Robert D. Simon

The biosynthesis of multi-l-arginyl-poly(l-aspartic acid) in the filamentous cyanobacterium Anabaena cylindrica

The cyanobacteria produce multi-L-arginyl-poly (aspartic acid), a high molecular weight (Mr=25 000-125 000) branched polypeptide consisting of a poly(aspartic acid) core with L-arginyl residues peptide bonded to each free carboxyl group of the poly(aspartic acid). An enzyme which will elongate Arg-poly(Asp) has been isolated and purified 92-fold from the filamentous cyanobacterium Anabaena cylindrica. The enzyme incorporates arginine and aspartic acid into Arg-poly(Asp) in a reaction which requires ATP, KCl, MgCl2, and a sulfhydryl reagent. The enzymatic incorporation of arginine is dependent upon the presence of L-aspartic acid but not visa versa, a finding which suggests the order of amino acid addition to the branched polypeptide-aspartic acid is added to the core followed by the attachment of an arginine branch. The elongation of Arg-poly(Asp) in-vitro is insensitive to the addition of protein synthesis inhibitors and to the addition of nucleases. These findings support the notion previosly suggested from in-vivo studies that Arg-poly(Asp) is synthesized via a non-ribosomal route and also demonstrate that amino-acetylated transfer-RNAs play no part in at least one step of the biosynthetic mechanism.

The effect of chloramphenicol on the production of cyanophycin granule polypeptide in the blue-green alga Anabaena cylindrica

SummaryThe cyanophycin or structured granule of the blue-green algae is composed of polypeptides which are copolymers of aspartic acid and arginine. The addition of chloramphenicol to an exponentially growing culture of the blue-green alga Anabaena cylindrica at concentrations which completely inhibit protein synthesis results both in the inhibition of growth and in the accumulation of the cyanophycin granule polypeptide (CGP). The chloramphenicol induced increase in CGP content is energy dependent. Removal of the chloramphenicol results in resumption of growth and the hydrolysis of the stored CGP. The data presented indicate that CGP is synthesized via a non-ribosomal system and are consistent with the idea that CGP serves as a cellular nitrogen reserve.

THE NORMAL AND INDUCED OCCURRENCE OF CYANOPHYCIN INCLUSION BODIES IN SEVERAL BLUE-GREEN ALGAE1

Multi‐L‐arginyl‐poly(L‐aspartic acid) [arg‐poly(asp)], the polypeptide component of the cyanophycin inclusion body, is found in cells of many blue‐green algae. Formation of this material can be induced by a variety of treatments including the addition of excess nitrogen‐containing compounds, the addition of specific inhibitors of macromolecular synthesis, and the exclusion of sulfur or phosphorus. Knowledge of the conditions that induce the synthesis of this polypeptide has made possible an ultrastructural survey to determine the presence and cellular location of cyanophycin bodies in a variety of cyanobacteria. Data presented show that certain strains of the unicellular genus Synechococcus Nägeli do not contain arg‐poly(asp) under environmental conditions that markedly increase the level of such material in other cyanobacteria. In addition many strains show spatial localization of the cyanophycin bodies under normal growth conditions, and moreover the normal pattern is retained even when massive synthesis of arg‐poly(asp) is induced. Finally there is no evidence that these inclusion bodies occur in certain beggiatoan gliding bacteria.

Morphology and Protein Composition of Gas Vesicles from Wild Type and Gas Vacuole Defective Strains of Halobacterium salinarium Strain 5

The morphology and protein composition of gas vesicles isolated from wild-type and gas vacuole defective (gvdef) isolates of Halobacterium salinarium strain 5 have been analysed. Two general vesicle shapes were found: spindles, and cylinders with short biconical ends. Wild-type strains contained a majority of spindle-shaped vesicles (94 %), while 69% of the vesicles in gvdef strains were cylindrical. Electrophoretic analysis of gas vesicle proteins showed that vesicle preparations contained only a single major protein species. In addition, the polypeptide in vesicles from the gvdef strain migrated more slowly than that from the wild-type strain. From the rates of electrophoretic migration, the molecular weights of gas vacuole proteins in the wild-type and gvdef strains were estimated as 16 800 and 18200, respectively. Data provided here are consistent with the suggestion that polypeptide processing may be involved in the formation and assembly of gas vesicles in Halobacterium.

Structural characterization of the cyanophycin granule polypeptide of Anabaena cylindrica by circular dichroism and raman spectroscopy

The cyanophycin granule polypeptide, or multi-L-arginyl-poly(L-aspartic acid), from the filamentous cyanobacterium Anabaena cylindrica has been examined by circular dichroism and laser Raman spectroscopy. The polypeptide exhibits defined secondary structure in acidic solution, but not in alkaline medium. Raman spectra show the structure of the insoluble (storage?) form of the polypeptide is similar but not identical to that of the acid soluble form. Analysis of the CD spectra suggests that beta-sheets exist in the acid soluble form, and by inference, in the insoluble cellular material.

The use of an ultrasonic bath to disrupt cells suspended in volumes of less than 100 μliters

Abstract A procedure is described for the efficient disruption of small (10–100 μl) volumes of algal and bacterial cell suspensions.

Sporulation in the filamentous cyanobacterium Anabaena cylindrica

Sporulation in the filamentous cyanobacterium Anabaena cylindrica involves the transformation of a vegetative cell into a thick-walled resistant structure. Because this process occurs at predictable loci in each filament and involves a significant increase in cell size, the course of sporulation in a culture can be quantitatively determined. Sporulation occurs during the late logarithmic phase of a culture, a time of slow but unbalanced growth. Under the conditions imployed here, sporulation is not a synchronous event either between or within filaments. The information in this paper provides an estimate of the rate of spore differentiation and supports the previous notion that in the formation of strings of more than one spore, a gradient of spore maturation exists.

The absence of a correlation between plasmids and luminescence in marine luminous bacteria

Members of four species of marine luminous bacteria were examined for the presence of plasmids using gel electrophoresis of purified alkaline extracts. One to four plasmids, with molecular weights ranging from 5 to 120 megadaltons, were found to occur in 43% of the 58 bioluminescent strains examined. There was, thus, no correlation between the presence and absence of plasmids and luminescence, nor was there any single size of plasmid common to the different bacterial species. Spontaneous dark (dim) mutants were selected from five strains ofVibrio (Beneckea) harveyi; in no case was there any difference in the plasmid content between the bright parent strain and the dim isolate.

Induction and Differentiation of Heterocysts in the Filamentous Cyanobacterium Cylindrospermum licheniforme

Summary: The fragmentation of Cylindrospermum licheniforme filaments resulted in the induction of a synchronous round of heterocyst differentiation. Young heterocysts (proheterocysts) appeared 12 to 15 h after fragmentation, and 12 to 15% of the cells became heterocysts within 24 h. NH4Cl or NaNO3 prevented the formation of new heterocysts, while differentiation was stimulated twofold by the absence of atmospheric N2. The sequence of appearance of heterocyst-specific characteristics was observed following filament fragmentation. Nitrogenase activity increased rapidly between 13 and 26 h, paralleling the increase in heterocyst frequency. Glycolipids unique to the heterocyst envelope were also synthesized between 13 and 26 h, and activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase increased after 10 h and 90 h respectively. Aged medium induced sporulation in intact filaments within 1 d. but sporulation could occur no earlier than 2 d after filament fragmentation, indicating that the induction of sporulation requires the presence of mature (but not necessarily functional) heterocysts.

Heterocyst differentiation in Cylindrospermum licheniforme: studies on the role of transcription.

Heterocysts of the cyanobacterium Cylindrospermum licheniforme occur at the ends of the filaments. These cells, specialized for aerobic N2 fixation, synchronously differentiate after the fragmentation of filaments grown in a medium free of combined nitrogen. This study has examined the role of transcription during and after heterocyst differentiation. Autoradiography of intact filaments pre-labelled for 3 h with [3H]uracil revealed that RNA synthesis occurred at similar rates in vegetative cells, developing (pro)heterocysts and mature heterocysts. Since mature heterocysts no longer divide and contain a similar amount of DNA as vegetative cells, transcription must continue under conditions where replication no longer occurs. Rifampicin, when added to fragmented filaments at concentrations that reduced RNA synthesis by 95% in all cell types, inhibited proheterocyst formation but not the final morphological step of differentiation (pore plug deposition). However, chloramphenicol blocked all stages of heterocyst differentiation. Since the average mRNA half-life was 29 min, and the rifampicin-insensitive stage lasted 3-6 h, the later stages of differentiation may depend on long-lived mRNAs.