Morphological characteristics and phylogenetic relationship of <Emphasis Type="Italic">Anabaena</Emphasis> species from Lakes Dianchi and Erhai,China

Although Anabaena is one of the most prevalent planktonic freshwater genus in China, there are few taxonomic reports of Anabaena strains by morphology and genetics. In this study, morphological characteristics and phylogenetic relationships of seven Anabaena strains isolated from two plateau lakes, Lakes Dianchi and Erhai, were investigated. Morphological characteristics such as morphology of filament, cellular shapes and sizes, relative position of heterocytes and akinetes, and presence or absence of aerotopes, were described for these seven strains. Phylogenetic relationships were determined by constructing 16S rRNA gene tree using the neighbor-joining algorithm. The seven strains were morphologically identified as three groups, and phylogenetic analysis based on 16S rRNA gene sequences also showed that these seven strains were in three groups. Strains EH-2, EH-3, and EH-4 were in group A belonging to the Anabaena circinalis and A. crassa group, and strains DC-1, DC-2, and EH-1 were in group B and identified as A. flos-aquae. Strain DC-3 without aerotopes was significantly different from the other isolated strains and was determined as A. cylindrica. Handling editor: J. Padisak     

Analysing diversity among Indian isolates of Anabaena (Nostocales, Cyanophyta) using morphological, physiological and biochemical characters

A set of 24 strains belonging to the genus Anabaena (Phylum Cyanobacteria), isolated from diverse geographic locations in India, were evaluated along with three International type strains of Anabaena (ATCC 29414, ATCC 29208 and ATCC 27899) for their morphological, physiological and biochemical diversity. The morphological dataset, consisting of 58 variants for 15 characters, and SDS-PAGE protein profiles comprising 17 polymorphic bands were utilized to differentiate the selected Anabaena strains and explore the patterns of diversity through cluster analysis. Physiological and biochemical characterization with respect to nitrogen fixation and accumulation of chlorophyll and phycobiliproteins led to the identification of some highly promising Anabaena strains for use as biofertilizers and source of pigments. The study highlighted the tremendous inter and intraspecific diversity within the Anabaena isolates and indicated the potential as well as constraints of the morphological and protein profiling datasets for unambiguous differentiation and analyses of diversity among the Anabaena strains.     

An assessment of soil enrichment by actinorhizal N2 fixation using δ15N values in a chronosequence of deglaciation at Glacier Bay,Alaska

The extent of transfer of fixed N between N₂-fixing and non-N₂-fixing plant species is largely unknown in successional studies. In order to redress this deficiency at a locale intensively studied ecologically, leaf tissue samples were collected from actinorhizal N₂-fixing (Alnus, Shepherdia, and Dryas) and two non-N₂-fixing (Salix) woody species within research plots located along a chronosequence of deglaciated fjord in Glacier Bay National Park, Alaska. The tissue samples were analyzed for ¹⁵N content, and the resulting data analyzed for trends in plant tissue N. Among the non-N₂-fixing Salix species, ¹⁵N values increased from the most recently deglaciated sites to converge with the temporally more-stable values for the symbiotic N₂-fixing species on sites at about 40 years after deglaciation. The lower ¹⁵N values of sequestered N in plant tissues suggested that N derived from N₂-fixing plants accounts for the major portion of N in associated plants up to 40 years after deglaciation. The ¹⁵N isotopic data also suggested that Shepherdia canadensis depends least on soil N, D. drummondii the most, and A. viridis ssp. sinuata somewhere between those two species. The presence of a sere dominated by dense thickets of A. viridis ssp. sinuata at the convergence of ¹⁵N values for the N₂-fixing and non-N₂-fixing species indicated that this species is most responsible for accumulation of fixed N in soil at Glacier Bay. This paper is dedicated to the memory of Steven J. Kohls who died prior to publication of this research.     

Phylogeny of symbiotic cyanobacteria within the genus <Emphasis Type="Italic">Nostoc</Emphasis> based on 16S rDNA sequence analyses

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.The GenBank accession numbers for the cyanobacterial 16S rRNA gene sequences determined in this study are AY742447-AY742454.     

Using reverse micelles as microreactor for hydrogen production by coupled systems of Nostoc / R. palustris and Anabaena / R. palustris

Uptake hydrogenase negative mutants of bloom forming cyanobacteria (Nostoc and Anabaena) and the fermentative bacteria Rhodopseudomonas palustris P₄ were used together for producing hydrogen within the reverse micelles fabricated by N-ethyl hexyl sodium sulfosuccinate (AOT) in isooctane and cetyl trimethyl ammonium bromide (CTAB) in benzene. The rate of H₂ production in AOT/isooctane reverse micellar system was found to be more promising in comparison to the CTAB/Benzene reverse micellar entrapment. After mutagenesis in 2.0% (v/v) ethyl methane sulphonate (EMS) mutants of Nostoc and Anabaena were selected on BG-11 plates (containing 2% agar) and then used for analysis of produced hydrogen. In comparison to the unmutated Nostoc with R. palustris (within AOT/isooctane) the coupled system of mutated Nostoc and R. palustris produced H₂ by 3.9-fold higher rate, which is 8.6 mmol H₂/h/mg protein. Whereas, mutated Anabaena coupled with R. palustris produced 4.8 times higher hydrogen production within (AOT)/isooctane reverse micelles in comparison to the unmutated Anabaena with R. palustris. Effect of nitrogen to carbon ratio (N/C) on hydrogen production was studied and Anabaena/R. palustris and Nostoc/R. palustris systems were, respectively, found to generate 11.2 and 9.8 mmol H₂/h/mg protein continuously for 3 days. Effects of temperature and light intensity were also investigated and we found that 32°C temperature and 1,000 Lux light intensity are the optimum values in these systems. Addition of sodium dithionite also resulted in further enhancement of the rate and duration of hydrogen production in both (mutated Nostoc/R. palustris and mutated Anabaena/R.␣palustris) systems.     

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacteriumAnabaena sp. strain PCC 7120

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacteriumAnabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of theAnabaena ADPGlc PPase gene and its expression inEscherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48347 Da which is in agreement with the molecular mass determined by SDS-PAGE for theAnabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and theE. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in theAnabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in anE. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the nativeAnabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be theAnabaena enzyme.     

Cyanobacteria dynamics in a restored Finnish lake: a long term simulation study

Several explanations have been presented for the formation of lacustrine cyanobacteria blooms. These include elevated water temperature, nutrient enrichment, low N/P ratios, low light energy requirements, high pH and/or low carbon dioxide concentration, selective zooplankton grazing, excretion of compounds which suppress the growth of competing algae, and migration. The impact of the first four were studied in Lake Tuusulanjärvi, a eutrophic Finnish lake, using deterministic simulation. The study, which covered 15 years, included a sequence of cases on the recovery of the lake after the implementation of two restoration measures: aeration of the hypolimnion, and termination of municipal waste discharge, both of which were done during the study period. The focus was on Microcystis and Oscillatoria, and on the N-fixing cyanobacteria, Anabaena and Aphanizomenon. Eukaryotic phytoplankton were also included in the study. Monitoring data were used. The roles of the hypotheses were discussed, from the point of view of the utility of the information in water resources management.     

迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白在蓝藻中的表达

在蓝藻中表达迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白。提取迟缓爱德华氏菌基因组DNA为模板,用PCR技术分别扩增两个已知具有较强免疫原性的基因eta1和gapdh,再采用重叠延伸PCR将这两个基因融合,获得目的融合基因eta1-L-gapdh。将目的基因连接到表达载体pRL489的两个Bam H I酶切位点之间构建表达载体,用质粒提取、PCR、酶切、测序等手段对表达载体进行验证。验证正确的表达载体通过三亲接合转化野生鱼腥藻PCC7120,用新霉素抗性筛选出转基因藻落,通过质粒提取和PCR验证转基因藻。用RT-PCR和Western-blot分别从转录水平和翻译水平对转基因藻中融合基因的表达进行了检测。结果表明,含目的基因的表达载体构建成功,目的基因在蓝藻中转录并表达蛋白,该蛋白在蓝藻中的表达量为2.46%。     

The use of a double isotope (15N and 34S) labelling technique to assess the suitability of various reference crops for estimating nitrogen fixation in Gliricidia sepium and Leucaena leucocephala

In this study, an approach involving a double isotope (¹⁵N and ³⁴S) labelling technique was used to examine which of five reference crops (Eucalyptus camaldulensis, Cassia siamea, Cassia spectabilis, Lolium perenne and Eucalyptus grandis) would be suitable for measuring N fixed by Gliricidia sepium and Leucaena leucocephala. The rationale is that the ratio of fertilizer-derived S to soil-derived S in a suitable reference crop is similar to that measured in the nitrogen fixing tree (NFT) since the N ratios in the two crop types cannot be measured directly. E. camaldulensis and E. grandis were found to be suitable reference crops because they absorbed fertilizer and soil S in the same ratio as G. sepium and L. leucocephala.     

Determination of root biomasses of three species grown in a mixture using stable isotopes of carbon and nitrogen

A method is evaluated that employs variation in stable C and N isotopes from fractionations in C and N acquisition and growth to predict root biomasses of three plant species in mixtures. Celtis laevigata Willd. (C₃), Prosopis glandulosa Torr. (C₃, legume) and Schizachyrium scoparium (Michx.) Nash (C₄), or Gossypium hirsutum L. (C₃), Glycine max (L.) Merr. (C₃ legume), and Sorghum bicolor (L.) Moench (C₄) were grown together in separate, three-species combinations. Surface roots (0–10 cm depth) of each species from each of the two combinations were mixed in various proportions, and the relative abundances of ¹⁵N and ¹⁴N and ¹³C and ¹²C in prepared mixtures, surface roots of single species, and roots extracted from the 80-cm soil profile in which each species combination was grown were analyzed by mass spectrometry. An algebraic determination which employed the δ ¹³C, % ¹⁵N, and C and N concentrations of root subsamples of individual species accounted for more than 95% of the variance in biomass of each species in prepared mixtures with G. max, G. hirsutum, and S. bicolor. A similar analysis demonstrated species-specific differences in rooting patterns. Root biomasses of the C₄ monocots in each combination, S. scoparium and S. bicolor, were concentrated in the upper 20 cm of soil, while those of G. hirsutum and the woody P. glandulosa were largest in lower soil strata. Analyses of stable C and N isotopes can effectively be used to distinguish roots of species which differ in ratios of ¹⁵N to ¹⁴N and ¹³C to ¹²C and thus to study belowground competition between or rooting patterns of associated species with different C and N isotope signatures. The method evaluated can be extended to quantify aboveground and belowground biomasses of component species in mixtures with isotopes of other elements or element concentrations that differ consistently among plants of interest.     

Taxonomic studies of planktic species of Anabaena based on morphological characteristics in cultured strains

Fifty (50) strains of planktic species of Anabaena (cyanobacteria), including collections from Japan and China and from different culture collections, were induced to form akinetes at low temperature (15 °C). Their morphologies were then observed and described. Fourty seven strains successfully formed akinetes and these were classified into 20 species comprising seven with straight trichomes and 13 with coiled trichomes. Three strains, which did not form akinetes, were separated into two taxonomic groups, but could not be identified to any described species. In addition, a key to the planktic species of Anabaena described in the study is presented.     

Daphnia vertical distribution and the presence of toxic cyanobacteria

Daphnia can alter its vertical position in the water column in response to chemical cues from predators. In this study we tested the hypothesis that a Daphnia pulex clone with little evolutionary exposure to cyanobacteria would move away from patches of cyanobacteria (Anabaena affinis and A. flos-aquae) which contain potent endotoxins. Daphnia was censused at 2 h intervals for 6 h in laboratory columns in which there was a steep vertical gradient of cyanobacteria. Data were analyzed by repeated-measures ANOVA. Control (no Anabaena) and experimental columns showed no significant differences in Daphnia distributions. Daphnia in experimental columns frequently moved into areas with dense concentrations of Anabaena and stayed there for long periods of time. Our results show that this D. pulex clone does not exhibit a rapid (within 6 h) avoidance response to toxic Anabaena.     

Genetic diversity and phylogeny analysis of Anabaena azollae based on RFLPs detected in Azolla-Anabaena azollae DNA complexes using nif gene probes

The cyanobacterium Anabaena has both symbiotic and free-living forms. The genetic diversity of Anabaena strains symbiotically associated with the aquatic fern Azolla and the evolutionary relationships among these symbionts were evaluated by means of RFLP (restriction fragment length polymorphism) experiments. Three DNA fragments corresponding to nif genes were cloned from the free-living cyanobacterium Anabaena PCC 7120 and used as probes. A mixture of Azolla, Anabaena and bacterial DNA was extracted from Azolla fronds and digested with two restriction enzymes. Single-copy RFLP signals were detected with two of the probes in all Azolla Anabaena examined. Multiple-copy RFLP signals were obtained from the third probe which corresponded to a part of the nif N gene. A total of 46 probe/enzyme combinations were scored as present or absent and used to calculate pairwise Nei's genetic distances among symbiotic Anaebaena strains. Phylogenetic trees summarizing phenetic and cladistic relationships among strains were generated according to three different evolutionary scenarios: parsimony, UPGMA and neighbour joining. All trees revealed identical phylogenetic relationships. Principal component analysis was also used to evaluate genetic similarities and revealed three groups: group one contains the cyanobacteria associated with plants from the Azolla section, group two contains those associated with plants from the pinnata species and group three contains those associated with plants from the nilotica species. The same groups had already been identified earlier in a random amplified polymorphic DNA (RAPD) analysis of Azolla-Anbaena DNA complexes, suggesting that the present Azolla taxonomy should be revised. We now suggest a taxonomy of Anabaena azollae that is parallel to such a revised Azolla taxonomy. An Azolla chloroplast DNA sequence derived from Oryza sativa was also used as an RFLP probe on Azolla DNA to confirm the presence of plant DNA in the total genomic DNA extracted from ferns with or without the symbiont. Our results also suggest that total DNA extracted from the Azolla-Anabaena complexes includes both plant and symbiont DNA and can be used equally well for RFLP analysis of host plant or symbiotic cyanobacteria.     

Growth interactions among blue-green (Anabaena Oscillarioides,Microcystis aeruginosa) and green (Chlorella sp.) algae

The growth interactions amongst the blue-green algal species Anabaena oscillarioides, Microcystis aeruginosa and the green alga, Chlorella sp. were studied both in mixed cultures and in filter cultures separated by a membrane filter in the two arms of an interaction U-tube. The role of nutrients especially phosphate upon the interaction has also been studied. Anabaena and Microcystis both inhibited the growth of Chlorella while Microcystis also inhibited the growth of Anabaena. The inhibitory effect of Microcystis was found to be dependent on high concentrations of the initial algal inocula and independent of the initial concentration of nutrients such as inorganic phosphate, indicating that the nature of the inhibition is probably due to the production of inhibitory extracellular products by Microcystis. On the other hand, the inhibitory effect of Anabaena on Chlorella is the consequence of nutrient competition with Anabaena competing more effectively for the available phosphate.     

Anatoxin-a concentration inAnabaena andAphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxicAnabaena-strains — a laboratory study

Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N₂ fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.     

Sporomusa,a new genus of gram-negative anaerobic bacteria including Sporomusa sphaeroides spec. nov. and Sporomusa ovata spec. nov.

A new genus of strictly anaerobic, gram-negative, banana-shaped bacteria is described. Cells formed spores and were motile by means of up to 15 laterally inserted flagella. Nitrate or sulfate were not used as electron acceptor. Organic substrates that were fermented included N-methyl compounds, such as betaine, N,N-dimethylglycine and sarcosine, primary alcohols, hydroxy fatty acids, and 2,3-butanediol. In addition, molecular hydrogen and carbon dioxide were fermented to acetate. The latter was the characteristic fermentation product in general. During growth on betaine, trimethylamine was formed in addition. The degradation of N,N-dimethylglycine yielded acetate, monomethylamine, and trimethylamine. The presence of cytochrome b and of ubiquinone in the cells was shown. The deoxyribonuleic acid base composition of the strains was between 41.3 and 47.4 mol% guanine plus cytosine. The name Sporomusa is proposed for this new genus. On the basis of the DNA-DNA homology values obtained, the shape of the spores and some other properties, the isolated strains were assigned to two species. Names proposed: Sporomusa sphaeroides and Sporomusa ovata. The type species is S. sphaeroides and the type strains are strain E, DSM 2875 (S. sphaeroides) and strain H1, DSM 2662 (S. ovata).Dedicated to Prof. H. G. Schlegel on the occasion of his 60th birthday     

Callus production from willow (Salix viminalis L.) protoplasts

Protoplasts were isolated from cell suspensions of Salix viminalis (basket willow) clone 78-0-90 and S. schwerinii clone 77-0-77, using cellulysin and macerase in modified Woody Plant medium. For clone 78-0-90, 6.3 · 10⁶ ± 1.9 · 10⁶ protoplasts were obtained per gram fresh weight. Cell divisions started two days after protoplast isolation and gave rise to callus which has been maintained in culture for up to four years. Protoplast yield from the clone 77-0-77 was lower (less than 10⁶ protoplasts per gram cells), cell division was infrequent and no callus was obtained. Protoplasts were also isolated from the leaves of willow shoot cultures using cellulysin and pectolyase, but these did not show cell divisions.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog (1962) medium - WP medium Woody Plant medium (Lloyd & McCown 1981)     

Expression of Anabaena ferredoxin genes in Escherichia coli

The genes for ferredoxin from heterocysts (fdx H) and vegetative cells (pet F) of Anabaena sp. strain 7120 were subcloned into plasmid pUC 18/19. Both genes were expressed in Escherichia coli at high levels (10% of total protein). Pet F could be expressed from its own promoter. The ferredoxins were correctly assembled to the holoprotein. Heterocyst ferredoxin was purified from E. coli extracts on a large scale. Its biochemical and biophysical properties were identical to those of the authentic ferredoxin, isolated from Anabaena heterocysts.This paper is dedicated to Prof. A. Trebst on the occasion of his 60th birthday.     

Electrophoretic and immunological comparisons of chloroplast and prokaryotic ribosomal proteins reveal that certain families of large subunit proteins are evolutionarily conserved

Summary Antibodies to individual chloroplast ribosomal (r-)proteins ofChlamydomonas reinhardtii synthesized in either the chloroplast or the cytoplasm were used to examine the relatedness ofChlamydomonas r-proteins to r-proteins from the spinach (Spinacia oleracea) chloroplast,Escherichia coli, and the cyanobacteriumAnabaena 7120. In addition,³⁵S-labeled chloroplast r-proteins from large and small subunits ofC. reinhardtii were coelectrophoresed on 2-D gels with unlabeled r-proteins from similar subunits of spinach chloroplasts,E. coli, andAnabaena to compare their size and net charge. Comigrating protein pairs were not always immunologically related, whereas immunologically related r-protein pairs often did not comigrate but differed only slightly in charge and molecular weight. In constrast, when³⁵S-labeled chloroplast r-proteins from large and small subunits of a closely related speciesC. smithii were coelectrophoresed with unlabeledC. reinhardtii chloroplast r-proteins, only one pair of proteins from each subunit showed a net displacement in mobility.Analysis of immunoblots of one-dimensional SDS and two-dimensional urea/SDS gels of large and small subunit r-proteins from these species revealed more antigenic conservation among the four species of large subunit r-proteins than small subunit r-proteins.Anabaena r-proteins showed the greatest immunological similarity toC. reinhardtii chloroplast r-proteins. In general, antisera made against chloroplast-synthesized r-proteins inC. reinhardtii showed much higher levels of cross-reactivity with r-proteins fromAnabaena, spinach, andE. coli than did antisera to cytoplasmically synthesized r-proteins. All spinach r-proteins that cross-reacted with antisera to chloroplast-synthesized r-proteins ofC. reinhardtii are known to be made in the chloroplast (Dorne et al. 1984b). FourE. coli r-proteins encoded by the S10 operon (L2, S3, L16, and L23) were found to be conserved immunologically among the four species. Two of the large subunit r-proteins, L2 and L16, are essential for peptidyltransferase activity. The third (L23) and two otherE. coli large subunit r-proteins (L5 and L27) that have immunological equivalents among the four species are functionally related to but not essential for peptidyltransferase activity.     

Nitrobacter winogradskyi cytochrome c oxidase genes are organized in a repeated gene cluster

Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation. The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library. Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster. Remarkably the cluster appears to be present in at least two copies per genome. It extends over a 5–6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene. Noteworthy is the new kind of gene order identified within the cluster. Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other -subdivision Proteobacteria, particularly the Rhizobiaceae. This confirms the phylogenetic relationships established only upon 16S rRNA data. Furthermore, interesting similarities exist between N. winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published -subdivision proteobacterial sequences.Abbreviations COI cytochrome oxidase subunit I - COII cytochrome oxidase subunit II - COIII cytochrome oxidase subunit III - HOS Heme O synthase - ORF open reading frame - SDS sodium dodecyl sulfate