Purification and properties of the membrane-bound hydrogenase from Proteus mirabilis.

The cytoplasmic membrane-bound hydrogenase of the facultative anaerobe, Proteus mirabilis, has been solubilized and purified to homogeneity. The purified enzyme exhibited a maximal specific activity of about 780 mumol H2 oxidized/min per mg protein (benzyl viologen reduction). The hydrogenase has a molecular weight of 205 000 and is composed of two subunits with a molecular weight of 63 000 and two of 33 000. The absorption spectrum of the enzyme was characteristic of non-heme iron proteins. The millimolar extinction coefficients at 400 and 280 nm are 106 and 390, respectively. The hydrogenase has about 24 iron atoms and 24 acid-labile sulfide atoms/molecule. Amino acid analyses revealed the presence of 39 half-cystine residues/molecule and a preponderance of acidic amino acids. The hydrogenase in its oxidized form exhibits an EPR signal of the HiPIP-type with g values at 2.025 and 2.018. Upon reduction with either dithionite or H2 the signal disappears; no other signals were detectable.

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans.

(1)H+ leads to/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO2- or N2O. Under optimal H+-translocation conditions, the ratios leads to H+/O, H+ leads to/N2O, H+ leads to/NO2- for reduction to N2 and H+ leads to/NO2- for reduction to N2O were 6.0-6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N,'N'-tetramethyl-p-phenylene-diamine as exogenous substrate, addition of NO2- or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. H+/N2O, H+/NO2- for reduction to N2 and H+/NO2- for reduction to N2O were -0.84, -2.33 and -1.90, respectively. (3) The H+/oxidant ratios, mentioned in item 2, were not altered in the presence of valinomycin/K+ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO2- and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO2-, N2O or O2 pass two H+-trans-locating sites. The H+ leads to/electron acceptor ratios predicted by this scheme are in good agreement with the experimental values.

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans.

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. leads to H+/2e- quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed leads to H+/O with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygen-and ferricyanide pulses, with endogenous substrates or added methanol as a substrate, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of 2H+/2e-. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis.

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with alpha-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, -75 and 187 mV, respectively. In addition, two very small contributions to the alpha-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c1 (lambda m (25 degrees C) = 553.5 nm; E'0 = 238 mV) and four cytochromes b (lambda m (25 degrees C) = 558.6, 561.2, 562.1, 566.1 nm and E'0 = -83, 26, 85, -60 mV).

The effect of multiple copies of the upstream region on expression of the Aspergillus niger glucoamylase-encoding gene.

The regulation of transcription of the glucoamylase-encoding gene (glaA) of Aspergillus niger was studied. To facilitate this study a reporter strain containing a fusion of the glaA promoter (PglaA) of A. niger to the beta-glucuronidase-encoding gene (uidA) of Escherichia coli was constructed. To analyze whether regulatory proteins are involved in the regulation of glaA, multiple copies of PglaA were introduced into this reporter strain. Analysis of the resulting strains revealed that introduction of an increasing number of PglaA copies resulted in lower expression of the uidA reporter gene and the endogenous glaA gene in cultures cultivated on different inducing carbon sources. However, repression by xylose was not influenced by the copy number of PglaA. These results indicate that the expression of genes under control of PglaA are regulated by specific trans-acting regulatory protein(s). Deletion analysis of PglaA indicated that regulatory proteins interact with DNA sequences within 0.5-kb upstream from the ATG, whereas sequences between about 0.8- and 0.5-kb upstream from the ATG are required for high-level expression of glaA.

Characterization of an efficient gene cloning strategy for Aspergillus niger based on an autonomously replicating plasmid: cloning of the nicB gene of A. niger.

The development of an improved gene cloning strategy by complementation of mutant alleles in Aspergillus niger is described. The strategy is based on the use of a fungal autonomously replicating vector, pAB4-ARp1. This vector was constructed by the introduction of a previously described sequence involved in autonomous replication (AMA1), into a pyrG integrative vector, pAB4-1. With vector pAB4-ARp1, a 10-100-fold increase in transformation frequency was obtained, as compared to pAB4-1. Furthermore, the transformation frequency of a co-transformed plasmid is also increased using pAB4-ARp1. A. niger transformants containing pAB4-ARp1 are mitotically unstable. Co-transformed plasmids strictly co-segregated with the autonomously replicating vector, as a result of recombination between both vectors. The use of pAB4-ARp1 in gene cloning was demonstrated by the complementation of two linkage group-VII-specific A. niger mutants. Complementation of a lysF mutant was achieved by co-transformation of pAB4-ARp1 with total genomic A. niger DNA ('instant bank'). A nicB-deficient A. niger was complemented by co-transformation with pAB4-ARp1 and an A. niger cosmid library. The complementing DNA was re-isolated from a Nic+ transformant by transforming Escherichia coli with total genomic DNA of this transformant. Gene disruption and genetic analysis was carried out to prove that the previously unknown A. niger nicB gene had been cloned.

Mutagenesis of the gene encoding amicyanin of Paracoccus denitrificans and the resultant effect on methylamine oxidation.

The gene encoding the blue-copper protein amicyanin was isolated from a genomic bank of Paracoccus denitrificans by using a synthetic oligonucleotide. It is located directly downstream of the gene encoding the small subunit of methylamine dehydrogenase. Amicyanin is transcribed as a precursor protein with a signal sequence, typical for periplasmic proteins. Specific inactivation of amicyanin by means of gene replacement techniques resulted in the complete loss of the ability to grow on methylamine.

Oxidation of methylamine by a Paracoccus denitrificans mutant impaired in the synthesis of the bc1 complex and the aa3-type oxidase. Evidence for the existence of an alternative cytochrome c oxidase in this bacterium.

A Paracoccus denitrificans fbcC-ctaDII double mutant strain impaired in the synthesis of both the bc1 complex and the aa3-type oxidase has been constructed. This mutant strain, which is still able to grow on methylamine as sole carbon and energy source, exhibits unimpaired oxygen consumption with succinate, methylamine and endogenous substrates as electron donors. From kinetic studies of the oxidation and reduction rates of cytochromes c, it can be concluded that P. denitrificans contains a second cytochrome c oxidase, different from the aa3-type.

Reversed electron transfer through the bc1 complex enables a cytochrome c oxidase mutant (delta aa3/cbb3) of Paracoccus denitrificans to grow on methylamine.

In Paracoccus denitrificans four classes of redox proteins are involved in the electron transfer from methylamine to oxygen:methylamine dehydrogenase (MADH), amicyanin, cytochrome c and cytochrome c oxidase. MADH and its electron acceptor amicyanin are indispensable for growth on methylamine. At least three different cytochromes c and two types of cytochrome c oxidase, cytochromes aa3 and cbb3, have previously been proposed to participate in the electron transfer pathways from methylamine to oxygen. In this study, participation of both cytochrome c oxidases and of the quinol oxidase (cytochrome bb3) has indeed been confirmed by analysis of a series of oxidase mutants. Interestingly, a P. denitrificans cytochrome c oxidase mutant (delta aa3/cbb3) retains the capacity to oxidise methylamine. It is demonstrated that the oxidation of the cytochrome c pool in this mutant does not proceed via an alternative cytochrome c oxidase, but rather via an 'uphill' electron transfer through the bc1 complex to ubiquinone, driven by the membrane potential. The subsequent oxidation of ubiquinol proceeds via the only remaining terminal oxidase, the bb3-type quinol oxidase.

Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators.

The nir and nor genes, which encode nitrite and nitric oxide reductase, lie close together on the DNA of Paracoccus denitrificans. We here identify an adjacent gene, nnr, which is involved in the expression of nir and nor under anaerobic conditions. The corresponding protein of 224 amino acids is homologous with the family of FNR proteins, although it lacks the N-terminal cysteines. A mutation in the nnr gene had a negative effect on the expression of nitrite and nitric oxide reductase. Synthesis of membrane bound nitrate reductase, of nitrous oxide reductase, and of the cbb3-type cytochrome c oxidase were not affected by mutation of this gene. These results suggest that denitrification in P. denitrificans may be governed by a signal transduction network that is similar to that involved in oxygen regulation of nitrogen metabolism in other organisms.

The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase.

Among aerobic prokaryotes, many different terminal oxidase complexes have been described. Sequence comparison has revealed that the aa3-type cytochrome c oxidase and the bo3-type quinol oxidase are variations on the same theme: the heme-copper oxidase. A third member of this family has recently been recognized: the cbb3-type cytochrome c oxidase. Here we give an overview, and report that nitric oxide (NO) reductase, a bc-type cytochrome involved in denitrification, shares important features with these terminal oxidases as well. Tentative structural, functional and evolutionary implications are discussed.

Evaluation of molecular and genetic approaches to generate glucoamylase overproducing strains of Aspergillus niger.

To evaluate the possibility of improving glucoamylase (GLA) production in Aspergillus niger strains carrying multiple copies of the GLA encoding gene (glaA), additional glaA copies were introduced either by genetic recombination or retransformation. For strains to be used in such experiments a genetic analysis was first carried out. The results of this analysis clearly revealed that in each transformant integration had occurred at a chromosome corresponding to a single linkage group (LG). The GLA production per gene copy showed considerable variation in these strains, indicating a clear effect of the site of integration on gene expression. Introduction of additional gene copies by genetic recombination experiments was carried out for different combinations of strains, carrying glaA copies in different chromosomes. The introduction of additional glaA gene copies by genetic recombination did not result in a considerable increase in GLA production compared to the parental strains. In some strains recombination resulted in genetic instability, observed by the frequent loss of glaA copies. Also, retransformation of multi-copy glaA strains did not result in an increase in GLA production. In several strains even a decrease in GLA production was found after retransformation. Southern analysis of these transformants suggested that newly introduced gene copies were heavily rearranged, which partly explains why GLA production was not increased. Further analysis of one such transformant provided evidence that the overexpression of the glaA gene is limited by the amount of trans-acting regulatory protein(s) available.