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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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Mutations associated with rifamycin resistance hinder AM1 growth. A) Spectrum of mutations to the RNA polymerase beta subunit (RpoB) during past and current selection for rifamycin resistance (RifR). Modern AM1 was selected for RifR in 1984 [45] and acquired two mutations to RpoB: Q521R, and Q1081R (denoted by an asterisk). By recapitulating selection for RifR in replicate Archival populations, we identified a number of other RpoB mutations putatively associated with RifR. B) The effect of RifR mutations on growth rate in the absence of antibiotic. Values represent the mean plus SEM of four biological replicates grown in 48-well plates with succinate. Strains that were significantly slower than Archival are marked with asterisks (p < 0.01, one-way ANOVA with Tukey post-hoc test).
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Figure 5: Mutations associated with rifamycin resistance hinder AM1 growth. A) Spectrum of mutations to the RNA polymerase beta subunit (RpoB) during past and current selection for rifamycin resistance (RifR). Modern AM1 was selected for RifR in 1984 [45] and acquired two mutations to RpoB: Q521R, and Q1081R (denoted by an asterisk). By recapitulating selection for RifR in replicate Archival populations, we identified a number of other RpoB mutations putatively associated with RifR. B) The effect of RifR mutations on growth rate in the absence of antibiotic. Values represent the mean plus SEM of four biological replicates grown in 48-well plates with succinate. Strains that were significantly slower than Archival are marked with asterisks (p < 0.01, one-way ANOVA with Tukey post-hoc test).

Mentions: The spectrum of rpoB mutations across RifR Archival isolates displayed highly variable effects on growth rate in the absence of antibiotic. Compared to their Archival ancestor, several RifR isolates show very little decrease in performance when grown on succinate, while other isolates slow to near Modern levels, and still others grow substantially worse than Modern (Figure 5B). Interestingly, the CM4022 isolate that perfectly recapitulated the change from Archival to Modern AM1 (Q521R) was slightly faster than Modern, suggesting perhaps that other mutations further hamper growth in the Modern lineage. We note, however, that a direct comparison of this strain is difficult given that many other mutations and mutational interactions were likely present in Modern AM1 during the original selection for RifR. Nevertheless, these results demonstrate that selection for RifR can substantially hinder growth of the Archival strain in the absence of antibiotic, and that this single researcher-imposed event – not laboratory domestication - is the major mechanism by which Modern AM1 became slower growing 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)

Mutations associated with rifamycin resistance hinder AM1 growth. A) Spectrum of mutations to the RNA polymerase beta subunit (RpoB) during past and current selection for rifamycin resistance (RifR). Modern AM1 was selected for RifR in 1984 [45] and acquired two mutations to RpoB: Q521R, and Q1081R (denoted by an asterisk). By recapitulating selection for RifR in replicate Archival populations, we identified a number of other RpoB mutations putatively associated with RifR. B) The effect of RifR mutations on growth rate in the absence of antibiotic. Values represent the mean plus SEM of four biological replicates grown in 48-well plates with succinate. Strains that were significantly slower than Archival are marked with asterisks (p < 0.01, one-way ANOVA with Tukey post-hoc test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Mutations associated with rifamycin resistance hinder AM1 growth. A) Spectrum of mutations to the RNA polymerase beta subunit (RpoB) during past and current selection for rifamycin resistance (RifR). Modern AM1 was selected for RifR in 1984 [45] and acquired two mutations to RpoB: Q521R, and Q1081R (denoted by an asterisk). By recapitulating selection for RifR in replicate Archival populations, we identified a number of other RpoB mutations putatively associated with RifR. B) The effect of RifR mutations on growth rate in the absence of antibiotic. Values represent the mean plus SEM of four biological replicates grown in 48-well plates with succinate. Strains that were significantly slower than Archival are marked with asterisks (p < 0.01, one-way ANOVA with Tukey post-hoc test).
Mentions: The spectrum of rpoB mutations across RifR Archival isolates displayed highly variable effects on growth rate in the absence of antibiotic. Compared to their Archival ancestor, several RifR isolates show very little decrease in performance when grown on succinate, while other isolates slow to near Modern levels, and still others grow substantially worse than Modern (Figure 5B). Interestingly, the CM4022 isolate that perfectly recapitulated the change from Archival to Modern AM1 (Q521R) was slightly faster than Modern, suggesting perhaps that other mutations further hamper growth in the Modern lineage. We note, however, that a direct comparison of this strain is difficult given that many other mutations and mutational interactions were likely present in Modern AM1 during the original selection for RifR. Nevertheless, these results demonstrate that selection for RifR can substantially hinder growth of the Archival strain in the absence of antibiotic, and that this single researcher-imposed event – not laboratory domestication - is the major mechanism by which Modern AM1 became slower growing 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