Cobalt binding in the photosynthetic bacterium R. sphaeroides by X-ray absorption spectroscopy

Cobalt is an important oligoelement required for bacteria; if present in high concentration, exhibits toxic effects that, depending on the microorganism under investigation, may even result in growth inhibition. The photosynthetic bacterium Rhodobacter (R.) sphaeroides tolerates high cobalt concentration and bioaccumulates Co²⁺ ion, mostly on the cellular surface. Very little is known on the chemical fate of the bioaccumulated cobalt, thus an X-ray absorption spectroscopy investigation was conducted on R. sphaeroides cells to gain structural insights into the Co²⁺ binding to cellular components. X-ray absorption near-edge spectroscopy and extended X-ray absorption fine structure measurements were performed on R. sphaeroides samples containing whole cells and cell-free fractions obtained from cultures exposed to 5 mM Co²⁺. An octahedral coordination geometry was found for the cobalt ion, with six oxygen-ligand atoms in the first shell. In the soluble portion of the cell, cobalt was found bound to carboxylate groups, while a mixed pattern containing equivalent amount of two sulfur and two carbon atoms was found in the cell envelope fraction, suggesting the presence of carboxylate and sulfonate metal-binding functional groups, the latter arising from sulfolipids of the cell envelope.     

Nickel uptake in Rhodopseudomonas capsulata

Nickel uptake system was investigated with a wild-type cell of Rhodopseudomonas capsulata and two mutants lacking uptake hydrogenase (Hup^-). Wild type cells grown photoheterotrophically incorporated ⁶³Ni²⁺ by a high affinity system. The uptake system had a pH of 7.0 and a temperature optimum of 28°C. Both Mg²⁺ and Co²⁺ ions severely repressed the uptake of Ni²⁺. Nickel transport was also inhibited by metabolic inhibitors including cyanide, azide, 2,4-dinitrophenol and m-chlorophenyl carbonylcyanidehydrazone. These data imply that Ni²⁺ uptake system occurs by the energy-linked system for Mg²⁺ transport. The intracellular distribution of ⁶³Ni²⁺ in Hup^- cells showed the same pattern as that of wild-type cells, indicate that the Hup^- strains have no deficiency in Ni²⁺ transport.Abbreviations CCCP m-chlorophenyl carbonylcyanidehydrazone - HEPES N-2-hydroxylethylpiperazine-N-2-ethane-sulfuric acid - HOQNO 2-n-nonly-4-hydroxyquinoline-N-oxide - TMA tetramethylammonium hydroxide     

Nickel resistance in fission yeast associated with the magnesium transport system

We isolated and characterized a nickel (Ni²⁺)-resistant mutant (GA1) of Schizosaccharomyces pombe. This mutant strain displayed resistance to both Ni²⁺ and Zn²⁺, but not to Cd²⁺, Co²⁺, and Cu²⁺. The growth rate of GA1 increased proportionally with increasing Mg²⁺ concentrations until 50 mM Mg²⁺. The GA1 mutation phenotype suggests a defect in Mg²⁺ uptake. Sequence analysis of the GA1 open reading frame (ORF) O13779, which is homologous to the prokaryotic and eukaryotic CorA Mg²⁺ transport systems, revealed a point mutation at codon 153 (ccc to acc) resulting in a Pro 153Thr substitution in the N-terminus of the CorA domain. Our results provide novel genetic information about Ni²⁺ resistance in fission yeast. Specifically, that reducing Mg²⁺ influx through the CorA Mg²⁺ transport membrane protein confers Ni²⁺ resistance in S. pombe. We also report that Ni²⁺ ion detoxification of the fission yeast is related to histidine metabolism and pH.     

Selective transport of divalent cations by transition metal permeases: the Alcaligenes eutrophus HoxN and the Rhodococcus rhodochrous NhlF

nhlF and hoxN, the genes encoding a cobalt transporter of Rhodococcus rhodochrous J1 and a nickel permease of Alcaligenes eutrophus H16, respectively, were expressed in Escherichia coli. ⁵⁷Co²⁺ and ⁶³Ni²⁺ transport of the recombinants was examined by means of a previously described physiological assay. Although the transporters are highly similar, different preferences for divalent transition metal cations were observed. HoxN was unable to transport ⁵⁷Co²⁺, but mediated ⁶³Ni²⁺ uptake. The latter activity was unaffected by a tenfold excess of other divalent cations, showing the specificity of HoxN for Ni²⁺. In contrast, NhlF transported both ⁵⁷Co²⁺ and ⁶³Ni²⁺ ion. NhlF-mediated ⁶³Ni²⁺ uptake was markedly reduced in the presence of Co²⁺, while ⁵⁷Co²⁺ uptake was only slightly lower in the presence of Ni²⁺. These results indicate different affinities of NhlF for Co²⁺ and Ni²⁺ and identified Co²⁺ ion as the preferred substrate. Received: 8 September 1998 / Accepted: 30 November 1998     

Cobalt transport in nickel-resistant strains ofNeurospora crassa

Uptake of Co²⁺ by three nickel-resistant strains (Ni^R1, Ni^R2, and Ni^R3) ofNeurospora crassa that differed in resistance to Co²⁺ has been studied. Uptake was linear with Co²⁺ concentration (up to 1 mM), with time (up to 6 h), and with pH between 3 and 6. Uptake rates were in the order Ni^R2>Ni^R1>Ni^R3. In all strains, there was gradual increase in Co²⁺ uptake between 10° and 28°C, with a much sharper increase between 28° and 40°C. Metabolic inhibitors decreased Co²⁺ uptake partially in all strains, except for KF in Ni^R3. About 50–80 g Co²⁺/100 mg dry weight was surface bound. Ni²⁺, Zn²⁺, and Mn²⁺ competed with Co²⁺, the effects being strain specific. Mg²⁺ inhibited Co²⁺ uptake in all strains with preformed mycelia. In Ni^R1 and Ni^R2 only with young mycelia (40 h old) was Mg²⁺ inhibitory to Co²⁺ uptake,during growth in the presence of Co²⁺. The results suggested the presence of two transport systems for Co²⁺ in Ni^R1 and Ni^R2, only one of which was sensitive to Mg²⁺; in contrast, Ni^R3 had a single system, which was sensitive to Mg²⁺.     

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Fe²⁺. This activation is based on the formation of Me²⁺-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me²⁺ in this transport process. Higher Me²⁺ concentrations inhibit calcium transport with various efficiencies. Mn²⁺ directly competes with Ca²⁺ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [γ-³²P]ATP is induced by Ca²⁺ while rapid dephosphorylation requires the presence of Mg²⁺. At higher concentrations Mn²⁺ and Ni²⁺ inhibit predominantly the formation of EP, while Co²⁺ and Fe²⁺ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [γ-³²P]ATP is significantly stimulated by Mn²⁺ and Co²⁺, as compared to that produced by Mg²⁺, Fe²⁺ and Ni²⁺. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.     

Ni2+-uptake inPseudomonas putida strain S4: a possible role of Mg2+-uptake pump

Essential metal ion homeostasis is based on regulated uptake of metal ions, both during its scarcity and abundance.Pseudomonas putida strain S4, a multimetal resistant bacterium, was employed to investigate Ni²⁺ entry into cells. It was observed that Mg²⁺ regulates the entry of Ni²⁺ and by this plays a protective role to minimize Ni²⁺ toxicity in this strain. This protection was evident in both growth as well as viability. Intracellular accumulation of Ni²⁺ varied in accordance with Mg²⁺ concentrations in the medium. It was hypothesized that Ni²⁺ enters the cell using a broad Mg²⁺ pump, i.e. the CorA system, as the CorA inhibitor, i.e. Co(III) Hex, also inhibits Ni²⁺ uptake. This led to the inference that Mg²⁺-based protection was basically due to competitive inhibition of Ni²⁺ uptake. We also show that Zn²⁺ can further regulate the entry of Ni²⁺     

Conjugative plasmid mediated inducible nickel resistance in<Emphasis Type="Italic"> Hafnia alvei</Emphasis> 5-5

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni²⁺ or 2 mM Co²⁺. The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni²⁺, and cobalt resistance to 2 mM Co²⁺. A 4.8-kb Sal–EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni²⁺) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni²⁺. By random TnphoA′-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A. Electronic Publication     

Isolation and characterization of nickel-resistant microflora from serpentine soils of Andaman

Serpentine soils of Andaman Islands, India characteristically contain high levels of nickel, cobalt and chromium and are colonized by indigenous nickel-hyperaccumulating plants. Attempts have been made to isolate and characterize nickel-resistant microorganisms from these hitherto unexplored naturally nickel-percolated soils. The majority of the nickel-resistant organisms showed a minimum inhibitory concentration (MIC) of Ni²⁺ ranging from 300 to 400 mg/l and about 3.4% of the total 89 isolates representing bacterial strains were able to grow at 400 mg/l Ni²⁺. The potent Ni²⁺-resistant strains AND305 and AND603 were tentatively identified as Pseudomonas spp. and strain AND408 as Bacillus sp. following detailed analysis of morphological and physio-biochemical characteristics. Growth kinetics of these Ni²⁺-resistant bacteria showed a prolonged lag phase in Ni²⁺-containing media, which extended with increasing nickel concentration. In addition to Ni²⁺, these isolates were also resistant to Co²⁺, Cd²⁺, Cr⁶⁺, Fe³⁺, Cu²⁺, Mg²⁺, Mn²⁺(50–200 mg/l) and Hg²⁺ (0.5–2.0 mg/l) and the multiple metal-resistance of the isolates were also associated with the resistance to antibiotics ampicillin, cycloserine and penicillin G.     

Nickel transport in Alcaligenes eutrophus

Transport of nickel ions was studied in Alcaligenes eutrophus. Two transport systems for nickel ions exist to satisfy the nickel demand for the lithotrophic hydrogen metabolism. A major nickel transport activity exhibited an apparent affinity constant (K _m) of 17 M nickel chloride. This activity was competitively inhibited by Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺. A minor nickel transport activity was determined in the presence of high (0.8 mM) magnesium. This activity was not inhibited by Zn²⁺ or Mn²⁺; its K _m was determined to be 0.34 M nickel chloride. These kinetics suggested a second transport system in A. eutrophus. The membrane potential of A. eutrophus was decreased upon the addition of ammonium ions leading to a decreased nickel transport. This inhibition could be reversed by fructose or by hydrogen indicating an energy dependent nickel transport. Protonophores inhibited the nickel transport. However, inhibitors of ATP synthase like dicyclohexylcabodimide or venturicidin had little or no effect on nickel transport. These data indicated that the transport was coupled to the proton motive force.     

Nickel Uptake by Pseudomonas aeruginosa: Role of Modifying Factors

Pseudomonas aeruginosa cells growing in minimal medium were 40-fold more sensitive to Ni²⁺ than cells growing in enriched medium, suggesting a possible protective role of medium ingredients. Likewise, cells pre-grown in enriched medium showed a high K _m (6.15 mM) and increased Ni²⁺ uptake (950 nmol mg⁻¹ protein, 1h) over cells pre-sown in minimal medium (K _m , 0.48 mM; 146 nmol mg⁻¹ protein, 1 h). The overall pattern indicates that cells pre-grown in enriched medium were characterized by having lowered affinity towards Ni²⁺ than those with minimal medium background. The enhanced Ni²⁺ uptake by enriched medium-grown cells can be correlated with the improved metabolic state of the cells. Ni²⁺ uptake was optimum at neutrality (pH 7.0). A major Ni²⁺ transport system was competitively inhibited by Mg²⁺, Zn²⁺, Cd²⁺, or Co²⁺ (400 μM each). Noticeably, a minor Ni²⁺ transport pathway was still operative even in the higher concentration range of Mg²⁺ (4 mM and 40 mM). The stimulation of Ni²⁺ uptake monitored in the presence of different carbon sources (0.5% wt/vol, each) showed the sequence: glucose (1.6-fold) > phenol = gallic acid (1.5-fold). Succinate, in comparison, reduced Ni²⁺ uptake (0.5-fold) possibly because of its acting as a metal chelator as well. Sensitivity of Ni²⁺ transport towards methyl viologen, azide, 2-4 DNP, and DCCD suggested that transport was energy-linked. Received: 13 January 1998 / Accepted: 21 May 1998     

News & Notes: Sorption and Desorption of Cobalt by Oscillatoria anguistissima

Oscillatoria anguistissima rapidly adsorbs appreciable amounts of cobalt from the aqueous solutions within 15 min of initial contact with the metal solution. O. anguistissima showed a high sequestration of cobalt at low equilibrium concentrations, and it followed the Freundlich model of adsorption. The adsorption is a strongly pH-dependent and temperature-independent phenomenon. The presence of Mg²⁺ and Ca²⁺ (100–200 ppm) resulted in decline in Co²⁺ adsorption capacity of Oscillatoria biomass. Sulphate and nitrate (0.75–10 mM) drastically reduced the extent of Co²⁺ biosorption. The biosorption of cobalt is an ion-exchange process as the Co²⁺ binding was accompanied by release of a large amounts of Mg²⁺ ions. Na₂CO₃ (1.0 mM) resulted in about 76% desorption of Co²⁺ from the loaded biomass. Received: 30 January 1999 / Accepted: 3 March 1999     

Inhibition of Urease from Seeds of Water-melon (Citrullus vulgaris) by Heavy Metal Ions

Urease from the seeds of water melon was found to be inhibited by heavy metal ions like copper, lead, nickel and cobalt. The order of effectiveness of these metals as inhibitors was Cu²⁺ > Pb²⁺ > Ni²⁺ > Co²⁺. The inhibition by these ions was noncompetitive. Time — dependent interaction of urease with nickel and cobalt exhibited a biphasic inhibition behaviour in which approximately half of the initial activity was lost rapidly (within 2 min) and remainder in a slow phase. The inhibition was largely irreversible, hence could not be reversed by dialysis. These observations are suggestive of half-and-half distribution of — SH groups on the native enzyme resulting urease into asymmetric oligomeric molecule.     

Inhibitor effects on the accumulation and efflux of nickel ions in plasmid pMOL28-harboring strains of Alcaligenes eutrophus

The accumulation and efflux of ⁶³Ni²⁺ ions were studied using two strains of the strictly respiratory bacterium Alcaligenes eutrophus, the wild type strain N9A and its transconjugant N9A-M243 which harbors plasmid pMOL28.1 encoding constitutive resistance to nickel. When 1 M ⁶³Ni²⁺ is added to respiring cells, strain N9A accumulates high, but M243 only negligibly small amounts of nickel. When the cells were preincubated for about 20 h under anoxic conditions and were then exposed to 1 M ⁶³Ni²⁺ anaerobically, both strains accumulated approximately the same amounts of nickel. Aeration of these preloaded cells resulted in rapid efflux by strain M243 but renewed uptake of nickel by N9A. ⁶³Ni²⁺ uptake and efflux are highly sensitive to protonophores such as CCCP, FCCP and TCS but insensitive to DCCD (each at 20 M concentrations). Measurements on the effects of the inhibitors on biosynthetic processes requiring ATP either from substrate phosphorylation or from electron transport phosphorylation made sure that at the concentrations used the inhibitor effects were specific. Thus the results suggest that in the nickelresistant strain M243 under normal aerobic conditions two constitutive energy-dependent nickel transport systems can function concomitantly, a chromosomally-determined specific uptake system and a plasmid-mediated specific nickel efflux system.     

Selective Inhibition of Flagellar Activity in Chlamydomonas by Nickel

Flagellar activity in the biflagellate chlorophyte Chlamydomonas reinhardtii is selectively inhibited by Ni²⁺ or by treatment with Ca²⁺-chelating agents. Inhibitions of swimming speed, geotaxis, phototaxis, and pattern swimming result from qualitative and quantitative losses in the activity of individual flagella and in the coordination of activity beween the 2 flagella of each cell. Addition of Ca²⁺ (a) prevents inhibition and (b) restores normal flagellar activity in inhibited cells. Mg²⁺ is partially effective in reversal of inhibition. Other ions do not cause similar inhibition or reversal of nickel inhibition. The characteristics of inhibition and reversal suggest that the prmary target for nickel is a component of the flagellar apparatus, and that this component uses Ca²⁺ to perform its normal function in the regulation of flagellar activity. A 2nd target for nickel is a Carequiring process specific to phototaxis (and not involved in the photophobic response).     

Interaction of divalent metal ions with the carboxyl-terminal domain of human voltage-gated proton channel Hv1

The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His²⁴⁴ or His²⁶⁶ residue. The binding of Zn²⁺ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co²⁺, Ca²⁺, Ni²⁺, Mg²⁺, and Mn²⁺. The strength of Ca²⁺, Co²⁺, Mg²⁺, Mn²⁺ and Ni²⁺ binding to the site close to His²⁴⁴ is stronger than that of these divalent metal ions binding to the site close to His²⁶⁶.     

Barrier function of the cell wall during uptake of nickel ions

Cell walls were isolated from roots of six plant species to study their ion-exchange capacity for nickel ions (S _Ni) at Ni²⁺ concentration of 10⁻³ M. The S _Ni values varied depending on the plant species from 50 to 150 μmol Ni²⁺ per gram dry wt; the sorption capacity increased in a row: Poaceae < Chenopodiaceae < Fabaceae. At pH 5 the sorption capacity of cell walls for nickel ions was determined by the presence of carboxyl groups of polygalacturonic acid in the polymeric cell-wall matrix. In all cases the ion-exchange capacity of cell walls was higher at pH 8 than at pH 5, indicating that Ni²⁺ binds also to a carboxyl group different from that of polygalacturonic acid. Irrespective of plant species, the presence of EDTA in the solution diminished drastically the absorption capacity of cell walls for Ni²⁺. It is concluded that the presence of 10⁻³ M EDTA weakens the defense properties of cell walls. The sequestration of Ni²⁺ in the cell wall can be considered as an effective means of plant cell defense against elevated concentrations of nickel ions in the external medium.     

Role of Hpn and NixA of Helicobacter pylori in Susceptibility and Resistance to Bismuth and Other Metal Ions

Background. Helicobacter pylori produces Hpn, a 60-amino acid, histidine-rich protein that avidly binds nickel and zinc ions, and NixA, a high-affinity nickel transporter in the cytoplasmic membrane. We tested the hypothesis that Hpn and NixA govern susceptibility to metal ions in H. pylori. Materials and Methods. Hpn-negative mutants of four H. pylori strains were constructed by standard allelic exchange techniques to yield isogenic Hpn⁺/Hpn-deficient pairs. A metal concentration that inhibited growth by 50% (IC₅₀) was calculated for Ni²⁺, Zn²⁺, Cu²⁺, and Co²⁺ by comparing OD₆₀₀ of cultures in metal-supplemented and control media. Results. Among all four pairs of isogenic strains, the tolerance for Ni²⁺ was reduced significantly (p < .001) in the Hpn mutants; the mean IC₅₀ value for wild-type strains was 1.9 mM; for the mutant, it was 0.8 mM. In  contrast, growth inhibition by Zn²⁺ was identical within the fours pairs, as was Cu²⁺ and Co²⁺ tolerance in one pair tested. We also found that deletion of the hpn gene increases susceptibility to therapeutic forms of bismuth by testing a mutant and wild-type pair with ranitidine bismuth citrate, bismuth citrate, and four antibiotics. Minimal inhibitory concentrations of ranitidine bismuth citrate dropped from 9.2 to 2.3 μg/ml, and those of bismuth citrate dropped from 7.4 to 3.2 μg/ml (p < .05 for both comparisons), while susceptibility to the antibiotics was unaffected. Disruption of the nixA gene encoding the specific Ni²⁺ transport protein of H. pylori did not change susceptibility to bismuth. Conclusion. We concluded that bacteria lacking Hpn, cultured in vitro, are more susceptible than is the wild type to bismuth and Ni²⁺.     

Inducible nickel resistance in a river isolate of India phylogenetically ascertained as a novel strain of Acinetobacter junii

Summary A gram negative, motile, short rod-shaped, and nickel resistant (tolerating 6.5 mM Ni²⁺) bacterium, strain BB1A, was isolated from the waters of the River Torsa in Hashimara, Jalpaiguri district, West Bengal, India. The isolate BB1A was identified as a strain of Acinetobacter junii following detailed analysis of morphological, physio-biochemical and 16S rRNA gene sequence. The expression of nickel resistance in BB1A was inducible by exposure to nickel chloride at a concentration as low as 50 μM Ni²⁺. The other metal ions, Cu²⁺, Zn²⁺, or Pb²⁺ at a concentration range of 20–30 μM, also induced the nickel resistance system in this bacterium. Southern hybridizations of BB1A genomic DNA with digoxigenin-dUTP labeled DNA probes specific for well known nickel resistance determinants, cnr, ncc or nre, resulted in no detectable signal, but nir specific probe yielded weak hybridization signal with restricted genomic DNA of BB1A. The isolate BB1A, therefore, carries out a novel induction phenomenon of nickel resistance and presumably with a nickel resistance genetic system different from that previously characterized in other bacteria.     

Identification and Characterization of a Putative Dihydroorotase,KPN01074, from <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Dihydroorotase (DHO; EC 3.5.2.3) is an essential metalloenzyme in the biosynthesis of pyrimidine nucleotides. Here, we identified and characterized DHO from the pathogenic bacterium Klebsiella pneumoniae (Kp). The activity of KpDHO toward l-dihydroorotate was observed with K _m = 0.04 mM and V _max = 8.87 μmol/(mg min). Supplementing the standard growth medium with Co²⁺, Mn²⁺, Mg²⁺, or Ni²⁺ increased enzyme activity. The catalytic activity of KpDHO was inhibited with Co²⁺, Zn²⁺, Mn²⁺, Cd²⁺, Ni²⁺, and phosphate ions. Substituting the putative metal binding residues His17, His19, Lys103, His140, His178, and Asp251 with Ala completely abolished KpDHO activity. However, the activity of the mutant D251E was fourfold higher than that of the wild-type protein. On the basis of these biochemical and mutational analyses, KpDHO (KPN01074) was identified as type II DHO.