Proteome analysis of Bacillus subtilis cells grown at low and high salinity revealed the induction of 16 protein spots and the repression of 2 protein spots, respectively. Most of these protein spots were identified by mass spectrometry. Four of the 16 high-salinity-induced proteins corresponded to DhbA, DhbB, DhbC, and DhbE, enzymes that are involved in the synthesis of 2,3-dihydroxybenzoate (DHB) and its modification and esterification to the iron siderophore bacillibactin. These proteins are encoded by the dhbACEBF operon, which is negatively controlled by the central iron regulatory protein Fur and is derepressed upon iron limitation. We found that iron limitation and high salinity derepressed dhb expression to a similar extent and that both led to the accumulation of comparable amounts of DHB in the culture supernatant. DHB production increased linearly with the degree of salinity of the growth medium but could still be reduced by an excess of iron. Such an excess of iron also partially reversed the growth defect exhibited by salt-stressed B. subtilis cultures. Taken together, these findings strongly suggest that B. subtilis cells grown at high salinity experience iron limitation. In support of this notion, we found that the expression of several genes and operons encoding putative iron uptake systems was increased upon salt stress. The unexpected finding that high-salinity stress has an iron limitation component might be of special ecophysiological importance for the growth of B. subtilis in natural settings, in which bioavailable iron is usually scarce.The soil bacterium Bacillus subtilis often experiences fluctuations in the osmolality of its habitat due to the drying and flooding of the upper layers of the soil (7,40). A rise in the external salinity and osmolality triggers the outflow of water from the cell, resulting in a reduction in turgor and dehydration of the cytoplasm. To cope with these unfavorable osmotic conditions, B. subtilis initiates a two-step adaptation response (7, 32). Initially, K ϩ is rapidly taken up (56) and subsequently replaced in part by proline (55), a member of the so-called compatible solutes (19). These osmolytes can be accumulated to high levels through either de novo synthesis or uptake of preformed osmoprotectants from the environment without interfering with central cellular functions. For B. subtilis, proline serves as the primary endogenously synthesized compatible solute (55), and large quantities are produced via a dedicated osmostress-responsive synthesis pathway (J. Brill and E. Bremer, unpublished results). In addition, B. subtilis can efficiently scavenge a wide variety of compatible solutes from environmental sources by means of five osmoregulated transport systems (OpuA to OpuE) (29-31, 41, 54) and can acquire choline for the production of the osmoprotectant glycine betaine (4, 30). The accumulation of compatible solutes offsets the detrimental effects of high osmolality on cell physiology and permits growth over a wide range of osmotic conditions (3,36).Under...

In conclusion, the whole-blood assay reveals a high prevalence of latent tuberculosis infection in renal transplant recipients. It may represent a valuable alternative to skin testing as the test result is not adversely affected by immunosuppression. Moreover, reactivity towards ESAT-6 allows the distinction of a latent infection from BCG-induced reactivity. The assay is well-suited for use in screening programmes and may facilitate the management of tuberculosis infection in immunocompromised individuals.

The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIA Glc , as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIA Glc phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIA Glc . Isopropyl-␤-D-thiogalactopyranoside-induced overexpression of EIIAGlc did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIA Glc . A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIA Glc is controlled by G3P and other phosphorylated sugars such as D-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Twodimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and D-tagatose-1,6-bisphosphate aldolase.Catabolite repression refers to the reduction in transcription of sensitive operons that is caused by certain carbon sources in the medium, most prominently by glucose (glucose effect). In Escherichia coli, phosphoenolpyruvate-dependent phosphotransferase system (PTS)-mediated uptake of glucose is crucial for this effect. The model largely accepted for E. coli focuses on the level of cyclic AMP (cAMP) synthesized by the membranebound adenylate cyclase (29,30,38). EIIA Glc , an intermediate in the phosphorylation cascade of the PTS for glucose, in its phosphorylated form is thought to stimulate adenylate cyclase. The basal level of adenylate cyclase activity would be present in the absence of EIIA Glc or in the presence of unphosphorylated EIIA Glc . As a consequence, CAP, the catabolite activator protein (or cAMP receptor protein [CRP]) that is needed for the transcription of sensitive operons (22) is linked in its activity to the PTS (31). The glucose PTS is also responsible for inducer exclusion, i.e., inhibition of the different transport systems by unphosphorylated EIIA Glc (31). Even though participation of the PTS in catabolite repression and inducer exclusion in E. coli has been documented very well, the effects of non-PTS sugars are less clear. Thus, gluc...

The pathway of the oxidation of propionate to pyruvate in Escherichia coli involves five enzymes, only two of which, methylcitrate synthase and 2-methylisocitrate lyase, have been thoroughly characterized. Here we report that the isomerization of (2S,3S)-methylcitrate to (2R,3S)-2-methylisocitrate requires a novel enzyme, methylcitrate dehydratase (PrpD), and the well-known enzyme, aconitase (AcnB), of the tricarboxylic acid cycle. AcnB was purified as 2-methylaconitate hydratase from E. coli cells grown on propionate and identified by its N-terminus. The enzyme has an apparent K m of 210 lM for (2R,3S)-2-methylisocitrate but shows no activity with (2S,3S)-methylcitrate. On the other hand, PrpD is specific for (2S,3S)-methylcitrate (K m ¼ 440 lM) and catalyses in addition only the hydration of cis-aconitate at a rate that is five times lower. The product of the dehydration of enzymatically synthesized (2S,3S)-methylcitrate was designated cis-2-methylaconitate because of its ability to form a cyclic anhydride at low pH. Hence, PrpD catalyses an unusual syn elimination, whereas the addition of water to cis-2-methylaconitate occurs in the usual anti manner. The different stereochemistries of the elimination and addition of water may be the reason for the requirement for the novel methylcitrate dehydratase (PrpD), the sequence of which seems not to be related to any other enzyme of known function. Northern-blot experiments showed expression of acnB under all conditions tested, whereas the RNA of enzymes of the prp operon (PrpE, a propionyl-CoA synthetase, and PrpD) was exclusively present during growth on propionate. 2D gel electrophoresis showed the production of all proteins encoded by the prp operon during growth on propionate as sole carbon and energy source, except PrpE, which seems to be replaced by acetyl-CoA synthetase. This is in good agreement with investigations on Salmonella enterica LT2, in which disruption of the prpE gene showed no visible phenotype.Keywords: 2-methylisocitrate; aconitase; methylcitrate dehydratase; propionate metabolism; prp operon. Several bacteria and fungi are able to oxidize propionate via methylcitrate to pyruvate. Initially propionyl-CoA condenses with oxaloacetate to (2S,3S)-methylcitrate, which isomerizes to (2R,3S)-2-methylisocitrate. Cleavage leads to pyruvate and succinate. The consecutive oxidative regeneration of oxaloacetate from succinate completes the methylcitrate cycle. Initially this cycle was discovered by growing a mutant strain of the yeast Candida lipolytica on odd-chain fatty acids. The accumulation of a tricarboxylic acid was observed during growth and identified as methylcitrate [1]. Further investigations revealed other enzymes necessary for a functional methylcitrate cycle. The enzymes, however, were only partially characterized and no genomic sequences were identified [2-6]. More recently it was discovered that propionate oxidation in aerobically growing Gram-negative bacteria, especially Escherichia coli [7] and Salmonella enterica serovar Thyphimurium L...