Proteus mirabilis urease: use of a ureA-lacZ fusion demonstrates that induction is highly specific for urea

Nicholson, Ellen; Concaugh, Elizabeth A.; Mobley, Harry L. T.

doi:10.1128/iai.59.10.3360-3365.1991

Cited by 31 publications

(10 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An in-frame translational fusion of ureD with gfp (S65T/ V68L/S72A) was successfully constructed and confirmed by nucleotide sequencing of the fusion junction. Using Western blotting and spectrophotofluorimetry, we found expression of UreD-GFP only in the samples with urea induction in E. coli; the expression level was correlated with urea concentration, with highest induction at 200 to 500 mM, an observation consistent with previous reports (37). We have learned that urease genes are indeed expressed in vivo by P. mirabilis during UTI, confirming an assertion for which significant circumstantial evidence exists.…”

Section: Discussionsupporting

confidence: 89%

“…1). It has been determined previously that ureD and ureA are transcribed on the same urea-inducible mRNA (18,21,22,37). Nevertheless, expression of the UreD-GFP fusion is an indirect measurement of urease enzyme expression.…”

Section: Discussionmentioning

confidence: 99%

“…Urease expression is regulated at the transcriptional level. ureD is upstream of structural subunits ureABC and is transcribed on the same mRNA as ureABC, under control of the same promoter (18,22,37) (Fig. 1).…”

Section: Construction Of Ured-gfp Translational Fusionmentioning

confidence: 99%

“…The structural genes, ure-ABC, which encode subunits of the enzyme, are flanked immediately upstream by the ureD and downstream by the ureEFG genes. These seven genes are transcribed on the same mRNA transcript (18,21,22,37). The four accessory genes (ureDEFG) are necessary for the insertion of nickel ions into the apoenzyme and required for assembly of a catalytically active urease.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic Proteus mirabilis during Experimental Ascending Urinary Tract Infection

Zhao¹,

Thompson²,

Lockatell

et al. 1998

Infect Immun

View full text Add to dashboard Cite

Proteus mirabilis, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of P. mirabilis urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene ureD, which is preceded by a urea-inducible promoter, were made withgfp (modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33–38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5α and P. mirabilis HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent E. coli and P. mirabilis bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, while aggregated bacterial clusters were observed in the bladder. Elongated swarmer cells were only rarely observed. These observations demonstrate that urease is expressed in vivo and that using GFP as a reporter protein is a viable approach to investigate in vivo expression ofP. mirabilis virulence genes in experimental urinary tract infection.

show abstract

Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Construction Of Ured-gfp Translational Fusionmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic Proteus mirabilis during Experimental Ascending Urinary Tract Infection

Zhao¹,

Thompson²,

Lockatell

et al. 1998

Infect Immun

View full text Add to dashboard Cite

show abstract

“…A main virulence factor is the urease, which cleaves urea into ammonium hydroxide and can promote stone formation or toxicity to the renal tubes by its strong alkalysing effects. A recent report demonstrates that urease production, controlled by the UreR repressor protein, is strongly inducible by urea, but not by closely related structural analogs [116]. P. mirabilis is an example of a pathogenic bacterium which exhibits a type of differentiation process, which can easily be seen on solid media as a swarming migration.…”

Section: Shigella Flexnerimentioning

confidence: 99%

Signal transduction and virulence regulation in human and animal pathogens

Gross¹

1993

FEMS Microbiology Letters

View full text Add to dashboard Cite

Pathogens have developed many strategies for survival in animals and humans which possess very effective defense mechanisms. Although there are many different ways, in which pathogenic bacteria solved the problem to overcome the host defense, some common features of virulence mechanisms can be detected even in phylogenetically very distant bacteria (Finlay and Falkow (1989) Microb. Rev. 6 1375–1383). One important feature is that the regulation of expression of virulence factors and the exact timing of their expression is very important for many of the pathogenic bacteria, as most of them have to encounter different growth situations during an infection cycle, which require a fast adaptation to the new situation by the expression of different factors. This review gives an overview about the mechanisms used by pathogenic bacteria to accomplish the difficult task of regulation of their virulence potential in response to environmental changes. In addition, the relationship of these virulence regulatory systems with other signal transduction mechanisms not involved in pathogenicity is discussed.

show abstract

Reporter genes in transgenic mice

Cui

Wani

Wight

et al. 1994

Transgenic Research

View full text Add to dashboard Cite

Although in vivo models utilizing endogenous reporter genes have been exploited for many years, the use of reporter transgenes to dissect biological issues in transgenic animals has been a relatively recent development. These transgenes are often, but not always, of prokaryotic origin and encode products not normally associated with eukaryotic cells and tissues. Some encode enzymes whose activities are detected in cell and tissue homogenates, whereas others encode products that can be detected in situ at the single cell level. Reporter genes have been used to identify regulatory elements that are important for tissue-specific gene expression or for development; they have been used to produce in vivo models of cancer; they have been employed for the study of in vivo mutagenesis; and they have been used as a tool in lineage analysis and for marking cells in transplantation experiments. The most commonly used in situ reporter gene is lacZ, which encodes a bacterial beta-galactosidase, a sensitive histochemical marker. Although it has been used with striking success in cultured cells and in transgenic mouse embryos, its postnatal in vivo expression has been unreliable and disappointing. Nevertheless, the ability to express reporter genes in transgenic mice has been an invaluable resource, providing insights into in vivo biological mechanisms. The development of new in vivo models, such as those in which expression of transgenes can be activated or repressed, should produce transgenic animal systems that extend our capacity to address heretofore unresolved biological questions.

show abstract

Proteus mirabilis urease: use of a ureA-lacZ fusion demonstrates that induction is highly specific for urea

Cited by 31 publications

References 21 publications

Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic Proteus mirabilis during Experimental Ascending Urinary Tract Infection

Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic Proteus mirabilis during Experimental Ascending Urinary Tract Infection

Signal transduction and virulence regulation in human and animal pathogens

Reporter genes in transgenic mice

Contact Info

Product

Resources

About