It has been identified previously as an osmolyte in methanogenic Archaea (Robertson et al., 1990, 1992; Selleckchem Epacadostat Lai et al., 1991; Roberts, 2005) and also in three genera of Bacteria: the marine gammaproteobacterium Alteromonas luteoviolacea (Henrichs & Cuhel, 1985), several species of the actinobacterial genus Nocardiopsis (Galinski & Trüper, 1994; DasSarma & Arora, 2002) and the anoxigenic phototrophic bacterium Thiohalocapsa halophila DSM 6210T (Severin et al., 1992). Anionic solutes, such as α-glutamate and β-glutamate, have been considered

to be counterions for elevated intracellular K+ concentration (Roberts, 2005). In addition, the most prominent 13C chemical shifts (at salinities >3%) in NMR data were consistent with the accumulation of a compound that was previously unknown as a compatible solute in Bacteria, tentatively identified as NeABL according to connectivities in 2D-NMR experiments and 13C DEPT-135 data. 1H–13C HMQC data allowed determining the correlation between 1H and 13C shifts detected on corresponding spectra for such compounds (Supporting Information, Fig. S1). Other connectivities derived from a 1H–1H COSY indicated both CH2-CH(N)-CH2 (asymmetric carbon in β-position) and -NCH2-CH2- moieties as well KU-60019 as the CH3CO group (Fig. S2), which was further assigned to the ɛ-position by means of 1H–13C HMBC experiments, because a cross-peak

was detected between the proton in the ɛ-position (CH2 resonance at 3.20 p.p.m.) and the carbonyl of the acetyl group (13C shift at 176.7 p.p.m.)

(Fig. S3). ESI-MS analyses from collected fractions of a desalted aqueous cell extract showed a molecular peak at m/z 188.5 for the suspected compound (Fig. S4). Therefore, the molecular formula of C8H16N2O3 was proposed. The observed MS signals proved to be consistent with the theoretical isotope pattern of this molecule. Subsequently, the proposed structure was confirmed through its chromatographic preparation and purification (Figs S5–S8). enough Both 13C and 1H chemical shifts of the purified compound as well as its 1H–1H (COSY) connectivities (Fig. 2) were also consistent with the proposed structure corresponding to NeABL. Both isolated and type GSB strains were cultured in media with different NaCl concentrations to determine differences in the composition of major compatible solutes from 13C-NMR spectra. Although GSB species had essentially the same cocktail of compatible solutes, changes in the relative intensity of signal in NMR results suggested that different ratios among these compounds occurred in different strains and salt concentrations. Thus, anionic solutes, either l-α-glutamate or β-glutamate, would be the main organic compounds accumulated for osmotic balance at a low NaCl content (3%) (Fig. 1).