Limits...
Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.

Clark IC, Melnyk RA, Youngblut MD, Carlson HK, Iavarone AT, Coates JD - MBio (2015)

Bottom Line: One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons.To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1.To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer.

View Article: PubMed Central - PubMed

Affiliation: Department of Civil and Environmental Engineering, University of California, Berkeley, California, USA.

No MeSH data available.


Related in: MedlinePlus

Investigating the roles of specific mutations in activating the chromosomal insertion. (A) Growth of single, double, and triple mutations made in the unevolved strain with 10 mM nitrate as a positive control. (B) Cld protein expression under nitrate-reducing conditions of strains ICC99, ICC225. and ICC228. (C) Investigating the role of SNP2 within the NarP binding sequence between narQP and nrfA. Growth of strains with mutations SNP1 plus SNP2 (ICC228), SNP1 plus ΔnrfA (ICC348), and SNP1 plus ΔnarP (ICC349) on 10 mM chlorate or 5 mM nitrate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4442138&req=5

fig3: Investigating the roles of specific mutations in activating the chromosomal insertion. (A) Growth of single, double, and triple mutations made in the unevolved strain with 10 mM nitrate as a positive control. (B) Cld protein expression under nitrate-reducing conditions of strains ICC99, ICC225. and ICC228. (C) Investigating the role of SNP2 within the NarP binding sequence between narQP and nrfA. Growth of strains with mutations SNP1 plus SNP2 (ICC228), SNP1 plus ΔnrfA (ICC348), and SNP1 plus ΔnarP (ICC349) on 10 mM chlorate or 5 mM nitrate.

Mentions: Changes in copy number represent a relatively rapid way to tune suboptimal expression, but harboring 600 to 900 kb of extra DNA represents a selective disadvantage. Under aerobic conditions, strains ICC121.1 and ICC121.2 experienced contraction in coverage and lost the ability to grow by chlorate reduction (strains ICC126.1 and ICC126) (Fig. 2). Even under selective conditions, large copy numbers are expected to give way to less frequent point mutations that are free from the cost of excessive DNA replication. One strain (ICC121.10) that contained an average of 2.5-fold chromosomal coverage grew more rapidly than the rate predicted by a regression of growth versus copy number (Fig. 2B, circled strain). This suggested that compensatory mutations allowed more robust growth in the absence of high gene dosage. This strain contained two SNPs and a single base pair insertion that could be responsible for this effect. The SNPs were located between cld and cytochrome c (SNP1) and between narQP (SO_3981 and SO_3982) and nrfA (SO_3980) (SNP2), while the indel was found between SO_3718 and SO_3719. To test the importance of the three mutations, we constructed single, double, and triple mutant combinations in the unadapted background (strain ICC99). The mutation upstream of cld (SNP1) was sufficient for growth on chlorate, while SNP2 and the indel alone were both insufficient. SNP2 improved growth in an SNP1 background but failed to have an effect in an indel background (Fig. 3A). This indicated that SNP1 was epistatic to SNP2.


Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.

Clark IC, Melnyk RA, Youngblut MD, Carlson HK, Iavarone AT, Coates JD - MBio (2015)

Investigating the roles of specific mutations in activating the chromosomal insertion. (A) Growth of single, double, and triple mutations made in the unevolved strain with 10 mM nitrate as a positive control. (B) Cld protein expression under nitrate-reducing conditions of strains ICC99, ICC225. and ICC228. (C) Investigating the role of SNP2 within the NarP binding sequence between narQP and nrfA. Growth of strains with mutations SNP1 plus SNP2 (ICC228), SNP1 plus ΔnrfA (ICC348), and SNP1 plus ΔnarP (ICC349) on 10 mM chlorate or 5 mM nitrate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4442138&req=5

fig3: Investigating the roles of specific mutations in activating the chromosomal insertion. (A) Growth of single, double, and triple mutations made in the unevolved strain with 10 mM nitrate as a positive control. (B) Cld protein expression under nitrate-reducing conditions of strains ICC99, ICC225. and ICC228. (C) Investigating the role of SNP2 within the NarP binding sequence between narQP and nrfA. Growth of strains with mutations SNP1 plus SNP2 (ICC228), SNP1 plus ΔnrfA (ICC348), and SNP1 plus ΔnarP (ICC349) on 10 mM chlorate or 5 mM nitrate.
Mentions: Changes in copy number represent a relatively rapid way to tune suboptimal expression, but harboring 600 to 900 kb of extra DNA represents a selective disadvantage. Under aerobic conditions, strains ICC121.1 and ICC121.2 experienced contraction in coverage and lost the ability to grow by chlorate reduction (strains ICC126.1 and ICC126) (Fig. 2). Even under selective conditions, large copy numbers are expected to give way to less frequent point mutations that are free from the cost of excessive DNA replication. One strain (ICC121.10) that contained an average of 2.5-fold chromosomal coverage grew more rapidly than the rate predicted by a regression of growth versus copy number (Fig. 2B, circled strain). This suggested that compensatory mutations allowed more robust growth in the absence of high gene dosage. This strain contained two SNPs and a single base pair insertion that could be responsible for this effect. The SNPs were located between cld and cytochrome c (SNP1) and between narQP (SO_3981 and SO_3982) and nrfA (SO_3980) (SNP2), while the indel was found between SO_3718 and SO_3719. To test the importance of the three mutations, we constructed single, double, and triple mutant combinations in the unadapted background (strain ICC99). The mutation upstream of cld (SNP1) was sufficient for growth on chlorate, while SNP2 and the indel alone were both insufficient. SNP2 improved growth in an SNP1 background but failed to have an effect in an indel background (Fig. 3A). This indicated that SNP1 was epistatic to SNP2.

Bottom Line: One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons.To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1.To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer.

View Article: PubMed Central - PubMed

Affiliation: Department of Civil and Environmental Engineering, University of California, Berkeley, California, USA.

No MeSH data available.


Related in: MedlinePlus