By their very nature, meeting reports are just snapshots of what occurred. A different angle would produce a very different view. The two reports below can only provide a flavor of some of the advances that have taken place.
The Vth Cyanobacterial Molecular Biology Workshop was held again at Asilomar, California, July 21-25. Asilomar has become the permanent home, as the next meeting in 1998 is also scheduled for the site on the Pacific Ocean. Many thanks to Don Bryant and Neil Straus for organizing the meeting.
Pradip Manna (Arizona State U.) reported the involvement of lumenal cytochromes on electron transport in a PSI-LESS MUTANT of Synechocystis PCC 6803. Of particular interest was the important but not essential role of cytochrome c553 in mediating the PSII-dependent flow of electrons to the terminal oxidase. Gaozhong Shen (Pennsylvania State U.) also reported PSII activity in a PSI-less mutant from Synechococcus PCC 7002. His results suggested that PSII-generated electrons can be transferred to NAD through NADH dehydrogenase and that this process seems to be regulated by light. On the PSI side, Shen also described his success in deleting psaC and psaD genes from Synechococcus PCC 7002. Both mutants showed hypersensitivity to light intensity and both mutants showed much lower PSI reaction center content. The construction of these mutants opens a new way to further analyze protein structure and function of PSI.
Jian-Ren Shen (RIKEN) presented evidence that CYTOCHROME C550 is a critical component of PSII located on the lumenal side of thylakoids in Synechocystis PCC 6803. When the gene encoding cytochrome C550 was deleted, a much lower growth rate as a result of slow down of PSII electron transport was observed. Dave Krogmann (Purdue U.) also reported cloning of the petK gene, encoding cytochrome C550. His results indicate that the cytochrome might be involved in H2 production.
Progress in understanding CYTB6/F FUNCTION was presented by Toivo Kallas (U. Wisconsin, Oshkosh). His group evaluated the function of specific amino acids in cytochrome b6 (encoded by petB) and subunit IV (encoded by petD) by introducing site-specific mutations into Synechococcus PCC 7002. Kallas also reported reconstitution of Rieske center from overproduced apoprotein. Functions of low potential cytochrome c were reported by two groups.
The regulation of CHLOROPHYLL-BINDING PROTEIN SYNTHESIS with chlL-mutant was described by Qingfang He (Arizona St. U.). When the chlL gene was deleted, chlorophyll synthesis became totally dependent on light. In darkness, protochlorophyllide accumulated. This mutant should provide a good system to study chlorophyll synthesis and its regulation.
Several presentations focused on CO2 FIXATION. Michael Zianni (Ohio State U.) described the disruption of the rca gene of Anabaena variabilis, encoding RubisCO activase. The resulting strain lacked activase and showed a marked change in RubisCO activity and growth under certain conditions. Dean Price and Dieter Sueltemeyer (both of Austral. Natl. U.) discussed different aspects of the CO2-concentrating mechanism encoded by ccm genes in Synechococcus PCC 7002. The genes have now been cloned. Curiously, a psaE- strain lacking cyclic electron flow around PSI was unable to induce high affinity transport system for bicarbonate under low CO2 conditions. The authors suggested that psaE-dependent cyclic electron flow may be important in energizing or inducing bicarbonate transport.
Carol Andersson (Texas A&M U.) presented the results of an international coalition of workers studying the molecular basis of CIRCADIAN RHYTHMS in Synechococcus PCC 7942. Synechococcus and other cyanobacteria remain the only prokaryotes with documented circadian rhythms. Using random transcriptional fusions to luxAB, encoding bacterial luciferase, and an automated image processing system, the group recorded the amplitude and periodicity expression of 6000 reporter fusions. Almost all 800 colonies that were bright enough to monitor showed rhythmic expression of luminescence, suggesting that circadian regulation is surprisingly common. A screen of integrational mutants for colonies defective in the rhythmic expression of psbAI yielded one with a low amplitude phenotype. The affected gene, which turned out to be a member of the sigma-70 family, affected some but not all circadian-regulated genes. Chemical mutagenesis yielded period length mutants that displayed a wider range of periodicity than previously observed in any other organism. Complementation analysis of chemically induced mutants indicates that 80% of these mutations lie in the same region of the chromosome, which may contain the gene(s) for the clock machinery.
Nick Mann (U. Warwick) presented the efforts of his group on the characterization of MEMBRANE ASSOCIATED KINASE activities in cyanobacteria. In Synechocystis PCC 6803, kinase activity is activated by dark or the presence of metabolizable carbon and appears to be related to the switch off of the carbon dioxide concentrating mechanism. The kinase has several targets, and characterization of an 18-kD target by protein sequencing indicated that it was beta-phycocyanin. The phosphorylated phycobiliprotein no longer fluoresces. The physiological role of phycobiliprotein phosphorylation is still obscure but may have to do with regulating energy flow through the photosystems. Characterization of other targets is currently underway.
Participants keenly felt the absence of David Laudenbach (U. Western Ontario), who died suddenly of complications from surgery just a month before the meeting. It was his presence, however, that was evident during the talk on SULFUR-CONTROLLED GENES given in his place by his student Mary Lou Nicholson. She described the isolation and localization of the regulatory gene cysR on a 50-Kb plasmid from Synechococcus PCC 7942. Several open reading frames (ORFs) were also found on this plasmid that are transcriptionally regulated by sulfate deprivation, mediated through CysR. These ORFs where characterized and identified as srpA, srpB, srpC (srp = sulfur regulated plasmid-encoded), and ggt, encoding, respectively, catalase, a Mg transport ATPase, a protein involved in chromate resistance, and gamma-glutamyl transferase (related to glutathione).
Ecology and Evolution
A couple of groups reported their results on responses by cyanobacteria to NUTRIENT DEPRIVATION. Jackie Collier and Brian Palenik (Scripps Inst. Oceanography) described the utilization of urea by a marine Synechococcus. They cloned and sequenced urease genes from several marine cyanobacteria and showed that its expression in Synechococcus WH7805 is not repressed by NO3- and NH4+. Neil Straus (U. Toronto) reported cloning and sequencing of a gene involved in iron repression of some genes. It was 41% similarity to regulatory protein Fur from E. coli and had a putative Fe-binding domain.
Jack Meek's group (U. California-Davis) found two transposon-generated mutants of Nostoc ATCC 29133 that have lost the ability to enter into SYMBIOSIS with the hornwort Anthoceros punctatus. Both mutants also are Fox-, i.e. unable to fix nitrogen in the presence of oxygen. It was previously thought that all Fox- mutants would be physiologically complemented by the anaerobic environment of the symbiotic cavity in the plant tissue and, therefore, be Sym+. One of the mutants, UCD307, is defective in heterocyst glycolipid production and the protein encoded by the interrupted ORF shows similarity to polyketide synthesis pathway enzymes and some similarity to HetO and HetQ. Most interesting is the high similarity to fix-23, a locus from Rhizobium meliloti involved in host-symbiont recognition. Perhaps the gene product of the ORF interrupted in UCD307 is involved in production of both heterocyst glycolipid and symbiotic recognition determinants. Both compounds may be synthesized by a common pathway.
Several interesting reports on the MOLECULAR EVOLUTION OF CYANOBACTERIA and chloroplasts appeared in this meeting, with gratifying agreement in their conclusions. Sean Turner (Louisiana St. U.) presented a statistical analysis of small subunit rRNA base composition and concluded that plastids are monophyletic. Nadia Dolganov (Stanford U.) told of the cloning of a gene from Synechococcus PCC 7942 whose product resembles chlorophyll a/b binding protein. This result supports the idea that there was only one original endosymbiosis event. Tanja Gruber (Pennsylvania St. U.) showed a phylogenetic tree based on the amino acid sequences of sigma factors. The tree agrees with Sean's 16S rRNA data. Vickie Stirewalt (Pennsylvania St. U.) reported that their long struggle of sequencing the whole cyanelle genome is finally over. The circular DNA is comprised of 135599 bp with a low G+C content (30.4%). It contains about 192 genes and ORFs and has two inverted repeats. The inverted repeats and gene organization of this genome also supports the idea that all plastids are monophyletic.
The situation is less clear with RBCL. Bob Tabita (Ohio St. U.) presented sequences from an oceanic strain Synechococcus WH7803. The deduced amino acid sequences indicated a close relationship to RbcL from purple bacteria. The importance of this finding in molecular evolution of photosynthetic bacteria remains to be elucidated.
Bob Haselkorn (U. Chicago) gave a talk concerning the role in heterocyst differentiation by Anabaena PCC 7120 of genes that bear similarity to those encoding response regulators and sensor kinases of TWO-COMPONENT REGULATORY SYSTEMS. PCR primers directed at conserved regions in the histidine kinase sensors of other two component systems amplified a series of products, denoted ask, with similarities to phoR, ntrB, pleC, and other sensory kinases. Preliminary results show that askA, which is most similar to sensory kinase phoR (which regulates phosphate deprivation genes), shows no phenotype when inactivated, and askC, most similar to pleC (required by Caulobacter for differentiation), alters heterocyst frequency when inactivated.
Jack Meeks (U. California, Davis) described a mutant (UCD311) evidently defective in the RESPONSE REGULATOR side of a two-component regulatory system. The gene, devR, was found by transposon mutagenesis of Nostoc ATCC 29133. The mutant is unable to fix nitrogen in the presence of oxygen (Fox-) but is symbiotically competent. The DevR gene product represents a different class of response regulators than PatA, a previously characterized gene from Anabaena, and is more closely related by sequence to response regulators CheY and SpoOF (involved in chemotaxis in E. coli and sporulation in Bacillus, respectfully).
Bill Buikema (U. Chicago) discussed results concerning the ROLE AND EXPRESSION OF HETR, a gene required early in heterocyst differentiation, using a fusion of hetR to green fluorescent protein (GFP) to examine cell-specific expression. In wild type Anabaena PCC 7120, a high level of fluorescence from hetR::GFP was seen in well-spaced cells prior to morphologically visible differentiation. When the fusion was placed in hetR, patA, or patB mutant strains, aberrant patterns of fluorescent cells are seen. The use of GFP does carry technical limitations: the protein is toxic and requires oxygen for proper folding. Detection of gene fusions in heterocysts is therefore problematic.
Terry Thiel (U. Missouri, St. Louis) presented a different approach to analyzing the cell-specific gene expression of TWO MOLYBDENUM-DEPENDENT NITROGENASES encoded by gene clusters nif1 and nif2 in Anabaena variabilis ATCC 29413. She and her co-workers utilized C12-fluorescein-beta-D-galacto- side, a substrate for beta-galactosidase, to localize expression of nifH1::lacZ and nifH2::lacZ fusions by fluorescence microscopy. The results indicate that the nif1 gene cluster is regulated developmentally and expressed only in heterocysts while the nif2 genes are expressed in all cells in response to nitrogen limitation and anoxia. Unpatterned expression of nif2 in vegetative cells, and, presumably, ammonia production in all cells did not prevent patterned heterocyst differentiation, suggesting that products of nitrogen fixation may not be involved in pattern formation.
This and other provocative results presented at the meeting prompted an informal roundtable discussion to consider approaches to studying heterocyst pattern formation. The discussion centered on the question of how to determine whether or not a PRE-PATTERN OF CELLS destined to become heterocysts exists in a nitrogen replete filament. Any pattern -- pre- or post-nitrogen stepdown -- that relies on the exchange of signal molecules should be disrupted in a strain that lacks intercellular communication. We should be able to detect in such a strain any intrinsic pattern (i.e. independent of interaction) that may exist. Does such a strain exist? Perhaps, and fluorescent dyes conceivably could be use to test putative communication-less strains.
- Tom Hanson & ZHAO Jindong
In 1994, Konstanze Mez and Beatrix Falch from the University of Zurich, Switzerland, were inspired to arrange a meeting of German-speaking PhD students. The very positive impressions from that meeting led to a second edition this past September in Vienna, Austria. Seventeen young scientists followed the invitation of Wolfgang Gregor, PhD student in the group of Georg Schmetterer, and discussed the results of their present work.
Toxicology and secondary metabolism
Juergen Steiner from the Loeffelhardt group (Wien) presented interesting results concerning the MEMBRANE INSERTION of the nuclear-encoded cytochrome c553 from cyanelles of Cyanophora paradoxa. They isolated the protein and the corresponding nuclear gene. Since the mature protein is located in the thylakoid lumen, it has to traverse three biological membranes (inner and outer envelope membranes, thylakoid membrane) and the peptidoglycan layer before it reaches its final subcellular locale. The transit sequence is composed of two different targeting signals, and this represents the first known bipartite transit sequence of a cyanelle protein.
Olaf Neuschaefer-Rube (Konstanz) studies a Synechocystis mutant isolated from the Bodensee that doesn't form normal PHYCOBILISOMES but rather contains a paracrystalline structure made up of phycocyanin and linker protein. Biochemical analysis of the crystal showed the presence not only of phycocyanin alpha and beta, but also of the rod linker LR35 C-PC. A colored polypeptide of 55 kD turned out to be a fusion protein of the rod linker at the n-terminus and a phycocyanin beta subunit. The protein is not predicted by the arrangement of genes in the mutant, indicating that a posttranscriptional event may be responsible for this strange fusion protein.
Stefan Schmitz (Bonn) investigated Anabaena FERREDOXIN-BINDING PROTEINS in E. coli in order to find out if they possess a common binding domain for ferredoxin. All negatively charged and conserved amino acid residues of ferredoxin from Anabaena were exchanged for neutral residues and the effects on binding to different redox partners were studied. Glu94 was identified as the most important among these residues, and for FNR and nitrite reductase an aromatic residue in position 65 also was essential. After having cloned petF gene (encoding FNR) from Anabaena variabilis, Stefan produced site specific mutations within that protein. He exchanged positively charged residues against neutral ones. Arg153, Lys209, Lys212, and Lys430 turned out to be very important, they are supposed to be lying in a cavern which binds ferredoxin. Such a cavern carrying a lot of positively charged residues could be found in several cyanobacterial nitrite and nitrate reductases.
Markus Geisler (Duesseldorf) characterized the p-type CALCIUM ATPASE from the same strain. The enzyme is localized in the cytoplasmic membrane and is more closely related to eukaryotic ATPases than to bacterial ones.
Josef Niederberger (Institut fur systematik Botanik, Zurich) reported on the TAXONOMY OF TOXIC CYANOBACTERIA in Swiss alpine lakes. He used RAPD-PCR for the classification of 16 toxic and non-toxic strains of Microcystis aeruginosa. The toxicity of the strains had been checked by other groups in a mouse bioassay, with HPLC and in a phosphatase inhibition assay. A phylogenetic tree was derived from the RAPD data, but the toxic strains did not cluster together.
Andrea Nowotny (Institut fur pharm. Biologie, Greifswald) detected ANTIVIRAL ACTIVITY in aqueous extracts from Microcystis waterblooms in the south Baltic Sea. The nature of the substance that inhibits replication of influenza virus A could not be clarified.
Egbert Hoiczyk (Max Planck Institut, Muenchen) related structural details of what may be the motor for GLIDING MOTILITY by cyanobacteria. Electron microscopical studies of the cell walls of strains from three different genera revealed that all species possess identical multilayered cell walls, which are covered with a complex double external layer, the surface of which is formed by a parallel array of helically arranged fibrils. The correlation of these structures with motion and their extracellular location indicates a possible role in gliding motility.
Alfred Hansel (Freiburg) presented his results concerning the major OUTER MEMBRANE PROTEINS of Synechococcus PCC 6301. After purification of the major outer membrane protein complex and its functional characterization, he cloned the gene coding for porin. Its sequence does not show any overall similarity with other porin sequences, but computer analysis indicates that the cyanobacterial porin has the same architecture as other porins. Downstream from this gene lay a second orf that shows great similarity (> 60%) to the porin gene, and reinvestigation of the protein led to the conclusion that Synechococcus has two different major outer membrane proteins, the monomers of which migrate almost identically in SDS gels. It is not yet clear if both function as porins.
-- Alfred Hansel
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