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Laboratory divergence of Methylobacterium extorquens AM1 through unintended domestication and past selection for antibiotic resistance.

Carroll SM, Xue KS, Marx CJ - BMC Microbiol. (2014)

Bottom Line: To explore the extent to which this lineage has diverged, we compared our own "Modern" stock of AM1 to a sample archived at a culture stock center shortly after the strain's discovery.Contrary to our expectations, Modern was both slower and less fit than Archival across a variety of growth substrates, and showed no improvement during long-term growth and storage.Recapitulating selection for rifamycin resistance in replicate Archival populations showed that mutations to RNA polymerase B (rpoB) substantially decrease growth in the absence of antibiotic, offering an explanation for slower growth in Modern stocks.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA. cmarx@oeb.harvard.edu.

ABSTRACT

Background: A common assumption of microorganisms is that laboratory stocks will remain genetically and phenotypically constant over time, and across laboratories. It is becoming increasingly clear, however, that mutations can ruin strain integrity and drive the divergence or "domestication" of stocks. Since its discovery in 1960, a stock of Methylobacterium extorquens AM1 ("AM1") has remained in the lab, propagated across numerous growth and storage conditions, researchers, and facilities. To explore the extent to which this lineage has diverged, we compared our own "Modern" stock of AM1 to a sample archived at a culture stock center shortly after the strain's discovery. Stored as a lyophilized sample, we hypothesized that this Archival strain would better reflect the first-ever isolate of AM1 and reveal ways in which our Modern stock has changed through laboratory domestication or other means.

Results: Using whole-genome re-sequencing, we identified some 29 mutations - including single nucleotide polymorphisms, small indels, the insertion of mobile elements, and the loss of roughly 36 kb of DNA - that arose in the laboratory-maintained Modern lineage. Contrary to our expectations, Modern was both slower and less fit than Archival across a variety of growth substrates, and showed no improvement during long-term growth and storage. Modern did, however, outperform Archival during growth on nutrient broth, and in resistance to rifamycin, which was selected for by researchers in the 1980s. Recapitulating selection for rifamycin resistance in replicate Archival populations showed that mutations to RNA polymerase B (rpoB) substantially decrease growth in the absence of antibiotic, offering an explanation for slower growth in Modern stocks. Given the large number of genomic changes arising from domestication (28), it is somewhat surprising that the single other mutation attributed to purposeful laboratory selection accounts for much of the phenotypic divergence between strains.

Conclusions: These results highlight the surprising degree to which AM1 has diverged through a combination of unintended laboratory domestication and purposeful selection for rifamycin resistance. Instances of strain divergence are important, not only to ensure consistency of experimental results, but also to explore how microbes in the lab diverge from one another and from their wild counterparts.

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Archival outperforms Modern AM1 under standard growth conditions. A) Representative growth curves showing the increase in OD600 over time of Modern (black circles) and Archival AM1 (gray squares) cultured using 3.5 mM succinate in 48-well plates. B) Growth rates calculated from the exponential phase of cultures grown on methanol (M), methylamine (Ma), or succinate (S) as a carbon source. Significant growth differences between Modern and Archival were calculated using a two-tailed, unpaired t test, and are marked by single (p < 0.05) and double asterisks (p < 0.01). C) Fitness of Archival AM1 measured via a head-to-head competition mixed in co-culture with a fluorescently labeled Modern reference. A control growth consisted of unlabeled Modern (black) versus the fluorescent Modern reference grown on M. All other bars (gray) show Archival fitness relative to Modern grown M, Ma, and S. Values are the mean plus SEM of growth rates or fitness values calculated from three or more biological replicates (see Methods).
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Figure 2: Archival outperforms Modern AM1 under standard growth conditions. A) Representative growth curves showing the increase in OD600 over time of Modern (black circles) and Archival AM1 (gray squares) cultured using 3.5 mM succinate in 48-well plates. B) Growth rates calculated from the exponential phase of cultures grown on methanol (M), methylamine (Ma), or succinate (S) as a carbon source. Significant growth differences between Modern and Archival were calculated using a two-tailed, unpaired t test, and are marked by single (p < 0.05) and double asterisks (p < 0.01). C) Fitness of Archival AM1 measured via a head-to-head competition mixed in co-culture with a fluorescently labeled Modern reference. A control growth consisted of unlabeled Modern (black) versus the fluorescent Modern reference grown on M. All other bars (gray) show Archival fitness relative to Modern grown M, Ma, and S. Values are the mean plus SEM of growth rates or fitness values calculated from three or more biological replicates (see Methods).

Mentions: Our initial hypothesis was that Modern AM1 would outperform the Archival strain, owing to an increased likelihood of mutations in Modern AM1 that could facilitate adaptation to laboratory conditions. However, contrary to our expectations, we found that the Archival strain was both faster and fitter under most conditions tested. The Archival strain was considerably faster growing on the C1 compounds methanol (42%) and methylamine (12%), as well as the multi-C substrate succinate (52%; Figure 2A and B). In head-to-head fitness competitions, the Archival strain showed roughly a 30% advantage across the substrates tested (Figure 2C), suggesting that analyses of growth rate and fitness are not entirely correlated. Nonetheless, these results show that the Archival strain outperforms Modern under most standard growth conditions, and suggest that the Modern lineage became slower and less fit during its time in the lab.


Laboratory divergence of Methylobacterium extorquens AM1 through unintended domestication and past selection for antibiotic resistance.

Carroll SM, Xue KS, Marx CJ - BMC Microbiol. (2014)

Archival outperforms Modern AM1 under standard growth conditions. A) Representative growth curves showing the increase in OD600 over time of Modern (black circles) and Archival AM1 (gray squares) cultured using 3.5 mM succinate in 48-well plates. B) Growth rates calculated from the exponential phase of cultures grown on methanol (M), methylamine (Ma), or succinate (S) as a carbon source. Significant growth differences between Modern and Archival were calculated using a two-tailed, unpaired t test, and are marked by single (p < 0.05) and double asterisks (p < 0.01). C) Fitness of Archival AM1 measured via a head-to-head competition mixed in co-culture with a fluorescently labeled Modern reference. A control growth consisted of unlabeled Modern (black) versus the fluorescent Modern reference grown on M. All other bars (gray) show Archival fitness relative to Modern grown M, Ma, and S. Values are the mean plus SEM of growth rates or fitness values calculated from three or more biological replicates (see Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926354&req=5

Figure 2: Archival outperforms Modern AM1 under standard growth conditions. A) Representative growth curves showing the increase in OD600 over time of Modern (black circles) and Archival AM1 (gray squares) cultured using 3.5 mM succinate in 48-well plates. B) Growth rates calculated from the exponential phase of cultures grown on methanol (M), methylamine (Ma), or succinate (S) as a carbon source. Significant growth differences between Modern and Archival were calculated using a two-tailed, unpaired t test, and are marked by single (p < 0.05) and double asterisks (p < 0.01). C) Fitness of Archival AM1 measured via a head-to-head competition mixed in co-culture with a fluorescently labeled Modern reference. A control growth consisted of unlabeled Modern (black) versus the fluorescent Modern reference grown on M. All other bars (gray) show Archival fitness relative to Modern grown M, Ma, and S. Values are the mean plus SEM of growth rates or fitness values calculated from three or more biological replicates (see Methods).
Mentions: Our initial hypothesis was that Modern AM1 would outperform the Archival strain, owing to an increased likelihood of mutations in Modern AM1 that could facilitate adaptation to laboratory conditions. However, contrary to our expectations, we found that the Archival strain was both faster and fitter under most conditions tested. The Archival strain was considerably faster growing on the C1 compounds methanol (42%) and methylamine (12%), as well as the multi-C substrate succinate (52%; Figure 2A and B). In head-to-head fitness competitions, the Archival strain showed roughly a 30% advantage across the substrates tested (Figure 2C), suggesting that analyses of growth rate and fitness are not entirely correlated. Nonetheless, these results show that the Archival strain outperforms Modern under most standard growth conditions, and suggest that the Modern lineage became slower and less fit during its time in the lab.

Bottom Line: To explore the extent to which this lineage has diverged, we compared our own "Modern" stock of AM1 to a sample archived at a culture stock center shortly after the strain's discovery.Contrary to our expectations, Modern was both slower and less fit than Archival across a variety of growth substrates, and showed no improvement during long-term growth and storage.Recapitulating selection for rifamycin resistance in replicate Archival populations showed that mutations to RNA polymerase B (rpoB) substantially decrease growth in the absence of antibiotic, offering an explanation for slower growth in Modern stocks.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA. cmarx@oeb.harvard.edu.

ABSTRACT

Background: A common assumption of microorganisms is that laboratory stocks will remain genetically and phenotypically constant over time, and across laboratories. It is becoming increasingly clear, however, that mutations can ruin strain integrity and drive the divergence or "domestication" of stocks. Since its discovery in 1960, a stock of Methylobacterium extorquens AM1 ("AM1") has remained in the lab, propagated across numerous growth and storage conditions, researchers, and facilities. To explore the extent to which this lineage has diverged, we compared our own "Modern" stock of AM1 to a sample archived at a culture stock center shortly after the strain's discovery. Stored as a lyophilized sample, we hypothesized that this Archival strain would better reflect the first-ever isolate of AM1 and reveal ways in which our Modern stock has changed through laboratory domestication or other means.

Results: Using whole-genome re-sequencing, we identified some 29 mutations - including single nucleotide polymorphisms, small indels, the insertion of mobile elements, and the loss of roughly 36 kb of DNA - that arose in the laboratory-maintained Modern lineage. Contrary to our expectations, Modern was both slower and less fit than Archival across a variety of growth substrates, and showed no improvement during long-term growth and storage. Modern did, however, outperform Archival during growth on nutrient broth, and in resistance to rifamycin, which was selected for by researchers in the 1980s. Recapitulating selection for rifamycin resistance in replicate Archival populations showed that mutations to RNA polymerase B (rpoB) substantially decrease growth in the absence of antibiotic, offering an explanation for slower growth in Modern stocks. Given the large number of genomic changes arising from domestication (28), it is somewhat surprising that the single other mutation attributed to purposeful laboratory selection accounts for much of the phenotypic divergence between strains.

Conclusions: These results highlight the surprising degree to which AM1 has diverged through a combination of unintended laboratory domestication and purposeful selection for rifamycin resistance. Instances of strain divergence are important, not only to ensure consistency of experimental results, but also to explore how microbes in the lab diverge from one another and from their wild counterparts.

Show MeSH
Related in: MedlinePlus