MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis

Ducret, Adrien; Quardokus, Ellen M.; Brun, Yves V.

doi:10.1038/nmicrobiol.2016.77

Cited by 892 publications

(896 citation statements)

References 18 publications

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“…We hypothesized that this represents basal expression. To quantify the expression, we measured the fluorescence intensity of Ͼ1,000 single cells using MicrobeJ (37) (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

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RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation

et al. 2017

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In the nosocomial opportunistic pathogen Acinetobacter baumannii, RecAdependent mutagenesis, which causes antibiotic resistance acquisition, is linked to the DNA damage response (DDR). Notably, unlike the Escherichia coli paradigm, recA and DDR gene expression in A. baumannii is bimodal. Namely, there is phenotypic variation upon DNA damage, which may provide a bet-hedging strategy for survival. Thus, understanding recA gene regulation is key to elucidate the yet unknown DDR regulation in A. baumannii. Here, we identify a structured 5= untranslated region (UTR) in the recA transcript which serves as a cis-regulatory element. We show that a predicted stem-loop structure in this 5= UTR affects mRNA half-life and underlies bimodal gene expression and thus phenotypic variation in response to ciprofloxacin treatment. We furthermore show that the stem-loop structure of the recA 5= UTR influences intracellular RecA protein levels and, in vivo, impairing the formation of the stem-loop structure of the recA 5= UTR lowers cell survival of UV treatment and decreases rifampin resistance acquisition from DNA damage-induced mutagenesis. We hypothesize that the 5= UTR allows for stable recA transcripts during stress, including antibiotic treatment, enabling cells to maintain suitable RecA levels for survival. This innovative strategy to regulate the DDR in A. baumannii may contribute to its success as a pathogen.IMPORTANCE Acinetobacter baumannii is an opportunistic pathogen quickly gaining antibiotic resistances. Mutagenesis and antibiotic resistance acquisition are linked to the DNA damage response (DDR). However, how the DDR is regulated in A. baumannii remains unknown, since unlike most bacteria, A. baumannii does not follow the regulation of the Escherichia coli paradigm. In this study, we have started to uncover the mechanisms regulating the novel A. baumannii DDR. We have found that a cisacting 5= UTR regulates recA transcript stability, RecA protein levels, and DNA damage-induced phenotypic variation. Though 5= UTRs are known to provide stability to transcripts in bacteria, this is the first example in which it regulates a bimodal DDR response through recA transcript stabilization, potentially enabling cells to have enough RecA for survival and genetic variability.KEYWORDS Acinetobacter, DNA damage, microbial genetics, RecA, gene expression, molecular biology A cinetobacter baumannii is a Gram-negative opportunistic pathogen that is a major problem in hospitals due to its ability to withstand desiccation (1), allowing it to ultimately reach immunocompromised individuals and cause diseases, including pneumonia and meningitis (2-4). Compounding this problem is the ability of A. baumannii to rapidly gain antibiotic resistances, such that there are now pan-resistant strains (5, 6). This may in part be due to A. baumannii's improved fitness during stress (7-10). However, fully understanding how A. baumannii rapidly acquires antibiotic resistances

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“…We hypothesized that this represents basal expression. To quantify the expression, we measured the fluorescence intensity of Ͼ1,000 single cells using MicrobeJ (37) (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

“…The white arrow points to a DDR High cell, while the gray arrow points to a DDR Low cell. The scale bar represents 10 m. (B) Single-cell quantification of mKate2 expression of each reporter strain with (ϩ) or without (Ϫ) Cip treatment using MicrobeJ (37). Each dot represents one cell.…”

Section: Resultsmentioning

confidence: 99%

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation

et al. 2017

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“…At least 2000 cells were visualized per sample by epifluorescence microscopy (Ti-E; Nikon, Tokyo, Japan) within 72 h of hybridization. Cell counting was partially automated using Microbe J (Ducret et al, 2016). E. coli frequency was determined from the number of ATTO550-positive cells per total number of Syto9-positive cells counted in the sample.…”

Section: Analytical Proceduresmentioning

confidence: 99%

Microbial mutualism dynamics governed by dose-dependent toxicity of cross-fed nutrients

et al. 2016

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Microbial interactions, including mutualistic nutrient exchange (cross-feeding), underpin the flow of energy and materials in all ecosystems. Metabolic exchanges are difficult to assess within natural systems. As such, the impact of exchange levels on ecosystem dynamics and function remains unclear. To assess how cross-feeding levels govern mutualism behavior, we developed a bacterial coculture amenable to both modeling and experimental manipulation. In this coculture, which resembles an anaerobic food web, fermentative Escherichia coli and photoheterotrophic Rhodopseudomonas palustris obligately cross-feed carbon (organic acids) and nitrogen (ammonium). This reciprocal exchange enforced immediate stable coexistence and coupled species growth. Genetic engineering of R. palustris to increase ammonium cross-feeding elicited increased reciprocal organic acid production from E. coli, resulting in culture acidification. Consequently, organic acid function shifted from that of a nutrient to an inhibitor, ultimately biasing species ratios and decreasing carbon transformation efficiency by the community; nonetheless, stable coexistence persisted at a new equilibrium. Thus, disrupting the symmetry of nutrient exchange can amplify alternative roles of an exchanged resource and thereby alter community function. These results have implications for our understanding of mutualistic interactions and the use of microbial consortia as biotechnology.

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“…Fluorescence images were quantified with the MicrobeJ plug-in for ImageJ (51). Bright-field and fluorescence images were arranged into a stack in ImageJ.…”

Section: Methodsmentioning

confidence: 99%

Cyclic AMP-Independent Control of Twitching Motility in Pseudomonas aeruginosa

et al. 2017

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FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through the activation of adenylate cyclase CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimV's cAMPdependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, requires both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds-lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH or expressing constitutively active CyaB-pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMPdependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimV's role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility.IMPORTANCE Pseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility and are involved in surface sensing, which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in the modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements-including the PilSR two-component system-to cell poles for optimal function.

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MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis

Cited by 892 publications

References 18 publications

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation

Microbial mutualism dynamics governed by dose-dependent toxicity of cross-fed nutrients

Cyclic AMP-Independent Control of Twitching Motility in Pseudomonas aeruginosa

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