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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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Equivalence of AM1 strains during tests of long-term growth and survival. Co-cultures were created by mixing either Modern (black circles) or Archival AM1 (gray squares) with a fluorescently labeled Modern reference, and the change in unlabeled versus fluorescent cells was monitored over time using flow cytometry. A) In continually shaken flasks with succinate, the Archival strain increased in frequency over the first two days of growth and maintained this advantage over Modern over time. B) Similarly, Archival increased in frequency during four days of growth on methylamine agar plates (not shown), and maintained this frequency during long-term storage at 4°C. Values represent the mean plus SEM of the percent unlabeled cells measured in three replicate co-cultures.
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Figure 4: Equivalence of AM1 strains during tests of long-term growth and survival. Co-cultures were created by mixing either Modern (black circles) or Archival AM1 (gray squares) with a fluorescently labeled Modern reference, and the change in unlabeled versus fluorescent cells was monitored over time using flow cytometry. A) In continually shaken flasks with succinate, the Archival strain increased in frequency over the first two days of growth and maintained this advantage over Modern over time. B) Similarly, Archival increased in frequency during four days of growth on methylamine agar plates (not shown), and maintained this frequency during long-term storage at 4°C. Values represent the mean plus SEM of the percent unlabeled cells measured in three replicate co-cultures.

Mentions: Here, Modern and Archival AM1 were roughly equivalent in terms of growth and survival during stationary phase. In flasks, the ratio of unlabeled Archival cells to labeled Modern cells was steady for 12 days after the initial 2 days of exponential growth (Figure 4A), while the unlabeled Modern control remained unchanged for the duration of the experiment. Archival held a similar advantage during storage at 4°C on agar plates for up to 60 days (Figure 4B). Collectively these results suggest that, at least under these conditions tested, the extent to which laboratory domestication improved the long-term growth and survival of AM1 is limited, and that the major difference between these strains lies in decreased exponential phase growth in Modern under standard conditions, and slightly increased growth on NB.


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

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

Equivalence of AM1 strains during tests of long-term growth and survival. Co-cultures were created by mixing either Modern (black circles) or Archival AM1 (gray squares) with a fluorescently labeled Modern reference, and the change in unlabeled versus fluorescent cells was monitored over time using flow cytometry. A) In continually shaken flasks with succinate, the Archival strain increased in frequency over the first two days of growth and maintained this advantage over Modern over time. B) Similarly, Archival increased in frequency during four days of growth on methylamine agar plates (not shown), and maintained this frequency during long-term storage at 4°C. Values represent the mean plus SEM of the percent unlabeled cells measured in three replicate co-cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Equivalence of AM1 strains during tests of long-term growth and survival. Co-cultures were created by mixing either Modern (black circles) or Archival AM1 (gray squares) with a fluorescently labeled Modern reference, and the change in unlabeled versus fluorescent cells was monitored over time using flow cytometry. A) In continually shaken flasks with succinate, the Archival strain increased in frequency over the first two days of growth and maintained this advantage over Modern over time. B) Similarly, Archival increased in frequency during four days of growth on methylamine agar plates (not shown), and maintained this frequency during long-term storage at 4°C. Values represent the mean plus SEM of the percent unlabeled cells measured in three replicate co-cultures.
Mentions: Here, Modern and Archival AM1 were roughly equivalent in terms of growth and survival during stationary phase. In flasks, the ratio of unlabeled Archival cells to labeled Modern cells was steady for 12 days after the initial 2 days of exponential growth (Figure 4A), while the unlabeled Modern control remained unchanged for the duration of the experiment. Archival held a similar advantage during storage at 4°C on agar plates for up to 60 days (Figure 4B). Collectively these results suggest that, at least under these conditions tested, the extent to which laboratory domestication improved the long-term growth and survival of AM1 is limited, and that the major difference between these strains lies in decreased exponential phase growth in Modern under standard conditions, and slightly increased growth on NB.

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