A simple plasmid-based system that allows rapid generation of tightly controlled gene expression in Staphylococcus aureus

Liew, Andrew T. F.; Theis, Torsten; Jensen, Slade O.; García‐Lara, Jorge; Foster, Simon J.; Firth, Neville; Lewis, Peter J.; Harry, Elizabeth J.

doi:10.1099/mic.0.045146-0

Cited by 44 publications

(50 citation statements)

References 38 publications

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“…We found that compound 1 inhibited the growth of permeable E. coli with an MIC 50 of 27±9 µM, while compound 2 was also inhibitory with an MIC 50 of 84 µM. 27 The inhibition curve for 2 against envA1 E. coli was unusually steep. Although this type of curve can indicate aggregation of a small molecule inhibitor, we note that none of the in vitro inhibition curves for 2 toward FtsZ show this behavior.…”

Section: Resultsmentioning

confidence: 76%

“…20 Liew et al described a S. aureus system for visualization of fluorescent Z-rings, but that system requires tight control of the expression of more than one plasmid making it more complex for screening purposes than an E. coli -based system. 27 Experiments were conducted to determine times of induction that would allow for expression of FtsZ-YFP and co-localization of fluorescent and wild-type FtsZ at mid-cell during division (detailed in Methods). Fluorescence micrographs showed that prior to arabinose induction there was no observable background fluorescence (data not shown).…”

Section: Resultsmentioning

confidence: 99%

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Chrysophaentins are competitive inhibitors of FtsZ and inhibit Z-ring formation in live bacteria

Keffer

Huecas

Hammill

et al. 2013

Bioorganic & Medicinal Chemistry

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The bacterial cell division protein FtsZ polymerizes in a GTP-dependent manner to form a Z-ring that marks the plane of division. As a validated antimicrobial target, considerable efforts have been devoted to identify small molecule FtsZ inhibitors. We recently discovered the chrysophaentins, a novel suite of marine natural products that inhibit FtsZ activity in vitro. These natural products along with a synthetic hemi-chrysophaentin exhibit strong antimicrobial activity toward a broad spectrum of Gram-positive pathogens. To define their mechanisms of FtsZ inhibition and determine their in vivo effects in live bacteria, we used GTPase assays and fluorescence anisotropy to show that hemi-chrysophaentin competitively inhibits FtsZ activity. Furthermore, we developed a model system using a permeable E. coli strain, envA1, together with an inducible FtsZ-yellow fluorescent protein construct to show by fluorescence microscopy that both chrysophaentin A and hemi-chrysophaentin disrupt Z-rings in live bacteria. We tested the E. coli system further by reproducing phenotypes observed for zantrins Z1 and Z3, and demonstrate that the alkaloid berberine, a reported FtsZ inhibitor, exhibits auto-fluorescence, making it incompatible with systems that employ GFP or YFP tagged FtsZ. These studies describe unique examples of non-nucleotide, competitive FtsZ inhibitors that disrupt FtsZ in vivo, together with a model system that should be useful for in vivo testing of FtsZ inhibitor leads that have been identified through in vitro screens but are unable to penetrate the Gram-negative outer membrane.

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Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Chrysophaentins are competitive inhibitors of FtsZ and inhibit Z-ring formation in live bacteria

Keffer

Huecas

Hammill

et al. 2013

Bioorganic & Medicinal Chemistry

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“…A more direct approach to demonstrate gene essentiality is to place the gene of interest under the control of an inducible promoter in the chromosome or in a low-copy plasmid and assess the essentiality based on survival in the presence or absence of the inducer (Fan et al , 2001; Jana et al , 2000; Liew et al , 2011; Zheng et al , 2005). The IPTG-inducible P spac promoter has been favoured in these systems.…”

Section: Gene Essentiality Testingmentioning

confidence: 99%

An update on the molecular genetics toolbox for staphylococci

2013

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Staphylococci are Gram-positive spherical bacteria of enormous clinical and biotechnological relevance. Staphylococcus aureus has been extensively studied as a model pathogen. A plethora of methods and molecular tools has been developed for genetic modification of at least ten different staphylococcal species to date. Here we review recent developments of various genetic tools and molecular methods for staphylococcal research, which include reporter systems and vectors for controllable gene expression, gene inactivation, gene essentiality testing, chromosomal integration and transposon delivery. It is furthermore illustrated how mutant strain construction by homologous or site-specific recombination benefits from sophisticated counterselection methods. The underlying genetic components have been shown to operate in wild-type staphylococci or modified chassis strains. Finally, possible future developments in the field of applied Staphylococcus genetics are highlighted.

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“…Gene deletions were made using the pMAD and pKFC allelic replacement systems (42,43). Complementation was performed using pLOW (44), and double mutants were created through ϕ85 transduction of a marked deletion into unmarked deletion backgrounds.…”

Section: Methodsmentioning

confidence: 99%

Compound-gene interaction mapping reveals distinct roles forStaphylococcus aureusteichoic acids

Maria

Sadaka

Moussa

et al. 2014

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

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Staphylococcus aureus contains two distinct teichoic acid (TA) polymers, lipoteichoic acid (LTA) and wall teichoic acid (WTA), which are proposed to play redundant roles in regulating cell division. To gain insight into the underlying biology of S. aureus TAs, we used a small molecule inhibitor to screen a highly saturated transposon library for cellular factors that become essential when WTA is depleted. We constructed an interaction network connecting WTAs with genes involved in LTA synthesis, peptidoglycan synthesis, surface protein display, and D-alanine cell envelope modifications. Although LTAs and WTAs are synthetically lethal, we report that they do not have the same synthetic interactions with other cell envelope genes. For example, D-alanylation, a tailoring modification of both WTAs and LTAs, becomes essential when the former, but not the latter, are removed. Therefore, D-alanine-tailored LTAs are required for survival when WTAs are absent. Examination of terminal phenotoypes led to the unexpected discovery that cells lacking both LTAs and WTAs lose their ability to form Z rings and can no longer divide. We have concluded that the presence of either LTAs or WTAs on the cell surface is required for initiation of S. aureus cell division, but these polymers act as part of distinct cellular networks.synthetic lethality | TraDIS | Tn-seq

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A simple plasmid-based system that allows rapid generation of tightly controlled gene expression in Staphylococcus aureus

Cited by 44 publications

References 38 publications

Chrysophaentins are competitive inhibitors of FtsZ and inhibit Z-ring formation in live bacteria

Chrysophaentins are competitive inhibitors of FtsZ and inhibit Z-ring formation in live bacteria

An update on the molecular genetics toolbox for staphylococci

Compound-gene interaction mapping reveals distinct roles forStaphylococcus aureusteichoic acids

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