Development of a novel continuous culture device for experimental evolution of bacterial populations

Crecy, Eudes de; Metzgar, David; Allen, Caitilyn; Pénicaud, M.; Lyons, Benjamin; Hansen, Christopher J.; Crécy‐Lagard, Valérie de

doi:10.1007/s00253-007-1168-5

Cited by 36 publications

(26 citation statements)

References 23 publications

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“…It is possible that a difference in strain background or growth condition, such as high temperature or low-osmolarity medium, led to the earlier conclusion that this factor is essential. In addition to Salmonella, EF-P has been successfully knocked out in E. coli, Agrobacterium, and Acinetobacter (5,17,47,59). In the case of the Acinetobacter efp mutant, suppressor mutations conveying enhanced growth were obtained after a few generations (17).…”

Section: Discussionmentioning

confidence: 99%

“…In addition to Salmonella, EF-P has been successfully knocked out in E. coli, Agrobacterium, and Acinetobacter (5,17,47,59). In the case of the Acinetobacter efp mutant, suppressor mutations conveying enhanced growth were obtained after a few generations (17). Taken together, these studies suggest that EF-P may be nonessential in many bacterial species.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, while a transposon insertion in the Agrobacterium homolog of efp, chvH, did not affect bacterial viability, the chvH mutant displayed reduced virulence and expression of the critical virulence factor VirE2, as well as increased sensitivity to detergents (47). Disruption of the efp gene in Acinetobacter also does not affect bacterial viability, although fitness is adversely affected (17). Given that poxA and yjeK are not essential for the viability of either E. coli or Salmonella, these findings indicate that efp is a nonessential gene and may instead be required for the synthesis of a subset of proteins necessary for stress tolerance.…”

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confidence: 99%

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Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity

et al. 2012

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Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits increased uptake of the hydrophobic dye 1-N-phenylnaphthylamine (NPN). Analysis of the membrane proteomes of wild-type and efp mutant Salmonella strains reveals few changes, including the prominent overexpression of a single porin, KdgM, in the efp mutant outer membrane. Removal of KdgM in the efp mutant background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious overexpression of an outer membrane porin.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity

et al. 2012

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“…More importantly, these results were achieved in only 8 months by using the Evolugator, less than half the time it took Rudolph et al by using traditional serial batch culture. The Evolugator is fully automated, is resistant to issues of contamination, and counterselects against wall growth, making it ideal for long-term experimental evolution (11,12). From a practical perspective, the ability to perform robust experimental evolution in the Evolugator could potentially reduce the time needed to develop strains with complex phenotypes.…”

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confidence: 99%

Experimental Evolution of a Facultative Thermophile from a Mesophilic Ancestor

Blaby¹,

Lyons²,

Wroclawska-Hughes³

et al. 2012

Appl Environ Microbiol

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Experimental evolution via continuous culture is a powerful approach to the alteration of complex phenotypes, such as optimal/ maximal growth temperatures. The benefit of this approach is that phenotypic selection is tied to growth rate, allowing the production of optimized strains. Herein, we demonstrate the use of a recently described long-term culture apparatus called the Evolugator for the generation of a thermophilic descendant from a mesophilic ancestor (Escherichia coli MG1655). In addition, we used whole-genome sequencing of sequentially isolated strains throughout the thermal adaptation process to characterize the evolutionary history of the resultant genotype, identifying 31 genetic alterations that may contribute to thermotolerance, although some of these mutations may be adaptive for off-target environmental parameters, such as rich medium. We undertook preliminary phenotypic analysis of mutations identified in the glpF and fabA genes. Deletion of glpF in a mesophilic wild-type background conferred significantly improved growth rates in the 43-to-48°C temperature range and altered optimal growth temperature from 37°C to 43°C. In addition, transforming our evolved thermotolerant strain (EVG1064) with a wild-type allele of glpF reduced fitness at high temperatures. On the other hand, the mutation in fabA predictably increased the degree of saturation in membrane lipids, which is a known adaptation to elevated temperature. However, transforming EVG1064 with a wildtype fabA allele had only modest effects on fitness at intermediate temperatures. The Evolugator is fully automated and demonstrates the potential to accelerate the selection for complex traits by experimental evolution and significantly decrease development time for new industrial strains.

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“…1(a) and 1(c). A similar device to ours but on the macroscale (volumetrically 10 6 times larger) was developed by de Crecy-Lagard et al 25,26 All compartments are cleaned for biofilm prevention before a new growth cycle begins. As a result of this biofilm-free constraint, 2 2 -fold dilution in the microbial population is executed, thus the dilution ratio g ¼ …”

Section: Introductionmentioning

confidence: 99%

A nanoliter microfluidic serial dilution bioreactor

Lee

Chiang

et al. 2015

Biomicrofluidics

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Bacterial culture is a basic technique in both fundamental and applied microbiology. The excessive reagent consumption and laborious maintenance of bulk bioreactors for microbial culture have prompted the development of miniaturized on-chip bioreactors. With the minimal choice of two compartments (N ¼ 2) and discrete time, periodic dilution steps, we realize a microfluidic bioreactor that mimics macroscopic serial dilution transfer culture. This device supports automated, long-term microbial cultures with a nanoliter-scale working volume and real-time monitoring of microbial populations at single-cell resolution. Because of the high surface-to-volume ratio, the device also operates as an effective biofilm-flow reactor to support cogrowth of planktonic and biofilm populations. We expect that such devices will open opportunities in many fields of microbiology. V C 2015 AIP Publishing LLC.

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Development of a novel continuous culture device for experimental evolution of bacterial populations

Cited by 36 publications

References 23 publications

Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity

Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity

Experimental Evolution of a Facultative Thermophile from a Mesophilic Ancestor

A nanoliter microfluidic serial dilution bioreactor

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