Morganella morganii urease: purification, characterization, and isolation of gene sequences

Li-Tai Hu; Eric B Nicholson; Bradley D Jones; Martha J Lynch; Harry L T Mobley

doi:10.1128/jb.172.6.3073-3080.1990

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Morganella morganii urease: purification, characterization, and isolation of gene sequences

Journal article

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Morganella morganii urease: purification, characterization, and isolation of gene sequences

Li-Tai Hu, Eric B Nicholson, Bradley D Jones, Martha J Lynch and Harry L T Mobley

Journal of bacteriology, Vol.172(6), pp.3073-3080

06/1990

DOI: 10.1128/jb.172.6.3073-3080.1990

PMCID: PMC209110

PMID: 2345135

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Abstract

Morganella morganii, a very common cause of catheter-associated bacteriuria, was previously classified with the genus Proteus on the basis of urease production. M. morganii constitutively synthesizes a urease distinct from that of other uropathogens. The enzyme, purified 175-fold by passage through DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 chromatography resins, was found to have a native molecular size of 590 kilodaltons and was composed of three distinct subunits with apparent molecular sizes of 63, 15, and 6 kilodaltons, respectively. Amino-terminal analysis of the subunit polypeptides revealed a high degree of conservation of amino acid sequence between jack bean and Proteus mirabilis ureases. Km for urea equalled 0.8 mM. Antiserum prepared against purified enzyme inhibited activity by 43% at a 1:2 dilution after 1 h of incubation. All urease activity was immunoprecipitated from cytosol by a 1:16 dilution. Antiserum did not precipitate ureases of other species except for one Providencia rettgeri strain but did recognize the large subunits of ureases of Providencia and Proteus species on Western blots (immunoblots). Thirteen urease-positive cosmid clones of Morganella chromosomal DNA shared a 3.5-kilobase (kb) BamHI fragment. Urease gene sequences were localized to a 7.1-kb EcoRI-SalI fragment. Tn5 mutagenesis revealed that between 3.3 and 6.6 kb of DNA were necessary for enzyme activity. A Morganella urease DNA probe did not hybridize with gene sequences of other species tested. Morganella urease antiserum recognized identical subunit polypeptides on Western blots of cytosol from the wild-type strain and Escherichia coli bearing the recombinant clone which corresponded to those seen in denatured urease. Although the wild-type strain and recombinant clone produced equal amounts of urease protein, the clone produced less than 1% of the enzyme activity of the wild-type strain.

Amino Acid Sequence

Immune Sera - immunology

Rabbits

Enterobacteriaceae - enzymology

Animals

Cloning, Molecular

Molecular Sequence Data

Urease - isolation & purification

Female

Neutralization Tests

Urease - genetics

Urease - immunology

Details

Title: Subtitle: Morganella morganii urease: purification, characterization, and isolation of gene sequences
Creators: Li-Tai Hu - Department of Medicine, University of Maryland School of Medicine, Baltimore 21201
Eric B Nicholson
Bradley D Jones
Martha J Lynch
Harry L T Mobley
Resource Type: Journal article
Publication Details: Journal of bacteriology, Vol.172(6), pp.3073-3080
DOI: 10.1128/jb.172.6.3073-3080.1990
PMID: 2345135
PMCID: PMC209110
NLM abbreviation: J Bacteriol
ISSN: 0021-9193
eISSN: 1098-5530
Grant note: AI23328 / NIAID NIH HHS
Language: English
Date published: 06/1990
Academic Unit: Microbiology and Immunology
Record Identifier: 9984083225102771

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