Statistical Package for Growth Rates Made Easy

Mira, Portia; Barlow, Miriam; Meza, Juan C.; Hall, Barry G.

doi:10.1093/molbev/msx255

Cited by 12 publications

(21 citation statements)

References 12 publications

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“…Overnight cultures (10 l) derived from different colonies were used to inoculate triplicate tubes, and absorbances at 600 nm (A 600 ) were read periodically in a test tube spectrophotometer. Lag and doubling times were calculated using the program GrowthRates (64). All statistical analyses were performed in GraphPad Prism 6.07.…”

Section: Methodsmentioning

confidence: 99%

Clinical Mutations That Partially Activate the Stringent Response Confer Multidrug Tolerance in Staphylococcus aureus

Bryson

Hettle

Boraston

et al. 2020

Antimicrob Agents Chemother

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Antibiotic tolerance is an underappreciated antibiotic escape strategy that is associated with recurrent and relapsing infections, as well as acting as a precursor to resistance. Tolerance describes the ability of a bacterial population to survive transient exposure to an otherwise lethal concentration of antibiotic without exhibiting an elevated MIC. It is detected in time-kill assays as a lower rate of killing than a susceptible strain and can be quantified by the metric minimum duration for killing (MDK). The molecular mechanisms behind tolerance are varied, but activation of the stringent response (SR) via gene knockouts and/or chemical induction has long been associated with tolerance. More recently, two Gram-positive clinical isolates from persistent bacteremias were found to bear mutations in the SR controller, Rel, that caused elevated levels of the alarmone (p)ppGpp. Here, we show that introduction of either of these mutations into Staphylococcus aureus confers tolerance to five different classes of antibiotic as a result of (p)ppGpp-mediated growth defects (longer lag time and/or lower growth rate). The degree of tolerance is related to the severity of the growth defect and ranges from a 1.5- to 3.1-fold increase in MDK. Two classes of proposed SR inhibitor were unable to reverse or reduce this tolerance. Our findings reveal the significance of SR-activating mutations in terms of tolerance and clinical treatment failures. The panel of strains reported here provide a clinically relevant model of tolerance for further investigation of its link to resistance development, as well as potential validation of high-throughput tolerance screens.

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Section: Methodsmentioning

confidence: 99%

Clinical Mutations That Partially Activate the Stringent Response Confer Multidrug Tolerance in Staphylococcus aureus

Bryson

Hettle

Boraston

et al. 2020

Antimicrob Agents Chemother

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show abstract

“…This is a fifteen-fold difference in the number of per milliliter in each overnight culture. This difference is important to consider when performing experiments that depend on the number of cellular divisions or cells present, such as cellular communication (11,12) and antibiotic susceptibility (13)(14)(15)(16) and biofilms (17).…”

Section: Discussionmentioning

confidence: 99%

Estimating Microbial Population Data from Optical Density

Mira

Yeh

Hall³

2021

Preprint

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The spectrophotometer has been used for decades to measure the density of bacterial populations using the turbidity expressed as optical density – OD. However, the OD alone is an unreliable metric and is only proportionately accurate to cell titers to about an OD of 0.6. The relationship between OD and cell titer depends on the configuration of the spectrophotometer, the length of the light path through the culture, the size of the bacterial cells, and the cell culture density. We demonstrate the importance of plate reader calibration to identify the exact relationship between OD and cells/ml. We use four bacterial genera and two sizes of micro-titer plates (96-well and 384-well) and show that the cell/ml per unit OD depends heavily on the bacterial cell size and plate size. We applied our calibration curve to real growth curve data and conclude the cells/ml – rather than OD – is a metric that can be used to directly compare results across experiments, labs, instruments, and species.

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“…Growth was monitored spectrophotometrically at 600 nm over time. Growth rates and other parameters such as lag and doubling times were analyzed using GrowthRates v3.0 ( Hall et al, 2014 ; Mira et al, 2017 ; Hall et al, 2020 ) as an unbiased way to identify datapoints comprising the linear part of the growth curve. Differences among the WY MED15 alleles the LAB allele and the deletion ( med15 Δ) were evaluated using ANOVA with Tukey post-hoc analysis ( Tukey, 1949 ).…”

Section: Methodsmentioning

confidence: 99%

Possible Role for Allelic Variation in Yeast MED15 in Ecological Adaptation

et al. 2021

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The propensity for Saccharomyces cerevisiae yeast to ferment sugars into ethanol and CO2 has long been useful in the production of a wide range of food and drink. In the production of alcoholic beverages, the yeast strain selected for fermentation is crucial because not all strains are equally proficient in tolerating fermentation stresses. One potential mechanism by which domesticated yeast may have adapted to fermentation stresses is through changes in the expression of stress response genes. MED15 is a general transcriptional regulator and RNA Pol II Mediator complex subunit which modulates the expression of many metabolic and stress response genes. In this study, we explore the role of MED15 in alcoholic fermentation. In addition, we ask whether MED15 alleles from wine, sake or palm wine yeast improve fermentation activity and grape juice fermentation stress responses. And last, we investigate to what extent any differences in activity are due to allelic differences in the lengths of three polyglutamine tracts in MED15. We find that strains lacking MED15 are deficient in fermentation and fermentation stress responses and that MED15 alleles from alcoholic beverage yeast strains can improve both the fermentation capacity and the response to ethanol stresses when transplanted into a standard laboratory strain. Finally, we find that polyglutamine tract length in the Med15 protein is one determinant in the efficiency of the alcoholic fermentation process. These data lead to a working model in which polyglutamine tract length and other types of variability within transcriptional hubs like the Mediator subunit, Med15, may contribute to a reservoir of transcriptional profiles that may provide a fitness benefit in the face of environmental fluctuations.

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Statistical Package for Growth Rates Made Easy

Cited by 12 publications

References 12 publications

Clinical Mutations That Partially Activate the Stringent Response Confer Multidrug Tolerance in Staphylococcus aureus

Clinical Mutations That Partially Activate the Stringent Response Confer Multidrug Tolerance in Staphylococcus aureus

Estimating Microbial Population Data from Optical Density

Possible Role for Allelic Variation in Yeast MED15 in Ecological Adaptation

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