FtsZ degradation in the cyanobacterium Anabaena sp. strain PCC 7120

In prokaryotes, cell division is normally achieved by binary fission, and the key player FtsZ is considered essential for the complete process. In cyanobacteria, much remains unknown about several aspects of cell division, including the identity and mechanism of the various components involved in the division process. Here, we report results obtained from a search of the players implicated in cell division, directly associating to FtsZ in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Histidine tag pull-downs were used to address this question. However, the main observation was that FtsZ is a target of proteolysis. Experiments using various cell-free extracts, an unrelated protein, and protein blot analyses further supported the idea that FtsZ is proteolytically cleaved in a specific manner. In addition, we show evidence that both FtsZ termini seem to be equally prone to proteolysis. Taken together, our data suggest the presence of an unknown player in cyanobacterial cell division, opening up the possibility to investigate novel mechanisms to control cell division in Anabaena PCC 7120.     

The ManR specifically binds to the promoter of a Nramp transporter gene in Anabaena sp. PCC 7120: a novel regulatory DNA motif in cyanobacteria

The Anabaena sp. PCC 7120 ManR and a homologous protein of MntH were identified by BLAST search. Recombinant ManR protein was overexpressed in Escherichia coli and purified by an immobilized metal (Ni) affinity chromatography. Electrophoretic mobility shift assays revealed that ManR specifically bound to the promoter region of the mntH gene. Site-directed mutagenesis experiments demonstrated that the specific recognition site for ManR is TATGAAAAGAATATGAGAA, which is composed of two direct repeats of the consensus sequence (T/A)ATGA(G/A)A(A/G). This is a novel regulatory DNA motif in cyanobacteria, indicating that the expression of mntH was regulated by a two-component Mn(2+)-Sensing System containing ManR in Anabaena sp. PCC 7120. To date, this specific pathway of regulating mntH expression has only been found in cyanobacteria.     

Isolation and characterization of heterocysts from Anabaena sp. strain CA

A comparative study has been made on the pigment composition and nitrogenase activity of whole filaments and isolated beterocysts from a mutant strain of Anabaena CA. The whole cell absorption spectra of intact filaments and isolated heterocysts showed close resemblance especially between 550–700 nm region. On a quantitative basis the chlorophyll a content was found almost equal between the vegetative cell and heterocyst but the c-phycocyanin content in the heterocyst was about 1/2 that of the vegetative cell. The purification of the phycobiliprotein on DEAE-cellulose showed the presence of c-phycocyanin (_max 615 nm) and allophycocyanin (_max 645 nm, shoulder 620 nm). Isolated heterocysts under H₂ showed acetylene reduction rates of 57 nmol C₂H₄/mg dry wt·min (342 mol C₂H₄/mg chl a·h), whereas intact filaments reduced at the rate of 18 nmol C₂H₄/mg dry wt·min (108 mol C₂H₄/mg chl a·h). This rate accounts for 30% recovery of nitrogenase activity in isolated heterocysts compared to whole filaments. The activity was strictly light dependent and was linear under H₂ for more than 3 h. Addition of as little as 5% H₂ under argon stimulated the C₂H₂ reductionseveral fold. The acetylene reduction (nitrogenase activity) also showed tolerance to 5% added O₂ either under H₂ or argon. The results suggest that the heterocyst of Anabaena CA-V is different in some characteristics (viz., higher endogenous C₂H₂ reduction rate, prolonged activity and higher levels of phycobiliproteins) than those reported in other Anabaena species.     

Characterization and structural properties of the major biliproteins of Anabaena sp.

Studies are presented of the biliproteins of Anabaena sp. This filamentous cyanobacterium contains three major biliproteins. Whereas two of these, C-phycocyanin and allophycocyanin, are common to all cyanobacteria, the third, phycoerythrocyanin (_max568nm) has hitherto not been described and its distribution among cyanobacteria appears to be limited. Anabaena variabilis and Anabaena sp. 6411 allophycocyanin, C-phycocyanin, and phycoerythrocyanin were purified to homogeneity and characterized with respect to molecular weight, isoelectric point, absorption spectrum and amino acid composition. The and subunits of each of these proteins were also purified to homogeneity and characterized in the same manner. The tetrapyrrole chromophore content was determined for each of the proteins and subunits. The subunit of phycoerythrocyanin carries a novel phycobiliviolin-like chromophore. This chromophore has not previously been detected in cyanobacterial biliproteins, but has been noted as a prosthetic group of a cryptophytan phycocyanin.Sedimentation equilibrium studies show that at pH 7.0, at protein concentrations of 0.2–0.6 mg/ml, allophycocyanin, C-phycocyanin and phycoerythrocyanin, each exists as a trimeric aggregate, ()₃, of molecular weight of approximately 105000. Structural studies of microcrystals of these three biliproteins by electron microscopy and X-ray diffraction reveal a common plan for the construction of higher assembly forms. The major building block appears to be the trimer ()₃. It is proposed that this is a dise-like structure about 3.0×12.0 nm. The individual or subunits are roughly spherical, 3 nm in diameter. Allophycocyanin trimers stack to form bundles of rods which form long needles. Both phycocyanin and phycoerythrocyanin form double dises ()₆ which are visible as ring-shaped structures by electron microscopy. The mode of assembly of the biliproteinstructures in the phycobilisome is, as yet, unknown.Abbreviation Used SDS sodium dodecyl sulfate Dedicated to Prof. Dr. Roger Y. Stanier on the occasion of his 60th birthday.     

Identification of genes controlled by the manganese response regulator, ManR, in the cyanobacterium, Anabaena sp. PCC 7120

A computational search was carried out to identify additional binding sites for the manganese response regulator, ManR, in the genome of Anabaena sp. PCC 7120. This approach predicted ManR binding sites: the promoter regions of the genes of all3575-alr3576 and the gene of alr5134 from Anabaena sp. PCC 7120. Electrophoretic mobility shift assays confirmed that the ManR of Anabaena sp. PCC 7120 specifically bound to the promoter regions of all3575-alr3576 and alr5134.     

Isolation of a solventogenic Clostridium sp. strain: Fermentation of glycerol to n-butanol,analysis of the bcs operon region and its potential regulatory elements

A new solventogenic bacterium, strain GT6, was isolated from standing water sediment. 16S-rRNA gene analysis revealed that GT6 belongs to the heterogeneous Clostridium tetanomorphum group of bacteria exhibiting 99% sequence identity with C. tetanomorphum 4474^T. GT6 can utilize a wide range of carbohydrate substrates including glucose, fructose, maltose, xylose and glycerol to produce mainly n-butanol without any acetone. Additional products of GT6 metabolism were ethanol, butyric acid, acetic acid, and trace amounts of 1,3-propanediol. Medium and substrate composition, and culture conditions such as pH and temperature influenced product formation. The major fermentation product from glycerol was n-butanol with a final concentration of up to 11.5 g/L. 3% (v/v) glycerol lead to a total solvent concentration of 14 g/L within 72 h. Growth was not inhibited by glycerol concentrations as high as 15% (v/v).     

Glucose-6-phosphate dehydrogenase of Anabaena sp.

The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three principal forms, with approximate molecular weights of 120 000 (M ₁), 240 000 (M ₂) and 345 000 (M ₃). The relative catalytic activities are: M ₁M ₂<M ₃. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M ₁ form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP⁺), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M ₁ form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M ₁ form is favored at pH values above 7; a pH of 6.5 is optimal for maintenace of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.Abbreviations G6PD glucose-6-phosphate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - RUDP ribulose-1,5-diphosphate - G6P glucose-6-phosphate - 6PG 6-phosphogluconate     

A novel,anchorless streptococcal surface protein that binds to human immunoglobulins

We have characterized a novel surface protein from urea extract of whole cells of group A Streptococcus pyogenes (GAS). A major protein band (35kD) was found to hybridize with human IgG by Western blotting. A search of the N-terminal amino acid sequence of this protein by using the GAS genome sequence database revealed an open reading frame that encoded a 38-kDa protein with a signal peptide sequence. We have named this protein streptococcal immunoglobulin-binding protein 35 (Sib35). It was found to be an anchorless protein with no LPXTG motif, distinct from the M protein superfamily exhibiting immunoglobulin-binding activity, and partially secreted in the culture supernatant. Recombinant Sib35 was also shown to bind human IgA and IgM. The sib35 gene was found in all GAS strains examined, but not in oral, group B, C, or G streptococcal strains. These results suggest that Sib35 is a unique immunoglobulin-binding protein in GAS.     

Utilization of the cyanobacteria Anabaena sp. CH1 in biological carbon dioxide mitigation processes

Before switching totally to alternative fuel stage, CO₂ mitigation process has considered a transitional strategy for combustion of fossil fuels inevitably. In comparison to other CO₂ mitigation options, such as oceanic or geologic injection, the biological photosynthetic process would present a far superior and sustainable solution under both environmental and social considerations. The utilization of the cyanobacteria Anabaena sp. CH1 in carbon dioxide mitigation processes is analyzed in our research. It was found that an original developed photobioreactor with internal light source exhibits high light utilization. Anabaena sp. CH1 demonstrates excellent CO₂ tolerance even at 15% CO₂ level. This enables flue gas from power plant to be directly introduced to Anabaena sp. CH1 culture. Double light intensity and increased 47% CO₂ bubble retention time could enhance CO₂ removal efficiencies by 79% and 67%, respectively. A maximum CO₂ fixation rate of 1.01 g CO₂ L⁻¹ day⁻¹ was measured experimentally.     

Characterization of two small cryptic plasmids from Pseudomonas sp. strain S-47

Two small cryptic plasmids, p47L and p47S, identified in Pseudomonas sp. S-47 were characterized by determination of DNA sequences and physical and functional maps. They are 3084 and 1782 bp in length, respectively, with GC contents of 63.55 and 65.21%. The detection of single-strand DNAs of both plasmids indicates that they replicate by a rolling-circle mechanism. The deduced polypeptide encoded by the rep gene of p47L is homologous with Rep proteins of plasmids belonging to the pIJ101/pJV1 family, which are known to replicate by the rolling-circle mechanism. Despite containing a homologous signature with Rep proteins of rolling-circle replicating (RCR) plasmids in the pT181 family, the Rep of p47S lacks significant homology with Rep proteins of this family and is missing a region similar to the family's replication origin (dso). Based on the rep sequence comparisons, p47L falls into a previously defined plasmid family whereas p47S defines a new family of RCR plasmid.     

LaRbp38: a Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.     

Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus

Myxococcus xanthus cells harbor two motility machineries, type IV pili (Tfp) and the A-engine. During reversals, the two machineries switch polarity synchronously. We present a mechanism that synchronizes this polarity switching. We identify the required for motility response regulator (RomR) as essential for A-motility. RomR localizes in a bipolar, asymmetric pattern with a large cluster at the lagging cell pole. The large RomR cluster relocates to the new lagging pole in parallel with cell reversals. Dynamic RomR localization is essential for cell reversals, suggesting that RomR relocalization induces the polarity switching of the A-engine. The analysis of RomR mutants shows that the output domain targets RomR to the poles and the receiver domain is essential for dynamic localization. The small GTPase MglA establishes correct RomR polarity, and the Frz two-component system regulates dynamic RomR localization. FrzS localizes with Tfp at the leading pole and relocates in an Frz-dependent manner to the opposite pole during reversals; FrzS and RomR localize and oscillate independently. The Frz system synchronizes these oscillations and thus the synchronous polarity switching of the motility machineries.     

Isolation and characterization of a temperate cyanophage for a tropical Anabaena strain

In this paper we describe the isolation and characterization of a temperate cyanophage N(S)1 of the genus cyanopodovirus which produces turbid plaques on the host Anabaena 77S15 isolated from tropical soil. Its properties have been compared to those of other well-characterized cyanophages. In addition, two strains of Anabaena 77S15 lysogenic for N(S)1 were isolated. N(S)1 seems to be integrated into the chromosome of the two lysogens, and a 2 kb plasmid present at a low copy number in the non-lysogenic strain is amplified significantly.