Interferon γ is a STAT1-dependent direct inducer of BCL6 expression in imatinib-treated chronic myeloid leukemia cells.

B-cell CLL/lymphoma 6 (BCL6) exerts oncogenic effects in several human hematopoietic malignancies including chronic myeloid leukemia (CML), where BCL6 expression was shown to be essential for CML stem cell survival and self-renewal during imatinib mesylate (IM) treatment. As several lines of evidence suggest that interferon γ (IFNγ) production in CML patients might have a central role in the response to tyrosine kinase inhibitor (TKI) therapy, we analyzed if IFNγ modulates BCL6 expression in CML cells. Although separate IFNγ or IM treatment only slightly upregulated BCL6 expression, combined treatment induced remarkable BCL6 upregulation in CML lines and primary human CD34+ CML stem cells. We proved that during combined treatment, inhibition of constitutive signal transducer and activator of transcription (STAT) 5 activation by IM allowed the specific enhancement of the STAT1 dependent, direct upregulation of BCL6 by IFNγ in CML cells. By using colony-forming assay, we found that IFNγ enhanced the ex vivo colony or cluster-forming capacity of human CML stem cells in the absence or presence of IM, respectively. Furthermore, inhibition of the transcriptional repressor function of BCL6 in the presence of IM and IFNγ almost completely blocked the cluster formation of human CML stem cells. On the other hand, by using small interfering RNA knockdown of BCL6, we demonstrated that in an IM-treated CML line the antiapoptotic effect of IFNγ was independent of BCL6 upregulation. We found that IFNγ also upregulated several antiapoptotic members of the BCL2 and BIRC gene families in CML cells, including the long isoform of MCL1, which proved to be essential for the antiapoptotic effect of IFNγ in an IM-treated CML line. Our results suggest that combination of TKIs with BCL6 and MCL1 inhibitors may potentially lead to the complete eradication of CML stem cells.

Rapid electron transfer to photosystem I and unusual spectral features of cytochrome c(6) in Synechococcus sp. PCC 7002 in vivo.

Cytochrome c(6) donates electrons to photosystem I (PS I) in Synechococcus sp. PCC 7002. In this work, we provide evidence for rapid electron transfer (t(1/2) = 3 micros) from cytochrome c(6) to PS I in this cyanobacterium in vivo, indicating prefixation of the reduced donor protein to the photosystem. We have investigated the cytochrome c(6)-PS I interaction by laser flash-induced spectroscopy of intact and broken cells and by redox titrations of membrane and supernatant fractions. Redox studies revealed the expected membrane-bound cytochrome f, b(6), and b(559) species and two soluble cytochromes with alpha-band absorption peaks of 551 and 553 nm and midpoint potentials of -100 and 370 mV, respectively. The characteristics and the symmetrical alpha-band spectrum of the latter correspond to typical cyanobacterial cytochrome c(6) proteins. Rapid oxidation of cytochrome c(6) by PS I in vivo results in a unique, asymmetric oxidation spectrum, which differs significantly from the spectra obtained for cytochrome c(6) in solution. The basis for the unusual cytochrome c(6) spectrum and possible mechanisms of cytochrome c(6) fixation to PS I are discussed. The occurrence of rapid electron transfer to PS I in cyanobacteria suggests that this mechanism evolved before the endosymbiotic origin of chloroplasts. Its selective advantage may lie in protection against photo-oxidative damage as shown for Chlamydomonas.

Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b(6) in Synechococcus sp. PCC 7002.

The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b(6) subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b(6) at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc(1) complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc(1) complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc(1) complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure-function relationships in the cytochrome bf complex.

Analysis of the nucleus-encoded and chloroplast-targeted rieske protein by classic and site-directed mutagenesis of Chlamydomonas.

Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the protein; two others carry substitutions Y87D and W163R that result in low accumulation of the protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske protein of photosynthesis.

Reconstitution of the 2Fe-2S center and g = 1.89 electron paramagnetic resonance signal into overproduced Nostoc sp. PCC 7906 Rieske protein.

The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp. PCC 7906. A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske protein. Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies. The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonance signals at 15 K. Two methods of reconstitution yielded Rieske preparations in which 20-60% of the protein contained 2Fe-2S clusters as determined by EPR spin quantitation. The reconstituted Rieske protein was soluble and stable at 4 degrees C in buffers containing nonionic detergents and showed a redox midpoint potential of +321 mV at pH 7.0 as determined by optical circular dichroism (CD) spectroscopy. These data demonstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cluster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis.

Plasmid and chromosomal DNA recovery by electroextraction of cyanobacteria.

High voltage electroporation has been investigated as a method for rapid recovery of plasmid and chromosomal DNA from the cyanobacteria Nostoc PCC 7121, Synechococcus PCC 7002, and Anabaena PCC 7120. Pulses of 18 kV/cm and higher applied to concentrated Nostoc cells carrying a shuttle plasmid (pRL25) resulted in copious release of nucleic acids and phycobiliproteins into the suspending medium. Small portions of these supernatants, when electroporated with Escherichia coli, gave rise to hundreds of E. coli transformants which contained pRL25. Electroporation of Synechococcus carrying plasmid pAQE19 did not cause detectable release of macromolecules but did reveal a low-level, voltage independent 'leakage' of pAQE19 into the medium. Electroextraction of Nostoc or Anabaena followed by addition of E. coli and delivery of a second high-voltage pulse permitted direct, one-cuvette transfer of shuttle plasmids from these cyanobacteria into E. coli. Electroextraction of single cyanobacterial colonies, as shown for Nostoc, also released sufficient chromosomal DNA for amplification of specific sequences by the polymerase chain reaction.

Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121.

We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121. A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extracts. Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC. Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site. These data further suggested that cleavage occurs after the first G (5'-G/GNCC-3') in this site to generate a three base 5' overhang. Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I. Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 10(3) viable cells. Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants. Conjugal transfer of pRL25 from E. coli into Nostoc was also possible but at much lower efficiency than by electroporation. These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.

Characterization of two operons encoding the cytochrome b6-f complex of the cyanobacterium Nostoc PCC 7906. Highly conserved sequences but different gene organization than in chloroplasts.

We have isolated and determined the nucleotide and derived protein sequences for the four genes, petCA and BD, which encode the cytochrome b6-f, electron-transfer complex of the filamentous cyanobacterium, Nostoc PCC 7906. The primary structure and cotranscription of the petCA genes encoding the Rieske-FeS (nuclear encoded in plants) and apocytochrome f proteins has been described previously (Kallas, T., Spiller, S., and Malkin, R. (1988) Proc. Natl. Acad. Sci. U.S.A., in press). The petBD genes (645 and 480 protein-coding nucleotides, respectively) for the apocytochrome b6 (24.3 kDa) and subunit-IV (17.5 kDa) proteins comprise a second operon located at least 12 kilobases (kb) from petCA. The Nostoc petBD genes are not closely linked to the psbB gene (encoding the 51-kDa photosystem II polypeptide) and do not contain introns as do the closely related chloroplast genes. DNA probes specific for each of the Nostoc cytochrome-complex genes hybridized to single bands in genomic DNA blots at intensities expected for single copy genes. These data suggest that a single set of cytochrome b6-f proteins function in the different types of membranes found in Nostoc vegetative and heterocyst cells. RNA blot hybridizations identified an 1.8-kb mRNA common to cytochrome b6 and subunit IV, and an intensely hybridizing 0.8-kb mRNA specific to the subunit IV gene probe. The role of the latter RNA is not clear but it may represent a transcript from the opposite strand. The deduced Rieske, apocytochrome f, apocytochrome b6, and subunit IV proteins exhibit 59, 58-63, 84-85, and 79-83% sequence identity with the proteins from chloroplast cytochrome b6-f complexes. The Nostoc proteins show lower but still significant sequences identity with the corresponding proteins of the mitochondrial-type b-c1 complexes. The four probable heme-liganding His residues, and the approximate spacings between them, have been conserved in all of the available cytochrome b6 and b sequences from divergent sources. The Nostoc apocytochrome b6 and subunit IV proteins, as well as the Rieske, appear to be translated and thus inserted into the membrane as mature forms without cleavable presequences. Hydropathy analyses revealed five potential membrane spans in cytochrome b6 and three in the subunit IV protein, consistent with the profiles observed for the chloroplast proteins and the related cytochrome b proteins of cytochrome b-c1 complexes.

Primary structure of cotranscribed genes encoding the Rieske Fe-S and cytochrome f proteins of the cyanobacterium Nostoc PCC 7906.

The thylakoid membrane cytochrome b6-f complex (plastoquinol:oxidized-plastocyanin oxidoreductase, EC 1.10.99.1) catalyzes electron-transfer and proton-translocation reactions essential for oxygenic photosynthesis. We have isolated and determined the nucleotide sequences of the petC and petA genes encoding the Rieske Fe-S and cytochrome f polypeptides from the filamentous cyanobacterium Nostoc PCC 7906. These genes occur as single genomic copies, are tightly linked, and, as indicated by hybridization of gene-specific probes to Nostoc RNA, are cotranscribed as a 2.0-kilobase message. The Rieske Fe-S/cytochrome f gene pair thus represents an example of clustering and cotranscription in cyanobacteria of functionally related genes that, in photosynthetic eukaryotes, reside on separate nuclear and plastid genomes. These data are consistent with the progressive degeneration of the modern chloroplast genome from the ancestral, cyanobacterial-like genome of an endosymbiont. The Rieske Fe-S and the mature cytochrome f apoproteins are encoded by 537 and 867 nucleotides and have molecular masses of 19.2 and 31.2 kDa, respectively. They show 59% and 60% protein sequence identity, respectively, relative to spinach. Forty-four amino acids (4.7 kDa) resembling a prokaryotic signal sequence precede apocytochrome f. In contrast, the Rieske Fe-S protein appears to be translated without a presequence. The 183 bases separating the Rieske Fe-S and preapocytochrome f genes contain two families of 7- to 9-base tandem repeats, and some part of this sequence is highly reiterated in the genome. The C terminus of the Rieske Fe-S protein contains cysteine and histidine residues (probable ligands for the Fe2S2 center) in two peptides, Cys-Thr-His-Leu-Gly-Cys-Val and Cys-Pro-Cys-His-Gly-Ser, which have been conserved in spinach and in the five available Rieske Fe-S sequences from the mitochondrial-type cytochrome b-c1 complexes. Cytochrome f shows the heme binding residues Cys-Xaa-Xaa-Cys-His near its N terminus. Single, long hydrophobic stretches occur near the N and C termini, respectively, of the Rieske Fe-S and cytochrome f proteins and may form membrane-spanning helices.

Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria.

Labeled probes carrying the Anabaena PCC 7120 nitrogenase (nifK and nifD) and nitrogenase reductase (nifH) genes were hybridized to Southern blots of DNA from diverse N2-fixing cyanobacteria in order to test a previous observation of different nif gene organization in nonheterocystous and heterocystous strains. The nif probes showed no significant hybridization to DNA from a unicellular cyanobacterium incapable of N2 fixation. All nonheterocystous cyanobacteria examined (unicellular and filamentous) had a contiguous nifKDH gene cluster whereas all of the heterocystous strains showed separation of nifK from contiguous nifDH genes. These findings suggest that nonheterocystous and heterocystous cyanobacteria have characteristic and fundamentally different nif gene arrangements. The noncontiguous nif gene pattern, as shown with two Het(-) mutants, is independent of phenotypic expression of heterocyst differentiation and aerobic N2-fixation. Thus nif arrangement could be a useful taxonomic marker to distinguish between phenotypically Het(-) heterocystous cyanobacteria and phylogenetically unrelated nonheterocystous strains.

The structural nif genes of the cyanobacteria Gloeothece sp. and Calothrix sp. share homology with those of Anabaena sp., but the Gloeothece genes have a different arrangement.

Probes carrying the Anabaena sp. strain PCC 7120 nitrogenase reductase (nifH) and nitrogenase (nifK and nifD) genes were hybridized to Southern blots of DNA from the unicellular, aerobic nitrogen-fixing cyanobacterium Gloeothece sp. strain PCC 6909 and from the filamentous cyanobacterium Calothrix sp. strain PCC 7601. These data suggest that the Gloeothece sp. nif structural proteins must be similar to those of other diazotrophs and that the ability for aerobic nitrogen fixation does not reside in the nif protein complex. We also found that the nif structural genes of Gloeothece sp. are clustered, whereas those of Calothrix sp. are arranged more like those of Anabaena sp.

Internal pH and ATP-ADP pools in the cyanobacterium Synechococcus sp. during exposure to growth-inhibiting low pH.

Y-7c-s Synechococcus thermophilic strain grew at its maximum rate at pH 8 and above. The growth rate of this strain was inhibited at pH 7.0 and below, and at pH 6.0 there was no sustained growth. At a suboptimal pH, high light intensity further depressed the growth rate. The inhibition of growth resulted neither from pheophytinization nor from a low chlorophyll content. At pH 5.0 a loss of viability preceded the appearance of pheophytin. Cells exposed to low, growth-inhibiting external pH levels continued to maintain a high internal pH (pH 7.1 to 7.3, as determined at moderate light intensities by 31P nuclear magnetic resonance spectroscopy). Even during exposure to pH 4.8, cells retained a relatively high internal pH. Thus, it appeared that the inhibition of growth at low pH was not caused by acidification of the cytoplasm. Darkened cells maintained a slightly lower internal pH than irradiated cells. The ATP/(ATP + ADP) ratio decreased from 0.80 to 0.82 at pH 8.0 to about 0.6 when growth was limited by exposure to pH 6.0 or by low light intensity. It is possible, but not likely, that a limitation of the energy supply may slow or stop growth when the external pH is lowered.

Rapid transient growth at low pH in the cyanobacterium Synechococcus sp.

The thermophilic cyanobacterium Synechococcus sp. strain Y-7c-s grows at its maximum rate at a high pH (pH 8 and above) the does not show sustained growth below pH 6.5. However, rapidly growing, exponential-phase cells from high-pH cultures continued to grow rapidly for several hours after transfer to pH 6.0 or 5.0. This transient growth represented increases in mass and protein, but cells failed to complete division. Viability loss commenced well before the cessation of growth, and cells at pH 5.0 showed no net DNA synthesis. When irradiated by visible light, cells at pH 6.0 and 5.0 maintained and internal pH of 6.9 to 7.1 (determined by 31P nuclear magnetic resonance spectroscopy) and an extremely high ATP/(ATP + ADP) ratio even after growth had ceased. Cells exposed to a low pH did not show an increase in the spontaneous mutation rate, as measured by mutation to streptomycin resistance. However, cells already resistant to streptomycin were more resistant to viability loss at a low pH than the parental type. Cultures that could grow transiently at a low pH had higher rates of viability loss than nongrowing cultures in light or darkness. The retention of a high internal pH by cells exposed to a low pH suggested that a low pH acted initially on the cell membrane, possibly on solute transport.

Phosphorus-31 nuclear magnetic resonance analysis of internal pH during photosynthesis in the cyanobacterium Synechococcus.

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectra were obtained from actively photosynthesizing and darkened suspensions of the unicellular cyanobacterium Synechococcus. These spectra show intracellular resonances belonging to inorganic phosphate (Pi), a sugar phosphate (sugar-P), nucleotide di- and triphosphates, and poly-phosphates. The pH-dependent chemical shifts of Pi and sugar-P allowed the estimation of intracellular pH. When irradiated with high-intensity tungsten-halogen light (100 x 10(4) ergs . cm-2 . s-1, measured in the visible range), concentrated cell suspensions in the NMR spectrometer incorporated NaH14CO3 at approximately two-thirds the rate shown by a dilute suspension of cells at saturating light intensity. On the basis of NaH14CO3 incorporation, the effective light intensity obtained under NMR conditions would support growth at approximately one-fourth the maximum rate in dilute suspensions of cells. Irradiated cells maintained a cytoplasmic pH of 7.1--7.3 when exposed to an external pH from 6.4 to 8.3. At an external pH of 6.7, a darkness to light shift caused a 0.4 pH unit alkalinization of the cytoplasm. Treatment of cell suspensions with the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in light or darkness, collapsed the internal pH to the level of the external pH. The results suggest a strong light- or energy-dependent buffering of the cytoplasm over a range of external pH. The study demonstrates that 31P NMR can be used to investigate intracellular events in an actively photosynthesizing microorganism.