Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria

Sörensen, Meike; Lippuner, Christoph; Kaiser, Toralf; Misslitz, Ana Clara; Aebischer, Toni; Bumann, Dirk

doi:10.1016/s0014-5793(03)00856-1

Cited by 109 publications

(89 citation statements)

References 18 publications

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“…Strains used in this study were S. enterica serovar typhimurium NCTC 12023 and its isogenic mutants ssaV::aphT (SPI-2 mutant) (24) and ΔrpoE::KmR (25) carrying pDiGc or pDiGi plasmids. Both plasmids encode a fluorescent-optimized DsRed protein whose expression is under the control of the arabinose-inducible P BAD promoter (26) and EGFP, either under the control of a constitutive promoter (Prpsm) in the case of pDiGc or the IPTG-inducible promoter Plac in the case of pDiGi. These plasmids were stable both in vitro and in vivo and, in agreement with a previous report (27), caused a minor increase in the bacterial cell division time in minimum medium and macrophages (by a factor of approximately 1.2).…”

Section: Methodsmentioning

confidence: 99%

Dynamics of intracellular bacterial replication at the single cell level

Hélaine

Thompson²,

Watson³

et al. 2010

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

275

314

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Several important pathogens cause disease by surviving and replicating within host cells. Bacterial proliferation is the product of both replication and killing undergone by the population. However, these processes are difficult to distinguish, and are usually assessed together by determination of net bacterial load. In addition, measurement of net load does not reveal heterogeneity within pathogen populations. This is particularly important in persistent infections in which slow or nongrowing bacteria are thought to have a major impact. Here we report the development of a reporter system based on fluorescence dilution that enables direct quantification of the replication dynamics of Salmonella enterica serovar Typhimurium (S. Typhimurium) in murine macrophages at both the population and single-cell level. We used this technique to demonstrate that a major S. Typhimurium virulence determinant, the Salmonella pathogenicity island 2 type III secretion system, is required for bacterial replication but does not have a major influence on resistance to killing. Furthermore, we found that, upon entry into macrophages, many bacteria do not replicate, but appear to enter a dormant-like state. These could represent an important reservoir of persistent bacteria. The approach could be extended to other pathogens to study the contribution of virulence and host resistance factors to replication and killing, and to identify and characterize nonreplicating bacteria associated with chronic or latent infections.

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Section: Methodsmentioning

confidence: 99%

Dynamics of intracellular bacterial replication at the single cell level

Hélaine

Thompson²,

Watson³

et al. 2010

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

275

314

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show abstract

“…As an additional reporter, the enhanced RFP gene (rfp) was cloned into pACYC184. Primer pair RFP59/RFP39 (Table 2) was used to amplify the DsRed T3_S4T gene (Sorensen et al, 2003). The resultant plasmid (pDW16) had single BamHI and KpnI sites 59 of the rfp+ gene, allowing promoters of interest to be cloned in-frame with the reporter gene.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the expression, regulation and export of NleA–E in Escherichia coli O157 : H7

Roe¹,

Tysall²,

Dransfield³

et al. 2007

Microbiology

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“…To label S. typhimurium fluorescently, a bacterial strain is used that carries the plasmid pBAD DsRED T3.S4T (a generous gift from D. Bumann, Max-Planck Institute, Berlin, Germany) (Sorensen et al, 2003), an expression vector that encodes the bacterially optimized fluorescent protein DsRED under the control of an arabinose-inducible promoter. Overnight but not subcultures of Salmonella are grown in the presence of ampicillin (100 μg/ml) to select for the plasmid-encoded marker.…”

Section: Reagentsmentioning

confidence: 99%

Investigating the Function of Rho Family GTPases during Salmonella/Host Cell Interactions

Patel¹,

Galán²

2008

Methods in Enzymology

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Salmonella enterica comprise a family of pathogenic gram-negative bacteria that have evolved sophisticated virulence mechanisms to enter non-phagocytic cells. The entry event is the result of a carefully orchestrated modulation of Rho family GTPase activity within the host cell, which in turn triggers localized remodeling of the actin cytoskeleton. These cytoskeletal rearrangements drive profuse membrane ruffling and lamellipodial extensions that envelop bacteria and trigger their internalization. This chapter describes a number of methods used to investigate the role of Rho family GTPases during Salmonella/host cell interactions. In particular, we detail a variety of complementary techniques, including affinity pull-down assays and bacterial-induced membrane ruffling and internalization assays to show that Salmonella-induced actin remodeling and entry require the Rho family members Rac and RhoG.

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Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria

Cited by 109 publications

References 18 publications

Dynamics of intracellular bacterial replication at the single cell level

Dynamics of intracellular bacterial replication at the single cell level

Analysis of the expression, regulation and export of NleA–E in Escherichia coli O157 : H7

Investigating the Function of Rho Family GTPases during Salmonella/Host Cell Interactions

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