Occurrence of secondary transpositions of Tn5 upon Tn5 mutagenesis inEscherichia coli

When Tn5 insertions were obtained in thehha gene ofEscherichia coli HB101 harboring the hemolytic multicopy plasmid pANN202-312, most of thehha mutants obtained that produced larger amounts of hemolysin than the wild-type cells segregated into 10 percent of clones, which did not further produce hemolysin. We demonstrate here that a secondary transposition of Tn5 intohlyA, the structural gene for hemolysin, was responsible for such phenotype.     

Purification and partial characterization of a bacteriocin produced by Eikenella corrodens

Aims: The purpose of this study was to purify and characterize a bacteriocin produced by Eikenella corrodens A32E2. Methods and Results: Peptostreptococcus anaerobius ATCC27337 was used as indicator strain in antagonistic assays for bacteriocin‐producing E. corrodens A32E2. Protein extraction was influenced by pH and buffer composition. The protein was active in the pH range 6–8. Inhibitory activity was lost by both heating and treatment with proteolytic enzymes and decreased with organic solvents. The substance is rather unstable but maintains 100% of its activity after being exposed to acetone and when stored at ?70°C. The antagonistic substance was first precipitated by ammonium sulfate and further partially purified by Mono‐Q FPLC and C‐18 HPLC. Mass spectrometry analysis showed that the molecular mass was 23 625 Da, and the sequence obtained for the N‐terminus was: Met‐Asn‐Phe‐Asp‐Glu‐Lys‐Val‐Gly‐Lys‐Val‐X‐Phe‐Lys‐Val‐Gly‐Asp. Conclusions: The evidence presented in this study supports the idea that an antagonistic substance produced by E. corrodens A32E2 isolated from a periodontal diseased site is a novel bacteriocin, which we designate corrodecin. Significance and Impact of the Study:  We anticipated that corrodecin might play an important role at the periodontal site. This compound could also be attractive in biotechnological applications as an interesting tool for oral ecosystem control.     

Analysis of the Structural Gene Encoding a Hemolysin in Vibrio mimicus

An environmental isolate of V. mimicus, strain E-33, has been reported to produce and secrete a hemolysin of 63 kDa. The hemolysin is enterotoxic in test animals. The nucleotide sequence of the structural gene of the hemolysin was determined. We found a 2,232 bp open reading frame, which codes a peptide of 744 amino acids, with a calculated molecular weight of 83,903 Da. The sequence for the structural gene was closely related to the V. cholerae el tor hlyA gene, coding an exocellular hemolysin. The amino terminal amino-acid sequence of the 63 kDa hemolysin, purified from V. mimicus, was determined by the Edman degradation method and found to be NH₂-S-V-S-A-N-N-V-T-N-N-N-E-T. This sequence is identical from S-152 to T-164 predicted from the nucleotide sequence. So, it seems that the mature hemolysin in V. mimicus is processed upon deleting the first 151 amino acids, and the molecular mass is 65,972 Da. Analyzing the deduced amino-acid sequence, we also found a potential signal sequence of 24 amino acids at the amino terminal. Our results suggest that, like V. cholerae hemolysin, two-step processing also exists in V. mimicus hemolysin.     

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS

The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli. Received. 18 October 1999 / Accepted: 21 December 1999     

Molecular cloning,characterization, and sequencing of the hemolysin gene from Edwardsiella tarda

Hemolysis is a major symptom of diseased eels infected by Edwardsiella tarda. The hemolysin gene of E. tarda strain ET16 was cloned into plasmid pSK and expressed in Escherichia coli. The mol. mass of the functional β-hemolysin was estimated to be approximately 34 kDa by gel filtration and by SDS-PAGE followed by in situ hemolysin activity analysis. The cloned fragment containing the β-hemolysin locus from E. tarda strain ET16 expressed in E. coli was estimated to be 5.3 kb in length; the deduced gene product was identical in mol. mass and properties to the extracellular products of E. tarda strain ET16. The presence of EcoRI and XbaI sites within the β-hemolysin gene of E. tarda was determined from the loss of hemolytic activity in subclones. Analysis of the DNA sequence of a 2,436-bp HaeIII-HindIII fragment that included EcoRI and XbaI sites revealed three ORFs organized as an operon that encoded three predicted polypeptides of 15,874, 7,055, and 34,804 Da. A 34-kDa polypeptide expressing hemolytic activity in cell lysates of the clone DH5α(pETH3E) is very likely the β-hemolysin encoded by the third ORF. The observation that hemolytic activity appeared in the culture medium of E. tarda, but not in that of E. coli strain DH5α(pETH3E) indicates the existence of a mechanism for transporting the hemolysin across the cell envelope in E. tarda that is different from that of E. coli. Received: 7 July 1995 / Accepted: 24 October 1995     

Purification and Identification of a Novel Leucine Aminopeptidase from <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65°C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65°C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase’s effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.     

Isolation of a novel promoter for efficient protein expression by <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> in solid-state culture

A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.     

Evidence for the production of a proteinaceous hemolytic exotoxin by wild-type strain of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 (Cyanobacteria)

A proteinaceous hemolysin produced by a wild-type strain of Synechocystis sp. PCC 6803 was purified from cell-free culture supernatants by successive column chromatography on DEAE-Sepharose Fast Flow and Sephacryl S-300 High Resolution. The molecular mass of the hemolysin, determined by SDS-PAGE, was approximately 81 kDa. The hemolysin was heat labile and showed potent hemolytic activity against rabbit and sheep erythrocytes. The hemolysin started to be secreted during the exponential growth phase and accumulated maximally at the stationary phase. The production of hemolysin varied with the amount of calcium present in modified BG-11 culture medium. Hemolysin production decreased in calcium-free medium, whereas it increased in medium containing 0.48 mM calcium. In contrast, the potency of hemolysin, as shown by hemolysis assay, was enhanced by deprivation of calcium (EDTA treatment) but decreased in the presence of calcium. Our results show that calcium stimulated production and secretion of hemolysin, but inhibited hemolytic potency.     

Processing by OmpT of fusion proteins carrying the HlyA transport signal during secretion by the Escherichia coli hemolysin transport system.

Summary A fusion gene (ces-hlyA _s) was constructed by ligating the genetic information for the C-terminal 60 amino acids (hlyA _s) ofEscherichia coli hemolysin (H1yA) to the ces gene for a cholesterol esterase/lipase (CE) from aPseudomonas species. Part (about 30 %) of the expressed fusion protein CE-H1yA_s was secreted inE. coli carryinghlyB andhlyD genes. Following the insertion between the reporter gene andhlyA _s of a linker sequence that contains the information for potential cleavage sites for the outer membrane protease OmpT, two different fusion proteins (PhoA-H1yA_s and CE-HlyA_s) were shown to be cleaved by OmpT between the two parts during H1yB/H1yD-mediated secretion. Processed PhoA and CE accumulated in the supernatant. The efficiency of cleavage by OmpT was considerably improved by increasedompT gene dose. It was further shown that OmpT preferentially recognizes potential cleavage sites within the linker sequence.     

Key Odorants in Cured Madagascar Vanilla Beans (Vanilla planiforia) of Differing Bean Quality

The effects of pressure on the enzyme activities of Lacto-bacillus helveticus LHE-511 were examined. Treatment at 400 MPa at 30°C for 10 min completely inhibited acid-producing activity, but the activities of aminopeptidase (AP) and X-prolyl dipeptidyl aminopeptidase (X-PDAP), which are important for acceleration of cheese ripening, increased. These results showed that pressure treatment of L. helveticus LHE-511 selectively inactivates its acid-producing activity.     

Dipeptidyl Aminopeptidase IV from Pseudomonas sp. WO24

Dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24 was purified as two molecular forms of 84 and 82-kDa by SDS–PAGE. Peptide mapping and N-terminal sequence analyses indicated that both proteins might be derived from the same protein, and that the 82-kDa molecule might be a truncated form from the 84-kDa molecule at least at the N-terminus. The DAP IV gene of Pseudomonas sp. WO24 was cloned and expressed in E. coli. The enzyme expressed in E. coli JM109 harboring a hybrid plasmid, pYO-6A, with about a 3-kbp fragment containing the DAP IV gene, was purified with an activity recovery of 24%. The recombinant enzyme also had the same two molecular forms, though the ratio of the two forms (about 1:1) was different from that of the native ones (about 1:4). The native and recombinant enzyme preparations had similar specific activities, suggesting that the 84 and 82-kDa molecules are in an active form and have almost the same specific activity. The molecular mass, the subunit number, the substrate specificity, and the effects of various inhibitors of the native enzyme indicated that this enzyme was a typical DAP IV and had properties similar to those of Flavobacterium meningosepticum rather than others.     

Genomic Recombination through Plasmid-Encoded Recombinase Enhances Hemolytic Activity and Adherence to Epithelial Cells in the Periodontopathogenic Bacterium Eikenella corrodens

The periodontopathogenic bacterium Eikenella corrodens has an N-acetyl-D-galactosamine (GalNAc)-specific lectin, that contributes significantly to the pathogenicity of the bacterium. Recently, we reported that plasmid-mediated genomic recombination enhances the activity of this lectin. In this study, we investigated the effects of genomic recombination on certain virulence factors. Introduction of the recombinase gene resulted in hemolysis and significantly increased bacterial adhesion to epithelial cells. It was suggested that the enhanced adhesion was attributable to increased lectin activity due to genomic recombination, because it was inhibited by the addition of GalNAc. In contrast, invasion of the epithelial cells was remarkably reduced by genomic recombination. Although we assumed that this decrease in invasion resulted from a loss of type-IV pili, the phase variant did not show any decrease in invasion activity. This suggests that type-IV pili do not contribute to the invasive ability of E. corrodens. Our results suggest that genomic recombination enhances the pathogenicity of E. corrodens.     

Efficient production of endotoxin depleted bioactive α-hemolysin of uropathogenic Escherichia coli

Abstract

Uropathogenic E. coli (UPEC), especially associated with severe urinary tract infections (UTI) pathologies, harbors an important virulence factor known as α-hemolysin (110?kDa). Hemolytic activity of α-hemolysin (HlyA) requires modification (acylation) of two lysine residues of HlyA by HlyC, part of operon hlyCABD. Most of the previous studies had used whole operon hlyCABD and gene tolC cloning for the production of active α-hemolysin. Studies involving α-hemolysin are limited due to the cumbersome and manual method of purification for this toxin. Here, we report a simple method for production of both active and inactive recombinant α-hemolysin by cloning only hlyA and hlyC genes of operon hlyCABD. Presence of both active and inactive α-hemolysin would be advantageous for functional characterization. After translation, the yield of the purified α-hemolysin was 1?mg/200?ml. Functionality of the recombinant α-hemolysin protein was confirmed using hemolytic assay. This is the first report of the production of active and inactive recombinant α-hemolysin for functional studies.     

Partial Purification and Characterization of Hemolysin from a Psychrotrophic Kanagawa-Positive Marine Vibrio

Psychrotrophic Kanagawa-positive marine vibrios were isolated from soft-shelled clams (Mya arenaria) collected in Yaquina Bay, Oreg. The 235 vibrio isolates obtained were screened for Gram reaction and morphology, Kanagawa reaction on Wagastsuma agar, and response to selected biochemical tests. The vibrio selected for further study was grown in broth, and the hemolysin was precipitated from a cleared supernatant with solid ammonium sulfate. The hemolytic substance was partially purified by DEAE-cellulose and Sephadex G-100 column chromatography. The hemolysin contained protein essential for activity, was thermolabile, and was more active against rabbit erythrocytes at 37°C than at lower temperatures. The molecular weight was estimated at 55,000 by using a Sephadex G-100 column. Hemolytic activity was partially inactivated by gangliosides and lowered against horse erythrocytes. The hemolysin did not react with antibody prepared against vibriolysin from Vibrio parahaemolyticus WP-1 by the Ouchterlony method. The hemolysin was high in aspartic and glutamic acids and low in arginine and histidine. Electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel gave three major bands. The hemolysin from a psychrotrophic vibrio and the hemolytic exotoxin of V. parahaemolyticus had some similar and dissimilar characteristics. The possibility that a Vibrio sp. other than V. parahaemolyticus might serve as the reservoir for the Kanagawa phenotype is discussed.     

Molecular cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. cremoris.

Lactococcus lactis subsp. cremoris P8-2-47 contains an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). A mixed-oligonucleotide probe prepared on the basis of the N-terminal amino acid sequence of the purified protein was made and used to screen a partial chromosomal DNA bank in Escherichia coli. A partial XbaI fragment cloned in pUC18 specified X-PDAP activity in E. coli clones. The fragment was also able to confer X-PDAP activity on Bacillus subtilis. The fact that none of these organisms contain this enzymatic activity indicated that the structural gene for X-PDAP had been cloned. The cloned fragment fully restored X-PDAP activity in X-PDAP-deficient mutants of L. lactis. We have sequenced a 3.8-kb fragment that includes the X-PDAP gene and its expression signals. The X-PDAP gene, designated pepXP, comprises 2,289 nucleotide residues encoding a protein of 763 amino acids with a predicted molecular weight of 87,787. No homology was detected between pepXP and genes that had been previously sequenced. A second open reading frame, divergently transcribed, was present in the sequenced fragment; the function or relationship to pepXP of this open reading frame is unknown.     

Molecular cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. cremoris.

Lactococcus lactis subsp. cremoris P8-2-47 contains an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). A mixed-oligonucleotide probe prepared on the basis of the N-terminal amino acid sequence of the purified protein was made and used to screen a partial chromosomal DNA bank in Escherichia coli. A partial XbaI fragment cloned in pUC18 specified X-PDAP activity in E. coli clones. The fragment was also able to confer X-PDAP activity on Bacillus subtilis. The fact that none of these organisms contain this enzymatic activity indicated that the structural gene for X-PDAP had been cloned. The cloned fragment fully restored X-PDAP activity in X-PDAP-deficient mutants of L. lactis. We have sequenced a 3.8-kb fragment that includes the X-PDAP gene and its expression signals. The X-PDAP gene, designated pepXP, comprises 2,289 nucleotide residues encoding a protein of 763 amino acids with a predicted molecular weight of 87,787. No homology was detected between pepXP and genes that had been previously sequenced. A second open reading frame, divergently transcribed, was present in the sequenced fragment; the function or relationship to pepXP of this open reading frame is unknown.     

Studies on the factors affecting the growth and hemolytic activity of <Emphasis Type="Italic">Anabaena variabilis</Emphasis>

The hemolytic activity of the cell-free culture supernatant of Anabaena variabilis OL S1 was investigated using the hemolysis of rabbit erythrocytes as an assay. The culture medium of A. variabilis started to exhibit hemolytic activity at the late exponential growth phase, and maximized at the stationary phase. The hemolytic toxin is heat-stable and can be extracted in dichloromethane. The hemolytic activities under different temperature, light intensity and pH showed a high correlation with the cell densities (r=0.965, 0.951, 0.865, respectively), and the optimum condition is 28~30°C, pH 7.5~8.0, light intensity 120 μmol photons m⁻²s⁻¹. The addition of 10~20 μg mL⁻¹ chloramphenicol, an inhibitor of protein synthesis, exhibited no marked suppression on the hemolytic activity. The supplement of 1~20 μg mL⁻¹ glycerol increased the hemolytic activity significantly, suggesting that synthesis of hemolysin was dependent on carbohydrate and lipid metabolism. The spectrum of erythrocyte sensitivity to the hemolysin indicated that rabbit erythrocytes were more sensitive to the hemolysin than were rat and human erythrocytes. Goldfish and cat erythrocytes were, however, insensitive to the hemolytic toxin of A. variabilis.     

地衣芽孢杆菌E7氨肽酶的异源表达及其在高效蛋白水解中的应用

【目的】将地衣芽孢杆菌(Bacilluslicheniformis)E7氨肽酶基因pepN克隆到大肠杆菌(Escherichia coli) BL21中,实现氨肽酶Ec PepN的异源表达,研究重组酶的酶学性质及其与碱性蛋白酶协同作用,高效水解大豆蛋白和酪蛋白,产生小分子活性肽和游离氨基酸。【方法】以地衣芽孢杆菌E7基因组DNA为模板,将氨肽酶基因pepN克隆到载体pET28a中,构建重组表达载体pET28-pepN,转化到大肠杆菌BL21感受态细胞中,经DNA测序验证,获得重组菌E. coli BL21/pET28-pepN。利用镍离子亲和层析柱对重组酶进行分离纯化,研究纯酶的pH和温度稳定性、半衰期和NaCl的耐受性等酶学性质。以商品化氨肽酶与碱性蛋白酶协同作用为对照,重组酶Ec PepN与碱性蛋白酶协同水解大豆蛋白和酪蛋白,测定水解产物中小分子活性肽和游离氨基酸的组成。【结果】Ec PepN在大肠杆菌BL21中可溶性表达,SDS-PAGE分析表明纯化的重组酶在52kDa左右显示单一条带。在7种测定底物中,Ec PepN的最适底物为Ala-pNA。在最适条件(pH 9.0和50°C...     

Efficient production of active Vibrio proteolyticus aminopeptidase in Escherichia coli by co-expression with engineered vibriolysin

The Vibrio proteolyticus aminopeptidase is synthesized as a preproprotein and then converted into an active enzyme by cleavage of the N-terminal propeptide. In recombinant Escherichia coli, however, the aminopeptidase is not processed correctly and the less-active form that has the N-terminal propeptide accumulates in the culture medium. Recently, we isolated a novel vibriolysin that was expressed as an active form in E. coli by random mutagenesis; this enzyme shows potential as a candidate enzyme for the processing of aminopeptidase. The E. coli cells were engineered to co-express the novel vibriolysin along with aminopeptidase. Co-expression of vibriolysin resulted in an approximately 13-fold increase in aminopeptidase activity, and a further increase was observed in the form lacking its C-terminal propeptide. The active aminopeptidase was purified from the culture supernatant including the recombinant vibriolysin by heat treatment and ion exchange and hydroxyapatite chromatography with high purity and 35% recovery rate. This purified aminopeptidase effectively converted methionyl-human growth hormone (Met-hGH) to hGH. Thus, this co-expression system provides an efficient method for producing active recombinant V. proteolyticus aminopeptidase.