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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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Modern outperforms Archival AM1 when grown on nutrient broth. A) Representative growth curves of Modern (black circles) and Archival (gray squares) AM1 grown on nutrient broth (NB). Note that growth - particularly for the Archival strain - slows considerably during late exponential phase, signifying density-dependent growth inhibition. B) Change in the proportion of either Modern or Archival AM1 mixed in co-culture with a fluorescently labeled Modern reference as measured by flow cytometry. Values represent the mean plus SEM of at least three biological replicates grown in 48-well plates (A) or flasks (B).
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Figure 3: Modern outperforms Archival AM1 when grown on nutrient broth. A) Representative growth curves of Modern (black circles) and Archival (gray squares) AM1 grown on nutrient broth (NB). Note that growth - particularly for the Archival strain - slows considerably during late exponential phase, signifying density-dependent growth inhibition. B) Change in the proportion of either Modern or Archival AM1 mixed in co-culture with a fluorescently labeled Modern reference as measured by flow cytometry. Values represent the mean plus SEM of at least three biological replicates grown in 48-well plates (A) or flasks (B).

Mentions: We found over multiple different growth experiments that Modern AM1 consistently outperformed Archival on NB, although both strains grew more poorly than on Hypho medium. Modern displayed a clear growth advantage on NB in 48-well plates (FigureĀ 3A), which could be traced in part to its relative insensitivity to perturbations arising during the later stages of NB growth. While monitoring the increase in OD600 over time, we observed that NB cultures tended to slow down during the later stages of exponential growth, and that the Archival strain was hindered to a greater extent than Modern. This suggested that Modern may have adapted to yet unknown components of growth in NB; however, due to this decrease during late exponential phase, we were unable to accurately assess differences in specific growth rate, and sought instead to quantify performance using a head-to-head competition of Modern and Archival co-cultures.


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

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

Modern outperforms Archival AM1 when grown on nutrient broth. A) Representative growth curves of Modern (black circles) and Archival (gray squares) AM1 grown on nutrient broth (NB). Note that growth - particularly for the Archival strain - slows considerably during late exponential phase, signifying density-dependent growth inhibition. B) Change in the proportion of either Modern or Archival AM1 mixed in co-culture with a fluorescently labeled Modern reference as measured by flow cytometry. Values represent the mean plus SEM of at least three biological replicates grown in 48-well plates (A) or flasks (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Modern outperforms Archival AM1 when grown on nutrient broth. A) Representative growth curves of Modern (black circles) and Archival (gray squares) AM1 grown on nutrient broth (NB). Note that growth - particularly for the Archival strain - slows considerably during late exponential phase, signifying density-dependent growth inhibition. B) Change in the proportion of either Modern or Archival AM1 mixed in co-culture with a fluorescently labeled Modern reference as measured by flow cytometry. Values represent the mean plus SEM of at least three biological replicates grown in 48-well plates (A) or flasks (B).
Mentions: We found over multiple different growth experiments that Modern AM1 consistently outperformed Archival on NB, although both strains grew more poorly than on Hypho medium. Modern displayed a clear growth advantage on NB in 48-well plates (FigureĀ 3A), which could be traced in part to its relative insensitivity to perturbations arising during the later stages of NB growth. While monitoring the increase in OD600 over time, we observed that NB cultures tended to slow down during the later stages of exponential growth, and that the Archival strain was hindered to a greater extent than Modern. This suggested that Modern may have adapted to yet unknown components of growth in NB; however, due to this decrease during late exponential phase, we were unable to accurately assess differences in specific growth rate, and sought instead to quantify performance using a head-to-head competition of Modern and Archival co-cultures.

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