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Metalloendopeptidase QG. Isolation from Escherichia coli and characterization.

ABSTRACT: A new proteinase, which preferentially cleaves the Gln-Gly bond, was isolated from Escherichia coli. Because of this narrow specificity, the enzyme was called metalloendopeptidase QG. The proteinase is a monomer and consists of a single polypeptide chain of Mr 67,000, which is significantly smaller than the other known metalloendopeptidases of E. coli. It is found in the cytoplasm, but not in the periplasm. The enzyme cleaves the substrate benzyloxycarbonyl-Gln-Gly-Pro 2-naphthylamide between the glutamine and glycine residues, as well as its extended homologues including a nonapeptide, but it does not hydrolyse either the oxidized A and B chains of insulin or azo-casein. The pH-dependence of substrate hydrolysis gives a bell-shaped curve with pK1 = 6.6 and pK2 = 8.8. The metallopeptidase is inhibited in Tris and imidazole buffers, the basic components of which are presumably liganded to the essential Zn2+ ion. 2-Aminobenzoyl-Gln-Gly-Pro 2-naphthylamide, designed as a fluorescent substrate for the metallopeptidase, proved to be a strong inhibitor. Bestatin, an inhibitor of aminopeptidases in the micromolar concentration range, inhibits the metalloendopeptidase only in the millimolar concentration range. Captopril, the widely used inhibitor of angiotensin-converting enzyme, is a fairly good inhibitor of the metalloendopeptidase. The simplest inhibitor that can be used to protect recombinant proteins from degradation by the metalloendopeptidase may be EDTA, which is effective at low millimolar concentration.

SUBMITTER: Polgar L

PROVIDER: S-EPMC1149823 | biostudies-other | 1991 Feb

REPOSITORIES: biostudies-other

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Project description:Escherichia coli O104:H4, an hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, involved in a major foodborne outbreak in Germany in 2011, has not been detected in cattle feces. Serogroup O104 with H type other than H4 has been reported to cause human illnesses, but their prevalence and characteristics in cattle have not been reported. Our objectives were to determine the prevalence of E. coli O104 in feces of feedlot cattle, by culture and PCR detection methods, and characterize the isolated strains. Rectal fecal samples from a total of 757 cattle originating from 29 feedlots were collected at a Midwest commercial slaughter plant. Fecal samples, enriched in E. coli broth, were subjected to culture and PCR methods of detection. The culture method involved immunomagnetic separation with O104-specific beads and plating on a selective chromogenic medium, followed by serogroup confirmation of pooled colonies by PCR. If pooled colonies were positive for the wzxO104 gene, then colonies were tested individually to identify wzxO104-positive serogroup and associated genes of the hybrid strains. Extracted DNA from feces were also tested by a multiplex PCR to detect wzxO104-positive serogroup and associated major genes of the O104 hybrid pathotype. Because wzxO104 has been shown to be present in E. coli O8/O9/O9a, wzxO104-positive isolates and extracted DNA from fecal samples were also tested by a PCR targeting wbdDO8/O9/O9a, a gene specific for E. coli O8/O9/O9a serogroups. Model-adjusted prevalence estimates of E. coli O104 (positive for wzxO104 and negative for wbdDO8/O9/O9a) at the feedlot level were 5.7% and 21.2%, and at the sample level were 0.5% and 25.9% by culture and PCR, respectively. The McNemar's test indicated that there was a significant difference (P < 0.01) between the proportions of samples that tested positive for wzxO104 and samples that were positive for wzxO104, but negative for wbdDO8/O9/O9a by PCR and culture methods. A total of 143 isolates, positive for the wzxO104, were obtained in pure culture from 146 positive fecal samples. Ninety-two of the 143 isolates (64.3%) also tested positive for the wbdDO8/O9/O9a, indicating that only 51 (35.7%) isolates truly belonged to the O104 serogroup (positive for wzxO104 and negative for wbdDO8/O9/O9a). All 51 isolates tested negative for eae, and 16 tested positive for stx1 gene of the subtype 1c. Thirteen of the 16 stx1-positive O104 isolates were from one feedlot. The predominant serotype was O104:H7. Pulsed-field gel electrophoresis analysis indicated that stx1-positive O104:H7 isolates had 62.4% homology to the German outbreak strain and 67.9% to 77.5% homology to human diarrheagenic O104:H7 strains. The 13 isolates obtained from the same feedlot were of the same PFGE subtype with 100% Dice similarity. Although cattle do not harbor the O104:H4 pathotype, they do harbor and shed Shiga toxigenic O104 in the feces and the predominant serotype was O104:H7.

Project description:BackgroundAntibiotic resistance in Escherichia coli, a member of the Enterobacteriaceae, is of particular concern because it is the most common (Gram-negative) pathogen causing nosocomial and community infections. Researchers are now considering the use of phages for the control of various antibiotic-resistant bacterial infections.ObjectivesThe purpose of this study was to isolate and characterize a novel pathogenic/lytic phage that targets multi-drug resistant (MDR) E. coli 3, and to investigate its effectiveness at lysing this bacterium.Materials and methodsA clinical strain of E. coli 3 was identified based on its 16S rRNA sequencing and its antibiotic resistance profile was determined by the disc diffusion method. A bacteriophage was isolated from wastewater and its various characteristics, such as host range, heat tolerance, pH stability, one step growth, total protein content, and genome size, were determined. The antibacterial property of the phage was determined against log-phase bacterial planktonic cells at 37°C.ResultsThe bacteriophage, designated MJ1, was isolated by testing against a clinical MDR E. coli 3 strain. The MJ1 phage showed a wide range of heat and pH stability. The phage morphology, determined by transmission electron microscopy, revealed a structure comprised of a head (108 ± 0.2 nm long by 128 ± 0.5 nm wide) and a contractile tail (123 ± 0.5 nm long by 15 - 26 nm wide). These features placed the MJ1 phage in the family Myoviridae and the order Caudovirales. Eleven structural proteins (17 to 200 kDa) for this phage were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A double stranded DNA, approximately 32 kb, in size was detected for this phage on agarose gels. The phage efficacy against E. coli 3 planktonic cells was also investigated. The MJ1 phage demonstrated a very good capability to reduce the numbers of E. coli 3 planktonic cells, as determined by a change in the bacterial growth (an optical density decrease at 600 nm from 0.40 to 0.12).ConclusionsMJ1 phage has much potential for use in phage therapy and other applications.

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Metalloendopeptidase QG. Isolation from Escherichia coli and characterization.

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