Export of Thermus thermophilus alkaline phosphatase via the twin-arginine translocation pathway in Escherichia coli.

The bacterial twin-arginine translocation (Tat) pathway is distinct from the Sec system by its remarkable capacity to export folded enzymes. To address the question whether the two systems are capable of translocating homologous enzymes catalyzing the same reaction, we cloned the tap gene encoding Thermus thermophilus alkaline phosphatase (Tap) and expressed it in Escherichia coli. Unlike the alkaline phosphatase of E. coli, which is translocated through the Sec system and then activated in the periplasm, Tap was exported exclusively via the Tat pathway and active Tap precursor was observed in the cytoplasm. These results demonstrate that two sequence and functional related enzymes are exported by distinct protein transport systems, which may play an integral role in the bacterial adaptation to their environment during the evolution.

Uptake of choline from salmon flesh and its conversion to glycine betaine in response to salt stress in Shewanella putrefaciens.

When cultured in M63 minimal medium plus 0.6 M NaCl, the growth of Shewanella putrefaciens was strongly inhibited. The addition of an extract from smoked salmon to this medium restored the growth almost to the unstressed level. A comparison of the 13C NMR spectra of intracellular solutes extracted from S. putrefaciens cells cultured in both conditions revealed the accumulation of glycine betaine (GB) from the smoked salmon extract (SSE). Analysis of the osmoprotective properties of this extract for several strains of Escherichia coli (which differ from each other in their ability to accumulate GB (i) from the surrounding environment, and (ii) from its hydroxylated precursor choline), demonstrated the absence of GB in the SSE. From the overall results, we inferred that salt-stressed S. putrefaciens cells accumulated GB from choline present in the SSE. Furthermore, the use of [14C]-labeled betaines gave evidence that S. putrefaciens (i) oxidised choline to GB, (ii) accumulated GB as a non-metabolisable osmolyte (up to 1300 nmol (mg dw)(-1) when cultured in a medium containing 0.5 M NaCl and either 1 mM choline or 1 mM GB), and (iii) both choline and GB uptake activities were osmotically upregulated (both activities were increased more than 50-fold in media containing 0.4 to 0.6 M NaCl). In all, our results suggest that in salted smoked salmon, S. putrefaciens imports and oxidises choline, leading to the intracellular accumulation of GB.

Osmoprotection by pipecolic acid in Sinorhizobium meliloti: specific effects of D and L isomers.

DL-Pipecolic acid (DL-PIP) promotes growth restoration of Sinorhizobium meliloti cells facing inhibitory hyperosmolarity. Surprisingly, D and L isomers of this imino acid supplied separately were not effective. The uptake of L-PIP was significantly favored in the presence of the D isomer and by a hyperosmotic stress. Chromatographic analysis of the intracellular solutes showed that stressed cells did not accumulate radiolabeled L-PIP. Rather, it participates in the synthesis of the main endogenous osmolytes (glutamate and the dipeptide N-acetylglutaminylglutamine amide) during the lag phase, thus providing a means for the stressed cells to recover the osmotic balance. (13)C nuclear magnetic resonance analysis was used to determine the fate of D-PIP taken into the cells. In the absence of L-PIP, the imported D isomer was readily degraded. Supplied together with its L isomer, D-PIP was accumulated temporarily and thus might contribute together with the endogenous osmolytes to enhance the internal osmotic strength. Furthermore, it started to disappear from the cytosol when the L isomer was no longer available in the culture medium (during the late exponential phase of growth). Together, these results show an uncommon mechanism of protection of osmotically stressed cells of S. meliloti. It was proved, for the first time, that the presence of the two isomers of the same molecule is necessary for it to manifest an osmoprotective activity. Indeed, D-PIP seems to play a major role in cellular osmoadaptation through both its own accumulation and improvement of the utilization of the L isomer as an immediate precursor of endogenous osmolytes.

Glycerol metabolism in Lactobacillus collinoides: production of 3-hydroxypropionaldehyde, a precursor of acrolein.

Lactobacillus collinoides is a lactic acid bacterium commonly found in fermenting apple juice. Although this bacterium is not particularly involved in malolactic conversion, the presence of L. collinoides in cider may have serious consequences on the product. L. collinoides is indeed considered to be responsible for the transformation of glycerol to 3-hydroxypropionaldehyde (3-HPA), a precursor of acrolein that spoils the product quality by generating bitter tastes. The purpose of our work was to evaluate the influence of environmental and culture conditions on the conversion of glycerol to 3-HPA in L. collinoides, and to obtain a DNA probe of the gene coding for glycerol dehydratase, the enzyme responsible for this conversion.

Is the accumulation of osmoprotectant the unique mechanism involved in bacterial osmoprotection?

Sucrose, trehalose, maltose, cellobiose, gentiobiose, turanose and palatinose are very unusual osmoprotectants for Sinorhizobium meliloti, because these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed S. meliloti cells. Rather, these compounds were catabolized during early exponential growth, and contributed to enhance the cytosolic levels of the two endogenously-synthesized osmolytes: glutamate and the dipeptide N-acetylglutaminylglutamine amide (NAGGN). Furthermore, all of the disaccharides that acted as powerful osmoprotectants shared the same uptake routes in S. meliloti. Here, we show that these disaccharides, in fact, belong to a new family of non-accumulated sinorhizobial osmoprotectants and that two mechanisms of osmoprotection coexist in S. meliloti.

Disaccharides as a new class of nonaccumulated osmoprotectants for Sinorhizobium meliloti.

Sucrose and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) are very unusual osmoprotectants for Sinorhizobium meliloti because these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed S. meliloti cells. Here, we show that, in fact, sucrose and ectoine belong to a new family of nonaccumulated sinorhizobial osmoprotectants which also comprises the following six disaccharides: trehalose, maltose, cellobiose, gentiobiose, turanose, and palatinose. Also, several of these disaccharides were very effective exogenous osmoprotectants for strains of Rhizobium leguminosarum biovars phaseoli and trifolii. Sucrose and trehalose are synthesized as endogenous osmolytes in various bacteria, but the other five disaccharides had never been implicated before in osmoregulation in any organism. All of the disaccharides that acted as powerful osmoprotectants in S. meliloti and R. leguminosarum also acted as very effective competitors of [14C]sucrose uptake in salt-stressed cultures of these bacteria. Conversely, disaccharides that were not osmoprotective for S. meliloti and R. leguminosarum did not inhibit sucrose uptake in these bacteria. Hence, disaccharide osmoprotectants apparently shared the same uptake routes in these bacteria. Natural-abundance 13C nuclear magnetic resonance spectroscopy and quantification of cytosolic solutes demonstrated that the novel disaccharide osmoprotectants were not accumulated to osmotically significant levels in salt-stressed S. meliloti cells; rather, these compounds, like sucrose and ectoine, were catabolized during early exponential growth, and contributed indirectly to enhance the cytosolic levels of two endogenously synthesized osmolytes, glutamate and the dipeptide N-acetylglutaminylglutamine amide. The ecological implication of the use of these disaccharides as osmoprotectants is discussed.

Sucrose is a nonaccumulated osmoprotectant in Sinorhizobium meliloti.

Intracellular accumulation of sucrose in response to lowered water activity seems to occur only in photosynthetic organisms. Here we demonstrate, for the first time, the potent ability of this common sugar, supplied exogenously, to reduce growth inhibition of Sinorhizobium meliloti cells in media of inhibitory osmolarity. Independently of the nature of the growth substrates and the osmotic agent, sucrose appears particularly efficient in promoting the recovery of cytoplasmic volume after plasmolysis. Surprisingly, sucrose is not accumulated by the bacteria at an osmotically efficient level. Instead, it strongly stimulates the accumulation of the main endogenous osmolytes glutamate and N-acetylglutaminylglutamine amide (NAGGN). Examining cell volume changes during the hyperosmotic treatment, we found a close correlation between the enhancement of the osmotically active solute pool and the increase in cell volume. Sucrose shares several features with ectoine, another nonaccumulated osmoprotectant for S. meliloti. Overall, osmoregulation in S. meliloti appears to be strongly divergent from that in most bacteria.

Transient Accumulation of Glycine Betaine and Dynamics of Endogenous Osmolytes in Salt-Stressed Cultures of Sinorhizobium meliloti.

The fate of exogenously supplied glycine betaine and the dynamics of endogenous osmolytes were investigated throughout the growth cycle of salt-stressed cultures of strains of Sinorhizobium meliloti which differ in their ability to use glycine betaine as a growth substrate, but not as an osmoprotectant. We present (sup13)C nuclear magnetic resonance spectral and radiotracer evidence which demonstrates that glycine betaine is only transiently accumulated as a cytoplasmic osmolyte in young cultures of wild-type strains 102F34 and RCR2011. Specifically, these strains accumulate glycine betaine as a preferred osmolyte which virtually prevents the accumulation of endogenous osmolytes during the lag and early exponential phases of growth. Then, betaine levels in stressed cells decrease abruptly during the second half of the exponential phase. At this stage, the levels of glutamate and the dipeptide N-acetylglutaminylglutamine amide increase sharply so that the two endogenous solutes supplant glycine betaine in the ageing culture, in which it becomes a minor osmolyte because it is progressively catabolized. Ultimately, glycine betaine disappears when stressed cells reach the stationary phase. At this stage, wild-type strains of S. meliloti also accumulate the disaccharide trehalose as a third major endogenous osmolyte. By contrast, glycine betaine is always the dominant osmolyte and strongly suppresses the buildup of endogenous osmolytes at all stages of the growth cycle of a mutant strain, S. meliloti GMI766, which does not catabolize this exogenous osmoprotectant under any growth conditions.