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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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Two distinct lineages of Methylobacterium extorquens AM1. Shortly after its discovery in 1960 (1), a sample of M. extorquens AM1 (“AM1”) was deposited to a culture stock center for storage and distribution (2; Archival AM1). Many researchers, however, use instead a working stock of AM1 that was maintained over fifty years in the lab (3; Modern AM1), and was at one point selected for rifamycin resistance (RifR) [45]. We hypothesized that these conditions may have fostered the accumulation of mutations and unintended evolutionary divergence in the Modern AM1 lineage, and sought to compare our Modern AM1 to the Archival strain. Dashes represent the accumulation of mutations in the Modern lineage.
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Figure 1: Two distinct lineages of Methylobacterium extorquens AM1. Shortly after its discovery in 1960 (1), a sample of M. extorquens AM1 (“AM1”) was deposited to a culture stock center for storage and distribution (2; Archival AM1). Many researchers, however, use instead a working stock of AM1 that was maintained over fifty years in the lab (3; Modern AM1), and was at one point selected for rifamycin resistance (RifR) [45]. We hypothesized that these conditions may have fostered the accumulation of mutations and unintended evolutionary divergence in the Modern AM1 lineage, and sought to compare our Modern AM1 to the Archival strain. Dashes represent the accumulation of mutations in the Modern lineage.

Mentions: To address this question, we sought to compare today’s AM1 to its ancestor isolated circa 1960, or a close descendent of this ancestor. Closely related strains of M. extorquens differ significantly in their gene content and metabolic capabilities [43], making these a less than ideal comparison to determine the ancestral “wild” AM1 state. Luckily, however, we realized that two major lineages of AM1 were established circa 1960: the stock archived circa 1961 at the culture stock center (herein referred to as the “Archival” strain); and the working line of AM1 that was propagated over many years from J.R. Quayle’s lab, to Mary Lidstrom’s group, to our own laboratory stock of “Modern” AM1 (Figure 1). We hypothesized that the Archival AM1 – which underwent longer periods of lyophilized storage with fewer growth cycles in between – might better-reflect the ancestral state of AM1 circa 1960, and offer an excellent reference with which to identify evolutionary divergence in the laboratory-maintained AM1 lineage. We document here the surprising extent to which our Modern AM1 has changed during its time in the lab using various assays of growth and fitness, long-term growth and storage, and whole-genome sequencing of the Archival AM1 strain. We then offer a discussion of specific laboratory practices and evolutionary processes that may have led to such divergence.


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

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

Two distinct lineages of Methylobacterium extorquens AM1. Shortly after its discovery in 1960 (1), a sample of M. extorquens AM1 (“AM1”) was deposited to a culture stock center for storage and distribution (2; Archival AM1). Many researchers, however, use instead a working stock of AM1 that was maintained over fifty years in the lab (3; Modern AM1), and was at one point selected for rifamycin resistance (RifR) [45]. We hypothesized that these conditions may have fostered the accumulation of mutations and unintended evolutionary divergence in the Modern AM1 lineage, and sought to compare our Modern AM1 to the Archival strain. Dashes represent the accumulation of mutations in the Modern lineage.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Two distinct lineages of Methylobacterium extorquens AM1. Shortly after its discovery in 1960 (1), a sample of M. extorquens AM1 (“AM1”) was deposited to a culture stock center for storage and distribution (2; Archival AM1). Many researchers, however, use instead a working stock of AM1 that was maintained over fifty years in the lab (3; Modern AM1), and was at one point selected for rifamycin resistance (RifR) [45]. We hypothesized that these conditions may have fostered the accumulation of mutations and unintended evolutionary divergence in the Modern AM1 lineage, and sought to compare our Modern AM1 to the Archival strain. Dashes represent the accumulation of mutations in the Modern lineage.
Mentions: To address this question, we sought to compare today’s AM1 to its ancestor isolated circa 1960, or a close descendent of this ancestor. Closely related strains of M. extorquens differ significantly in their gene content and metabolic capabilities [43], making these a less than ideal comparison to determine the ancestral “wild” AM1 state. Luckily, however, we realized that two major lineages of AM1 were established circa 1960: the stock archived circa 1961 at the culture stock center (herein referred to as the “Archival” strain); and the working line of AM1 that was propagated over many years from J.R. Quayle’s lab, to Mary Lidstrom’s group, to our own laboratory stock of “Modern” AM1 (Figure 1). We hypothesized that the Archival AM1 – which underwent longer periods of lyophilized storage with fewer growth cycles in between – might better-reflect the ancestral state of AM1 circa 1960, and offer an excellent reference with which to identify evolutionary divergence in the laboratory-maintained AM1 lineage. We document here the surprising extent to which our Modern AM1 has changed during its time in the lab using various assays of growth and fitness, long-term growth and storage, and whole-genome sequencing of the Archival AM1 strain. We then offer a discussion of specific laboratory practices and evolutionary processes that may have led to such divergence.

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