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It has been shown previously that starvation of a mid4kgarithmic-phase culture of Escherickia coil B/r for an essential nutrient results in the methylation of a membrane-sociated protein (P-43) (C. C. Young and R. W. Bernlohr, J. Bacteriol. 172:5147-5153, 1990 Bacterial cells respond to environmental change through the differential expression of families of proteins that are required for survival during periods of stress. During starvation, these proteins allow the cell to utilize nutrients more efficiently and confer upon the cell a stress-resistant phenotype. The degradation of intracellular proteins and RNA presumably provides the necessary precursors for the cell to synthesize approximately 40 proteins that are regulated in response to starvation (20). Of these, approximately 13 proteins are common to nitrogen, carbon, and phosphate starvation and have been designated Pex proteins. Many of the 40 proteins induced by starvation are also induced during heat shock and oxidative stress, indicating that a single response may have evolved that can be activated by various factors. This is supported by studies showing that starvation can confer a strong cross-protection against heat shock, osmotic shock, oxidative stress, and antibiotics (for a review, see reference 20).Our laboratory has previously reported that a membraneassociated protein in Escherichia coli (25), Bacillus subtilis (9), and Bacillus licheniformis (2) is differentially methylated in response to the availability of nutrients in the medium. The Bacillus protein (P-40) has a molecular mass of approximately 40 kDa and is methylated when nutrients are added to a starving culture. The methyl groups on P-40 turn over with a half-life of 10 to 15 min. The protein is not involved with chemotaxis, and the demethylation process is defective in some stage 0 mutants that are unable to sporulate and are, therefore, constitutive for growth (9). In contrast, the E. coli protein (P-43) is methylated in response to depletion of nutrients in the medium (25). The protein is not involved in chemotaxis and is labeled on lysine residues. When E. coli is starved for an essential nutrient, P-43 is methylated with a doubling time of approximately 5 min, whereas introduction of the missing nutrient to the medium results in an immediate cessation of P-43 methylation followed by a slower demethylation process.This communication describes the purification and identification of the P-43 molecule as elongation factor Tu (EF-Tu) and discusses the possible implications for bacterial nutrient sensing.* Corresponding author. MATERIALS AND METHODSBacterial strains and growth conditions. Cultures of E. coli B/r were grown at 37°C as described previously (25), except that 100 mM morpholinepropanesulfonate (MOPS) buffer (pH 7.5) was used instead of potassium phosphate buffer in the growth medium.Preparation of crude extracts of P43. P-43 was labeled and extracted from membrane fragments by the procedure described by Young and Bernlohr (25), except that 250 ,Ci (60 nM) of [methyl-3H]meth...

The enzymes in the arginine breakdown pathway (arginase, ornithine-6-transaminase, and A'-pyrroline-5-carboxylate dehydrogenase) were found to be present in Bacillus licheniformis cells during exponential growth on glutamate. These enzymes could be coincidentally induced by arginine or ornithine to a very high level and their synthesis could be repressed by the addition of glucose, clearly demonstrating catabolite repression control of the arginine degradative pathway. The strongest catabolite repression control of arginase occurred when cells were grown on glucose and this control decreased when cells were grown on glycerol, acetate, pyruvate, or glutamate. The proline catabolite pathway was present in B. licheniformis during exponential growth on glutamate. The proline oxidation and the A'-pyrroline-5-carboxylate dehydrogenase in this breakdown pathway were induced by L-proline to a high level. The A'-pyrroline-5-carboxylate dehydrogenase was found to be under catabolite repression control. Arginase could be induced by proline and arginine addition induced proline oxidation, suggesting a common in vivo inducer for these convergent pathways.

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Changes in the endogenous intracellular amino acid pool and total free amino acid production in Bacillus licheniformis grown in minimal media were investigated. The total intracellular pool increased during exponential growth and then decreased rapidly after the end of growth. Most of the amino acids were present at low concentrations, but glutamate and alanine comprised 60 to 90% of the total intracellular free amino acid at most times during the growth cycle. It was concluded that, in addition to providing monomers for protein synthesis, the intracellular amino acid pool may be maintained for the storage of energy-providing metabolic intermediates and possibly as a balance to the ionic strength of the medium. The total free amino acid production by the cell was found to be dependent upon the composition of the salts medium as well as the culture age under conditions in which the carbon and nitrogen sources were the same. A 10-fold increase in extracellular amino acid was observed as the cells changed from vegetative to sporulation metabolism, mostly due to the extrusion of intracellular amino acid. The impact of this increase upon amino acid uptake and pulse-labeling studies using unwashed cells is discussed.

Cell-free extracts of Bacillus licheniformis and B. cereus were found to contain high specific activities of nicotinamide adenine dinucleotide phosphate (NADP)-dependent- l -glutamate dehydrogenase [EC 1.4.1.4; l -glutamate: NADP oxidoreductase (deaminating)]. Maximum specific activities were found in extracts of cells during the late exponential phase of growth when ammonium ion served as the sole source of nitrogen. Extremely low specific activities were detected throughout the growth cycle when l -glutamate or Casamino Acids served as the source of carbon and nitrogen. The enzyme was purified 55-fold from crude extracts of B. licheniformis , and apparent kinetic constants were determined. Sigmoidal saturation kinetics were not observed, and various adenylates had no effect on the enzyme. Repression of enzyme synthesis during growth on l -glutamate or Casamino Acids was partially overcome by additions of glucose or pyruvate, and this apparent derepression was totally abolished by inhibitors of ribonucleic acid and protein synthesis. Similarly, additions of l -glutamate or Casamino Acids to cells growing on glucose-ammonium ion resulted in strong repression of enzyme synthesis. It is suggested that the enzyme serves an anabolic role in metabolism. Nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase activity was not detected in five species of Bacillus , irrespective of nutritional conditions or of the physiological age of cells.

othermophilus (55) and a partially purified GS preparation from Bacillus licheniformis ATCC 9945A (22,23) indicate that the glutamine synthetases of Bacillus spp. are regulated differently from the GS of E. coli (49). Most significant are the findings that the GS enzymes of Bacillus spp. are not regulated by covalent modification and are more susceptible to feedback regulation than is the Escherichia coli GS. There are also reports of more than one form of GS in some systems (5,19,29,50), including the 589 on July 16, 2020 by guest http://jb.asm.org/ Downloaded from 590 DONOHUE AND BERNLOHR GS assays. Biosynthetic GS (L-glutamate-ammonia ligase, EC 6.3.1.2) activity was assayed by a modification of the radiochemical method of Prusiner and J. BACTERIOL.on July 16, 2020 by guest

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