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

ABSTRACT 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 dat...

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Autores principales:	Esha Atolia, Spencer Cesar, Heidi A. Arjes, Manohary Rajendram, Handuo Shi, Benjamin D. Knapp, Somya Khare, Andrés Aranda-Díaz, Richard E. Lenski, Kerwyn Casey Huang
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	density-dependent growth glycerol lag phase long-term evolution experiments teichoic acids Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/7ba8cdbef49643fca248f598ea6e92fd
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spelling	oai:doaj.org-article:7ba8cdbef49643fca248f598ea6e92fd2021-11-15T16:19:08ZEnvironmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth10.1128/mBio.01378-202150-7511https://doaj.org/article/7ba8cdbef49643fca248f598ea6e92fd2020-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01378-20https://doaj.org/toc/2150-7511ABSTRACT 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.Esha AtoliaSpencer CesarHeidi A. ArjesManohary RajendramHanduo ShiBenjamin D. KnappSomya KhareAndrés Aranda-DíazRichard E. LenskiKerwyn Casey HuangAmerican Society for Microbiologyarticledensity-dependent growthglycerollag phaselong-term evolution experimentsteichoic acidsMicrobiologyQR1-502ENmBio, Vol 11, Iss 5 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	density-dependent growth glycerol lag phase long-term evolution experiments teichoic acids Microbiology QR1-502
spellingShingle	density-dependent growth glycerol lag phase long-term evolution experiments teichoic acids Microbiology QR1-502 Esha Atolia Spencer Cesar Heidi A. Arjes Manohary Rajendram Handuo Shi Benjamin D. Knapp Somya Khare Andrés Aranda-Díaz Richard E. Lenski Kerwyn Casey Huang Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth
description	ABSTRACT 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.
format	article
author	Esha Atolia Spencer Cesar Heidi A. Arjes Manohary Rajendram Handuo Shi Benjamin D. Knapp Somya Khare Andrés Aranda-Díaz Richard E. Lenski Kerwyn Casey Huang
author_facet	Esha Atolia Spencer Cesar Heidi A. Arjes Manohary Rajendram Handuo Shi Benjamin D. Knapp Somya Khare Andrés Aranda-Díaz Richard E. Lenski Kerwyn Casey Huang
author_sort	Esha Atolia
title	Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth
title_short	Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth
title_full	Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth
title_fullStr	Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth
title_full_unstemmed	Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth
title_sort	environmental and physiological factors affecting high-throughput measurements of bacterial growth
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/7ba8cdbef49643fca248f598ea6e92fd
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Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth

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