Use of genetic recombination as a reporter of gene expression.

Camilli, Andrew; Beattie, David T.; Mekalanos, John J.

doi:10.1073/pnas.91.7.2634

Cited by 160 publications

(151 citation statements)

References 21 publications

(11 reference statements)

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“…An appreciation of the limitations of studying surrogate in vitro signals as the cues for controlling gene expression has led the field to explore genetic approaches that could define ''in vivo-induced genes'' for pathogens (29)(30)(31). However, in vivo induction is an arbitrary parameter to measure because assumptions must be made about which in vitro condition is appropriate for comparison to any in vivo condition.…”

Section: Discussionmentioning

confidence: 99%

Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro

Dziejman

Mekalanos

2003

Proc. Natl. Acad. Sci. U.S.A.

217

177

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Vibrio cholerae is the etiologic bacterial agent of cholera, a severe diarrheal disease endemic in much of the developing world. The V. cholerae genome contains 3,890 genes distributed between a large and a small chromosome. Although the large chromosome encodes the majority of recognizable gene products and virulence determinants, the small chromosome carries a disproportionate number of hypothetical genes. Thus, little is known about the role of the small chromosome in the biology of this organism or other Vibrio species. We have used the rabbit ileal loop model of V. cholerae infection to obtain in vivo-grown cells under near midexponential conditions in the small-intestinal environment. We compared the global transcriptional pattern of these in vivo-grown cells to those grown to midexponential phase in rich medium under aerobic conditions. Under both conditions, the genes showing the highest levels of expression reside primarily on the large chromosome. However, a shift occurs in vivo that results in many more small chromosomal genes being expressed during growth in the intestine. Our analysis further suggests that nutrient limitation (particularly iron) and anaerobiosis are major stresses experienced by V. cholerae during growth in the rabbit upper intestine. Finally, relative to in vitro growth, the intestinal environment significantly enhanced expression of several virulence genes, including those involved in phenotypes such as motility, chemotaxis, intestinal colonization, and toxin production.

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Section: Discussionmentioning

confidence: 99%

Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro

Dziejman

Mekalanos

2003

Proc. Natl. Acad. Sci. U.S.A.

217

177

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“…This allowed us to examine two levels of gene expression in the different V. cholerae biotypes, whereby the El Tor biotype reporter strain was more sensitive to low levels of tnpR expression than the classical biotype reporter strain. Expression of resolvase in each strain background catalysed excision of tet out of the bacterial chromosome (Camilli et al, 1994). The excised tet gene is present on a non-replicating DNA circle which is segregated by cell division leading to a Tc S phenotype in descendent bacteria (Camilli et al, 1994).…”

Section: Construction Of V Cholerae Transcriptional Fusion Librariesmentioning

confidence: 99%

“…Accordingly, we have designed a genetic 'screen' to identify bacterial genes that are transcriptionally silent during growth in the laboratory but which are then transiently or constitutively induced within the host to either low or high levels of transcription. Our approach is based on the recently developed method of assaying gene expression via transcriptional fusions to the site-specific recombinase resolvase (Camilli et al, 1994). In this report, we describe the application of this approach to identify genes of V. cholerae which are transcriptionally induced in an infant mouse model of cholera (Wachsmuth et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection

Camilli

Mekalanos

1995

Molecular Microbiology

258

240

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SummaryA complete understanding of host-parasite interactions must necessarily include the identification and characterization of gene products expressed by both parties during the infectious process. We have developed a new screen to identify bacterial genes that are transcriptionally induced during infection of a host animal. The method is based on pre-selection of strains carrying tnpR operon fusions (encoding resolvase, a site-specific DNA recombinase) which are not expressed in vitro, followed by screening for a subset of these strains that subsequently express resolvase within the host environment. The latter subset was recognized as recombinants that had deleted a resolvasespecific reporter construct. Thirteen transcription units of Vibrio cholerae were identified that were induced during infection in an infant mouse model of cholera. Five of these were predicted to encode polypeptides with diverse functions in metabolism, biosynthesis and motility; one encoded a secreted lipase; two appear to be antisense to genes involved in motility; and five are predicted to encode polypeptides of unknown function. Three of the transcripts were shown to be required for full virulence in infant mice, as assessed by competition experiments.

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“…One of these technologies, which uses site-specific recombination as a reporter of gene activation, is especially suitable to detect the in vivo induction of genes that are only transiently expressed at a certain stage of infection (19). In C. albicans, the development of similar reporter techniques has been hampered by the unusual codon usage of this organism (20), which is responsible for the failure to express most heterologous genes (21)(22)(23).…”

mentioning

confidence: 99%

Differential activation of a Candida albicans virulence gene family during infection

Staib¹,

Kretschmar²,

Nichterlein³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

147

138

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The yeast Candida albicans is a harmless commensal in most healthy people, but it causes superficial as well as life-threatening systemic infections in immunocompromised patients. C. albicans can colonize or infect virtually all body sites because of its high adaptability to different host niches, which involves the activation of appropriate sets of genes in response to complex environmental signals. We have used an in vivo expression technology that is based on genetic recombination as a reporter of gene expression to monitor the differential activation of individual members of a gene family encoding secreted aspartic proteinases (Saps), which have been implicated in C. albicans virulence, at various stages of the infection process. Our results demonstrate that SAP expression depends on the type of infection, with different SAP isogenes being activated during systemic disease as compared with mucosal infection. In addition, the activation of individual SAP genes depends on the progress of the infection, some members of the gene family being induced immediately after contact with the host, whereas others are expressed only after dissemination into deep organs. In the latter case, the number of invading organisms determines whether induction of a virulence gene is necessary for successful infection. The in vivo expression technology allows the elucidation of gene expression patterns at different stages of the fungus-host interaction, thereby revealing regulatory adaptation mechanisms that make C. albicans the most successful fungal pathogen of humans and, at the same time, identifying the stage of an infection at which certain virulence genes may play a role.

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Use of genetic recombination as a reporter of gene expression.

Cited by 160 publications

References 21 publications

Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro

Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro

Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection

Differential activation of a Candida albicans virulence gene family during infection

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