Differential Regulation of Ota and Otb, Two Primary Glycine Betaine Transporters in the Methanogenic Archaeon <i>Methanosarcina mazei</i> Gö1

Abstract

Methanogenic archaea accumulate glycine betaine in response to hypersalinity, but the regulation of proteins involved, their mechanism of activation and regulation of the corresponding genes are largely unknown. Methanosarcina mazei differs from most other methanoarchaea in having two gene clusters both encoding a potential glycine betaine transporter, Ota and Otb. Western blot as well as quantitative real-time PCR revealed that Otb is not regulated by osmolarity. On the other hand, cellular levels of Ota increased with increasing salt concentrations. A maximum was reached at 300–500 mM NaCl. Ota concentrations reached a maximum 4 h after an osmotic upshock. Hyperosmolarity also caused an increase in cellular Ota concentrations. In addition to osmolarity Ota expression was regulated by the growth phase. Expression of Ota as well as transport of betaine was downregulated in the presence of glycine betaine.

Canonical and ECF-type ATP-binding cassette importers in prokaryotes: diversity in modular organization and cellular functions

“…The Ota system is composed of a substrate-binding protein, OtaC, a transmembrane subunit, OtaB, and the ATPase, OtaA (Spanheimer et al, 2008). The Ota system is composed of a substrate-binding protein, OtaC, a transmembrane subunit, OtaB, and the ATPase, OtaA (Spanheimer et al, 2008).…”

A novel limb in the osmoregulatory network of Methanosarcina mazei Gö1: Nε‐acetyl‐β‐lysine can be substituted by glutamate and alanine

“…Cells were grown at different salinities to late exponential growth phase, harvested and transport was studied in cell suspensions using [ 14 C]‐alanine and [ 14 C]‐glycine betaine as a control. As shown before, cells of the wild type took up glycine betaine (Roeßler et al ., 2002; Spanheimer et al ., 2008) and the same was true for M. mazei Δ abl::pac . However, we did not detect any uptake of alanine at various salinities tested either in the wild type or in the mutant (data not shown).…”

“…The internal pool of compatible solutes of M. mazei cells was analysed by NMR spectroscopy. Cells were grown in minimal medium to an OD 578 of about 0.8, harvested and freeze dried, and the quantification was performed in ethanolic cell extracts as previously described (Pflüger et al ., 2003; Spanheimer et al ., 2008). Minimal medium contained different NaCl concentrations (38.5, 400 or 800 mM), 150 mM methanol and betaine (1 mM) as indicated.…”

“…NaCl concentration was added to the medium as indicated. Harvesting and preparation of cell suspension were performed as previously described (Spanheimer et al ., 2008).…”

“…Cell suspensions for transport studies were always prepared fresh. Determination of alanine uptake in M. mazei was determined as previously described for glycine betaine (Spanheimer et al ., 2008). Radioactive labelled [ 14 C]‐alanine (Hartmann Analytik, Braunschweig, Germany) was added to the assay buffer in a final concentration of 100 μM (1 μCi, specific activity: 110 μCi μmol −1 ).…”