Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria

Shiver, Anthony L.; Culver, Rebecca; Deutschbauer, Adam M.; Huang, Kerwyn Casey

doi:10.1101/780593

Cited by 12 publications

(26 citation statements)

References 25 publications

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“…The final library contains 315,668 uniquely barcoded mutants with insertions that we could confidently map to a single location in the genome (Figure 1A). To enable follow-up studies, we generated a 40-plate archived collection of individual transposon-insertion mutants in a 96-well microplate format by sorting cells using flow cytometry (Shiver et al, 2019).…”

Section: A Gene-phenotype Map Of B Thetaiotaomicronmentioning

confidence: 99%

Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments

et al. 2021

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Section: A Gene-phenotype Map Of B Thetaiotaomicronmentioning

confidence: 99%

Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments

et al. 2021

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“…The creation of genome-wide knockout libraries ( 23 , 24 ) has motivated high-throughput measurements of population growth—a commonly used proxy for fitness—in microtiter plates, such as the systems biological characterization of essential gene knockdowns in Bacillus subtilis ( 5 ) and of Escherichia coli nonessential gene knockouts in liquid ( 25 , 26 ) or embedded in a gel ( 26 , 27 ). As the development of advanced genetic tools simplifies the creation of strain libraries ( 28 – 30 ), it is critical to ensure that OD measurements in multiwell plates provide reliable estimates of population-level growth parameters.…”

Section: Introductionmentioning

confidence: 99%

Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth

Atolia

Cesar

Arjes

et al. 2020

mBio

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Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth. IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli. Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

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“…First, our study was conducted in a defined medium to avoid introduction of molecules from another species into the two-species system, such as culture media compositions that contain whole cell lysates, and it is important to consider that culture and growth conditions may affect either secreted molecule production and/or sensory responses. Next, the species being studied must have the necessary genetic tools available, such as reporter expression systems and random mutagenesis protocols, however these are becoming more widely accessible (96)(97)(98). The identification of sensed products using our approach was more straightforward when there was a primary sensory molecule driving the response, such as in the case of StP, citrate, or acetoin, although fractionation of WT and mutant supernatants can reveal the molecules underlying multifactordriven responses as well.…”

Section: S6 and Tablementioning

confidence: 99%

Systematic identification of molecular mediators underlying sensing ofStaphylococcus aureusbyPseudomonas aeruginosa

Zarrella

Khare

2021

Preprint

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Bacteria typically exist in dynamic, multispecies communities where polymicrobial interactions influence fitness. Elucidating the molecular mechanisms underlying these interactions is critical for understanding and modulating bacterial behavior in natural environments. While bacterial responses to foreign species are frequently characterized at the molecular and phenotypic level, the exogenous molecules that elicit these responses are understudied. Here we outline a systematic strategy based on transcriptomics combined with genetic and biochemical screens of promoter-reporters to identify the molecules from one species that are sensed by another. We utilized this method to study interactions between the pathogens Pseudomonas aeruginosa and Staphylococcus aureus that are frequently found in co-infections. We discovered that P. aeruginosa senses diverse staphylococcal exoproducts including the metallophore staphylopine, intermediate metabolites citrate and acetoin, and multiple molecules that modulate its iron starvation response. Further, we show that staphylopine inhibits biofilm formation and that P. aeruginosa can utilize citrate and acetoin for growth, revealing that these interactions have both antagonistic and beneficial effects. Our screening approach thus identified multiple S. aureus secreted molecules that are sensed by P. aeruginosa and affect its physiology, demonstrating the efficacy of this approach, and yielding new insight into the molecular basis of interactions between these two species.

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Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria

Cited by 12 publications

References 25 publications

Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments

Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments

Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth

Systematic identification of molecular mediators underlying sensing ofStaphylococcus aureusbyPseudomonas aeruginosa

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