the purified 62,000-molecular-weight protein confirmed that it was the mature form of the ShlB protein initially synthesized as a precursor (65,000-molecular-weight protein). By using polyclonal antisera raised against the purified proteins, ShlA and ShlB were identified in the outer membrane of S. marcescens. The shlA gene product was shown to interact with erythrocyte membranes, confirming it as the hemolysin proper. Both hemolysis and the interaction of ShlA with erythrocyte membranes did, however, require the ShlB function. Progressive deletion of the C terminus of the ShlA protein gradually reduced hemolytic activity until 37% of the amino acids had been removed. Elimination of 54% of the amino acids produced a nonhemolytic protein which, however, was still capable of associating with erythrocyte membranes. 相似文献
11.
Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli. 总被引:12,自引:5,他引:7
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The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated. No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes. However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR. Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain. For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX. Four cysteine residues in the amino-terminal region are also conserved. The promoter region of hlyX is very similar to that of fnr. It has a putative -10 sequence which closely resembles the E. coli -10 consensus sequence. This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence. Functional homology between HlyX and FNR was also demonstrated. Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E. coli. These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E. coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded. 相似文献
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13.
RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria. 总被引:24,自引:21,他引:3
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The nonstructural protein NS3 of the prototypic flavivirus, yellow fever virus, was investigated for possession of an NTPase activity. The entire NS3 protein coding sequence and an amino-terminal truncated version thereof were engineered into Escherichia coli expression plasmids. Bacteria harboring these plasmids produced the expected polypeptides, which upon cell disruption were found in an insoluble aggregated material considerably enriched for the NS3-related polypeptides. Solubilization and renaturation of these materials, followed by examination of their ability to hydrolyze ATP, revealed an ATPase activity present in both the full-length and amino-terminal truncated NS3 preparations but not in a similarly prepared fraction from E. coli cells engineered to express an unrelated polypeptide. The amino-terminal truncated NS3 polypeptide was further enriched to greater than 95% purity by ion-exchange and affinity chromatography. Throughout the purification scheme, the ATPase activity cochromatographed with the recombinant NS3 polypeptide. The enzymatic activity of the purified material was shown to be a general NTPase and was dramatically stimulated by the presence of particular single-stranded polyribonucleotides. These results are discussed in view of similar activities identified for proteins of other positive-strand RNA viruses. 相似文献
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15.
Campylobacter concisus is an opportunistic pathogen commonly found in the human oral cavity. It has also been isolated from clinical sources including gastroenteritis cases. Both secreted and cell-associated hemolytic activities were detected in C. concisus strains isolated from children with gastroenteritis. The secreted hemolytic activity of C. concisus strains was labile and was detected in variable levels from fresh-culture filtrates only. In addition, another secreted hemolysin/cytotoxin with a molecular weight < 10 kDa was detected in a single C. concisus strain (RCH 12). A C. concisus genomic library, constructed from strain RCH 3 in Escherichia coli XL1-Blue, was screened for hemolytic clones. Subcloning and sequence analysis of selected hemolytic clones identified ORFs for genes that enhance hemolytic activity but do not appear to be related to any known hemolysin genes found in Gram-negative bacteria. In a previous study, a stable cell-associated hemolysin was identified as an outer-membrane phospholipase A (OMPLA) encoded by the pldA gene. In this study, we report cloning of the pldA gene of the clinical strain C. concisus RCH 3 and the complementation of phospholipase A activity in an E. coli pldA mutant. 相似文献
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17.
Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli. 总被引:31,自引:13,他引:18
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We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes O4, O6, O18, and O75. The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further subcloned either as SalI fragments in pACYC184 or as BamHI-SalI fragments in a recombinant plasmid (pANN202) containing cistron C (hlyC) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure. These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency. 相似文献
18.
Three cDNA clones encoding the closely related glutamine synthetase (GS) alpha, beta and gamma polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli. The GS expression plasmids correctly synthesised the recombinant alpha, beta and gamma polypeptides which then assembled into catalytically active homo-octameric isoenzymes. These isoenzymes behaved similarly to their native homologues on ion-exchange and gel-filtration chromatography. Furthermore, the alpha and gamma isoenzymes complemented a GS(glnA)-deficient mutant, thus demonstrating their physiological activity in E. coli. Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E. coli mutant. These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression. The production of active GS isoenzymes in E. coli facilitates the isolation and characterisation of the individual P. vulgaris homo-octameric GS isoenzymes. 相似文献
19.
Secretion of the Bordetella pertussis adenylate cyclase from Escherichia coli containing the hemolysin operon 总被引:12,自引:0,他引:12
The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H.R., & Storm, D.R. (1989) biochemistry 28, 438-442]. The gene encoding this enzyme, cyaA, is part of the cya operon that also includes the genes cyaB, cyaD, and cyaE. A comparison of the predicted amino acid sequences encoded by cyaA, cyaB, and cyaD with the amino acid sequences encoded by hlyA, hlyB, and hlyD genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997-4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of HlyA (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, CyaB, CyaD, and CyaE are required for the secretion of CyaA from Bordetella pertussis. We placed the cyaA gene and a truncated cyaA gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cyaA gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
Expression and characterization of HlyX hemolysin from Leptospira interrogans serovar Copenhageni: potentiation of hemolytic activity by LipL32 总被引:2,自引:0,他引:2
Hauk P Negrotto S Romero EC Vasconcellos SA Genovez ME Ward RJ Schattner M Goméz RM Ho PL 《Biochemical and biophysical research communications》2005,333(4):1341-1347
The HlyX, a putative hemolysin identified from the Leptospira genomes, was cloned, expressed in Escherichia coli, purified, and its hemolytic activity was confirmed. Mouse polyclonal antiserum against the recombinant HlyX recognized HlyX-related antigens in a panel of Leptospira species extracts and it was also able to abolish the hemolytic activity of HlyX. A mixture of HlyX and LipL32, a known hemolysin from Leptospira, induced hemolysis in a synergistic way that was fully inhibited by antiserum against either protein. Moreover, sera from patients with leptospirosis also recognized the recombinant HlyX, showing that it is presented to the host immune system during Leptospira infection. 相似文献