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Large subunits of NiFe-hydrogenases undergo a unique maturation process in which the last step consists of the endoproteolytic cleavage of the C-terminal extension after the Ni-Fe metal center has been assembled. To assess in vivo the influence of alteration of the C-terminal extension on the processing, green fluorescence protein (GFP) was fused to the C-terminus of the large subunit (HybC) of the Escherichia coli hydrogenase 2. Interestingly, no processing of HybC-GFP was observed. In addition, the chromophore of GFP was not formed, implying a nonproductive folding of the upstream HybC moiety. These results strongly suggest that the alteration of the C-terminus of the hydrogenase 2 large subunit interferes with the folding and processing of HybC.

We have cloned the multigenic hydC locus of Escherichia coli on a 7.1-kb BamHI-EcoRI fragment. Since its gene products are likely to be involved in the specific nickel transport [Wu et al., Mol. Microbiol. 3 (1989) 1709-1718], we propose a new gene designation, nik, to replace hydC. In vivo gene expression studies and complementation analysis support the notion that the nik locus is a multi-cistronic operon consisting of two to five genes. The first two genes, arranged in the order nikA and nikB, encode two proteins of 59 and 27.5 kDa, respectively. The downstream genes direct the biosynthesis of three polypeptides of 30, 28 and 25.5 kDa. Southern-blot analysis showed that mutant HYD720 carries a chromosomal deletion of 5.1-kb covering both nikA and nikB, whereas a 0.8-kb deletion in mutant HYD790 includes the sole nikB region. The cloned nik operon and the deletion mutants will aid greatly in the further molecular characterization of the multicomponent-specific transport of nickel in E. coli.

NikA, a periplasmic nickel-binding protein involved in nickel-repellent chemotaxis has been crystallized. The crystals are hexagonal with space group P6(2) (or its enantiomorph) with a = 160.3 A and c = 138.4 A and they diffract to at least 3.0 A resolution in the laboratory. NikA presents sequence homology with several periplasmic solute-binding proteins from Gram-negative bacteria.

Using natural inducers of beta-glucuronidase, methyl-glucuronide and fructuronate, under gratuitous conditions (without metabolic conversion of these two compounds), we corroborate the fact that both molecules are required simultaneously in order to derepress the enzyme synthesis to a maximum level. Structurally related analogs of the natural inducers, thiophenyl-glucuronide and mannonic amide respectively, were assayed in the wild type and suitable mutant strains of E. coli. The results are in agreement with the model where the dual negative regulation of the enzyme synthesis is exerted by two regulatory genes uidR and uxuR. The concerted action of mannonic amide and thiophenyl-glucuronide, which alone fail to induce significantly beta-glucuronidase synthesis, reveals that a cooperative effect of the two repressor molecules responsible for the complete blocking of the enzyme synthesis is occuring.

d -mannonate:NAD oxidoreductase ( d -mannonate:NAD+ 5-oxidoreductase, EC 1.1.1.57) from Escherichia coli normally catalyzes the reaction: d - fructuronate + NADH ⇌ d -mannonate + NAD + [4,5]. It can also catalyze the reduction of d -glucuronate according to the equation: d - glucuronate + NADH → l -gulonate + NAD + . Kinetic evidence is given for the second reaction to proceed in two steps: (i) NADH binds reversibly the free enzyme to form a binary complex E · NADH; (ii) d -glucuronate reacts subsequently with the binary complex to give products, without formation of an intermediary ternary complex E · NADH · glucuronate. The rate of the overall reaction obeys the classical Michaelis-Menten law with respect to NADH concentration (K1 = 0.03 mM) but is proportional to d -glucuronate concentration in the range 0–100 mM. Results concerning the action of several inhibitors on d -glucuronate reduction were interpreted satisfactorily by developing models based on mechanism involving an enzyme carrying one binding site for the alternative substrate d -mannonate and one binding site for the coenzyme NADH (Table I). In any case the experimental values of the dissociation constants for the complexes enzyme-effector were deduced. NAD and ATP act as strict competitive inhibitors (K2 = 0.60 mM and 20 mM) able to bind the free enzyme only. In the case of ATP, a second binding site with a high affinity (K3 = 0.02 mM) is unmasked when the first binding site is saturated. Mannonate and altronate behave as strictly non-competitive inhibitors able to bind equally the free enzyme and the binary complex E · NADH. l -Gulonate acts as a mixed non-competitive inhibitor able to bind much easily the free enzyme (K4 = 0.41 mM) than the complex E · NADH (K2 = 131 mM). In no case was d -glucuronate shown to be able to react with complexes other than E · NADH. Such an unusual mechanism for a two-substrate enzyme looks similar to the sequence found by Chance for horse-radish peroxidase [6].

The Mu dl (ApR lac) bacteriophage was used to generate mutants of Escherichia coli which were defective in formate hydrogenlyase. Three mutants were chosen for further analysis: they lacked hydrogenase (hydrogen: benzyl viologen oxidoreductase) activity, but produced normal levels of fumarate reductase activity and two- to three-fold reduced levels of benzyl viologen (BV)-dependent formate dehydrogenase activity. Two of them (hydC) were shown to contain about 4-fold reduced amounts of formate hydrogenlyase and fumarate-dependent H2 uptake activities. The third one (hydD) was totally devoid of both activities. Their insertion sites were located at 77 min on the E. coli map. Subdivision of these mutants into two classes was subsequently based on the restoration capacity of hydrogenase activity with high concentration of nickel in the growth media. Addition of 500 microM NiCl2 led to a complete recovery of hydrogenase activity, and to the concomitant restoration of normal BV-linked formate dehydrogenase, formate hydrogenlyase and fumarate-dependent H2 uptake activities in the hydC mutants. The hydD mutant was insensitive to the effect of nickel. Expression of the lac operon in hydC and hydD mutants was induced by anaerobiosis. It was not increased by the addition of formate under anaerobic conditions. The presence of nitrate resulted in slightly reduced beta-galactosidase activities in the hydC mutants, whereas those found in the hydD mutant reached only one third of the level obtained in its absence. Fumarate had no effect on both classes. Moreover, in contrast to the hydD locus, the hydC::Mu dl fusions were found to be dependent upon the positive control exerted by the nirR gene product and were totally repressed by an excess of nickel. In addition, the low levels of overall hydrogenase-dependent activities found in a nirR strain were also relieved by the presence of nickel. Our results strongly suggest that the pleiotropic regulatory gene nirR is essential for the expression of a gene (hydC) involved in either transport or processing of nickel in the cell, whose alteration leads to a loss of hydrogenase activity.