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The sequence of a genomic clone encoding a 100-kDa stress protein of Plectonema boryanum (p-ClpB) was determined. The predicted polypeptide contains two putative ATPase regions located within two highly conserved domains (N1 and N2), a spacer region that likely forms a coiled-coil domain, and a highly conserved consensus CK2 phosphorylation domain. The coiled-coil region and the putative site of phosphorylation are not unique to p-ClpB; they are present in all ClpB sequences examined and are absent from the ClpB paralogs ClpA, ClpC, ClpX, and ClpY. Small quantities of a 4.5-kb p-clpB transcript and 110-kDa cytosolic p-ClpB protein were detected in cells grown under optimal conditions; however, increases in the quantities of the transcript and protein were observed in cells grown under excess light and low temperature conditions. Finally, we analyzed ClpA, ClpB, and ClpC sequences from 27 organisms in order to predict phylogenetic relationships among the homologs. We have used this information, along with an identity alignment, to redefine the Clp subfamilies.

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A eubacterial homolog of a kinesin light chain gene has been isolated and characterized from the cyanobacterium Plectonema boryanum. Although the eubacterial and eukaryotic kinesin light chains are highly similar in amino acid sequence, the eubacterial sequence differs in several distinguishing structural features, including the absence of a putative PEST domain and the presence of additional highly conserved imperfect tandem repeats. Two soluble kinesin light chain antigens have been identified from whole-cell lysates by immunoblot analysis. Attempts to identify a canonical kinesin heavy-chain gene or protein were unsuccessful, suggesting that a kinesin heavy chain may be absent or unnecessary for kinesin light-chain function in this eubacterium. Our findings establish that certain basal elements of eukaryotic cellular transport appear to be resident in eubacteria. We discuss the possibility that the eukaryotic kinesin light chain was acquired by lateral gene transfer.

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Fungal fimbriae are surface appendages that were first described on the haploid cells of the smut fungus, Microbotryum violaceum. They are long (1-20 microm), narrow (7 nm) flexuous structures that have been implicated in cellular functions such as mating and pathogenesis. Since the initial description, numerous fungi from all five phyla have been shown to produce fimbriae on their extracellular surfaces. The present study analyses the protein component of M.violaceum fimbriae. The N-terminus and three internal amino acid sequences were determined. All four show a strong similarity to sequences which are characteristic of the collagen gene family. Enzymatic digests and immunochemical analyses support this finding. Based on these results, it is suggested that the proteinaceous subunits of fimbriae should be termed fungal collagens. Previously, collagen has been found only among members of the kingdom Animalia where it is the principal component of the animal extracellular matrix and is the most abundant animal protein. The unexpected finding of collagen in the members of the Mycota suggests that it may have evolved from a common ancestor that existed before the divergence of fungi and animals. Further, native fungal fimbriae can function as a mammalian extracellular matrix component. They can act as a substratum which permits animal cells to adhere, spread, and proliferate in a manner similar to animal collagens. The implications of this finding to both phylogeny and pathology are discussed.

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We demonstrate that photosynthetic adjustment at the level of the light-harvesting complex associated with photosystem II (LCHII) in Dunaliella salina is a response to changes in the redox state of intersystem electron transport as estimated by photosystem II (PSII) excitation pressure. To elucidate the molecular basis of this phenomenon, LHCII apoprotein accumulation and cab mRNA abundance were examined. Growth regimes that induced low, but equivalent, excitation pressures (either 13[deg]C/20 [mu]mol m-2 s-1 or 30[deg]C/150 ([mu]mol m-2 s-1) resulted in increased LHCII apoprotein and cab mRNA accumulation relative to algal cultures grown under high excitation pressures (either 13[deg]C/150 [mu]mol m-2 s-1 or 30[deg]C/2500 [mu]mol m-2 s-1). Thermodynamic relaxation of high excitation pressures, accomplished by shifting cultures from a 13 to a 30[deg]C growth regime at constant irradiance for 12 h, resulted in a 6- and 8-fold increase in LHCII apoprotein and cab mRNA abundance, respectively. Similarly, photodynamic relaxation of high excitation pressure, accomplished by a shift from a light to a dark growth regime at constant temperature, resulted in a 2.4- to 4-fold increase in LHCII apoprotein and cab mRNA levels, respectively. We conclude that photosynthetic adjustment to temperature mimics adjustment to high irradiance through a common redox sensing/signaling mechanism. Both temperature and light modulate the redox state of the first, stable quinone electron acceptor of PSII, which reflects the redox poise of intersystem electron transport. Changes in redox poise signal the nucleus to regulate cab mRNA abundance, which, in turn, determines the accumulation of light-harvesting apoprotein. This redox mechanism may represent a general acclimation mechanism for photosynthetic adjustment to environmental stimuli.

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The cyanobacterium Synechococcus sp. PCC 7942 contains two endogenous, genetically cryptic plasmids of 8.0 and 48.5 kb, which have been designated pANS and pANL, respectively. Characterization of the 3.8 kb Ba6 BamHI fragment of pANL identified three open reading frames which were transcriptionally regulated by sulfur availability and the protein CysR. One of these genes, designated srpG, encodes a protein which exhibits 67% amino acid identity to the Escherichia coli enzyme O-acetyl-L-serine (thio)-lyase A. Overlapping the 3' end of srpG is a second gene, designated srpH, which encodes a protein with similarity to the amino-terminal region of serine acetyltransferase enzymes. DNA hybridization results indicate that there is second copy of srpG in Synechococcus sp. PCC 7942, which is consistent with previous isoenzyme studies on O-acetyl-L-serine (thiol)-lyase in cyanobacteria. The introduction of srpG and srpH into E. coli cysKcysM and cysE mutant strains, respectively, results in the complementation of the lesion in cysteine biosynthesis. Additionally, the E. coli cysK cysM strain containing srpG is able to utilize sulfate more efficiently than thiosulfate, indicating that SrpG is probably a type A O-acetyl-L-serine (thiol)-lyase. The possible function of these genes in the adaptation of cyanobacteria to sulfur stress is discussed.

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A cyanobacterial sulfur-regulated gene (cysR), which encodes a protein with similarity to the Crp family of prokaryotic regulatory proteins, has recently been isolated and characterized. Polyacrylamide gel electrophoresis of periplasmic protein extracts reveals that a cysR mutant fails to synthesize a 36-kDa polypeptide that is normally induced in wild-type cells that have been grown under sulfur-deficient conditions. The amino-terminal sequence of this protein was obtained, and a synthetic oligonucleotide was used to isolated a clone containing a 1.9-kb NruI-KpnI fragment from a Synechococcus sp. strain PCC 7942 genomic library. RNA blot analysis indicates that this fragment encodes a transcript that is detectable in wild-type but not cysR mutant cells that have been starved for sulfur. DNA blot analysis revealed that the 1.9-kb NruI-KpnI fragment is contained within the Ba4 BamHI fragment of the endogenous 50-kb plasmid pANL. RNA blot studies indicate that the accumulation of a large number of pANL transcripts is regulated by sulfur levels and CysR. DNA sequence analysis confirmed that the gene encoding the sulfur-regulated 36-kDa periplasmic protein is encoded on the Ba4 fragment of pANL. The sequence of the 36-kDa protein displays sequence similarity to the enzyme catalase, and two downstream proteins exhibit 25 and 62% identity to a subunit of a P-type ATPase complex involved in Mg2+ transport and a chromate resistance determinant, respectively. Surprisingly, a strain in which the putative chromate resistance gene was interrupted by a drug resistance marker exhibited increased resistance to chromate when grown in media containing low sulfate concentrations. The possible role of this protein in the acclimation of cyanobacteria to conditions of low sulfur availability is discussed.

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By using an oligonucleotide probe constructed from a conserved region of amino acids located in the carboxyl-terminal end of superoxide dismutase (SOD) proteins, four SOD genes were cloned from the cyanobacterium Plectonema boryanum UTEX 485. One of these genes, designated sodB, encoded an FeSOD enzyme, while the remaining three genes, designated sodA1, sodA2, and sodA3, encoded MnSOD enzymes. To investigate the expression of these four genes, total cellular RNA was isolated from P. boryanum UTEX 485 cells grown under various conditions and RNA gel blot analysis was carried out. Results indicated that sodB and sodA1 were constitutively expressed, although sodB expression was partially repressed in cells grown under conditions of iron stress. sodA2 transcripts, which were not detectable in control cells, accumulated to high levels in cells treated with methyl viologen or in cells grown under conditions of iron or nitrogen stress. However, under microaerobic conditions, iron and nitrogen stress failed to induce sodA2, indicating that multiple factors affect the regulation of sodA2. While discrete transcripts were not detected for sodA3, hybridization was observed under a number of conditions, including those which increased the accumulation of sodA2 transcripts. Additionally, there were high levels of the sodA3 transcript detected in a P. boryanum UTEX 485 mutant strain resistant to methyl viologen treatment.

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Under conditions of iron-stress, the Photosystem II associated chlorophyll a protein complex designated CP 43', which is encoded by the isiA gene, becomes the major pigment-protein complex in Synechococcus sp. PCC 7942. The isiB gene, which is located immediately downstream of isiA, encodes the protein flavodoxin, which can functionally replace ferredoxin under conditions of iron stress. We have constructed two cyanobacterial insertion mutants which are lacking (i) the CP 43' apoprotein (designated isiA (-)) and (ii) flavodoxin (designated isiB (-)). The function of CP 43' was studied by comparing the cell characteristics, PS II functional absorption cross-sections and Chl a fluorescence parameters from the wild-type, isiA (-) and isiB (-) strains grown under iron-stressed conditions. In all strains grown under iron deprivation, the cell number doubling time was maintained despite marked changes in pigment composition and other cell characteristics. This indicates that iron-starved cells remained viable and that their altered phenotype suggests an adequate acclimation to low iron even in absence of CP 43' and/or flavodoxin. Under both iron conditions, no differences were detected between the three strains in the functional absorption crossection of PS II determined from single turnover flash saturation curves of Chl a fluorescence. This demonstrates that CP 43' is not part of the functional light-harvesting antenna for PS II. In the wild-type and the isiB (-) strain grown under iron-deficient conditions, CP 43' was present in the thylakoid membrane as an uncoupled Chl-protein complex. This was indicated by (1) an increase of the yield of prompt Chl a fluorescence (Fo) and (2) the persistence after PS II trap closure of a fast fluorescence decay component showing a maximum at 685 nm.

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The cells of the fungus Microbotryum violaceum produce many long, fine surface hairs that are similar in size and morphology to bacterial pili or fimbriae. These fungal fimbriae are assembled from 74 kDa glycoprotein subunits. We now present evidence that these fimbriae also have a RNA component. Isopycnic centrifugation of fimbriae in caesium chloride produced one band at a density intermediate to that of protein and nucleic acid. The absorbance spectrum of the intact fimbriae was consistent with that of a nucleoprotein. After extraneous RNAs were enzymically removed from the purified fimbrial preparation, disruption of the fibrils resulted in the release of not only the 74 kDa glycoprotein subunits, but also a 30 base single-stranded RNA species. To our knowledge, this is the first example of extracellular RNA as a component of a surface appendage.

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The acclimation of the photosynthetic apparatus to growth irradiance in a mutant strain of Synechococcus sp. PCC 7942 lacking detectable iron superoxide dismutase activity was studied. The growth of the mutant was inhibited at concentrations of methyl viologen 4 orders of magnitude smaller than those required to inhibit the growth of the wild-type strain. An increased sensitivity of photosynthetic electron transport near photosystem I (PSI) toward photooxidative stress was also observed in the mutant strain. In the absence of methyl viologen, the mutant exhibited similar growth rates compared with those of the wild type, even at high growth irradiance (350 [mu]E m-2 s-1) where chronic inhibition of photosystem II (PSII) was observed in both strains. Under high growth irradiance, the ratios of PSII to PSI and of [alpha]-phycocyanin to chlorophyll a were less than one-third of the values for the wild type. In both strains, cellular contents of chlorophyll a, [alpha]-phycocyanin, and [beta]-carotene, as well as the length of the phycobilisome rods, declined with increasing growth irradiance. Only the cellular content of the carotenoid zeaxanthin seemed to be independent of growth irradiance. These results suggest an altered acclimation to growth irradiance in the sodB mutant in which the stoichiometry between PSI and PSII is adjusted to compensate for the loss of PSI efficiency occurring under high growth irradiance. Similar shortening of the phycobilisome rods in the sodB mutant and wild-type strain suggest that phycobilisome rod length is regulated independently of photosystem stoichiometry.

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The enzyme superoxide dismutase is ubiquitous in aerobic organisms where it plays a major role in alleviating oxygen-radical toxicity. An insertion mutation introduced into the iron superoxide dismutase locus (designated sodB) of the cyanobacterium Synechococcus sp. PCC 7942 created a mutant strain devoid of detectable iron superoxide dismutase activity. Both wild-type and mutant strains exhibited similar photosynthetic activity and viability when grown with 17 mumol.m-2.s-1 illumination in liquid culture supplemented with 3% carbon dioxide. In contrast, the sodB mutant exhibited significantly greater damage to its photosynthetic system than the wild-type strain when grown under increased oxygen tension or with methyl viologen. Although damage occurs at both photosystems I and II, it is primarily localized at photosystem I in the sodB mutant. Growth in 100% molecular oxygen for 24 hr decreased photoacoustically measured energy storage in 3-(3,4-dichlorophenyl)-1,1-dimethylurea and abolished the fluorescence state 2 to state 1 transition in the sodB mutant, indicating interruption of cyclic electron flow around photosystem I. Analysis of the flash-induced absorption transient at 705 nm indicated that the interruption of cyclic electron flow occurred in the return part of the cycle, between the two [4 Fe-4 S] centers of photosystem I, FA and FB, and cytochrome f. Even though the sodB mutant was more sensitive to damage by active oxygen than wild-type cells, both strains were equally sensitive to the photoinhibition of photosystem II caused by exposure to strong light.

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A sulfur-regulated gene (cysA) that encodes the membrane-associated ATP-binding protein of the sulfate transport system of the cyanobacterium Synechococcus sp. strain PCC 7942 was recently isolated and sequenced. Adjacent to cysA and transcribed in the opposite direction is a gene encoding the sulfate-binding protein (sbpA). Two other genes, cysT and cysW, encode proteins that may form a channel for the transport of sulfate across the cytoplasmic membrane. A fourth gene, cysR, located between cysT, and cysW, encodes a polypeptide that has some homology to a family of prokaryotic regulatory proteins. Mutant strains in which cysA, cysT, or cysW was interrupted by a drug resistance marker were not viable when grown with sulfate as the sole sulfur source and exhibited essentially no sulfate uptake. In contrast, sbpA and cysR mutants grew on sulfate, although they did not exhibit the 20-fold increase in the Vmax (concentration of sulfate at half-maximal transport rate) for sulfate transport characteristic of wild-type cells grown under sulfur-limiting conditions. Three of the sulfur-regulated genes in Synechococcus sp. strain PCC 7942 are similar to genes encoded by the chloroplast genome of the primitive plant Marchantia polymorpha. These data suggest that a sulfate transport system similar to that of Synechococcus sp. strain PCC 7942 may exist in the chloroplast envelope of photosynthetic eukaryotes.

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During sulfur-limited growth, the cyanobacterium Synechococcus sp. strain PCC 7942 loses most of its photosynthetic pigments and develops an increased capacity to acquire sulfate. Sulfur deprivation also triggers the synthesis of several soluble polypeptides. We have isolated a prominent polypeptide of 33 kDa that accumulates specifically under sulfur-limiting conditions. This polypeptide was localized to the periplasmic space. The gene for this protein (designated rhdA) was isolated and discovered to lie within a region of the Synechococcus sp. strain PCC 7942 genome that encodes components of the sulfate permease system. The mRNA for the 33-kDa protein accumulates to high levels within an hour after the cells are deprived of sulfur and drops rapidly when sulfur is added back to the cultures. The amino acid sequence of the protein has similarity to bovine liver rhodanese, an enzyme that transfers the thiol group of thiosulfate to a thiophilic acceptor molecule, and a rhodaneselike protein of Saccharopolyspora erythraea. A strain in which rhdA was interrupted by a drug resistance marker exhibited marginally lower levels of rhodanese activity but was still capable of efficiently utilizing a variety of inorganic sulfur sources. The possible role of this protein in the transport of specific sulfur compounds is discussed.

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In cyanobacteria, the water-soluble cytochrome c-553 functions as a mobile carrier of electrons between the membrane-bound cytochrome b6-f complex and P-700 reaction centers of Photosystem I. The structural gene for cytochrome c-553 (designated cytA) of the cyanobacterium Synechococcus sp. PCC 7942 was cloned, and the deduced amino acid sequence was shown to be similar to known cyanobacterial cytochrome c-553 proteins. A deletion mutant was constructed that had no detectable cytochrome c-553 based on spectral analyses and tetramethylbenzidine-hydrogen peroxide staining of proteins resolved by polyacrylamide gel electrophoresis. The mutant strain was not impaired in overall photosynthetic activity. However, this mutant exhibited a decreased efficiency of cytochrome f oxidation. These results indicate that cytochrome c-553 is not an absolute requirement for reducing Photosystem I reaction centers in Synechococcus sp. PCC 7942.

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The E. coli iron superoxide dismutase gene (sodB) was utilized as a heterologous probe to isolate a superoxide dismutase (sod) gene from Anacystis nidulans R2. Nucleotide sequence analysis revealed a 603 bp open reading frame with deduced amino acid sequence similar to other sod genes and to cyanobacterial superoxide dismutase amino-terminal sequences. Assuming proteolytic cleavage of the initial methionine residue, the molecular mass of the mature A. nidulans R2 sodB polypeptide is 22,000 daltons. Only a single copy of the superoxide dismutase sequence was detected in the A. nidulans R2 genome using Southern hybridization. Northern hybridization analysis indicated a single, monocistronic RNA transcript of approximately 720 bases. Primer extension mapping localized the transcription start site to 46 bases upstream from the initial methionine residue. A single orientation of a 2.1 kb PstI fragment containing the entire sod gene cloned into pUC18 was able to complement E. coli sodAsodB mutants. Complementation of the E. coli mutants was based on the ability of the cells to grow aerobically on minimal glucose medium. Growth curves of the complemented E. coli sodAsodB mutants showed that these cells exhibited levels of resistance to paraquat comparable to that of the wild-type E. coli phenotype.

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Using the cysA locus of Salmonella typhimurium as a heterologous probe, we have cloned a region of the Anacystis nidulans R2 (Synechococcus PCC 7942) genome involved in sulfate assimilation. The 8.3-kilobase-pair region encodes at least five transcripts that cannot be detected unless the cells are deprived of sulfur. One of the genes in this region has been sequenced, and the protein that it encodes is homologous to a polypeptide component of other permease systems of Escherichia coli and Salmonella. Insertional inactivation of the putative sulfate permease gene, designated cysA, as well as of other genes within this region, results in cysteine auxotrophy, reduced sulfate uptake, and altered expression of soluble and cytoplasmic-membrane polypeptides associated with sulfur starvation.

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Recently we have reported that the flavodoxin gene from the cyanobacterium Anacystis nidulans R2 is transcribed as part of an iron stress-induced operon containing multiple mRNA species (D. E. Laudenbach, M. E. Reith, and N. A. Straus, J. Bacteriol. 170: 258-265, 1988). Here we report that nucleotide sequence analyses of DNA located immediately upstream of the flavodoxin gene revealed an open reading frame of 1,026 bases (designated isiA; iron stress inducible) with a deduced amino acid sequence showing similarity to that of the psbC polypeptide of higher plants and cyanobacteria. Assuming proteolytic cleavage of the initial methionine residue, the open reading frame encodes a 341-amino-acid polypeptide with a molecular mass of 36,824 daltons. Amino acid sequence comparisons with known psbC polypeptides from spinach and A. nidulans R2 showed extensive similarity, especially in the proposed membrane-spanning regions. Mung bean nuclease mapping and primer extension experiments have localized a transcriptional start site to a position 19 bases upstream from the first methionine codon of the isiA gene product. The upstream region contains an Escherichia coli-like -10 sequence but lacks the typical -35 consensus sequence. Approximately 15, 25, and 150 bases upstream from the isiA transcription start site are 17 base sequences which resemble the operator sequences of iron-regulated genes of E. coli.

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The nonheme, iron-sulfur protein ferredoxin is the terminal constituent of the photosynthetic electron transport chain. Under conditions of iron stress, many cyanobacteria and eucaryotic algae replace ferredoxin with the flavoprotein flavodoxin. The gene for flavodoxin was cloned from the cyanobacterium Anacystis nidulans R2 by using three mixed oligonucleotide probes derived from the partial Synechococcus sp. strain PCC 6301 amino acid sequence. Nucleotide sequence analysis revealed a 513-base-pair open reading frame with a deduced amino acid sequence having homology to other long-chain flavodoxins. Assuming proteolytic cleavage of the initial methionine residue, the molecular weight of the A. nidulans R2 flavodoxin is 18,609. Southern blot hybridization under conditions of reduced stringency detected only one copy of the flavodoxin sequence in the A. nidulans R2 genome. Northern (RNA) blot hybridization analyses by using cloned flavodoxin gene probes indicated that no transcripts are detectable under conditions of iron saturation. However, under iron-deficient growth conditions the flavodoxin gene appeared to be transcribed as part of a larger operon. The operon yielded at least three transcripts. The first was of approximately 1,100 bases (designated RNA 1) and terminated immediately upstream from the 5' end of the flavodoxin open reading frame. A second, less abundant transcript of approximately 1,900 bases (designated RNA 2) encoded all of RNA 1 as well as the flavodoxin polypeptide. Analysis indicated that both transcripts initiate in close proximity to each other. A third, minor transcript of approximately 1,100 bases (designated RNA 3) was detectable downstream of the flavodoxin gene sequence. Addition of iron-stressed A. nidulans R2 cells resulted in almost total loss of detectable mRNA transcripts within 60 min of the addition. The ferredoxin gene transcript has previously been characterized as a monocistronic message of approximately 430 bases (M. E. Reith, D. E. Laudenbach, and N. A. Straus, J. Bacteriol. 168: 1319-1324, 1986). Here we show that the ferredoxin message is detectable under all iron regimes tested is quantitatively unaffected by decreases in iron availability to the cells.

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Two mixed oligonucleotide probes derived from conserved regions of the Synechocystis sp. strain PCC 6714 ferredoxin amino acid sequence were utilized to isolate an Anacystis nidulans R2 clone containing the ferredoxin I gene. Nucleotide sequence analysis revealed a 297-base-pair (bp) open reading frame with a deduced amino acid sequence having high homology to other cyanobacterial ferredoxins. Assuming proteolytic cleavage of the initial methionine residue, the molecular weight of the mature A. nidulans R2 ferredoxin was 10,370. The initial methionine residue was preceded by a probable ribosome-binding site sequence, AGGA. Northern hybridization analysis with the cloned ferredoxin gene indicated an RNA transcript of approximately 450 bp. S1 nuclease mapping localized the transcription start site to a position 64 bases upstream from the initial methionine residue. The nucleotide sequence 14 to 8 bp preceding the transcription start site resembled a typical Escherichia coli promoter, but the sequence in the -35 region did not. Southern hybridization detected only a single copy of the ferredoxin sequence in the A. nidulans R2 genome.

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