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The oxidized recombinant flavodoxin from the cyanobacterium Anabaena 7120 has been crystallized in a trigonal form. The recombinant protein has an identical primary structure to that purified directly from Anabaena, which functions as a substitute for ferredoxin in an iron-deficient environment for electron transfer from photosystem I to ferredoxin-NADP(+) reductase. X-ray data to 1.40 A were collected on a Siemens area detector. Of the 311 379 reflections collected, 36069 reflections were unique in space group P3(1)21 (a = 55.36, c = 102.59 A) with an R(merge) of 3.8%. The structure was solved by molecular replacement using coordinates from the wild-type monoclinic structure previously solved in this laboratory [Rao, Shaffie, Yu, Satyshur, Stockman & Markley (1992). Protein Sci. 1, 1413-1427]. The structure was refined with X-PLOR and SHELXL93 to a crystallographic R-factor of 13.9% for 32963 reflections with I> 2sigma(I). The final structure contains 2767 atoms including 31 flavin mononucleotide (FMN) atoms, 299 water molecules, and one sulfate ion. The protein is comprised of a central five-stranded beta-sheet surrounded by five helices and binds a single molecule of FMN at the C-terminus of the sheet. The trigonal protein structure and the crystal packing are compared with the monoclinic wild-type protein. Helix alpha3 in this structure is less distorted than in the monoclinic structure and shows additional hydrogen bonds in the N-terminal portion of the helix. The trigonal structure is extensively hydrogen bonded in three major areas with neighboring molecules compared with five regions in the monoclinic structure, but using significantly fewer hydrogen bonds to stabilize the lattice. There are several hydrogen bonds to the amide groups from water molecules several of which stabilize and extend the ends of the beta-sheet.

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The nucleotide sequence of the 3-chlorobenzoate 3,4-dioxygenase genes, designated cbaAB, from the transposon Tn5271 was determined. The function of the two sequenced open reading frames was evaluated by mutagenesis and expression in vivo to show that the cbaA and cbaB genes code for dioxygenase and reductase proteins, respectively. Comparison of the deduced amino acid sequences of the cbaAB genes with sequences for other oxygenases revealed a clearly defined lineage among the class IA oxygenases that shows several unique features. This lineage includes phthalate 4,5-dioxygenase (pht23), and based on the available NH3-terminal sequence of component A, also includes 4-sulphobenzoate 3,4-dioxygenase. Vanillate demethylase, encoded by the vanAB genes and formally a monooxygenase enzyme catalysing an oxidative demethylation, is also included in this lineage. The terminal chlorobenzoate dioxygenase (CbaA) component is characterized by a conserved Rieske-type [2Fe-2S]R ligand centre. The reductase component (CbaB) contains a plant-type ferredoxin [2Fe-2S]Fd, FMN-isoalloxazine and NAD-ribose-binding domains and the orientation of these domains is conserved in all known class IA reductases. These results support the hypothesis that alternative fusions of the electron transfer modules of the reductases arose early in the divergence of oxygenase systems. The over-riding evolutionary constraint acting on the divergence of the class IA oxygenases would appear to be the requirement for a carboxyl group para to the site of oxygen insertion into the aromatic ring.

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In some cyanobacteria and eukaryotic algae, cytochrome c-553 (c-552) and plastocyanin function as alternative electron carriers between the cytochrome b6-f complex and Photosystem I. In these organisms plastocyanin is the electron carrier under copper-replete conditions, and cytochrome c-553 is the electron carrier during copper deprivation. In this paper we report the cloning, sequencing and transcriptional analysis of the genes for cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120. The gene for cytochrome c-553 encodes a preprotein containing 111 amino acids with a predicted N-terminal transit peptide sequence of 25 amino acids. The gene for plastocyanin encodes a preprotein containing 139 amino acids with a N-terminal transit peptide sequence of 34 amino acids. RNA transcript analyses indicate that the expression of the genes for cytochrome c-553 (petJ) and plastocyanin (petE) are regulated in reciprocal ways in response to copper concentration. In copper-replete conditions, petJ is expressed at very low levels, but is transcribed at high levels under copper deprivation; petE is down-regulated in the absence of copper, but is rapidly up-regulated when copper is added back to the medium.

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Under conditions of iron deprivation cyanobacteria produce flavodoxin to replace ferredoxin as the terminal electron acceptor of photosynthesis. In unicellular cyanobacteria, the gene for flavodoxin is the second open reading frame in a dicistronic operon whose transcription is tightly regulated by iron. The first gene, isiA, produces a protein that is very similar to CP43, a chlorophyll-binding, antenna protein of the photosystem II reaction center. In the filamentous, heterocystous cyanobacterium Anabaena sp. PCC 7120, isiA and the gene for flavodoxin are located in separate operons with independent promoters. In this paper, we report on the sequence of isiA and show that it is found in a monocistronic operon that is transcriptionally regulated to be expressed under iron stress but does not produce detectable transcripts under conditions of iron repletion. We also report on the sequence, organization and expression of the gene that codes for CP43, psbC. In Anabaena sp. PCC 7120, psbC has a genetic organization similar to that of other cyanobacteria and higher plants; the 5' end of psbC overlaps the 3' end of psbDI. Transcriptional analysis of the psbDC operon showed that it is constitutively expressed in both iron-repleted and iron-stressed conditions; however, a new monocistronic transcript was detected that contains psbC and is preferentially expressed under iron stress conditions.

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The internal transcribed spacer region (ITS1 and ITS2) of the 18S-25S nuclear ribosomal DNA sequence and the intervening 5.8S region were sequenced from three individuals in each of eight taxa of the Mimulus guttatus species complex. Three discrete variants, or "types," of ITS sequences were found, among which 30%-40% of sites differed, compared with 1%-2% within types. Dot plots indicate that these types were not related by conspicuous rearrangements or inversions. More than one ITS type was often found in the same taxon, and two of three ITS types span species boundaries, indicating their presence prior to speciation. These ITS sequences showed essentially no positional homology with the nearest sequenced relative, tomato. In contrast, the 5.8S region was relatively unvaried, with 8 of 162 sites varied in the sample among all eight taxa. The phylogeny inferred by the most common ITS sequence type, rooted by the two other ITS types, agreed with isozymes in showing the distinctness of M. nudatus, M. laciniatus, and M. tilingii from the other five taxa.

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Ribosomal DNA sequences for the ITS 1, 5.8S, ITS 2 and adjoining regions of the 18S and 25S were obtained from Mimulus glaucescens (Scrophulariaceae) via cloned PCR products. The spacer sequences were completely unrelated to other plant taxa, although spacer lengths were approximately the same. Interestingly, the Mimulus 5.8S sequence was much more divergent than other higher-plant rDNA sequences. Consideration of the secondary structure of the 5.8S rRNA shows that most of the changes in Mimulus are compensatory and preserve the basic secondary structure of the mature RNA molecule.

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The iron-stress-induced genes isiA and isiB have been cloned and sequenced from the marine unicellular cyanobacterium Synechococcus sp. PCC 7002. These genes code for a photosystem II chlorophyll-binding protein and flavodoxin respectively. The genes form a dicistronic operon that is transcriptionally activated under iron-stress conditions to produce an abundant monocistronic message containing isiA and a much less abundant dicistronic message that also contains isiB. The arrangement of these genes, their transcriptional control and the relative abundance of the monocistronic and dicistronic messages produced under iron stress parallels the pattern shown by the freshwater cyanobacterium Synechococcus sp. PCC 7942. The genes for the corresponding proteins found under iron-replete conditions, CP-43 and ferredoxin, have also been cloned and sequenced. Northern blot analysis indicates that both of these genes are constitutively expressed under both iron-stress and iron-replete conditions.

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A method has been developed for the preparation of large single-stranded DNA sequencing templates from primary cloning plasmids or cosmids. The method utilizes the separate action of T7 Gene 6 exonuclease and exonuclease III to generate large quantities of single-stranded template for each strand of a large-cloned fragment. Since the procedure is intended for use on primary clones, it avoids the time-consuming subcloning steps associated with most sequencing programs. The procedure also has the advantage of avoiding clone instability problems associated with subcloning in M13.

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Sequence-specific 1H and 13C NMR assignments have been made for residues that form the five-stranded parallel beta-sheet and the flavin mononucleotide (FMN) binding site of oxidized Anabaena 7120 flavodoxin. Interstrand nuclear Overhauser enhancements (NOEs) indicate that the beta-sheet arrangement is similar to that observed in the crystal structure of the 70% homologous long-chain flavodoxin from Anacystis nidulans [Smith et al. (1983) J. Mol. Biol. 165, 737-755]. A total of 62 NOEs were identified: 8 between protons of bound FMN, 29 between protons of the protein in the flavin binding site, and 25 between protons of bound FMN and protons of the protein. These constraints were used to determine the localized solution structure of the FMN binding site. The electronic environment and conformation of the protein-bound flavin isoalloxazine ring were investigated by determining 13C chemical shifts, one-bond 13C-13C and 15N-1H coupling constants, and three-bond 13C-1H coupling constants. The carbonyl edge of the flavin ring was found to be slightly polarized. The xylene ring was found to be nonplanar. Tyrosine 94, located adjacent to the flavin isoalloxazine ring, was shown to have a hindered aromatic ring flip rate.

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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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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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Cyanelles are photosynthetic organelles which are considered as intermediates between cyanobacteria and chloroplasts, and which have been found in unicellular eukaryotes such as Cyanophora paradoxa. The nucleotide sequence of a 667-bp region of the cyanelle genome from Cyanophora paradoxa containing genes coding for tRNA(UUCGlu) and tRNA(UAALeu) has been determined. The gene coding for tRNA(UAALeu) is split by a 232-bp intron which has a secondary structure typical for class-I structured introns and which is closely related to the intron located in the corresponding gene from liverwort and higher plant chloroplasts. It appears therefore that these tRNA(UAALeu) genes are all derived from one common ancestral gene which already contained a class-I intron.

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A mixed community of bacteria from surface runoff waters of the Hyde Park industrial landfill was enriched on 3-chlorobenzoate. Alcaligenes and Pseudomonas species were dominant in the community. Alcaligenes sp. BR60 carried an unstable plasmid specifying 3-chlorobenzoate catabolism. Metabolites detected in culture supernatants included chlorocatechol and chloro-cis, cismuconic acid. Oxygen uptake in the presence of 3- and 4-substituted methyl-catechols revealed a catechol-1,2-oxygenase activity specific for substituted catechols with very limited activity for catechol. The isolate grew very slowly on benzoate. Alcaligenes sp. BR60 was isolated in co-culture with Pseudomonas fluorescens NR52. The latter contained no detectable plasmids and did not grow on benzoate or any of the chlorobenzoates in pure culture. Growth of the co-culture in Bloody Run Creek water supplemented with 3-chlorobenzoate indicated that phosphate concentrations in the water severely limited biodegradation. Under phosphate limited conditions in continuous culture, Pseudomonas fluorescens NR52 effectively scavenged available phosphate when it was present at a ratio of 1 cell to 20 of Alcaligenes sp. BR60. Under these conditions the growth of Alcaligenes sp. BR60 on 3-chlorobenzoate was reduced 5 fold, the frequency of plasmid deletion mutants increased, and 96% of the contaminant remained in the outflow in the form of the starting material or metabolites. No evidence was found for conjugation of the plasmid determining chlorobenzoate catabolism in Alcaligenes sp. BR60 to P. fluorescens NR52.

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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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An Alcaligenes sp. BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation. The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation. The deletion mutant BR 40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10(-10) cell-1 generation-1. Transformation or conjugation of pBR60 into cured strains restored catabolic activity. An EcoRI, BglII, HindIII and SalI restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18. Conjugation of resistance plasmid R68.45 into Alcaligenes sp. BR 60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45. In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains. Hybridization of deletion region fragments to BglII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants. Hybridization also revealed a repeated sequence flanking the deletion region of pBR60. Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba+ mutants of Alcaligenes sp. BR60.

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The nucleotide sequence for the psbA gene from a triazine resistant cultivar of B. napus (cv 'Triton') has been determined. This gene encodes an open reading frame of 353 amino acids that is highly homologous to other higher plant psbA genes at both the nucleotide and amino acid levels. As has been found for other triazine resistant psbA genes, the 'Triton' psbA contains an A to G nucleotide change which results in a serine to glycine amino acid substitution at position 264. The B. napus psbA gene also has a G insertion at position -9 resulting in a ribosome binding site sequence (AGGA) just before the initial methionine and suggesting that the entire open reading frame is translated. A large (72 bp) insertion is also found upstream of the B. napus psbA gene which resembles a similar insertion in the mustard psbA. The "uncloneable" nature of the entire gene is further investigated through reconstruction experiments and the implications discussed.

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The Vicia faba chloroplast genome lacks inverted repeat sequences and contains only one set of ribosomal RNA genes. The genetic organization has been altered by inversions, relative to the typical arrangement of most higher plant chloroplast genomes. The Vicia faba plastid genome thus represents one of the more interesting results of chloroplast genomic evolution. The present study employs small DNA probes and Southern blot hybridizations to investigate the steps involved in the evolution of the Vicia faba chloroplast genome. The data from heterologous hybridizations between chloroplast DNA of Brassica napus (a conserved genome) and of Vicia faba led to three observations: 1) The inverted repeat segment closest to the psbA gene was deleted prior to the rearrangements. 2) A quarter of the ancestral small single copy region was lost during the deletion. 3) The genetic organization observed in Vicia faba resulted from three inversions after the deletion event. Our findings, combined with previous observations, helped devise a stepwise model for the evolution of the Vicia faba chloroplast genome. The area of the small single copy region absent from the Vicia faba chloroplast chromosome lacks in vivo transcription activity in Brassica napus.