Dataset Information

Uropathogenic specific protein gene, highly distributed in extraintestinal uropathogenic Escherichia coli, encodes a new member of H-N-H nuclease superfamily.

ABSTRACT:

Background

The uropathogenic specific protein (Usp) and three OrfU proteins (OrfU1, OrfU2 and OrfU3) are encoded in the putative small pathogenicity island which is closely associated with Uropathogenic Escherichia coli. Although homology search revealed that Usp and OrfUs have a homology with nuclease-type bacteriocins, which possess H-N-H nuclease motif, and immunity proteins respectively, the molecular activity of these proteins was never investigated. In this study, we try to over-express Usp in E. coli, purify Usp and characterize its molecular activity.

Method

Recombinant Usp protein was expressed in E. coli BL21(DE3) cells together with 6× Histidine tagged OrfU1 (OrfU1-His) protein, and purified with affinity chromatography using Ni2+ chelating agarose. The nuclease activity of the purified Usp was examined in vitro by using plasmid DNA as a substrate. The importance of H-N-H motif in nuclease activity of Usp was examined by site-directed mutagenesis study.

Results

We revealed that pET expression vector encoding Usp alone could not be maintained in E. coli BL21(DE3), and insertion of the orfUs as well as usp in the constructed plasmid diminished the toxic effect, suggesting that co-expressed OrfUs masked the activity of Usp. To purify Usp protein, we employed the expression vector encoding untagged Usp together with OrfU1-His. A tight complex formation could be observed between Usp and OrfU1-His, which allowed the purification of Usp in a single chromatographic step: binding of Usp/OrfU1-His complex to Ni2+ chelating agarose followed by elution of Usp from the complex with denaturing reagent. The purified free Usp was found to have the nuclease activity, and the activity was constitutively higher than Usp/OrfU1-His complex. H-N-H motif, which is found in various types of nucleases including a subfamily of nuclease-type bacteriocin, had been identified in the C-terminal region of Usp. Site-directed mutagenesis study showed that the H-N-H motif in Usp is indispensable for its nuclease activity.

Conclusion

This is the first evidence of the molecular activity of the new member of H-N-H superfamily and lays the foundation for the biological characterization of Usp and its inhibitor protein, OrfUs.

SUBMITTER: Zaw MT

PROVIDER: S-EPMC3685522 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Uropathogenic specific protein gene, highly distributed in extraintestinal uropathogenic Escherichia coli, encodes a new member of H-N-H nuclease superfamily.

Zaw Myo Thura MT Yamasaki Eiki E Yamamoto Shingo S Nair G Balakrish GB Kawamoto Keiko K Kurazono Hisao H

Gut pathogens 20130610 1

<h4>Background</h4>The uropathogenic specific protein (Usp) and three OrfU proteins (OrfU1, OrfU2 and OrfU3) are encoded in the putative small pathogenicity island which is closely associated with Uropathogenic Escherichia coli. Although homology search revealed that Usp and OrfUs have a homology with nuclease-type bacteriocins, which possess H-N-H nuclease motif, and immunity proteins respectively, the molecular activity of these proteins was never investigated. In this study, we try to over-ex ...[more]

PMID: 23759109

Similar Datasets

Project description:The impact of the Gram-negative bacterium Escherichia coli (E. coli) on the microbiomic and pathogenic phenomena occurring in humans and other warm-blooded animals is relatively well-recognized. At the same time, there are scant data concerning the role of E. coli strains in the health and disease of cold-blooded animals. It is presently known that reptiles are common asymptomatic carriers of another human pathogen, Salmonella, which, when transferred to humans, may cause a disease referred to as reptile-associated salmonellosis (RAS). We therefore hypothesized that reptiles may also be carriers of specific E. coli strains (reptilian Escherichia coli, RepEC) which may differ in their genetic composition from the human uropathogenic strain (UPEC) and avian pathogenic E. coli (APEC). Therefore, we isolated RepECs (n = 24) from reptile feces and compared isolated strains' pathogenic potentials and phylogenic relations with the aforementioned UPEC (n = 24) and APEC (n = 24) strains. To this end, we conducted an array of molecular analyses, including determination of the phylogenetic groups of E. coli, virulence genotyping, Pulsed-Field Gel Electrophoresis-Restriction Analysis (RA-PFGE) and genetic population structure analysis using Multi-Locus Sequence Typing (MLST). The majority of the tested RepEC strains belonged to nonpathogenic phylogroups, with an important exception of one strain, which belonged to the pathogenic group B2, typical of extraintestinal pathogenic E. coli. This strain was part of the globally disseminated ST131 lineage. Unlike RepEC strains and in line with previous studies, a high percentage of UPEC strains belonged to the phylogroup B2, and the percentage distribution of phylogroups among the tested APEC strains was relatively homogenous, with most coming from the following nonpathogenic groups: C, A and B1. The RA-PFGE displayed a high genetic diversity among all the tested E. coli groups. In the case of RepEC strains, the frequency of occurrence of virulence genes (VGs) was lower than in the UPEC and APEC strains. The presented study is one of the first attempting to compare the phylogenetic structures of E. coli populations isolated from three groups of vertebrates: reptiles, birds and mammals (humans).

Dataset Information

Uropathogenic specific protein gene, highly distributed in extraintestinal uropathogenic Escherichia coli, encodes a new member of H-N-H nuclease superfamily.

Background

Method

Results

Conclusion

Publications

Uropathogenic specific protein gene, highly distributed in extraintestinal uropathogenic Escherichia coli, encodes a new member of H-N-H nuclease superfamily.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets