6. Background: Phosphate, essential for life? 2. Growth in arsenic-rich environment 1. Isolation of GFAJ-1 3. Arsenic within GFAJ-1?
7. Background: Phosphate, essential for life? 2. Growth in arsenic-rich environment 1. Isolation of GFAJ-1 Conclusions: GFAJ-1 incorporates arsenic into DNA, other biomolecules 3. Arsenic within GFAJ-1? 4. Arsenic within DNA? Criticisms: Dr. Rosie Redfield Dr. Alex Bradley
8.
9. Metabolic pathways confuse Arsenate (AsO43-) and Phosphate (PO43-) 3. 4. Can arsenate be biochemically substituted for phosphate in an organism specialized for coping with the presence of arsenic?
10. 1. Isolation of GFAJ-1 2. 1. 3. 4. Image courtesy of Ed Yong Placed lake sediments into medium containing increasing AsO43- (100 uM-5mM), trace amounts of PO43- (3.1 uM) In 5 mM AsO43- media shows growth rate of 0.1/day (double every 7 days)
11. 2. Growth in arsenic-rich environment 2. 1. Media grown in 5 mM AsO43- plated onto agar dishes containing 40 mM AsO43-, 3.1 uM of PO43- (+As/-P) -As/+P 3. 4. +As/-P -As/-P GFAJ-1 both increase in number and size in +As/-P conditions -As/+P +As/-P -As/-P
12. 3. Arsenic within GFAJ-1? 2. 1. Analyzed whole GFAJ-1 cells for arsenic content using Inductively Coupled Plasma Mass Spectrometry 3. 4. GFAJ-1 cultured in +As/-P conditions contain larger intracellular amounts of arsenic
13. 4. Arsenic within DNA? 2. 1. Gel purified DNA from +As/-P and –As/+P GFAJ-1 3. Tested the identities of arsenic’s chemical neighbors using Extended X-ray Absorption Fine Structure 4. Tested whether isolated DNA contained arsenic using high-resolution secondary ion mass spectrometry (NanoSIMS) Arsenate is incorporated into DNA backbone Purified GFAJ-1DNA contains arsenic
14. Conclusions 1. Bacteria isolated from Mono Lake, CA can grow in conditions with high arsenic and minimal phosphate 2. GFAJ-1 contain arsenic, localized to protein, metabolite, lipid, and DNA fractions GFAJ-1 can incorporate arsenic into DNA, as well as other biomolecules (NADH, ATP, glucose, acetyl-CoA, other proteins) 3. DNA isolated from GFAJ-1 contains arsenic, little phosphate. 4. The chemical bond lengths of arsenic are similar to those estimated by models where arsenate replaced phosphate in DNA backbone
15. Criticism 2. Were GFAJ-1 exposed to phosphate concentrations sufficient to support growth? 1. Unexplained presence of vacuoles. May contain arsenic, sequestered using membrane pumps implicated in the arsenic resistance of a closely related microbe All solutions contained a background of 3.1 uM PO43- Sargasso Sea contains <10 nM PO43-, contains microbes with phosphate-based DNA 4. Arsenic chemical bonds are inherently unstable. 3. Assimilation into DNA has not been conclusively demonstrated. Arsenic bonds hydrolyze after ~10 minutes. Isolation of GFAJ-1 DNA involved submersion in water and should therefore have fragmented. How does organism overcome instability? EXAFS primarily measures neighboring molecules, doesn’t show complete incorporation into helical DNA structure Alternate (and classical) method for direct analysis of nucleotides: mass spectrum of DNA sequences
16. Conclusions 1. Bacteria isolated from Mono Lake, CA can grow in conditions with high arsenic and minimal phosphate 1. Bacteria isolated from Mono Lake, CA can survive in conditions rich in arsenic and poor in phosphate. 2. GFAJ-1 contain arsenic, localized to protein, metabolite, lipid, and DNA fractions 2. GFAJ-1 grown in the presence of arsenic will contain intracellular arsenic. GFAJ-1 can incorporate arsenic into DNA, as well as other biomolecules (NADH, ATP, glucose, acetyl-CoA, other proteins) At best, GFAJ-1 can assimilate arsenic into DNA and other biomolecules, perhaps to be used as a functional replacement of phosphate. Alternatively, GFAJ-1 can prioritize phosphate usage while avoiding arsenic poisoning. 3. DNA isolated from GFAJ-1 contains arsenic, little phosphate. 3. Arsenic may be associated with arsenic-grown DNA, may be a thorough contaminate 4. The chemical bond lengths of arsenic are similar to those estimated by models where arsenate replaced phosphate in DNA backbone 4. Intracellular arsenic is in the form of arsenate, is not present as ionic species.
17. Further Reading Dr. Rosie Renfield’s (UBC) criticism (available online, coming soon to Science) Dr. Alex Bradley’s (Harvard) criticism (available at Science Blogs) Carl Zimmer’s article for Slate (with interviews with several eminent scientists) Felicia Wolfe-Simons response to criticism Faculty of 1000 Evaluations Ed Yong’s timeline, from the NASA press release on Dec. 2nd through his post-mortem evaluation on Dec. 10th (available at Discover Magazine blog) Bora Zivkovic’s (PLoS) link dump (available at A Blog Around the Clock) Felicia Wolfe-Simons’ TED Talk on March 2nd.
Editor's Notes
Casting back to the end of 2010, some of you may remember reading headlines similar to the following from the Atlanta Journal-ConstitutionThis is referring.
During the course of this talk, I will first discuss…Then, I will describe the major methods and results…Note: I will not be describing all of the experiments included in the paper, as there were several which overlapped. I have therefore picked the most relevant.
To begin, I will describe the isolation…Strain ofHalomonadaceae
Next, I will describe the culture of GFAJ-1 in an arsenic rich enivornment
I will show you evidence that GFAJ-1 contains arsenic
And (via the use of two separate methods) that GFAJ-1 DNA contains arsenic.All together, I will present Wolfe-Simons’ evidence that…Finally, I will present a few of the criticisms raised, in particular…RR: University of British Columbia, Vancouver
Note: normal Mono Lake concentrations are 200 uM As, 1 mM P (more than nearly any other environment on earth)
Growth rate in 40 mM arsenate: one doubling every 2 days. Grows faster if arsenate replaced with 1.5 mM phosphateCell counts via acridine orange direct cell count (acridine orange unable to differentiate between viable and non-viable cells)Note Vacuoles: likely carbon/energy storage material polyhydroxybutyrate (PHB): bacterial cells produce it when their carbon/energy supply is good but other nutrients needed for growth are in short supply.
Note: variability in arsenic measures: they claims its due to cells breaking apartICP-MS: A droplet of nebulized sample enters the inductively coupled plasma stream, evaporates, and any solid that was dissolved in the liquid vaporizes and then breaks down into atoms. At the temperatures prevailing in the plasma, a significant proportion of the atoms of many chemical elements are ionized, each atom losing its most loosely-bound electron to form a singly charged ion. These ions are extracted into the mass spec, and separated on the basis fo their mass-to-charge ratio, with a detector receiving an ion signal proportional to the concentration of the ions.
NanoSIMS: analyze the composition of solid surfaces. Shoot a ion beam at the surface, collect ejected secondary ions.EXAFS: Shoot x-rays at a sample, detect reflected and transmitted x-ray intensities. Gives info on # and chemical identities off near neighbors and average inter-atomic distance (up to 5-6 angstroms). Occurs due to interference effects as photoelectrons leave the surface of a material. The interference depends on the inter-atomic distance between the atom that ejected the photo-electrode and the nearest neighbor atom.
Questions for the future: How does the switch affect the functionality of the molecules? Within the bacteria will arsenlyation take the place of phosphorylation? How do these cells avoid the main problem of arsenic poisoning, namely that arsenic replaces phosphate, blocking metabolic pathways?
#2: what did NanoSIMS measure: arsenate in gel, stuck to cellular material. Control: grow in both high phosphate and arsenate: see if similar high levels.Note: EXAFS shown in study is similar to that of unbound sodium arsenate – need to do better controls.Closely related microbe: Halomonaselongata strain DSM 2581 (94% identity on rRNA level)Phosphate transporterArsenate bonds reinfoced by specialized molecules? Arsenic-based life simply has higher turnover for molecules disintegration/assembly than conventional life.
Prioritizing phosphate usage to allow viability. The third option being that GFAJ-1 uses phosphate for critical biochemical events (eg DNA), using arsenic as an alternative for less critical processes. Need to test to see whether arsenic gums up metabolic pathways as is seen in classical arsenic poisoning.
Carl Zimmer interviewed Roger Summons (MIT), Bradley’s Ph.D advisor, who backed up Alex.Note: Arsenic is very rare extraterrestriallly
