Gisèle Le Bras

The NADH oxidase of Streptococcus pneumoniae : its involvement in competence and virulence

A soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase‐deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence‐regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox+ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O2. Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O2 in the regulation of pneumococcal transformability and virulence.

Rétro-inhibition et répression de l'homosérine déshydrogénase d'Escherichia coli

Abstract The homoserine dehydrogenase of Escherichia coli K12 is under the control of the two types of known regulatory negative feedback mechanisms: repression and end-product inhibition. The regulatory agent is l -threonine. End-product inhibition by l -threonine is highly specific and strictly non-competitive. The inhibition site of the enzyme can be selectively destroyed by heat. The stability of the enzyme and of its capacity to be inhibited have been studied. The sedimentation constant of the inhibitable enzyme is higher than that of the enzyme having lost its inhibition site. The synthesis of E. coli homoserine dehydrogenase is greatly and specifically repressed by l -threonine in the culture medium. The genetic control of the synthesis of the aspartic acid family of amino acids is discussed.

Regulation by methionine of the synthesis of third aspartokinase and of a second homoserine dehydrohenase in Escherichia coli K 12

Abstract 1. 1. Evidence is presented that, in addition to the lysine-sensitive aspartokinase III and to the threonine-sensitive association of aspartokinase I and homoserine dehydrogenase I, there exist in Escherichia coli K 12 two heretofore undetected enzymes: aspartokinase II and homoserine dehydrogenase II. The synthesis of these two enzymes is under repressive control by methionine. 2. 2. Some of their properties are described: it should be noted that their activity is not end-product regulated. 3. 3. A constitutive mutant for the two novel activities has been isolated. 4. 4. In E. coli B, homoserine dehydrogenase II is absent. 5. 5. The overall features of repression and end-product inhibition in branched biosynthetic pathways are discussed.

Crystal structures of the small G protein Rap2A in complex with its substrate GTP, with GDP and with GTPgammaS.

The small G protein Rap2A has been crystallized in complex with GDP, GTP and GTPγS. The Rap2A–GTP complex is the first structure of a small G protein with its natural ligand GTP. It shows that the hydroxyl group of Tyr32 forms a hydrogen bond with the γ‐phosphate of GTP and with Gly13. This interaction does not exist in the Rap2A–GTPγS complex. Tyr32 is conserved in many small G proteins, which probably also form this hydrogen bond with GTP. In addition, Tyr32 is structurally equivalent to a conserved arginine that binds GTP in trimeric G proteins. The actual participation of Tyr32 in GTP hydrolysis is not yet clear, but several possible roles are discussed. The conformational changes between the GDP and GTP complexes are located essentially in the switch I and II regions as described for the related oncoprotein H‐Ras. However, the mobile segments vary in length and in the amplitude of movement. This suggests that even though similar regions might be involved in the GDP–GTP cycle of small G proteins, the details of the changes will be different for each G protein and will ensure the specificity of its interaction with a given set of cellular proteins.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli: III. Inactivation at pH 9

1. 1. Homoserine dehydrogenase I (l-homoserine:NADP+ oxidoreductase, EC 1.1.1.3) of Escherichia coli is inactivated with apparent first-order kinetics by exposure at pH 9 in Tris buffer. This inactivation is accompanied by desensitization of the enzyme towards threonine. 2. 2. L-Aspartate and ATP, the substrates of the associated activity, β-aspartokinase I (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) protect against the desensitization of homoserine dehydrogenase.

The role of Glu187 in the regulation of phosphofructokinase by phosphoenolpyruvate.

In bacterial phosphofructokinases, either a glutamic or an aspartic residue is present at position 187, and the mechanism of inhibition by phosphoenolpyruvate seems to be correlated to the nature of residue 187. Upon binding phosphoenolpyruvate, only the enzymes with a Glu187 would undergo a major allosteric conformational change from an active into an inactive state, whereas the enzymes with an Asp187 would only show a simple upward shift in their pH-profile of activity. The phosphofructokinase from Spiroplasma citri, which has an Asp187, has been purified and its properties follow this pattern. The behaviour of mutants of the enzyme from Escherichia coli in which Glu187 is replaced by either aspartate or leucine confirms the importance of residue 187. The major allosteric transition of E. coli phosphofructokinase is abolished by the substitution Glu187-->Asp, suggesting that a glutamate at position 187 is necessary (but not sufficient) for the protein to undergo the change from the active into the inactive state induced by phosphenolpyruvate. In addition, the presence of an acidic residue, aspartate or glutamate, at position 187 is required (but not sufficient) for the binding of ADP (or GDP). This requirement of a negative charge for ADP binding could explain the striking conservation of an aspartate residue at position 187 in all the eukaryotic phosphofructokinases.

Crystallization of D-lactate dehydrogenase from Lactobacillus bulgaricus.

The D-lactate dehydrogenase (D-LDH) from Lactobacillus bulgaricus has been purified and co-crystallized with its cofactor NAD+. Crystals suitable for X-ray diffraction experiments have been obtained from an ammonium sulfate solution by the hanging-drop method. The crystals belong to the orthorhombic space group C222 (or C222(1)) with cell dimensions a = 76.5 A, b = 93.3 A, c = 118.4 A and one monomer of 37,000 daltons per asymmetric unit. They diffract beyond 3.0 A resolution. Sequence comparison suggests that D-LDHs have no evolutionary relationship to L-LDHs and belong instead to the family of the D-2-hydroxyacid dehydrogenases. The X-ray crystallographic structure of the D-LDH from Lactobacillus bulgaricus will be a decisive test of this hypothesis.