Downstream of flhA, the Paracoccus denitrificans gene encoding glutathione-dependent formaldehyde dehydrogenase, an open reading frame was identified and called fghA. The gene product of fghA showed appreciable similarity with human esterase D and with the deduced amino acid sequences of open reading frames found in Escherichia coli, Haemophilus influenzae, and Saccharomyces cerevisiae. Mutating fghA strongly reduced S-formylglutathione hydrolase activity. The mutant was unable to grow on methanol and methylamine, indicating that the enzyme is essential for methylotrophic growth. S-Formylglutathione hydrolase appears to be part of a formaldehyde detoxification pathway that is universal in nature.In Paracoccus denitrificans, the oxidation of methanol, methylamine, and choline leads to the transient synthesis of formaldehyde. This poses a special regulation problem, as the concentration of this toxic compound must be kept within the limits imposed by its toxicity on the one hand and the concentration required for rapid further oxidation on the other hand. The oxidation of methanol and methylamine is catalyzed by the periplasmic quinoproteins methanol dehydrogenase and methylamine dehydrogenase, respectively (11). Formaldehyde formed during these reactions is transported to the cytoplasm by a protein-mediated mechanism (14). Choline is oxidized in several steps to glycine. In this process, formaldehyde is released in the cytoplasm. In the cytoplasm, formaldehyde is coupled to reduced glutathione (GSH) to yield S-hydroxymethyl-glutathione. The latter compound is oxidized by NADdependent formaldehyde dehydrogenase (GD-FALDH) to S-formylglutathione (27). The P. denitrificans gene (flhA) encoding this enzyme has been isolated and sequenced (21). An flhA mutant was unable to grow on methanol, methylamine, or choline, indicating that GD-FALDH is essential for methylotrophic growth of P. denitrificans. S-formylglutathione is hydrolyzed to formate and GSH by S-formylglutathione hydrolase (FGH). This enzyme has not yet been isolated from P. denitrificans, but FGH activity has been demonstrated in human tissues (26) and in the methylotrophic yeasts Candida boidinii (20) and Kloeckera sp. strain No2201 (13). FGH isolated from human liver and Kloeckera sp. strain No2201 is a homodimer with a molecular mass of 58 kDa. The C. boidinii FGH is a heterodimer with subunits of 35 and 25 kDa. In these organisms, genes encoding FGH have not been identified. Studies on the polymorphism of FGH in human erythrocytes revealed that the enzyme is identical to human esterase D (7). Human esterase D is a member of a group of nonspecific esterases. The native enzyme has a molecular weight of 70,000 and consists of two identical subunits (16). Esterase D has been found in most human tissues, but the highest activities were found in placenta, kidney, liver, and erythrocytes. The gene encoding this protein has been isolated and sequenced (15).The capacity to detoxify formaldehyde is an important feature for every organism. Both GD-FALDH and FGH ar...
NAD-and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) ofParacoccus denitrificans has been purified as a tetramer with a relative molecular mass of 150 kDa. The gene encoding GD-FALDH (flhA) has been isolated, sequenced, and mutated by insertion of a kanamycin resistance gene. The mutant strain is not able to grow on methanol, methylamine, or choline, while heterotrophic growth is not influenced by the mutation. This finding indicates that GD-FALDH of P. denitrificans is essential for the oxidation of formaldehyde produced during methylotrophic growth.Paracoccus denitrificans is a gram-negative, aerobic soil bacterium which is able to grow on methanol, methylamine, and choline. The oxidation of methanol and methylamine to formaldehyde is catalyzed by the periplasmically located enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. During growth on methylamine, formaldehyde is transported to the cytoplasm by a transport mechanism in which a transport protein is involved (16). During the oxidation of choline to glycine, several molecules of formaldehyde are produced. In the presence of formaldehyde and reduced glutathione, the compound S-hydroxymethylglutathione is nonenzymatically formed. NAD-and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) oxidizes S-hydroxymethylglutathione to S-formylglutathione (14,19,20,24,27), which is oxidized further via formate to carbon dioxide. In this report, we describe the purification of GD-FALDH of P. denitrificans and the isolation and mutagenesis of the gene encoding this enzyme. From growth characteristics of the mutant strain, it can be concluded that GD-FALDH of P. denitrificans is essential for methylotrophic growth.Purification and biochemical analysis of GD-FALDH. GD-FALDH was purified approximately 50-fold from methylamine-grown P. denitrificans cells, as shown in Table 1. By this method, 65 g (wet weight) of cells was harvested and washed with 50 mM Tris hydrochloride (pH 7.5). The cells were disrupted with a French pressure cell, yielding the cell extract. Enzyme activity was measured routinely by determining the rate of NADH formation at 340 nm at room temperature. The assay mixture contained (final concentrations) 0.1 M Na 4 P 2 O 7 -HCl (pH 9.0), 2 mM (reduced) glutathione, and 2.5 mM NAD. After 30 s of incubation with enzyme solution, the reaction was started by adding formaldehyde to a final concentration of 5 mM. Activities were calculated by using a molar absorption coefficient for NADH at 340 nm of 6,220 M Ϫ1 cm Ϫ1 (4).(NH 4 )SO 4 was added to the cell extract. The enzyme precipitated between 25 and 60% saturation. The precipitate was dissolved in 10 mM potassium phosphate buffer (KPB; pH 7.0) and applied to a Phenyl-Sepharose HP column (12.4 by 2.6 cm) equilibrated with 1.5 M (NH 4 )SO 4 in 10 mM KPB (pH 7.0). After washing of the column with the same buffer, elution occurred with a gradient of 1.5 to 0 M (NH 4 )SO 4 in 10 mM KPB in 3 h at a flow rate 3 ml/min. GD-FALDH eluted when the (NH 4 )SO 4 concentration ...
A new suicide vector, pRVSl, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denit4ficans. The vector was derived from suicide vector pGRPdl, which was equipped with the lacZ gene encoding 13-galactosidase. The reporter gene was found to be a successl screening marker for the discrimnation between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPdl and pRVSl were used in gene replacement tchniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the perAplasmic cytochromes c..., cssl1, and c5531, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the corrtnss of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type'levels of methylamine dehydrogenase activities. cycA mutant stan, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow, on met,hmol and showed wdid-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum spific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes.Paracoccus denitrificans is a gram-negative soil bacterium, which is well adapted for aerobic and anaerobic growth on a variety of carbon sources. During aerobic heterotrophic growth, electrons can be passed from reducing compounds to oxygen via at least three different routes. One of these routes has typical mitochondrionlike characteristics, involving NADH dehydrogenase, the bc1 complex, and the aa3-type oxidase (1,5,6,12,19,43,44,46). Electron transport from the bc1 complex to the aa3-type oxidase is mediated by the constitutively formed cytochrome c550, which operates in the periplasm (32,41). An alternative route between the latter two proton-translocating complexes is suggested to involve membrane-bound cytochrome c552 (4,21). Besides the two routes to the aa3-type oxidase, P. denitrificans has the disposal of a third aerobic electron transport route in which oxygen reduction is mediated by a quinol oxidase (10,28).For growth on methanol or methylamine, additional periplasmically located dehydrogenases and electron carriers are induced to enable the bacterium to use the Cl compounds concerned as carbon and energy sources (2,6,(15)(16)(17)(18). Methylamine is oxidized by methylamine dehydrogenase, and electrons are transferred to their natural electron acceptor, amicyanin (16,18,42). This blue copper enzyme seems to be the branching point of tw...
The periplasmically located cytochrome c5531 of Paracoccus denitrificans was purified from cells grown aerobically on choline as the carbon source. The purified protein was digested with trypsin to obtain several protein fragments. The N-terminal regions of these fragments were sequenced. On the basis of one of these sequences, a mix of 17-mer oligonucleotides was synthesized. By using this mix as a probe, the structural gene encoding cytochrome c553, (cycB) was isolated. The nucleotide sequence of this gene was determined from a genomic bank. The N-terminal region of the deduced amino acid sequence showed characteristics of a signal sequence. Based on the deduced amino acid sequence of the mature protein, the calculated molecular weight is 22,427. The gene encoding cytochrome c553. was mutated by insertion of a kanamycin resistance gene. As a consequence of the mutation, cytochrome c553, was absent from the periplasmic protein fraction. The mutation in cycB resulted in a decreased maximum specific growth rate on methanol, while the molecular growth yield was not affected. Growth on methylamine or succinate was not affected at all. Upstream of cycB the 3' part of an open reading frame (ORF1) was identified. The deduced amino acid sequence of this part of ORF1 showed homology with methanol dehydrogenases from P. denitrificans and Methylobacterium extorquens AM1. In addition, it showed homology with other quinoproteins like alcohol dehydrogenase from Acetobacter acedi and glucose dehydrogenase from both Acinetobacter calcoaceticus and Escherichia coli. Immediately downstream from cycB, the 5' part of another open reading frame (ORF2) was found. The deduced amino acid sequence of this part of ORF2 showed homology with the moxJ gene products from P. denitficans and M. extorquens AM1.
E. coli ST131 was the predominant clone, which accumulated a high number of chromosomal mutations. The I529L SNP in parE was a signature of most, but not all, ST131 strains. In contrast to E. coli, fluoroquinolone resistance mechanisms were predominantly plasmid-encoded in K. pneumoniae.
By oligonucleotide-directed mutagenesis, stop codon mutations were introduced at various sites in the pCloDF13-derived bacteriocin release protein (BRP) structural gene. The expression, lipid modification (incorporation of [3H]palmitate), and processing (in the presence and absence of globomycin) of the various carboxyl-terminal shortened BRPs were analyzed by a special electrophoresis system and immunoblotting with an antiserum raised against a synthetic BRP peptide, and their functioning with respect to release of cloacin DF13, lethality, and apparent host cell lysis were studied in Sup-, supF, and supP strains of Escherichia coli. All mutant BRPs were stably expressed, lipid modified, and processed by signal peptidase II, albeit with different efficiencies. The BRP signal peptide appeared to be extremely stable and accumulated in induced cells. Full induction of the mutant BRPs, including the shortest containing only 4 amino acid residues of the mature polypeptide, resulted in phospholipase A-dependent and Mg2 -suppressible apparent cell lysis. The extent of this lysis varied with the mutant BRP used. Induction of all mutant BRPs also prevented colony formation, which appeared to be phospholipase A independent. One shortened BRP, containing 20 amino acid residues of the mature polypeptide, was still able to bring about the release of cloacin DF13. The results indicated that the 8-amino-acid carboxyl-terminal segment of the BRP contains a strong antigenic determinant and that a small segment between amino acid residues 17 and 21, located in the carboxyl-terminal half of the BRP, is important for release of cloacin DF13. Either the stable signal peptide or the acylated amino-terminal BRP fragments (or both) are involved in host cell lysis and lethality.Bacteriocins are plasmid-encoded toxic proteins which are among the few proteins released into the extracellular medium by Escherichia coli. The mechanism of bacteriocin release is unusual in two ways. First, the bacteriocin is synthesized without a cleavable signal peptide which could trigger its secretion across the cytoplasmic membrane. Second, a helper protein, the so-called bacteriocin release protein (BRP), is essential for translocation of the bacteriocin across both the cytoplasmic and outer membranes (6,26).The genes encoding the bacteriocin and its corresponding BRP are coordinately transcribed from a common promoter which is regulated by the SOS response (6, 15). Expression of the bacteriocin operon results in the synthesis and more or less specific release of the bacteriocin and also in inhibition of colony formation on broth agar plates (cell lethality), whereas full induction results in a marked decline in culture turbidity, called "lysis" (6). Initially, this "lysis" is not caused by a degradation of peptidoglycan but results from alterations in the bacterial outer membrane which are host strain dependent. The elevated level of BRP expression is responsible for the observed lethality and "lysis" of fully induced host cells. Divalent cations, such a...
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