Sucrose synthase and RuBisCo expression is similarly regulated by the nitrogen source in the nitrogen-fixing cyanobacterium Anabaena sp.

In higher plants and cyanobacteria, sucrose (Suc) metabolism is carried out by a similar set of enzymes. The function and regulation of Suc metabolism in cyanobacteria has begun to be elucidated. In strains of Anabaena sp., filamentous nitrogen-fixing cyanobacteria, Suc synthase (SuS, EC 2.4.1.13) controls Suc cell level through the cleavage of the disaccharide. The present work shows that there are two sus genes in Anabaena (Nostoc) sp. that are co-regulated regarding the nitrogen source; however, only susA accounts for the extractable SuS activity and for the control of the Suc level. Primer extension analysis has uncovered the sequence of the Anabaena susA and susB ammonium-activated putative promoters, which share a high sequence similarity with that of rbcLS encoding ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and other ammonium up-regulated genes. Moreover, susA and rbcLS expression is developmentally co-localized to the vegetative cells of the nitrogen-fixing cyanobacterial filaments. Our results strongly suggest the existence of a regulatory network that would coordinate the expression of key genes for Suc and nitrogen metabolism, carbon fixation, and development in Anabaena sp.     

Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a newin vivo amino acid biosynthesis pathway and improved wool growth

It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. As mammals can only synthesis cysteine indirectly from methionine via trans-sulphuration, expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the sheep. If expressed in the rumen epithelium, the abundant sulphide, produced by ruminal microorganisms and normally excreted, could be captured for conversion to cysteine. This paper describes the characterisation of expression of the cysteine biosynthesis genes ofSalmonella typhimurium, cysE,cysM andcysK, and linkedcysEM,cysME andcysKE genes as transgenes in mice and sheep. The linked transgenes were constructed with each gene driven by a separate promoter, either with the Rous sarcoma virus long terminal repeat (RSVLTR) promoter or the mouse phosphoglycerate kinase-1 (mPgk-1) promoter, and with human growth hormone (hGH) polyadenylation sequences. Transgenesis of mice with the RSVLTR-cysE gene afforded tissue-specific, heritable expression of the gene. Despite high levels of expression in a number of tissues, extremely low levels of expression occurred in the stomach and small intestine. Results of a concurrent sheep transgenesis experiment using the RSVLTR-cysEM and-cysME linked transgenes revealed that the RSVLTR promoter was inadequate for expression in the rumen. Moreover, instability of transgenes containing the RSVLTR sequence was observed. Expression of mPgk-cysME and-cysKE linked transgenes in most tissues of the mice examined, including the stomach and small intestine, suggested this promoter to be a better candidate for expression of these transgenes in the analogous tissues of sheep. However, a subsequent sheep transgenesis experiment indicated that use of the mPgk-1 promoter, active ubiquitously and early in development, may be inappropriate for expression of the cysteine biosynthesis transgenes. In summary, these results indicate that enzymically active bacterial cysteine biosynthesis gene products can be coexpressed in mammalian cellsin vivo but that expression of the genes should be spatio-temporally restricted to the adult sheep rumen epithelium.     

Genetic variation at theapcAB,cpcAB,gvpA1, andnifH loci and in DNA methylation among N2-fixing cyanobacteria designatedNostoc punctiforme

Genetic similarity among cyanobacteria of a morphological subgroup ofNostoc was evaluated through a comparison of several specific genes and the extent of DNA methylation. Four of six cyanobacteria were originally cultured from facultative symbioses with higher plants (Gunnera andEncephalartos); these and one free-living isolate had been identified or reputed to beN. punctiforme. No consistent correlation to species or symbiotic history was found from DNA hybridizations to genes coding for phycocyanin (cpcAB), allophycocyanin (apcAB), gas vesicle protein (gvpA1), and dinitrogenase reductase (nifH). One gene (gvpC) was not present, andgvpA1 was a single-copy gene in all strains. The gas vesicle genes were concluded to be potentially useful for broadly characterizingNostoc or at least this subgroup. Incubations ofNostoc genomic DNA with 22 restriction endonucleases indicated a high degree of methylation and similarity of its methylated DNA to that of other heterocystous cyanobacteria. The genetic variation of theNostoc isolates was judged to reflect primarily different soil origins.     

Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli

Summary In bacteria 5-aminolevulinate, the universal precursor in the biosynthesis of the porphyrin nucleus of hemes, chlorophylls and bilins is synthesised by two different pathways: in non-sulphur purple bacteria (Rhodobacter) or Rhizobium 5-aminolevulinate synthase condenses glycine and succinyl-CoA into 5-aminolevulinate as is the case in mammalian cells and yeast. In cyanobacteria, green and purple sulphur bacteria, as in chloroplasts of higher plants and algae a three step pathway converts glutamate into 5-aminolevulinate. The last step is the conversion of glutamate 1-semialdehyde into 5-aminolevulinate. Using a cDNA clone encoding glutamate 1-semialdehyde aminotransferase from barley, genes for this enzyme were cloned from Synechococcus PCC6301 and Escherichia coli and sequenced. The popC gene of E. coli, previously considered to encode 5-aminolevulinate synthase, appears to be a structural gene for glutamate 1-semialdehyde aminotransferase. Domains with identical amino acid sequences comprise 48% of the primary structure of the barley, cyanobacterial and putative E. coli glutamate 1-semialdehyde aminotransferases. The cyanobacterial and barley enzymes share 72% identical residues. The peptide containing a likely pyridoxamine phosphate binding lysine is conserved in all three protein sequences.     

Overexpression of jasmonic acid carboxyl methyltransferase increases tuber yield and size in transgenic potato

Jasmonates control diverse plant developmental processes, such as seed germination, flower, fruit and seed development, senescence and tuberization in potato. To understand the role of methyl jasmonate (MeJA) in potato tuberization, the Arabidopsis JMT gene encoding jasmonic acid carboxyl methyltransferase was constitutively overexpressed in transgenic potato plants. Increases in tuber yield and size as well as in vitro tuberization frequency were observed in transgenic plants. These were correlated with JMT mRNA level––the higher expression level, the higher the tuber yield and size. The levels of jasmonic acid (JA), MeJA and tuberonic acid (TA) were also higher than those in control plants. Transgenic plants also exhibited higher expression of jasmonate-responsive genes such as those for allene oxide cyclase (AOC) and proteinase inhibitor II (PINII). These results indicate that JMT overexpression induces jasmonate biosynthesis genes and thus JA and TA pools in transgenic potatoes. This results in enhanced tuber yield and size in transgenic potato plants.     

Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium

In order to study DNA replication control elements in cyanobacteria we cloned and sequenced the dnaA gene from the marine cyanobacterium Prochlorococcus marinus. The dnaA gene is ubiquitous among bacteria and encodes the DNA replication initiation factor DnaA. The deduced amino acid sequence of the P. marinus DnaA protein shows highest similarity to the DnaA protein from the freshwater cyanobacterium Synechocystis sp. PCC6803. Using a solid-phase DNA binding assay we demonstrated that both cyanobacterial DnaA proteins specifically recognize chromosomal origins, oriC, of Escherichia coli and Bacillus subtilis in vitro. The genetic environment of dnaA is not conserved between the two cyanobacteria. Upstream of the P. marinusdnaA gene we identified a gene encoding a putative ATP-binding cassette (ABC) transport protein. The gor gene encoding glutathione reductase lies downstream of dnaA. Comparison of the genetic structure of dnaA regions from 15 representative bacteria shows that the pattern of genes flanking dnaA is not universally conserved among them. Received: 20 July 1997 / Accepted: 7 October 1997     

Genetics of microcystless mutants of polysphondylium pallidum

Mutants were selected that are incapable of differentiating microcysts, a resting stage formed in response to high osmotic conditions. In the selection procedure amebae that failed to encyst were removed by flotation in 46% Percoll. Genetic crosses among 15 mutant strains were made by means of the macrocyst sexual cycle. Eleven of the strains mapped to three loci. Mutations at two of these loci (cysA and cysB) produced no observable alteration in the aggregation-fruiting pathway, although one set of strains altered at the cysA locus carried defects at a second unlinked site which blocked aggregation. The single strain that defined the third locus (cysC) is aggregateless. These results confirm the conclusion that there are several genes whose function is essential to microcyst development and is exclusive to this pathway. It remains uncertain whether there are other genes whose action is crucial to both encystment and to aggregation/fruiting.     

Molecular cloning and nucleotide sequence of the psaA and psaB genes of the cyanobacterium Synechococcus sp. PCC 7002

The psaA and psaB genes, which encode the P700 chlorophyll a apoproteins of the Photosystem I complex, have been cloned from the unicellular, transformable cyanobacterium Synechococcus sp. PCC 7002. The nucleotide sequence of these genes and of their flanking sequences have been determined by the chain termination method. As found in the chloroplast genomes of higher plants, the psaA gene lies 5 to the psaB gene; however, the cyanobacterial genes are separated by a greater distance (173 vs. 25–26 bp). The psaA gene is predicted to encode a polypeptide of 739 amino acid residues (81.7 kDa), and the psaB gene is predicted to encode a polypeptide of 733 residues (81.4 kDa). The cyanobacterial psa gene products are 76% to 81% identical to their higher plant homologues; moreover, because of conservative amino acid replacements, the cyanobacterial sequences are more than 95% homologous to those determined for higher plants. These results provide the basis for a genetic analysis of Photosystem I, and are discussed in relationship to structural and functional aspects of the Photosystem I complexes of both cyanobacteria and higher plants.     

Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.Abbreviations AK aspartokinase - HSDH homoserine dehydrogenase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.     

GENETIC CHARACTERIZATION OF STRAINS OF CYANOBACTERIA USING PCR-RFLP OF THE cpcBA INTERGENIC SPACER AND FLANKING REGIONS1

Oligonucleotide primers, specific for conserved regions of the genes encoding the β- and α-phycocyanin subunits of phycobilisomes (cpcB and cpcA) of cyanobacteria, were used to amplify a DNA fragment containing the intervening intergenic spacer region (cpcBA-IGS) of 19 strains of three morphospecies of cyanobacteria. Six Australian strains were identified as Anabaena circinalis Rabenhorst, six strains were identified as Microcystis aeruginosa Kützing, and seven strains were identified as Nodularia spumigena Mertens. Restriction enzyme digestion of the amplification products from the strains revealed restriction fragment length polymorphism (RFLP) within all three morphospecies. Strains corresponding to M. aeruginosa were highly polymorphic: 11 of the 14 restriction enzymes used displayed RFLPs. The A. circinalis and N. spumigena strains were less variable: three of 14 enzymes and seven of 14 enzymes, respectively, showed RFLPs. The presence of genetic variation between strains within these three divergent morphospecies, which span two orders of cyanobacteria (Chroococcales Wettstein and Nostocales (Borzi) Geitler), show that the cpcBA- IGS fragment has broad application as a molecular marker for intrageneric studies of cyanobacteria systematics and genetics.     

Insights into eukaryotic Rubisco assembly — Crystal structures of RbcX chaperones from Arabidopsis thaliana

Background

Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6 billion years ago. During evolution most of the cyanobacterial genes were transferred from the chloroplast to the nuclear genome. The rbcX gene, encoding an assembly chaperone required for Rubisco biosynthesis in cyanobacteria, was duplicated. Here we demonstrate that homologous eukaryotic chaperones (AtRbcX1 and AtRbcX2) demonstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures.

Methods

Three-dimensional structures of AtRbcX1 and AtRbcX2 were resolved by the molecular replacement method. Equilibrium binding constants of the C-terminal RbcL peptide by AtRbcX proteins were determined by spectrofluorimetric titration. The binding mode of RbcX–RbcL was predicted using molecular dynamic simulation.

Results

We provide crystal structures of both chaperones from Arabidopsis thaliana providing the first structural insight into Rubisco assembly chaperones form higher plants. Despite the low sequence homology of eukaryotic and cyanobacterial Rubisco chaperones the eukaryotic counterparts exhibit surprisingly high similarity of the overall fold to previously determined prokaryotic structures. Modeling studies demonstrate that the overall mode of the binding of RbcL peptide is conserved among these proteins. As such, the evolution of RbcX chaperones is another example of maintaining conserved structural features despite significant drift in the primary amino acid sequence.

General significance

The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.     

Mutation and cloning of clustered Streptomyces genes essential for sulphate metabolism

Summary A range of mutants auxotrophic for cysteine (cys) and resistant to selenate (sel) were isolated from many Streptomyces strains but chiefly from S. coelicolor A3(2) and S. lividans 66. Two of the classes of sel/cys mutants probably contained simple biochemical lesions of sulphate permease (selC) and ATP sulphurylase (selA) activities, while a further two classes (selD and selE) were pleiotropic and possibly regulatory. Most classes of sel mutations were clustered around the cysD locus of S. coelicolor. Segments of chromosomal DNA cloned from S. coelicolor, S. cattleya and S. clavuligerus and able to complement various sel/cys mutations allowed the relative positions of these mutations and the cysC and cysD mutations of S. coelicolor to be determined. The sel/cys DNA can be used for two-way selection: Cys⁺Sel^sCys^-Sel^r.     

Application of ribulose-1,5-bisphosphate carboxylase/oxygenase genes as molecular markers for assessment of the diversity of autotrophic microbial communities inhabiting the upper sediment horizons of the saline and soda lakes of the Kulunda Steppe

The genes encoding the key metabolic reactions are often used as functional markers for phylogenetic analysis and microbial ecology studies. The composition and structure of the genes encoding ribulose-1,5-bisphosphate carboxylase (RuBisCO) of various photoautotrophic bacteria, representatives of the order Chromatiales, including collection strains and the strains isolated from saline and soda lakes, were studied in detail. The green-like form I RuBisCO was detected in the majority of the studied strains. In some strains, the genes encoding both form I and form II RuBisCO were present, which has not been previously known for the representatives of this group of bacteria. Moreover, RuBisCO genes were used as functional markers to investigate the autotrophic microbial community inhabiting the upper horizons of bottom sediments of two saline soda lakes and two hypersaline neutral lakes of the Kulunda Steppe. In general, the diversity of autotrophic bacteria in the studied sediment horizons was low. In soda lakes, haloalkaliphilic cyanobacteria and sulfuroxidizing bacteria (SOB) of the genus Halorhodospira were predominant. In saline lakes, halophilic chemoautotrophic SOB Halothiobacillus and Thioalkalivibrio were found, as well as photoautotrophic bacteria of the genus Ectothiorhodosinus and cyanobacteria. Many phylotypes remained unidentified, which indicates the presence of groups of microorganisms with an unknown type of metabolism.