The molecular basis of salt adaptation in Methanosarcina mazei Gö1.

The study on the molecular basis of salt adaptation and its regulation in archaea is still in its infancy, but genomics and functional genome analyses combined with classical biochemistry shed light on the processes conferring salt adaptation in the methanogenic archaeon Methanosarcina mazei Gö1. In this article, we will review discoveries made within the last years that will culminate in the description of the overall cellular response of M. mazei Gö1 to elevated salinities. This response includes accumulation of solutes and export of Na+ as well as potential uptake/export of K+ but also a restructuring of the cell surface.

The salt-induced ABC transporter Ota of the methanogenic archaeon Methanosarcina mazei Gö1 is a glycine betaine transporter.

The genes encoding the three subunits of the primary ABC transporter Ota of the methanogenic archaeon Methanosarcina mazei Gö1 were cloned in an expression vector (pBAD24) and transformed into the glycine betaine transport-negative mutant Escherichia coli MKH13. Ota was produced as demonstrated by Western blotting. Uptake studies revealed that Ota catalyzed the transport of glycine betaine in E. coli MKH13(pBAD-Ota) with a K(m) of 10+/-5 microM and a maximal velocity of 1.5+/-0.5 nmol min(-1) mg protein(-1). Transport was ATP dependent. Ota was activated by salinity gradients, but only marginally by sugar gradients across the membrane. Glycine betaine transport was inhibited to a small extent by an excess of dimethylglycin or proline betaine, but not by sarcosine or glycine.

Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides.

Organisms, which grow on organic substrates that are metabolized via acetyl-CoA, are faced with the problem to form all cell constituents from this C(2)-unit. The problem was solved by the seminal work of Kornberg and is known as the glyoxylate cycle. However, many bacteria are known to not contain isocitrate lyase, the key enzyme of this pathway. This problem was addressed in acetate-grown Rhodobacter sphaeroides. An acetate-minus mutant identified by transposon mutagenesis was affected in the gene for beta-ketothiolase forming acetoacetyl-CoA from two molecules of acetyl-CoA. This enzyme activity was missing in this mutant, which grew on acetoacetate and on acetate plus glyoxylate. A second acetate/acetoacetate-minus mutant was affected in the gene for a putative mesaconyl-CoA hydratase, an enzyme which catalyses the hydration of mesaconyl-CoA to beta-methylmalyl-CoA. Beta-methylmalyl-CoA is further cleaved into glyoxylate and propionyl-CoA. These results as well as identification of acetate-upregulated proteins by two-dimensional gel electrophoresis lead to the proposal of a new pathway for acetate assimilation. In a first part, affected by the mutations, two molecules of acetyl-CoA and one molecule CO(2) are converted via acetoacetyl-CoA and mesaconyl-CoA to glyoxylate and propionyl-CoA. In a second part glyoxylate and propionyl-CoA are converted with another molecule of acetyl-CoA and CO(2) to l-malyl-CoA and succinyl-CoA.

Stress response by solute accumulation in archaea.

The accumulation of organic solutes is a prerequisite for osmotic adjustment of all organisms. Archaea synthesize unusual solutes such as beta-amino acids, Nepsilon-acetyl-beta-lysine, mannosylglycerate and di-myo-inositol phosphate but, as in other cells, uptake of solutes such as glycine betaine is preferred over de novo synthesis. Study of the molecular basis of osmoadaptation and its regulation in archaea is still in its infancy, but genomics and functional genome analyses combined with classical biochemistry shed light on the processes that confer osmoadaptation in archaea. Most interestingly, some solutes are not only produced in response to salt but also to temperature stress.