Sunday, 13 October 2013

A light-driven sodium ion pump in marine bacteria

A light-driven sodium ion pump in marine bacteria

Rhodopsins are common in microorganisms; they are used to create a membrane potential for ATP synthesis. Proteorhodopsins are light driven proton pumps common in Marine Prokaryotes (convert energy of light to a proton gradient, which is used as the energy source of the cell). Sodium ion pumps are separate, and help drive the uptake of nutrients. No hybrid of these had been discovered, but this was considered reasonable at a chemical level.

The authors of this paper have discovered a bacterium containing the first light-driven sodium ion pump, a Marine Flavobacterium K.eikastus. This has two pumps, KR1 which is a typical proton pump, and KR2 which pumps sodium outward. Through testing they highlighted that:

·        KR2 and KR1 are major products at 48 and 96 hours respectively in the growth of K.eikastus. Pumps were discovered and verified using both K.eikastus and E.coli.
·        KR1 is a light driven proton pump, shown by measuring pH in cell suspension which, upon illumination, decreased at the 96h mark.
·        pH increased at 48 hours, suggesting  the KR2 pump has a different role. Further tests in anion and cation solutions showed that KR2 is an outward pump for sodium and lithium but pumps proton in salts of K+, RB+ and Cs+.

K.eikastus binds sodium ions.
Analysis of spectral change showed that different spectra were observed between NaCl (Sodium Chloride) and KCl (Potassium Chloride) exchange. Na+ to K+ yielded identical spectral changes as Na+ to Rb+ and Na+ to Cs+. This suggests that none of K+, Rb+ or Cs+ binds to the sodium ion receptor (NaR), meaning that sodium must be the binding agent.

Mutational analysis of K.eikastus KR2.
Carried out by removing or replacing several key amino acids (e.g. R190, D116). The results of this experiment were:

·        R109, D116 and D215 have some effect on sodium ion and proton pumping activities, as upon removal, these abilities were lost.
·        R109 is a prerequisite for sodium ion proton pumps binding. When removed, sodium ion binding is lost.
·        D116 and D215 are key residues for the pump, but are not necessary for sodium ion binding. Sodium ion binding is weak but substantial for D116N and D215N.
·        D116 is a prerequisite for the sodium ion pump. Shown by the formation of blue-shifted intermediate (as opposed to red-shifted).
·        D116 is suggested to function as the proton acceptor, allowing uptake of a sodium ion from cytoplasmic side. D116 constitutes selectivity filter of ions.
·        R109, N112 and carboxylates (found in the BC loop mutant) constitute sodium ion binding site. Ability lost when these were removed.
·        Sodium ion binding is not the requirement of the sodium ion pump.
·        H30 in alpha-helix is required for proton pumping. This is unique for the KR2 pump.

K.eikastus is a hybrid outward sodium ion-proton pump.
This pump is less efficient than other prototypical proton pumps. Sodium ion and proton transport pathways are mostly common in KR2. KR2 also keeps lithium ion concentration low (excreted by light) which is useful as lithium is poisonous to the cells.

This paper details the first classified bacteria which have a light-driven sodium ion pump. It has been shown that just a few (as little as 5 or 6) amino acids found in the organism can constitute this ability. The authors believe that not only will this discovery develop our knowledge of marine bacterial physiology, but that the pump could become a key tool in optogenetics. This is a method by which we can control the activity of individual neurons in living tissue, even within freely-moving animals, and precisely measure the effects of those manipulations in real-time. Personally I think that this study also highlights how small changes in amino acid sequences and organisation can have vast impacts on organism function. If you view this sequence in the light of Horizontal Gene Transfer, it is not impossible to imagine that this ability could be shared between many marine bacteria.

Keiichi Inoue, Hikaru Ono, Rei Abe-Yoshizumi, Susumu Yoshizawa, Hiroyasu Ito, Kazuhiro Kogure & Hideki Kandori. (2013). A light-driven sodium ion pump in marine bacteria. Nature Communications. 4, 1678.

1 comment:

  1. Thanks Ethan - an interesting paper. I think it's unfortunate that the authors used the term "bacteria" in their title instead of the domain name "Bacteria" This is because the naming of these rhodopsins is confusing - bacteriorhodopsin was the first type - discovered in Archaea from hypersaline environments and is known to be a sodium pump. Subsequently, proteorhodopsins were shown to be widespread in Archaea and Bacteria (due to extensive horizontal gene transfer). [It's not helped by continued use of the term "prokaryotes"]. There are various ways in which micro-organisms maintain low sodium conc. in the cell, which requires energy. I think the significance of this is that it seems to be the first time that light has been shown to provide the energy. The optogentics idea sounds fascinating - how would this work?