Green fluorescent protein as a novel marker and reporter system in<i>Helicobacter</i>sp.

Josenhans, Christine; Friedrich, Susanne; Suerbaum, Sebastian

doi:10.1111/j.1574-6968.1998.tb12957.x

Cited by 27 publications

(7 citation statements)

References 21 publications

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“…1c). The gfp system was also compromised by high autofluorescence of the H. pylori strains, especially in later phases of growth (Josenhans et al, 1998 Note that the flaA/flaB ratio is lower in the early to mid-exponential phases (up to approx. 20 h growth, OD 600 of about 0n5) in all systems and increases from midexponential phase on (OD 600 of approx.…”

Section: Construction and Evaluation Of Transcriptional Reporter Fusimentioning

confidence: 99%

“…The cat cassette was chosen to serve the two purposes of providing i) a selective marker for recombinant H. pylori flaAΩcat strains and ii) a reporter gene, whose expression under the control of the flaA promoter can be assayed. For the weakly transcribed flaB and flhA genes, a shuttle transposon mutagenesis approach was used in addition to a simple insertion mutagenesis (Josenhans et al, 1998). For constructing transposon insertions in E. coli, a km-catTn3 derivative, which contains a kmcassette with a strong promoter and an additional promoterless cat-cassette, was utilized, which creates a transcriptional fusion of the cat gene to the gene it is inserted into.…”

Section: Construction and Evaluation Of Transcriptional Reporter Fusimentioning

confidence: 99%

“…Few studies describe environmentally and growth phase-regulated expression of virulencerelated genes in H. pylori (Karita et al, 1996 ;Spohn et al, 1997 ;van Vliet et al, 2001 ;Allan et al, 2001 ;Delany et al, 2001 ; Joyce et al, 2001). Reporter genes that have been used so far in Helicobacter are xylE, cat, gfp and lacZ (Josenhans et al, 1995b(Josenhans et al, , 1998Karita et al, 1996 ;van Vliet et al, 2001, de Vries et al, 2001. Recently, a lacZ reporter transposon strategy has been devised to identify stress-regulated genes in H. pylori (de Vries et al., 2001).…”

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confidence: 99%

“…For quantitative standardization of CAT expression, the Roche purified CAT standard was used. The reporter gene fusions with the red-shifted and enhanced gfp variant gfpmut2 (Cormack et al, 1996) were constructed as described previously (Josenhans et al, 1998). The phenotypes were checked by the same methods as for the cat fusion mutants and were shown to be similar (see above, data not shown).…”

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confidence: 99%

“…Transposon insertions in flaB and flhA were integrated into the H. pylori chromosome by allelic exchange using the mutagenized suicide plasmids. flaAcat as well as flaB-cat and flhA-cat reporter gene fusions led to detectable chloramphenicol resistance in the respective H. pylori mutants.As a second reporter gene fusion system, mainly for subsequent use in visualization of gene regulation in vivo and in cell culture models, flaA and flaB fusions with a promoterless gfp gene (gfpmut2 ; Cormack et al, 1996) were generated as previously described (Josenhans et al, 1998). Finally, since the results of the experiments with fla-gfp and fla-cat reporter gene assays were ambiguous in some aspects (see below), we constructed flaA and flaB transcriptional fusions with the luxAB operon of V. harveyi, which has been used as a highly sensitive reporter system in different bacterial species and eukaryotes, and is a good reporter for use in vivo and in infected cultures of eukaryotic cells (Baldwin et al, 1984 ; Kirchner et al, 1989 ;Heuner et al, 1999).…”

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Growth phase-dependent and differential transcriptional control of flagellar genes in Helicobacter pylori

Niehus

Suerbaum

et al. 2002

Microbiology

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INTRODUCTIONThe eubacterial genus Helicobacter comprises the gastric Helicobacter species that infect the stomach mucosa of many different kinds of mammals. All of these have developed specific adaptation mechanisms to permit persistent life in the inhospitable environment of the stomach. Helicobacter pylori, the causative agent of chronic gastritis and peptic ulcers in humans, like other Helicobacter species depends on its strong motility to move through the viscous mucus gel lining the epithelium of the human stomach, to establish chronic infection of the human gastric mucosa and to persist in the gastric mucus (Hazell et al., 1986 ; Worku et al., 1999). Flagellar filaments contain FlaA and FlaB subunits in a stoichiometric ratio of approximately ten to one (Kostrzynska et al., 1991 ;Suerbaum et al., 1993 ;Josenhans et al., 1995aJosenhans et al., , 1999. Mutants lacking the major flagellin FlaA show strongly impaired motility in vitro and possess only truncated flagella, whereas flaB mutants have moderately diminished motility and apparently normal flagella. In the gnotobiotic piglet animal model and the H. mustelae-ferret model, isogenic mutants lacking either the FlaB or the FlaA subunits were both severely impaired in their ability to colonize (Andrutis et al., 1997 ;Eaton et al., 1996). Those results demonstrated the importance of both flagellins in vivo. The H. pylori flaA and flaB genes are unlinked on the chromosome and their transcription is regulated by two different promoters (Leying et al., 1992 ;Suerbaum et al., 1993). Expression of flaA is controlled by a σ#)-dependent promoter, a class of promoters responsible for the transcriptional activation of late flagellar genes in many flagellated bacteria (class 3 ;Aizawa, 2000 ;Josenhans et al., 2002). flaB and several other flagellar (Spohn & Scarlato, 1999). Dependency of a number of flagellar genes on the ' environmentally regulated ' σ&% factor has also been observed in Caulobacter crescentus, Vibrio cholerae and Campylobacter species, (Anderson et al., 1995 ;Jagannathan et al., 2001 ;Prouty et al., 2001). However, the evolutionary, environmental and functional basis for the importance of σ&% promoters in flagellar regulation in all these bacterial species is not well established.The genetic background and global regulation of flagellar motility and signal transduction in Helicobacter species have not been thoroughly investigated yet. The complete genome sequences of two H. pylori strains have confirmed that both strains possess most motility genes known from other bacteria, but that these are much less frequently organized in operons than in most other bacteria (Alm et al., 1999 ; Tomb et al., 1997). Based on homology searches, some genes known from other bacteria to be important in motility regulation have not been identified in H. pylori (Josenhans & Suerbaum, 2001 Labigne-Roussel et al., 1988).H. pylori strains were cultured on blood agar plates (Columbia agar base II, Oxoid), supplemented with 10 % sheep blood and the following antibiotics : v...

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Section: Construction and Evaluation Of Transcriptional Reporter Fusimentioning

confidence: 99%

Section: Construction and Evaluation Of Transcriptional Reporter Fusimentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Growth phase-dependent and differential transcriptional control of flagellar genes in Helicobacter pylori

Niehus

Suerbaum

et al. 2002

Microbiology

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Molekulare Mikrobiologie von Helicobacter pylori: Die postgenomische Ära

Josenhans¹,

Suerbaum²

1999

Ökosystem Darm VIII

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Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila

et al. 2000

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The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella pneumophila. To analyze GFP expression in Legionella, transcriptional fusions of gfp with the Legionella-specific mip (Macrophage Infectivity Potentiator) promoter (P(mip)) and the sod (SuperOxide Dismutase) promoter (P(sod)) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was associated with a 1ten-fold higher intensity. No fluorescence was observed in L. pneumophila transformed with the promoterless gfp gene. Comparison of fluorescence yields between various L. pneumophila strains that differ in their virulence characteristics and were transformed with the P(mip)-gfp carrying plasmid revealed no differences in GFP expression. Infection studies using Acanthamoeba castellanii as host and recombinant L. pneumophila strains carrying the P(mip)-gfp and P(sod)-gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly. GFP expression was also used to monitor, in infected A. castellanii cells, the intracellular survival of, and incidence of host-cell killing by. L. pneumophila strains that vary in their virulence properties. As quantified by flow cytometry the highly virulent L. pneumophila strain Corby was twice as infectious to A. castellanii as the Philadelphia strain JR 32. Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed. In addition, we examined by flow cytometry the influence of cytochalasin D, cycloheximide, and methylamine on the uptake of Legionella by A. castellanii. In conclusion, gfp appears to be a convenient reporter gene whose expression in Legionella can be followed in real time and allows analysis of promoter activities in Legionella and monitoring of the infection process.

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Green fluorescent protein as a novel marker and reporter system inHelicobactersp.

Cited by 27 publications

References 21 publications

Growth phase-dependent and differential transcriptional control of flagellar genes in Helicobacter pylori

Growth phase-dependent and differential transcriptional control of flagellar genes in Helicobacter pylori

Molekulare Mikrobiologie von Helicobacter pylori: Die postgenomische Ära

Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila

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