Intracytoplasmic Membranes Research Articles

Is intracytoplasmic membrane structure a generic criterion? It does not coincide with phylogenetic interrelationships among phototrophic purple nonsulfur bacteria.

The 16S rRNA or rRNA gene sequences of the type strains of 5 species of Rhodobacter, Rhodopseudomonas blastica and Paracoccus denitrificans were determined. The sequence analysis revealed that Rhodobacter species, whose intracytoplasmic membrane systems were characteristically vesicular, composed a sole cluster. Rhodopseudomonas blastica, whose intracytoplasmic membrane system was lamellar, was included in the cluster of Rhodobacter. The phylogenetic co-clustering of these bacteria conformed to their possessing of the identical types of carotenoids. Paracoccus denitrificans, which is nonphototrophic, is a right member of the Rhodobacter cluster. Rhodobacter species, Rhodopseudomonas blastica and Paracoccus denitrificans are apart from the other phototrophic bacteria and have the common deletions of 21 bases at the positions 1258 to 1278 (Escherichia coli numbering system). It was demonstrated that the morphological character "intracytoplasmic membrane structure", that has been regarded as a generic criterion does not reflect the phylogeny in the phototrophic bacteria. The transfer of Rhodopseudomonas blastica to the genus Rhodobacter is proposed.

Cooperation of plasma and thylakoid membranes for the biosynthesis of chlorophyll in cyanobacteria: the role of the thylakoid centers

Summary Discontinuous sucrose density gradient centrifugation was used to isolate four distinct membrane fractions from cell-free extracts of Synechocystis PCC 6803, viz. two chlorophyll-free plasma membrane fractions, a dark-green thylakoid membrane fraction and a light-green thylakoid membrane fraction with buoyant densities of 1.066, 1.092, 1.208 and 1.162g·cm −3 , respectively. The four types of membranes could not be further resolved by recentrifugation on continuous sucrose density gradients; each of them had its genuine complement of cytochrome c oxidase, NADPH-protochlorophyllide reductase, P700, protochlorophyllide and chlorophyllide, which could not be explained by superposition (mixture) of different membrane fractions. Due to the high concentration (activity) of cytochrome c oxidase, NADPHprotochlorophyllide reductase, P700, protochlorophyllide and chlorophyllide in the light-green thylakoid membrane fraction it is tempting to suggest that these membranes are derived from thylakoid centers that appear to represent contact sites between cytoplasmic (plasma) and intracytoplasmic (thylakoid) membranes where the chlorophyllide synthesized from protochlorophyllide in the plasma membrane is esterified with phytol to chlorophyll and the chlorophyll is transferred to the thylakoid membranes. Our findings will be discussed with respect to the long-standing question of continuity between plasma and thylakoid membranes in cyanobacteria.

5-Aminolevulinic acid availability and control of spectral complex formation in hemA and hemT mutants of Rhodobacter sphaeroides.

In the photosynthetic bacterium Rhodobacter sphaeroides, two genes, hemA and hemT, each encode a distinct 5-aminolevulinic acid (ALA) synthase isozyme (E. L. Neidle and S. Kaplan, J. Bacteriol. 175:2292-2303, 1993). This enzyme catalyzes the first and rate-limiting step in a branched pathway for tetrapyrrole formation, leading to the biosynthesis of hemes, bacteriochlorophylls, and corrinoids. In an attempt to determine the functions of hemA and hemT, mutant strains were constructed with specific chromosomal disruptions. These chromosomal disruption allowed hemA and hemT to be precisely localized on the larger and smaller of two R. sphaeroides chromosomes, respectively. Mutants carrying a single hemA or hemT disruption grew well without the addition of ALA, whereas a mutant, HemAT1, in which hemA and hemT had both been inactivated required exogenous ALA for growth. The growth rates, ALA synthase enzyme levels, and the amounts of bacteriochlorophyll-containing intracytoplasmic membrane spectral complexes of all strains were compared. Under photosynthetic growth conditions, the levels of bacteriochlorophyll, carotenoids, and B800-850 and B875 light-harvesting complexes were significantly lower in the Hem mutants than in the wild type. In the mutant strains, available bacteriochlorophyll appeared to be preferentially targeted to the B875 light-harvesting complex relative to the B800-850 complex. In strain HemAT1, the amount of B800-850 complex varied with the concentration of ALA added to the growth medium, and under conditions of ALA limitation, no B800-850 complexes could be detected. In the Hem mutants, there were aberrant transcript levels corresponding to the puc and puf operons encoding structural polypeptides of the B800-850 and B875 complexes. These results suggest that hemA and hemT expression is coupled to the genetic control of the R. sphaeroides photosynthetic apparatus.

Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph, Methylomonas methanica 68-1

A methanotroph (strain 68-1), originally isolated from a trichloroethylene (TCE)-contaminated aquifer, was identified as the type I methanotroph Methylomonas methanica on the basis of intracytoplasmic membrane ultrastructure, phospholipid fatty acid profile, and 16S rRNA signature probe hybridization. Strain 68-1 was found to oxidize naphthalene and TCE via a soluble methane monooxygenase (sMMO) and thus becomes the first type I methanotroph known to be able to produce this enzyme. The specific whole-cell sMMO activity of 68-1, as measured by the naphthalene oxidation assay and by TCE biodegradation, was comparatively higher than sMMO activity levels in Methylosinus trichosporium OB3b grown in the same copper-free conditions. The maximal naphthalene oxidation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 551 +/- 27 and 321 +/- 16 nmol h mg of protein , respectively. The maximal TCE degradation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 2,325 +/- 260 and 995 +/- 160 nmol h mg of protein, respectively. The substrate affinity of 68-1 sMMO to naphthalene (K(m), 70 +/- 4 muM) and TCE (K(m), 225 +/- 13 muM), however, was comparatively lower than that of the sMMO of OB3b, which had affinities of 40 +/- 3 and 126 +/- 8 muM, respectively. Genomic DNA slot and Southern blot analyses with an sMMO gene probe from Methylosinus trichosporium OB3b showed that the sMMO genes of 68-1 have little genetic homology to those of OB3b. This result may indicate the evolutionary diversification of the sMMOs.

Preparation and characterization of highly pure fractions of outer membrane, cytoplasmic and intracytoplasmic membranes from Ectothiorhodospira mobilis

Outer membrane (OM), cytoplasmic membrane (CM) and intracytoplasmic membranes (ICM) from the halophilic phototrophic purple sulphur bacterium Ectothiorhodospira mobilis 9903 were purified and characterized. The three membrane fractions were significantly different in regard to protein profiles on SDS-PAGE, and to the composition of amino acids, fatty acids and lipids. The presence of lipoproteins, the occurrence of lyso-phosphatidyl-ethanolamine and an increased content of saturated and short-chain fatty acids are characteristic properties of the OM. CM and ICM fractions are different on the basis of buoyant density, of protein profiles and amino acid composition, and due to the presence of succinate dehydrogenase activity in CM. In addition, CM and ICM showed significant differences in pigment content and absorption spectra.

Mycoplasma membranes as models in membrane research.

The mycoplasmas, lacking a cell wall and intracytoplasmic membranes, have only one type of membrane, the plasma membrane. The ease with which this membrane can be isolated and the fact that controlled alterations in its composition are intraducibie have made mycoplasma membranes most effective and popular tools in biomembrane research. The aim of this chapter is to introduce the reader to the mycoplasmas and provide a historical background of the various phases in mycoplasma membrane research. Focus will be on the advantages of mycoplasma membranes as models in studying structure and function of biological membranes, and on the development of concepts that have arisen since the beginning of mycoplasma membrane research, about 30 years ago.

Translocation of precytochrome C2 into intracytoplasmic membrane vesicles of Rhodobacter capsulatus requires a peripheral membrane protein

Rhodobacter capsulatus is a member of the group of alpha-purple bacteria which are closely related to the ancestral endosymbiont that gave rise to mitochondria. It has therefore been hypothesized that the molecular mechanisms governing protein export in alpha-purple bacteria have been conserved during the evolution of mitochondria. To enable analysis of protein export in alpha-purple bacteria we describe here the development of a homologous cell-free synthesis/export system consisting entirely of components of R. capsulatus. Translocation of precytochrome c2 into intracytoplasmic membrane vesicles of this organism was found to require the proton-motive force and proceed at a significantly higher efficiency when membranes were present during protein synthesis. Furthermore, we show that, in this cell-free system, translocation depends on a preparation of peripheral membrane proteins which do not possess detectable SecA- and SecB-like activities.

Nitrification of NH 4-N polluted sea water by immobilized acclimated marine nitrifying sludge (AMNS)

Acclimated marine nitrifying sludge (AMNS) prepared from activated sludge obtained from a night soil treatment plant equipped with a sea water dilution system for controlling the reactor's temperature was successfully immobilized using a polyvinyl alcohol (PVA) freezing method. About 2 weeks of recovery culture was required before continuous treatment could be carried out. Transmittance electron microscopic observations of sliced immobilized AMNS pellets indicated that bacteria with an intracytoplasmic membrane dominated the AMNS colony. Continuous nitrification experiments of NH 4-N containing synthetic sea water were carried out in a 1.2 l bioreactor containing 57.2 g (wet weight) of immobilized AMNS pellets. The NH 4-N removal rate reached a saturation level above an NH 4-N loading rate of 1.5 mg-NH 4-N/g-pellet/d. The maximum allowable NH 4-N loading rate necessary to obtain 90% NH 4-N removal was found to be 1.0 mg-NH 4-N/g-pellet/d. It was possible to store the immobilized AMNS pellets in a refrigerator for at least 1 week without loss of nitrifying capability. Inorganic carbon source was shown to be a limiting factor in the continuous nitrification experiments. The maximum allowable NH 4-N loading rate needed to obtain 90% NH 4-N removal increased to 2.0 mg-NH 4-N/g-pellet/d through supplementation of an inorganic carbon (IC) source to the influent synthetic sea water.

Evidence for Methylotrophic Symbionts in a Hydrothermal Vent Mussel (Bivalvia: Mytilidae) from the Mid-Atlantic Ridge

Symbioses between chemolithoautotrophic bacteria and the major macrofaunal species found at hydrothermal vents have been reported for numerous sites in the Pacific Ocean. We present microscopical and enzymatic evidence that methylotrophic bacteria occur as intracellular symbionts in a new species of mytilid mussel discovered at the Mid-Atlantic Ridge hydrothermal vents. Two distinct ultrastructural types of gram-negative procaryotic symbionts were observed within gill epithelial cells by transmission electron microscopy: small coccoid or rod-shaped cells and larger coccoid cells with stacked intracytoplasmic membranes typical of methane-utilizing bacteria. Methanol dehydrogenase, an enzyme diagnostic of methylotrophs, was detected in the mytilid gills, while tests for ribulose-1,5-bisphosphate carboxylase, the enzyme diagnostic of autotrophy via the Calvin cycle, were negative. Stable carbon isotope values (deltaC) of mytilid tissue (-32.7 and -32.5% for gill and foot tissues, respectively) fall within the range of values reported for Pacific vent symbioses but do not preclude the use of vent-derived methane reported to be isotopically heavy relative to biogenically produced methane.

A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides. II. Analysis of a region of the genome encoding hemF and the puc operon.

The puc operon of Rhodobacter sphaeroides encoding polypeptides of the major light-harvesting complex, LH2, has been found to be linked to hemF, a gene encoding a putative anaerobic coproporphyrinogen III oxidase. The puc-hemF region of the R. sphaeroides genome has been investigated by insertional mutagenesis, complementation analysis of these insertional mutants and DNA sequencing. A third gene, designated pucC, has been found immediately downstream of pucA and has been shown to be essential for LH2 expression. pucC is cotranscribed with pucB and pucA; however, hemF and the pucBAC operon were found not to be transcriptionally linked. Ultrastructural studies indicated that the morphology of the intracytoplasmic membrane may depend upon expression of pucC as well as pucBA.

An electron microscopic study of picoplanktonic organisms from a Small Lake

Picoplankton, both prokaryotic and eukaryotic, are distinguished from other aquatic organisms by their small size (0.1-2.0 μm). Such organisms were recovered from waters of a small oligotrophic lake using screens, filters, and high-speed centrifugation. The majority of the picoplankton were unable to form visible colonies on common media. Cells examined in thin sections by electron microscopy showed that 60-75% of the cells had an average diameter after dehydration of 0.48-0.51 μm. The maximum dimensions of the rest of the cells ranged from 0.56-1.81 μm. Using details of ultrastructure, cells were classified as prokaryotic or eukaryotic. Phototrophs present included two cyanobacterial morphotypes (5-6%) and two eukaryotic algae (less than I%). The arrays of intracytoplasmic membranes in 18-20% of the cells were suggestive of methanotrophic rods and chemoautotrophs. Relatively few prosthecate bacteria were observed in the water column samples. The smallest cells (1-2%) contained magnetosomes, the presence of which were confirmed by x-ray spectroscopy. Iron was also detected in the envelopes of some rod shaped cells by the same technique. The study of in situ picoplankton populations using TEM coupled with other techniques may provide better understanding of picoplankton biomass.

Studies on the expression of the pufX polypeptide and its requirement for photoheterotrophic growth in Rhodobacter sphaeroides.

The puf operon in Rhodobacter sphaeroides contains the genes for the light-harvesting antenna complex I (LHI), the reaction centre (RC) L and M subunits and an additional small open reading frame identified as pufX. It has been demonstrated before that a photosynthetically incompetent pufLMX deletion strain was not complemented by a plasmid-borne truncated puf operon version lacking only pufX, although expression of the pufL and pufM gene products was restored. We demonstrate here that the functional reinsertion of only the pufX open reading frame into the same construct is sufficient and necessary for complementation of the non-photosynthetic phenotype. We also demonstrate that the observed lack of photoheterotrophic growth in the absence of pufX is not the result of decreased light-harvesting ability, but rather the result of an impairment in light-driven cyclic electron transfer. Western blots using polyclonal antibodies against a synthetic peptide corresponding to a portion of the DNA-derived pufX amino acid sequence showed that the pufX open reading frame is expressed and that the gene product has an M(r) of 8-10,000 on SDS gels; a value close to the predicted mass of 9 kDa. The pufX polypeptide was localized to the intracytoplasmic membrane fraction and appeared to co-purify with the RC-LHI complex. It is suggested that the pufX polypeptide is associated with the RC-LHI complex and that it may play a critical role in facilitating the interaction between this complex and other components required for light-driven cyclic electron transfer.

Presence of methyl sterol and bacteriohopanepolyol in an outer-membrane preparation from Methylococcus capsulatus (Bath).

Cytoplasmic/intracytoplasmic and outer membrane preparations of Methylococcus capsulatus (Bath) were isolated by sucrose density gradient centrifugation of a total membrane fraction prepared by disruption using a French pressure cell. The cytoplasmic and/or intracytoplasmic membrane fraction consisted of two distinct bands, Ia and Ib (buoyant densities 1.16 and 1.8 g ml-1, respectively) that together contained 57% of the protein, 68% of the phospholipid, 73% of the ubiquinone and 89% of the CN-sensitive NADH oxidase activity. The only apparent difference between these two cytoplasmic bands was a much higher phospholipid content for Ia. The outer membrane fraction (buoyant density 1.23 - 1.24 g ml-1) contained 60% of the lipopolysaccharide-associated, beta-hydroxypalmitic acid, 74% of the methylsterol, and 66% of the bacteriohopanepolyol (BHP); phospholipid to methyl sterol or BHP ratios were 6:1. Methanol dehydrogenase activity and a c-type cytochrome were also present in this outer membrane fraction. Phospholipase A activity was present in both the cytoplasmic membrane and outer membrane fractions. The unique distribution of cyclic triterpenes may reflect a specific role in conferring outer membrane stability in this methanotrophic bacterium.

Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium.

A novel bacterium designated strain MV1 was isolated from a sludge enrichment taken from the wastewater treatment plant at a plastics manufacturing facility and shown to degrade 2,2-bis(4-hydroxyphenyl)propane (4,4'-isopropylidenediphenol or bisphenol A). Strain MV1 is a gram-negative, aerobic bacillus that grows on bisphenol A as a sole source of carbon and energy. Total carbon analysis for bisphenol A degradation demonstrated that 60% of the carbon was mineralized to CO2, 20% was associated with the bacterial cells, and 20% was converted to soluble organic compounds. Metabolic intermediates detected in the culture medium during growth on bisphenol A were identified as 4-hydroxybenzoic acid, 4-hydroxyacetophenone, 2,2-bis(4-hydroxyphenyl)-1-propanol, and 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Most of the bisphenol A degraded by strain MV1 is cleaved in some way to form 4-hydroxybenzoic acid and 4-hydroxyacetophenone, which are subsequently mineralized or assimilated into cell carbon. In addition, about 20% of the bisphenol A is hydroxylated to form 2,2-bis(4-hydroxyphenyl)-1-propanol, which is slowly biotransformed to 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Cells that were grown on bisphenol A degraded a variety of bisphenol alkanes, hydroxylated benzoic acids, and hydroxylated acetophenones during resting-cell assays. Transmission electron microscopy of cells grown on bisphenol A revealed lipid storage granules and intracytoplasmic membranes.

Pleiotropic effects of pufX gene deletion on the structure and function of the photosynthetic apparatus of Rhodobacter capsulatus

By deletion of the pufX gene of Rhodobacter capsulatus from a plasmid carrying the puf operon and complementation of a chromosomal puf operon deletion, we created pufX mutants and used them to characterize possible functions of the pufX gene product. The pufX mutants were incapable of photosynthetic growth in a minimal medium, or in a rich medium at low light intensities, although second-site mutations suppressed this phenotype. Measurements made in vitro with intact and solubilized chromatophore preparations indicated that the individual complexes of the photosynthetic unit seemed to function normally, but electron transfer from the reaction center to the cytochrome bc1 complex was impaired. The structures of the photosynthetic apparatus of pseudo-wild type and mutant strains were evaluated using absorption spectroscopy and electron microscopy. The pufX mutants had intracytoplasmic membrane invaginations about 50% larger in diameter than those of the pseudo-wild type and higher levels of B870 light-harvesting complex. It is concluded that the PufX protein plays an important role in the structure of the functional photosynthetic unit, and its absence results in loss of efficient electron transfer from the QB site of the reaction center to the QZ site of the cytochrome bc1 complex.

Characterization of LHI- and LHI+ Rhodobacter capsulatus pufA mutants.

The NH2 termini of light-harvesting complex I (LHI) polypeptides alpha and beta of Rhodobacter capsulatus are thought to be involved in the assembly of the LHI complex. For a more detailed study of the role of the NH2-terminal segment of the LHI alpha protein in insertion into the intracytoplasmic membrane (ICM) of R. capsulatus, amino acids 6 to 8, 9 to 11, 12 and 13, or 14 and 15 of the LHI alpha protein were deleted. Additionally, the hydrophobic stretch of the amino acids 7 to 11 was lengthened by insertion of hydrophobic or hydrophilic amino acids. All mutations abolished the ability of the mutant strains to form a functional LHI antenna complex. All changes introduced into the LHI alpha protein strongly reduced the stability of its LHI beta partner protein in the ICM. The effects on the mutated protein itself, however, were different. Deletion of amino acids 6 to 8, 9 to 11, or 14 and 15 drastically reduced the amount of the LHI alpha protein inserted into the membrane or prevented its insertion. Deletion of amino acids 12 and 13 and lengthening of the stretch of amino acids 7 to 11 reduced the half-life of the mutated LHI alpha protein in the ICM in comparison with the wild-type LHI alpha protein. Under the selective pressure of low light, revertants which regained a functional LHI antenna complex were identified only for the mutant strain deleted of amino acids 9 to 11 of the LHI alpha polypeptide [U43 (pTPR15)]. The restoration of the LHI+ phenotype was due to an in-frame duplication of 9 bp in the pufA gene directly upstream of the site of deletion present in strain U43(pTPR15). The duplicated nucleotides code for the amino acids Lys, Ile, and Trp. Membranes purified from the revertants were different from that of the reaction center-positive LHI+ LHII- control strain U43(pTX35) in doubling of the carotenoid content and increase of the size of the photosynthetic unit. By separating the reaction center and LHI complexes of the revertants by native preparative gel electrophoresis, we confirmed that the higher amount of carotenoids was associated with the LHI proteins.

Pleiotropic effects of pufX gene deletion on the structure and function of the photosynthetic apparatus of Rhodobacter capsulatus

By deletion of the pufX gene of Rhodobacter capsulatus from a plasmid carrying the puf operon and complementation of a chromosomal puf operon deletion, we created pufX mutants and used them to characterize possible functions of the pufX gene product. The pufX mutants were incapable of photosynthetic growth in a minimal medium, or in a rich medium at low light intensities, although second-site mutations suppressed this phenotype. Measurements made in vitro with intact and solubilized chromatophore preparations indicated that the individual complexes of the photosynthetic unit seemed to function normally, but electron transfer from the reaction center to the cytochrome bc 1 complex was impaired. The structures of the photosynthetic apparatus of pseudo-wild type and mutant strains were evaluated using absorption spectroscopy and electron microscopy. The pufX mutants had intracytoplasmic membrane invaginations about 50% larger in diameter than those of the pseudo-wild type and higher levels of B870 light-harvesting complex. It is concluded that the PufX protein plays an important role in the structure of the functional photosynthetic unit, and its absence results in loss of efficient electron transfer from the Q B site of the reaction center to the Q Z site of the cytochrome bc 1 complex.

Rhodobacter capsulatus mutants lacking the Rieske FeS protein form a stable cytochrome bc1 subcomplex with an intact quinone reduction site.

The ubiquinol-cytochrome c oxidoreductase (or bc1 complex) of Rhodobacter capsulatus consists of three subunits: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, encoded by the fbcF, fbcB, and fbcC genes, respectively. In the preceding paper [Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F. (1992) Biochemistry (preceding paper in this issue)], we have observed that the apoproteins for cytochromes b and c1 are fully present in the intracytoplasmic membrane of R. capsulatus mutants containing low amounts of, or no, Rieske apoprotein. Here we present evidence that the redox midpoint potentials of cytochromes b and c1, as well as their ability to bind antimycin and stabilize a semiquinone at the Qi site, are unaffected by the absence of the Rieske subunit. This is the first report describing a mutant containing a stable bc1 subcomplex with an intact Qi site in the chromatophore membranes, and provides further evidence that a functional quinone reduction site can be formed in the absence of a quinol oxidation (Qo) site. Additional mutants carrying fbc deletions expressing the remaining subunits of the cytochrome bc1 complex were constructed to investigate the relationship among these subunits for their stability in vivo. Western blot analysis of these mutants indicated that cytochromes b and c1 protect each other against degradation, suggesting that they form a two-protein subcomplex in the absence of the Rieske protein subunit.

Reinvestigation of regulation of biosynthesis and subunit composition of nickel-dependent Hup-hydrogenase of Rhodospirillum rubrum

Regulation of biosynthesis and structural properties of the membrane-bound ‘hydrogen uptake’ (Hup)-hydrogenase of Rhodospirillum rubrum were studied. Under photoheterotrophic conditions, the highest activities were recorded in media with L-lactate, the lowest activities with malate. At high concentrations (20–40 mM), NH 4 + or glutamate suppressed biosynthesis of hydrogenase. In culture media supplemented with 0.5 mM EDTA, normal Hup activities were only obtained in the presence of high concentrations of Ni 2+ (80–150 μM). Activation of hydrogenase biosynthesis by Ni 2+ was completely inhibited by chloramphenicol. The enzyme, solubilized from intracytoplasmic membranes by Triton X-114 and partially purified by gelfiltration, had a M r of about 100 000. When analysed by SDS-PAGE, the native enzyme yielded three different protein bands with M r values of 40 000, 34 000 and 29 000.

Reinvestigation of regulation of biosynthesis and subunit composition of nickel-dependent Hup-hydrogenase ofRhodospirillum rubrum

Regulation of biosynthesis and structural properties of the membrane-bound ‘hydrogen uptake’ (Hup)-hydrogenase of Rhodospirillum rubrum were studied. Under photoheterotrophic conditions, the highest activities were recorded in media with L-lactate, the lowest activities with malate. At high concentrations (20–40 mM), NH4+ or glutamate suppressed biosynthesis of hydrogenase. In culture media supplemented with 0.5 mM EDTA, normal Hup activities were only obtained in the presence of high concentrations of Ni2+ (80–150 μM). Activation of hydrogenase biosynthesis by Ni2+ was completely inhibited by chloramphenicol. The enzyme, solubilized from intracytoplasmic membranes by Triton X-114 and partially purified by gelfiltration, had a Mr of about 100 000. When analysed by SDS-PAGE, the native enzyme yielded three different protein bands with Mr values of 40 000, 34 000 and 29 000.