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PubMed1983

Plasmid transformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties.

Biphasic, chimeric plasmids for the transformation of Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain 7002) were constructed by splicing the 3.0-megadalton cryptic plasmid from strain PR-6 into plasmids pBR322 and pBR325 from Escherichia coli. Transformants of either E. coli or strain PR-6 by these plasmids could be detected on the basis of the drug resistance marker(s) carried by the chimeric plasmids. Plasmid DNA isolated from a PR-6 transformant transformed PR-6 much more efficiently than plasmid DNA prepared from E. coli. Plasmids from which the AvaI recognition site was deleted (AvaI is an isoschizomer of the AquI restriction endonuclease of strain PR-6) also transformed strain PR-6 much more efficiently than did plasmids containing the AvaI recognition site. These and other results suggest that AquI strongly effects plasmid transformation when the donor plasmid contains an unmodified AquI recognition site. Multimeric forms of the chimeric plasmids are also much more efficient at transforming strain PR-6 than are the analogous monomeric forms.

Chimera↗
PubMed1986

Heterospecific transformation among cyanobacteria.

Heterospecific transformation occurred between cyanobacteria currently classified in either the genus Synechococcus or Synechocystis. Cyanobacterial strains 73109 and 6906 were capable of physiological transformation.

Cyanobacteria↗
PubMed1987

Functional expression of plastid allophycocyanin genes in a cyanobacterium.

In Cyanophora paradoxa, the allophycocyanin apoprotein subunits, alpha and beta, are encoded in the cyanelle (plastid) genome. These genes were transferred to the cyanobacterium Synechococcus sp. PCC 7002 on a plasmid replicon. Phycobilisomes isolated from transformed cyanobacteria were found to contain C. paradoxa allophycocyanin subunits. Thus, these plastid genes are expressed in the cyanobacterium as polypeptides which become linked to a chromophore and are incorporated into the light-harvesting apparatus.

Cloning, Molecular↗
PubMed1988

Isolation, sequence analysis, and transcriptional studies of the flavodoxin gene from Anacystis nidulans R2.

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.

Amino Acid Sequence↗
PubMed1988

Restriction analysis and quantitative estimation of methylated bases of filamentous and unicellular cyanobacterial DNAs.

The DNAs of strains of three cyanobacterial genera (Anabaena, Plectonema, and Synechococcus) were found to be partially or fully resistant to many restriction endonucleases. This could be due to the absence of specific sequences or to modifications, rendering given sequences resistant to cleavage. The latter explanation is substantiated by the content of N6-methyladenine and 5-methylcytosine in these genomes, which is high in comparison with that in other bacterial genomes. dcm- and dam-like methylases are present in the three strains (based on the restriction patterns obtained with the appropriate isoschizomeric enzymes). Their contribution to the overall content of methyladenine and methylcytosine in the genomes was calculated. Partial methylation of GATC sequences was observed in Anabaena DNA. In addition, the GATC methylation patterns might not have been random in the three cyanobacterial DNA preparations, as revealed by the appearance of discrete fragments (possibly of plasmid origin) withstanding cleavage by DpnI (which requires the presence of methyladenine in the GATC sequence).

5-Methylcytosine↗
PubMed1990

Chromosomal transformation in the cyanobacterium Agmenellum quadruplicatum.

Chromosomal transformation of Agmenellum quadruplicatum PR-6 (= Synechococcus sp. strain 7002) was characterized for phenotypic expression, for exposure time to DNA, and for dependence on DNA concentration with regard to Rifr donor DNA. Exponentially growing cells of PR-6 were competent for chromosomal transformation. Competence decreased in cells in the stationary phase of growth or in cells deprived of a nitrogen source. Dark incubation of cells before exposure to donor DNA also decreased competence. Homologous Rifr and Strr DNA and heterologous Escherichia coli W3110 DNA were used in DNA-DNA competition studies, which clearly showed that DNA binding by PR-6 was nonspecific. DNA binding and uptake by PR-6 exhibited single-hit kinetics. Single-stranded DNA failed to transform competent cells of PR-6, and DNA eclipse was not observed, suggesting that double-stranded DNA was the substrate for the binding and uptake reactions during the transformation of PR-6. A significant improvement in transformation frequency was achieved by increasing the nitrate content of the culture medium and by lowering the temperature at which cells were exposed to donor DNA from 39 degrees C (the optimal temperature for growth) to 30 degrees C.

Chromosomes↗
PubMed1991

Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.

The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms.

Animals↗
PubMed1993

Anabaena sp. strain PCC 7120 bifA gene encoding a sequence-specific DNA-binding protein cloned by in vivo transcriptional interference selection.

VF1 is a DNA-binding protein from the cyanobacterium Anabaena sp. strain PCC 7120. VF1 was originally identified on the basis of its binding affinity to the upstream region of xisA, which encodes a heterocyst-specific site-specific recombinase. VF1 also binds to the glnA, rbcL, and nifH promoters in vitro, suggesting that VF1 interacts with genes expressed in both vegetative cells and heterocysts. The role of VF1 in regulating gene expression in PCC 7120 is unknown. As a step towards the goal of understanding the role of VF1 in regulating gene expression, we have cloned the bifA gene by using a genetic selection strategy. bifA encodes a protein, BifA, that has chromatographic and DNA-binding properties indistinguishable from those of VF1. The cloning strategy was based on a transcriptional interference assay in which a strong synthetic promoter, conII, interferes with the expression of an aadA gene, which provides resistance to spectinomycin and streptomycin (S. J. Elledge, P. Sugiono, L. Guarente, and R. W. Davis, Proc. Natl. Acad. Sci. USA 86:3689-3693, 1989). A selection plasmid, pAM994, which has the conII promoter negatively regulated by a VF1-binding site, was used to enrich for VF1-producing clones from an expression library containing PCC 7120 DNA fragments. Mobility shift assays were used to identify a 672-bp open reading frame that encoded VF1-like binding activity. The deduced BifA amino acid sequence shows 77% identity to NtcA, which is a global regulator involved in nitrogen control in Synechococcus sp. strain PCC 7942. Both BifA and NtcA belong to the cyclic AMP receptor protein (CRP) family of prokaryotic regulatory proteins. Genes similar to envM, hisB, and ORF60-5 were found near the bifA gene.

Amino Acid Sequence↗
PubMed1993

The glnA gene of the cyanobacterium Agmenellum quadruplicatum PR-6 is nonessential for ammonium assimilation.

The glnA gene of the cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002) was isolated by complementing an Escherichia coli strain auxotrophic for glutamine (YMC11) with a PR-6 cosmid library. PR-6 glnA is a single-copy gene that encodes a deduced amino acid sequence that is highly homologous to the deduced glnA amino acid sequences reported for other bacteria. No homology was found between the PR-6 glnA flanking sequences and the ntrB, ntrC, or glnB genes of other bacteria. Northern (RNA) and primer extension analyses of PR-6 RNA revealed one predominant and several minor glnA transcripts of about 1.5 to 1.7 kb. The steady-state amounts of these transcripts increased three- to fivefold when the cells were starved for nitrogen. However, we found that mutant PR-6 cells lacking glnA were still able to use nitrate or ammonium as a sole nitrogen source. Although no RNA homologous to an internal fragment of the glnA gene could be detected in the mutant cells, they retained about 60% of wild-type glutamine biosynthetic activity. The mutant cells were more sensitive than the wild-type cells to methionine sulfoximine, a transition state analog of glutamate, a result that might indicate the presence of an additional glutamine synthetase; however, cell extracts of wild-type PR-6 cells and those lacking glnA were both able to use carbamyl phosphate instead of ammonium as a nitrogen donor for the synthesis of glutamine, a result that indicates the use of carbamyl phosphate synthetase to assimilate ammonium and produce glutamine.

Amino Acid Sequence↗
PubMed1993

Eubacterial components similar to small nuclear ribonucleoproteins: identification of immunoprecipitable proteins and capped RNAs in a cyanobacterium and a gram-positive eubacterium.

Small nuclear ribonucleoprotein (snRNP) particles play an important role in the processing of pre-mRNA. snRNPs have been identified immunologically in a variety of cells, but none have ever been observed in prokaryotic systems. This report provides the first evidence for the presence of snRNP-like components in two types of prokaryotic cells: those of the cyanobacterium Synechococcus leopoliensis and those of the gram-positive eubacterium Bacillus subtilis. These components consist of snRNP-immunoreactive proteins and RNAs, including some with the snRNP-unique 5' m2,2,7G (m3G) cap. Immunoreactivity was determined by immunoprecipitation procedures, with either antinuclear-antibody-positive (RNP- and Sm-monospecific) patient sera or a m3G monoclonal antibody, with radiolabelled cell extracts that were preadsorbed with antinuclear-antibody-negative sera. S. leopoliensis immunoprecipitates showed the presence of high-molecular-mass proteins (14 to 70 kDa) and RNAs (138 to 243 nucleotides) that are analogous in size to proteins and RNAs found in human (HEp-2) cell immunoprecipitates but absent in Escherichia coli immunoprecipitates. Thin-layer chromatography of S. leopoliensis immunoprecipitates confirmed the presence of a capped nucleotide similar to a capped nucleotide in HEp-2 immunoprecipitates; no such nucleotide was observed in E. coli immunoprecipitates. Immunoreactive RNAs (117-170 nucleotides) were identified in a second eubacterium, B. subtilis, as well. This work suggests that snRNPs or their evolutionary predecessors predate the emergence of eukaryotic cells.

Antibodies, Antinuclear↗
PubMed1994

Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence.

Reproducible circadian rhythms of bioluminescence from individual colonies of cyanobacteria (Synechococcus sp. strain PCC 7942) has been observed. Phenotypic monitoring of colonies on agar plates will enable us to genetically analyze the molecular mechanism of the circadian clock of cyanobacteria by screening for clock mutants. By the introduction of a bacterial luciferase gene, we previously developed a transformed cyanobacterial strain (AMC149) that expresses luciferase as a bioluminescent reporter of the circadian clock. In liquid culture, AMC149 expresses a rhythm of bioluminescence that displays the same behavior as circadian rhythms in higher eukaryotes. Improvements in the technique for administering the reporter enzyme's substrate (decanal) and a highly sensitive photon-counting camera allow monitoring the bioluminescence of single colonies. Individual colonies on agar plates displayed a rhythmicity which is essentially the same as that previously reported for liquid cultures.

Biological Clocks↗
PubMed1995

Disruption of a gene encoding a novel thioredoxin-like protein alters the cyanobacterial photosynthetic apparatus.

A gene that may encode a novel protein disulfide oxidoreductase, designated txlA (thioredoxin-like), was isolated from the cyanobacterium Synechococcus sp. strain PCC7942. Interruption of txlA near the putative thioredoxin-like active site yielded cells that grew too poorly to be analyzed. In contrast, a disruption of txlA near the C terminus that left the thioredoxin-like domain intact yielded two different mutant phenotypes. One type, designated txlXb, exhibited a slightly reduced growth rate and an increased cellular content of apparently normal phycobilisomes. The cellular content of phycobilisomes also increased in in the other mutant strain, designated txlXg. However, txlXg also exhibited a proportionate increase in chlorophyll and other components of the photosynthetic apparatus and grew as fast as wild-type cells. Both the txlXb and txlXg phenotypes were stable. The differences between the two strains may result from a genetic polymorphism extant in the original cell population. Further investigation of txlA may provide new insights into mechanisms that regulate the structure and function of the cyanobacterial photosynthetic apparatus.

Amino Acid Sequence↗
PubMed1995

A novel nitrite reductase gene from the cyanobacterium Plectonema boryanum.

The gene (nirA) for nitrite reductase was cloned from the nonheterocystous, filamentous cyanobacterium Plectonema boryanum. The predicted protein consists of 654 amino acids and has a calculated molecular weight of 72,135. The deduced amino acid sequence from positions 1 to 511 is strongly similar to the entire sequence of the ferredoxin-dependent nitrite reductases from other phototrophs, while the remainder of the protein is unique to the Plectonema nitrite reductase. The C-terminal portion of the protein (amino acids 584 to 654) is 30 to 35% identical to [2Fe-2S] ferredoxins from higher plants and cyanobacteria, with all of the four Cys residues involved in binding of the [2Fe-2S] cluster in the ferredoxins being conserved. Immunoblotting analysis of the extracts of P. boryanum cells showed that the NirA polypeptide has an apparent molecular mass of 75 kDa. An insertional mutant of nirA lacked the 75-kDa polypeptide, had no nitrite reductase activity, and failed to grow on nitrate and nitrite, indicating that the novel nirA is the sole nitrite reductase gene in P. boryanum and that the NirA polypeptide with the ferredoxin-like domain is the apoprotein of the functional nitrite reductase. As in Synechococcus sp. strain PCC7942, nirA is the first gene of a large transcription unit (> 7 kb in size) and is repressed by ammonium and derepressed simply by deprivation of ammonium from the medium. The development of nitrite reductase activity was, however, found to require the presence of nitrate in the medium.

Amino Acid Sequence↗
PubMed1995

Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster.

A fragment of the Salmonella typhimurium ethanolamine utilization operon was cloned and characterized. The 6.3-kb nucleotide sequence encoded six complete open reading frames, termed cchA, cchB, eutE, eutJ, eutG, and eutH. In addition, the nucleotide sequences of two incomplete open reading frames, termed eutX and eutI, were also determined. Comparison of the deduced amino acid sequences and entries in the GenBank database indicated that eutI encodes a phosphate acetyltransferase-like enzyme. The deduced amino acid sequences of the EutE and EutG proteins revealed a significant degree of homology with the Escherichia coli alcohol dehydrogenase AdhE sequence. Mutations in eutE or eutG completely abolished the ability of mutants to utilize ethanolamine as a carbon source and reduced the ability to utilize ethanolamine as a nitrogen source. The product of eutE is most probably an acetaldehyde dehydrogenase catalyzing the conversion of acetaldehyde into acetyl coenzyme A. The product of the eutG gene, an uncommon iron-containing alcohol dehydrogenase, may protect the cell from unconverted acetaldehyde by converting it into an alcohol. The deduced amino acid sequence of cchA resembles that of carboxysome shell proteins from Thiobacillus neapolitanus and Synechococcus sp. as well as that of the PduA product from S. typhimurium. CchA and CchB proteins may be involved in the formation of an intracellular microcompartment responsible for the metabolism of ethanolamine. The hydrophobic protein encoded by the eutH gene possesses some characteristics of bacterial permeases and might therefore be involved in the transport of ethanolamine. Ethanolamine-utilization mutants were slightly attenuated in a mouse model of S. typhimurium infection, indicating that ethanolamine may be an important source of nitrogen and carbon for S. typhimurium in vivo.

Alcohol Dehydrogenase↗
PubMed1995

Bacterial luciferase as a reporter of circadian gene expression in cyanobacteria.

To allow continuous monitoring of the circadian clock in cyanobacteria, we previously created a reporter strain (AMC149) of Synechococcus sp. strain PCC 7942 in which the promoter of the psbAI gene was fused to Vibrio harveyi luciferase structural genes (luxAB) and integrated into the chromosome. Northern (RNA) hybridization and immunoblot analyses were performed to examine changes in abundance of the luxAB mRNA, the native psbAI mRNA, and the luciferase protein to determine whether bioluminescence is an accurate reporter of psbAI promoter activity in AMC149. Under constant light conditions, the mRNA abundances of both luxAB and psbAI oscillated with a period of approximately 24 h for at least 2 days. The expression of these two genes following the same pattern: both mRNAs peaked in the subjective morning, and their troughs occurred near the end of the subjective night. The amount of luciferase protein also oscillated with a period of approximately 24 h, and the protein rhythm is in phase with the bioluminescence rhythm. The rhythm of the luciferase mRNA phase-leads the rhythms of luciferase protein and in vivo bioluminescence by several hours. Comparable results were obtained with a short-period mutant of AMC149. Together, these results indicate that the bioluminescence rhythm in AMC149 is due primarily to circadian oscillation of psbAI promoter activity in this cyanobacterium.

Circadian Rhythm↗
PubMed1995

Genes encoded on a cyanobacterial plasmid are transcriptionally regulated by sulfur availability and CysR.

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.

Amino Acid Sequence↗
PubMed1996

Use of PCR to isolate genes encoding sigma54-dependent activators from diverse bacteria.

Degenerate PCR probes were used to amplify gene fragments encoding the catalytic domain of sigma54-dependent transcription activators. The procedure should be widely applicable, as it recovered both known and novel gene fragments: 5 from Rhizobium meliloti, 13 from Myxococcus xanthus, and 3 from Bacillus subtilis. No fragments were obtained from Synechococcus sp. strain PCC 7002 or Saccharomyces cerevisiae.

Amino Acid Sequence↗