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Oscillin, an extracellular, Ca2+-binding glycoprotein essential for the gliding motility of Cyanobacteria
Abstract
Electron microscopic studies have demonstrated that various gliding filamentous cyanobacteria have trichome surfaces with a common structural organization. They contain an S-layer attached to the outer membrane and an array of parallel fibrils on top of the S-layer. In all species studied, the helical arrangement of these fibrils corresponds to the sense of rotation of the organism during the gliding movement. We have investigated the surface fibrils of Phormidium uncinatum using electron microscopic, spectroscopic and biochemical techniques. The fibrils consist of a single rod-shaped protein, which we refer to as oscillin. Oscillin is a 646 amino acid residue protein (Mr 65 807; pI 3.63) and appears to be glycosylated. Sequence analysis reveals a two-domain structure: a 554 residue domain contains 46 repeats of a Ca2+-binding motif; it is followed by a 92 residue C-terminal domain, which might mediate its export. Filaments that do not express oscillin lose their ability to move. Homology studies suggest that similar proteins play comparable roles in other motile cyanobacteria. The structure of oscillin appears to favour a passive role in gliding.
Supplementary resource (1)
... Based on Ca 2+ chelation and calcium ionophore studies, the authors concluded that phototactic orientation was likely caused by the uptake of extracellular Ca 2+ , whereas gliding motility was dependent on internal Ca 2+ mobilization or release from Ca 2+ -binding proteins [28]. Using genetic and biochemical approaches, proteins containing Ca 2+ -binding domains were discovered293031. A key step to understand gliding motility in cyanobacteria was to isolate surface fibrils. ...
... A key step to understand gliding motility in cyanobacteria was to isolate surface fibrils. Fibrils in some cyanobacteria are composed of a single type of protein called oscillin, which contains multiple Ca 2+ -binding motifs [29]. Phormidium uncinatum filaments that did not accumulate oscillin were not able to glide [29]. ...
... Fibrils in some cyanobacteria are composed of a single type of protein called oscillin, which contains multiple Ca 2+ -binding motifs [29]. Phormidium uncinatum filaments that did not accumulate oscillin were not able to glide [29]. Similar cell surface-associated glycoproteins function in this manner in other cyanobacteria [29]. ...
Second messengers are intracellular substances regulated by specific external stimuli globally known as first messengers. Cells rely on second messengers to generate rapid responses to environmental changes and the importance of their roles is becoming increasingly realized in cellular signaling research. Cyanobacteria are photooxygenic bacteria that inhabit most of Earth's environments. The ability of cyanobacteria to survive in ecologically diverse habitats is due to their capacity to adapt and respond to environmental changes. This article reviews known second messenger-controlled physiological processes in cyanobacteria. Second messengers used in these systems include the element calcium (Ca2+), nucleotide-based guanosine tetraphosphate or pentaphosphate (ppGpp or pppGpp, represented as (p)ppGpp), cyclic adenosine 3',5'-monophosphate (cAMP), cyclic dimeric GMP (c-di-GMP), cyclic guanosine 3',5'-monophosphate (cGMP), and cyclic dimeric AMP (c-di-AMP), and the gaseous nitric oxide (NO). The discussion focuses on processes central to cyanobacteria, such as nitrogen fixation, light perception, photosynthesis-related processes, and gliding motility. In addition, we address future research trajectories needed to better understand the signaling networks and cross talk in the signaling pathways of these molecules in cyanobacteria. Second messengers have significant potential to be adapted as technological tools and we highlight possible novel and practical applications based on our understanding of these molecules and the signaling networks that they control.
... It is also shown that the organization of the fibrils in the sheaths of various gliding cyanobacteria always correlate with the motion of the species. Finally, it is suggested that specific surface glycoproteins such as oscillin (12) act as platforms for the assembly and attachment of these extracellular carbohydrate structures. ...
... The preparations also allowed visualization of the contact zone between the sheath and the underlying cell wall surface, which could not be observed in thin sections. As seen in the inset of Fig. 6, the sheath fibril orientation corresponds to the orientation of the oscillin glycoprotein surface fibrils previously described (11,12). Electron microscopy of metal-shadowed, air-dried preparations showed the fibrillar texture of the sheaths (Fig. 7). ...
... SDS-PAGE of isolated sheaths revealed only a weakly stained band with a molecular mass of 130 kDa (Fig. 9), suggesting that this sheath-associated protein is the surface fibrilforming protein oscillin (12). Oscillin is a 66-kDa protein which not only forms the contact zone between the filament surface and the sheath but is arranged helically just as the sheath fibrils are (see also Fig. 5A and Fig. 6). ...
The sheath of the filamentous, gliding cyanobacterium Phormidium uncinatum was studied by using light and electron microscopy. In thin sections and freeze fractures the sheath was found to be composed of helically arranged carbohydrate fibrils, 4 to 7 nm in diameter, which showed a substantial degree of crystallinity. As in all other examined motile cyanobacteria, the arrangement of the sheath fibrils correlates with the motion of the filaments during gliding motility; i.e., the fibrils formed a right-handed helix in clockwise-rotating species and a left-handed helix in counterclockwise-rotating species and were radially arranged in nonrotating cyanobacteria. Since sheaths could only be found in old immotile cultures, the arrangement seems to depend on the process of formation and attachment of sheath fibrils to the cell surface rather than on shear forces created by the locomotion of the filaments. As the sheath in P. uncinatum directly contacts the cell surface via the previously identified surface fibril forming glycoprotein oscillin (E. Hoiczyk and W. Baumeister, Mol. Microbiol. 26:699-708, 1997), it seems reasonable that similar surface glycoproteins act as platforms for the assembly and attachment of the sheaths in cyanobacteria. In P. uncinatum the sheath makes up approximately 21% of the total dry weight of old cultures and consists only of neutral sugars. Staining reactions and X-ray diffraction analysis suggested that the fibrillar component is a homoglucan that is very similar but not identical to cellulose which is cross-linked by the other detected monosaccharides. Both the chemical composition and the rigid highly ordered structure clearly distinguish the sheaths from the slime secreted by the filaments during gliding motility.
... In an attempt to explain gliding motility in cyanobacteria, we recently proposed a model [4] based on structural studies that accounts for the characteristic features of gliding motility [5]. The so-called 'surface-mucilage' model is based on the structural comparison of the filament surfaces of four different gliding Oscillatoriaceae species and the biochemical characterization of oscillin, the major outer surface protein that forms the cell surface of one of these species [4]. ...
... In an attempt to explain gliding motility in cyanobacteria, we recently proposed a model [4] based on structural studies that accounts for the characteristic features of gliding motility [5]. The so-called 'surface-mucilage' model is based on the structural comparison of the filament surfaces of four different gliding Oscillatoriaceae species and the biochemical characterization of oscillin, the major outer surface protein that forms the cell surface of one of these species [4]. According to the model, specifically arranged surface proteins such as oscillin create surface topographies that guide the observed path of the filaments -helical topographies in rotating species such as Phormidium or radial topographies in non-rotating species such as Anabaena. ...
... A first clue for the role of the pores in motility came from the investigation of cell walls of old non-motile and spontaneously induced non-motile Phormidium filaments [4]. Even with the use of various extraction protocols and staining procedures, no pore complexes could be isolated or visualized in the cell walls of filaments which no longer showed gliding motility. ...
Whereas most bacteria move by means of flagella, some prokaryotes move by gliding. In cyanobacteria, gliding motility is a slow uniform motion which is invariably accompanied by a continuous secretion of slime. On the basis of these characteristics, a model has been proposed in which the gliding motility of cyanobacteria depends on the steady secretion of slime using specific pores, as well as the interaction of the slime with the filament surface and the underlying substrate.
The structures of the pore apparatus of two different filamentous cyanobacteria have been characterized. In both species, pores are formed by a hitherto uncharacterized type of prokaryotic organelle that spans the entire multilayered cell wall and possesses structural properties expected for an organelle that is involved in the rapid secretion of extracellular carbohydrates. Light microscopic observations of the secretion process provided direct evidence that the pore complexes are the actual sites of slime secretion, that the secreted slime fibrils are elongated at about the same rate as the filament glides (up to 3 micrometer s-1), and that gliding movements are caused directly by the secretion of slime.
It has been known for a long time that carbohydrate secretion has an important role in the gliding motility of various prokaryotes. Our results strongly suggest that slime secretion is not only a prerequisite for this peculiar type of motility in cyanobacteria, but also directly generates the necessary thrust for locomotion.
... The filamentous cyanobacteria show gliding motility, which requires contact with a solid surface and occurs in a direction parallel to the long axis of the filament. Interestingly, nonmotile Phormidium uncinatum cells are deficient in components of the cell surface, including extracellular slime and helically arrayed fibrils that are comprised of a rod-shaped glycoprotein called oscillin (3,4). Oscillin has been proposed to play a passive role in motility with propulsion resulting from shear forces generated between the oscillin fibrils on the cell surface and slime as it is exuded from cells (''surface mucilage'' hypothesis). ...
... Neither P. uncinatum nor the marine Synechoccus species that have been examined have obvious cell appendages, such as flagella or pili (3)(4)(5), that have been shown to facilitate movement in other microbes. However, pili-like structures have been observed on the surface of certain unicellular and filamentous cyanobacteria (8)(9)(10)(11). ...
... This protein class is characterized by tandem arrays of a 9-aa motif rich in glycine, aspartic acid, and asparagine [LXGGXG(N͞D)DX], which is involved in Ca 2ϩ binding. The Synechocystis HlyA protein also shows similarity to the motility-related glycoproteins oscillin (30% identity and 60% similarity), and SwmA (25% identity and 50% similarity), both of which contain several repeats of the 9-aa motif and are cell-surface associated (3,4,6). The elevated levels of extracellular pigments and HlyA polypeptide in the growth medium of cultures of the sigF mutant suggested that this strain had a defect in structuring or synthesizing components of its extracellular surface layers. ...
Disruption of a gene for an alternative sigma factor, designated sigF, in the freshwater, unicellular cyanobacterium Synechocystis sp. strain PCC6803 resulted in a pleiotropic phenotype. Most notably, this mutant lost phototactic movement with a concomitant loss of pili, which are abundant on the surface of wild-type cells. The sigF mutant also secreted both high levels of yellow-brown and UV-absorbing pigments and a polypeptide that is similar to a large family of extracellular proteins that includes the hemolysins. Furthermore, the sigF mutant had a dramatically reduced level of the transcript from two tandemly arranged pilA genes (sll1694 and sll1695), which encode major structural components of type IV pili. Inactivation of these pilA genes eliminated phototactic movement, though some pili were still present in this strain. Together, these results demonstrate that SigF plays a critical role in motility via the control of pili formation and is also likely to regulate other features of the cell surface. Furthermore, the data provide evidence that type IV pili are required for phototactic movement in certain cyanobacteria and suggest that different populations of pili present on the Synechocystis cell surface may perform different functions.
... It remains to be determined whether SwmA in fact binds calcium. SwmA is also similar to oscillin, a 646-aa Ca 2+ -binding glycoprotein that forms an array of parallel fibrils on the surface of the gliding filamentous cyanobacterium Phormidium uncinatum (Hoiczyk and Baumeister, 1997), as well as to HlyA, a 322-aa partial ORF of unknown function from the non-motile filamentous cyanobacterium Anabaena sp. strain PCC7120 (Brahamsha, 1996a). ...
... Both oscillin and HlyA also contain multiple repeats of the Ca 2+ binding nonapeptide, and the regions of similarity to SwmA are for the most part confined to regions containing these repeats. These three cyanobacterial proteins have been proposed to be homologous on the basis of a stretch of similarity at their C-terminal 47 amino acids, a region that does not contain multiple repeats of the Ca 2+ -binding motif (Hoiczyk and Baumeister, 1997). Although this is possible, this region does contain one copy of the nonapeptide within the parameters used by Hoyczyk and Baumeister (1997) (GNLYFDTNG for oscillin, GALFFDVDG for HlyA, and GVLSFDADG for SwmA). ...
... Although this is possible, this region does contain one copy of the nonapeptide within the parameters used by Hoyczyk and Baumeister (1997) (GNLYFDTNG for oscillin, GALFFDVDG for HlyA, and GVLSFDADG for SwmA). It has been suggested that SwmA and oscillin are functionally related (Hoiczyk and Baumeister, 1997). This raises the intriguing proposition that swimming in Synechococcus and gliding in Phormidium share a common mechanism. ...
Certain marine unicellular cyanobacteria of the genus Synechococcus exhibit a unique type of swimming motility characterized by the absence of flagella and of any other obvious organelle of motility. Although the mechanism responsible for this phenomenon remains mysterious, recent advances have included the development of testable models as well as the identification of a cell-surface polypeptide that is required for the generation of thrust. These developments, as well as the future research directions they suggest, are discussed.
... The mechanism of this phenomenon is difficult to explain due to many unresolved questions regarding Cyanobacteria movements and sheath formation (Adams et al., 1999; Hoiczyk, 2000; Wolgemuth & Oster, 2004; Read et al., 2007 ). The most helpful seem to be the findings of Hoiczyk and Baumeister, who described two factors most likely responsible for gliding motility in Phormidium (Hoiczyk & Baumeister, 1997). One is the presence of protein fibrils on the surface of trichomes that according to the authors play a passive role in gliding (Hoiczyk & Baumeister, 1997). ...
... The most helpful seem to be the findings of Hoiczyk and Baumeister, who described two factors most likely responsible for gliding motility in Phormidium (Hoiczyk & Baumeister, 1997). One is the presence of protein fibrils on the surface of trichomes that according to the authors play a passive role in gliding (Hoiczyk & Baumeister, 1997). The other is slime secretion through special organelles situated in the cell wall (Hoiczyk & Baumeister, 1998). ...
The nature of stimuli evoking cyanobacterium defence was investigated in experiments on Phormidium sp. strain able to defend itself against ciliate grazers. Limited dispersion of trichomes in reaction to Pseudomicrothorax dubius separated from cyanobacterium with a mesh-insert, indicates the existence of a chemical cue originating from the ciliates. Grazers released into the wells where trichomes' dispersion was already limited by the cue, initially had no difficulty finding food, but started to starve 24h later. Similar situation was observed in control wells. Direct observations of trichomes attacked by the ciliates showed a distinct difference between the trichomes previously subjected to mesh- separated ciliate and the control ones. The former withdrew more frequently into a rigid sheath, whereas the latter usually withdrew into elastic tubes. This suggests that both chemical and mechanical stimuli are necessary to express cyanobacterium defence to the fullest extent. Further investigations showed that ciliates specialised in ingesting filamentous cyanobacteria limit trichomes' dispersion, whereas filter-feeding Euplotes and cyanobacteria-feeding rotifer do not. The cyanobacterium can detect grazer presence even without direct contact, and modify its morphology in a way enabling full expression of defence reaction. This is the first report on ciliate-cyanobacterium chemical mediation. This article is protected by copyright. All rights reserved.
... The surface fibrils on the top of P. uncinatum S-layer consist of a single rod-shaped RTX protein of 646 residues with 46 repeats, the oscillin. Its structure appears to favor gliding, a relatively slow and smooth surface-associated translocation (Hoiczyk & Baumeister, 1997). As proposed by Hoiczyk and colleagues, the highly glycosylated surface of oscillin fibrils possesses ideal physicochemical properties for the temporary adhesion of the slime necessary for the generation of thrust and the helical arrangement of oscillin fibrils might guide the rotation of the P. uncinatum filament (Hoiczyk & Baumeister, 1997; Hoiczyk, 2000). ...
... Its structure appears to favor gliding, a relatively slow and smooth surface-associated translocation (Hoiczyk & Baumeister, 1997). As proposed by Hoiczyk and colleagues, the highly glycosylated surface of oscillin fibrils possesses ideal physicochemical properties for the temporary adhesion of the slime necessary for the generation of thrust and the helical arrangement of oscillin fibrils might guide the rotation of the P. uncinatum filament (Hoiczyk & Baumeister, 1997; Hoiczyk, 2000). The hemolysin-like RTX protein Sll1951 of 1741 residues produced by a unicellular freshwater cyanobacterium Synechocystis appears to be related to the elimination of motility, although the mechanism of its action remains largely unknown (Sakiyama et al., 2006). ...
Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca(2+) ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest.
... The recent molecular dissection of gliding in the cyanobacterium Phormidium unicatum [26] suggests that there may be another mechanism of gliding, in addition to the social and adventurous mechanisms. This photosynthetic glider has an 'S-layer' sheathing its envelope, overlaid by parallel fibrils comprising a single rod-shaped protein, oscillin, which is essential for gliding. ...
... Its glycoprotein product has a two-domain structure, with an amino terminus comprising multiple repeats of an 'EFhand' motif for binding Ca 2+ . Although oscillin likely plays a passive role in motility, oscillin filaments may have sliding-filament partners [26]. ...
Many bacteria glide over surfaces without the aid of flagella. Gliding is still somewhat mysterious, but recent studies show that it involves specialized secretory systems that assemble membrane-associated filaments, and the recognition of extracellular components that trigger movement via transmembrane transducers.
... In this model, the frequent reversal of movements of the filaments can be explained by the alternation of the set of junctional pores used. The importance of the protein fibrils and the secretion of slime for motility are substantiated by the observation that spontaneous mutants lacking these features are nonmotile (57). ...
... Only in Phormidium have the protein fibrils been characterized at the molecular level (57). They are formed by oscillin, a 646-aa glycoprotein (GenBank accession no. ...
... O9.13F genome. Genes encoding the S-layer gliding motility protein (oscillin) [91], as well as genes encoding type IV pilusrelated proteins involved in twitching motility (pilA, pilC-like, pilD and pilT2 genes) are present in the Arthrospira sp. O9.13F genome. ...
Cyanobacteria from the genus Arthrospira/Limnospira are considered haloalkalotolerant organisms with optimal growth temperatures around 35 °C. They are most abundant in soda lakes in tropical and subtropical regions. Here, we report the comprehensive genome-based characterisation and physiological investigation of the new strain O9.13F that was isolated in a temperate climate zone from the winter freezing Solenoye Lake in Western Siberia. Based on genomic analyses, the Siberian strain belongs to the Arthrospira/Limnospira genus. The described strain O9.13F showed the highest relative growth index upon cultivation at 20 °C, lower than the temperature 35 °C reported as optimal for the Arthrospira/Limnospira strains. We assessed the composition of fatty acids, proteins and photosynthetic pigments in the biomass of strain O9.13F grown at different temperatures, showing its potential suitability for cultivation in a temperate climate zone. We observed a decrease of gamma-linolenic acid favouring palmitic acid in the case of strain O9.13F compared to tropical strains. Comparative genomics showed no unique genes had been found for the Siberian strain related to its tolerance to low temperatures. In addition, this strain does not possess a different set of genes associated with the salinity stress response from those typically found in tropical strains. We confirmed the absence of plasmids and functional prophage sequences. The genome consists of a 4.94 Mbp with a GC% of 44.47% and 5355 encoded proteins. The Arthrospira/Limnospira strain O9.13F presented in this work is the first representative of a new clade III based on the 16S rRNA gene, for which a genomic sequence is available in public databases (PKGD00000000).
... We have detected junction pores in the cells of P. tunnelli with TEM. The finding of large pores on the sheath surface near the cross walls where junction pores occur should be investigated more thoroughly with freeze substitution and freeze fracture methods to resolve interacting proteins (Hoiczyk & Baumeister 1997;Hoiczyk & Hansel 2000). It is hoped that this work will help resolve questions related to the unique motility of this cyanobacteria. ...
The Laguna Madre-Tamaulipas is the world’s largest hypersaline lagoon and is equally located in south Texas, USA, and Tamaulipas, Mexico. Over 40% of its American surface area is covered by wind-tidal sand flats containing cyanobacteria as the major autotrophic component of the microbial community. A recent laboratory examination of these microbial mats revealed the presence of a boring, filamentous cyanobacterium which, in culture, ‘drilled’ through an agar substrate. The observed clockwise/counterclockwise
rotation, gliding, and boring ability of the filaments was atypical for any known cyanobacterium. The isolate from Laguna Madre produces nine toxins that were originally described from both marine and freshwater habitats. Presence of a diverse array of bioactive metabolites in our unialgal culture agrees with earlier work demonstrating diverse toxin production by marine cyanobacteria. Initial comparison of this new taxon with its polyphasically closest genus, Oxynema, showed several morphological,
physiological, and phylogenetic differences, warranting establishment of a new family
Laspinemaceae, containing Perforafilum tunnelli gen. & sp. nov.
... Dilution series from 100 µg ml −1 to 5 ng ml −1 were prepared for the geosmin standard to test the response and sensitivity of the GC-MS method. Geosmin was identified using the mass spectra libraries, NIST 21,27,107,147 and Wiley 229. ...
Cyanobacteria are ubiquitous organisms with a relevant contribution to primary production in all range of habitats. Cyanobacteria are well known for their part in worldwide occurrence of aquatic blooms while producing a myriad of natural compounds, some with toxic potential, but others of high economical impact, as geosmin. We performed an environmental survey of cyanobacterial soil colonies to identify interesting metabolic pathways and adaptation strategies used by these microorganisms and isolated, sequenced and assembled the genome of a cyanobacterium that displayed a distinctive earthy/musty smell, typical of geosmin, confirmed by GC-MS analysis of the culture’s volatile extract. Morphological studies pointed to a new Oscillatoriales soil ecotype confirmed by phylogenetic analysis, which we named Microcoleus asticus sp. nov. Our studies of geosmin gene presence in Bacteria, revealed a scattered distribution among Cyanobacteria, Actinobacteria, Delta and Gammaproteobacteria, covering different niches. Careful analysis of the bacterial geosmin gene and gene tree suggests an ancient bacterial origin of the gene, that was probably successively lost in different time frames. The high sequence similarities in the cyanobacterial geosmin gene amidst freshwater and soil strains, reinforce the idea of an evolutionary history of geosmin, that is intimately connected to niche adaptation.
... The cell-surface processes for Fe-siderophore uptake may accelerate this dissolution of Fe from dust (Basu and Shaked 2018;Polyviou et al. 2018a). Proteins with a HTCaBs domain, which occur in tandem repeats to form a parallel β-roll structure, have been reported to aid the mobilization of Fe from dust (Brahamsha 1996;Hoiczyk and Baumeister 1997;Polyviou et al. 2018a). In the present study, the acquisition systems of both organically complexed Fe and particulate Fe (e.g., TonB-ExbBD, Fec/FhuD, Tery_3823-3826, and HTCaBs proteins) were significantly upregulated in response to OA in Fe-limited T. erythraeum (Fig. 5). ...
Growth and dinitrogen (N2) fixation of the globally important diazotrophic cyanobacteria Trichodesmium are often limited by iron (Fe) availability in surface seawaters. To systematically examine the combined effects of Fe limitation and ocean acidification (OA), T. erythraeum strain IMS101 was acclimated to both Fe-replete and Fe-limited concentrations under ambient and acidified conditions. Proteomic analysis showed that OA affected a wider range of proteins under Fe-limited conditions compared to Fe-replete conditions. OA also led to an intensification of Fe deficiency in key cellular processes (e.g., photosystem I and chlorophyll a synthesis) in already Fe-limited T. erythraeum. This is a result of reallocating Fe from these processes to Fe-rich nitrogenase to compensate for the suppressed N2 fixation. To alleviate the Fe shortage, the diazotroph adopts a series of Fe-based economic strategies (e.g., upregulating Fe acquisition systems for organically complexed Fe and particulate Fe, replacing ferredoxin by flavodoxin, and using alternative electron flow pathways to produce ATP). This was more pronounced under Fe-limited-OA conditions than under Fe limitation only. Consequently, OA resulted in a further decrease of N2- and carbon-fixation rates in Fe-limited T. erythraeum. In contrast, Fe-replete T. erythraeum induced photosystem I (PSI) expression to potentially enhance the PSI cyclic flow for ATP production to meet the higher demand for energy to cope with the stress caused by OA. Our study provides mechanistic insight into the holistic response of the globally important N2-fixing marine cyanobacteria Trichodesmium to acidified and Fe-limited conditions of future oceans.
... HTCaB domains occur in tandem repeats in proteins that can form a parallel β roll structure (Baumann et al., 1993;Lilie et al., 2000) and are exported from the cell to function as haemolysins, cyclolysins, leukotoxins and metallopeptidases (Boehm et al., 1990;Duong et al., 1992;Rose et al., 1995). Further they may have adhesive properties (Sánchez-Magraner et al., 2007) and roles in motility (Brahamsha and Haselkorn, 1996;Hoiczyk and Baumeister, 1997;Pitta et al., 1997). These characteristics, alongside the differential regulation observed here ( Figure 5A) suggest an important function of the proteins in Fe metabolism and a putative role in attachment to particles or mobilization of Fe from the dust. ...
The marine cyanobacterium Trichodesmium sp. accounts for approximately half of the annual ‘new’ nitrogen introduced to the global ocean but its biogeography and activity is often limited by the availability of iron (Fe). A major source of Fe to the open ocean is Aeolian dust deposition in which Fe is largely comprised of particles with reduced bioavailability over soluble forms of Fe. We report that Trichodesmium erythraeum IMS101 has improved growth rate and photosynthetic physiology and down-regulates Fe-stress biomarker genes when cells are grown in the direct vicinity of, rather than physically separated from, Saharan dust particles as the sole source of Fe. These findings suggest that availability of non-soluble forms of dust-associated Fe may depend on cell contact. Transcriptomic analysis further reveals unique profiles of gene expression in all tested conditions, implying that Trichodesmium has distinct molecular signatures related to acquisition of Fe from different sources. Trichodesmium thus appears to be capable of employing specific mechanisms to access Fe from complex sources in oceanic systems, helping to explain its role as a key microbe in global biogeochemical cycles.
... However, genomic studies showed the presence of genes encoding proteins containing an RTX-motif, that is characteristic for hemolysins (Fujisawa et al., 2010;Cheevadhanarak et al., 2012), though toxicity analysis proved that Arthrospira is safe to consume (FDA, 2002). RTXmotif proteins are also thought to play a role in plant nodulation and cyanobacteria motility, which may explain their presence in Arthrospira genomes (Hoiczyk and Baumeister, 1997;Linhartová et al., 2010). ...
The cyanobacterial genus Arthrospira appears very conserved and has been divided into five main genetic clusters on the basis of molecular taxonomy markers. Genetic studies of seven Arthrospira strains, including genome sequencing, have enabled a better understanding of those photosynthetic prokaryotes. Even though genetic manipulations have not yet been performed with success, many genomic and proteomic features such as stress adaptation, nitrogen fixation, or biofuel production have been characterized. Many of above-mentioned studies aimed to optimize the cultivation conditions. Factors like the light intensity and quality, the nitrogen source, or different modes of growth (auto-, hetero-, or mixotrophic) have been studied in detail. The scaling-up of the biomass production using photobioreactors, either closed or open, was also investigated to increase the production of useful compounds. The richness of nutrients contained in the genus Arthrospira can be used for promising applications in the biomedical domain. Ingredients such as the calcium spirulan, immulina, C-phycocyanin, and γ-linolenic acid (GLA) show a strong biological activity. Recently, its use in the fight against cancer cells was documented in many publications. The health-promoting action of “Spirulina” has been demonstrated in the case of cardiovascular diseases and age-related conditions. Some compounds also have potent immunomodulatory properties, promoting the growth of beneficial gut microflora, acting as antimicrobial and antiviral. Products derived from Arthrospira were shown to successfully replace biomaterial scaffolds in regenerative medicine. Supplementation with the cyanobacterium also improves the health of livestock and quality of the products of animal origin. They were also used in cosmetic preparations.
... In related filamentous cyanobacteria where ultrastructure studies are available (Hoiczyk and Baumeister, 1995, 1997, 1998, the outermembrane exit channels for PilA on either side of a cell junction are within approximately 50 nm, and oriented at approximately a 458 angle with respect to the long axis of the filament, away from the source cell and toward the adjacent cell. Because of this close proximity and orientation, it is not technically feasible to definitively determine whether PilA is emanating from the leading or lagging cell poles using conventional fluorescence microscopy. ...
Many cyanobacteria exhibit surface motility powered by type 4 pili (T4P). In the model filamentous cyanobacterium Nostoc punctiforme, the T4P systems are arrayed in static, bipolar rings in each cell. The chemotaxis-like Hmp system is essential for motility and the coordinated polar accumulation of PilA on cells in motile filaments, while the Ptx system controls positive phototaxis. Using transposon mutagenesis, a gene, designated hmpF, was identified as involved in motility. Synteny among filamentous cyanobacteria and the similar expression patterns for hmpF and hmpD imply that HmpF is part of the Hmp system. Deletion of hmpF produced a phenotype distinct from other hmp genes, but indistinguishable from pilB or pilQ. Both an HmpF-GFPuv fusion protein, and PilA, as assessed by in situ immunofluorescence, displayed coordinated, unipolar localization at the leading pole of each cell. Reversals were modulated by changes in light intensity and preceded by the migration of HmpF-GFPuv to the lagging cell poles. These results are consistent with a model where direct interaction between HmpF and the T4P system activates pilus extension, the Hmp system facilitates coordinated polarity of HmpF to establish motility, and the Ptx system modulates HmpF localization to initiate reversals in response to changes in light intensity. This article is protected by copyright. All rights reserved.
... The cell wall of cyanobacteria is generally composed of the external layer, outer membrane, and peptidoglycan layer (for more details see: Hoiczyk and Baumeister, 1995; Lee, 2008; Baulina, 2012). Particular components have various functions (Benz and Bauer, 1988; Hoiczyk and Baumeister, 1997). As far as filament stiffness is concerned, the most important component of the cell wall seems to be the peptidoglycan layer consisting of N-acetylmuramic acid, Nacetylglucosamine , and amino acids (for more details see Woitzik et al., 1988). ...
Recent studies have shown that the filamentous cyanobacterium Aphanizomenon gracile Lemmermann, strain SAG 31.79, consists of two types of filaments that differ in thickness. These two types are known to vary in resistance to Daphnia magna grazing: thin filaments (<2.5 µm) are more vulnerable to grazing than the thick ones (>2.5 µm). In this study, we investigated whether the difference in the vulnerability to grazing of thin and thick filaments is a result of different thickness of their cell walls, a filament stiffness determinant. We expected thick filaments to have thicker cell walls than the thin ones. Additionally, we analysed whether cell wall thickness correlates with filament thickness regardless of the filament type. A morphometric analysis of cell walls was performed using transmission electron micrographs of ultra-thin sections of the batch-cultured cyanobacterial material. Our study revealed that the thin type of filaments had thinner cell walls than the thick filaments. Moreover, cell wall thickness was positively correlated with filament thickness. TEM (transmission electron microscopy) observations also revealed that the thin type of filaments was often at different stages of autocatalytic cell destruction, which was mainly manifested in the increase in cell vacuolization and degradation of the cytoplasm content. Based on our findings, we assume that previously reported higher resistance of thick filaments to Daphnia grazing results from greater stiffness and excellent physiological conditions of thick filaments.
... Gliding motility and cell-cell contacts in cyanobacteria are also associated with secreted and/or cell surface proteins. The surface of the gliding cyanobacterium Phormidium uncinatum contains fi brils on top of its S-layer surface (Smarda et al. 2002) that are comprised of the rodshaped 66 kDa protein oscillin (Hoiczyk and Baumeister 1997) . Evidence supports the role of this protein as a calcium-binding glycoprotein; strains that do not produce the protein lose motility. ...
The region of space at the periphery of cyanobacterial cells is the interface between the environment and intracellular processes. This metaspace may include a structure appressed to the outer wall and membrane, such as an extracellular polysaccharide (EPS), a structural and/or physiological discontinuity in the control of flow metabolites, as well as a temporal flux that accompanies stress or cell division. The functional framework within this region is designed to recognize environmental perturbations and relay physical and biochemical information to the cell interior, and perhaps to the cell community, for the appropriate physiological response. Communication between the environment and the cells is thus initiated within this extracellular milieu, which is therefore an important spatial domain in the cyanobacteria. The ECM of cyanobacterial cells is multifaceted. It is not only a complex and dynamic mixture of polysaccharides, proteins, cell remnants and lower molecular weight secondary metabolites, but a hyperspace that tunes seasonal as well as short-term stochastic modulations in environmental conditions. Such stresses result in changes in both the composition and organization of the matrix as cyanobacterial cells adjust to the environmental perturbations. This chapter provides a critical appraisal of the ecology and evolution of the cyanobacterial ECM compared with other prokaryotes. Emphasis is placed on how little is understood about this “occupied space” and several hypotheses and examples are presented in an effort to promote additional investigations of this oft-ignored interface.
... Thus, the protein content of CPS was higher under lower light intensity. Additionally, cyanobacterial extracellular glycoprotein or polypeptide may influence the motility of algal filament (Brahamsha 1996;Hoiczyk & Baumeister 1997). Microcoleus vaginatus often vertically migrates within BSCs according to UV (Bebout & Garcia-Pichel, 1995). ...
Extracellular polymeric substances (EPS) acting as cementing substance (chelator) of biological soil crusts (BSCs) are a considerable amount of metabolic investment for cyanobacteria. However, their partition mechanism has long been overlooked. In this paper, we investigated the effects of light intensity on production, distribution pattern, carbohydrate and protein contents, monosaccharide composition, and topographical structures of EPS from Microcoleus vaginatus, the first dominant cyanobacterium of BSCs. The results showed that the amounts of released polysaccharides (RPS), capsular polysaccharides (CPS), and total EPS increased with enhanced light intensity. The weaker the light, the higher the ratio of CPS to EPS and the lower the ratio of RPS to EPS. Although the highest light intensity significantly reduced the charge distribution in RPS, it had no effect on the charge distribution of CPS. For monosaccharide composition, although light intensity did not influence either RPS or CPS, the proportions of main components were still different. Carbohydrate, glucose, galactose, and arabinose were most abundant in RPS; whereas, glucose and galactose were highest in CPS. Comparison with protein content in RPS, carbohydrate was dominant in water eluates under 40 mu E m(-2) s(-1), whereas protein was dominant in 1.0 M NaCl eluates. Carbohydrate was dominant in both eluates under 80 mu E m(-2) s(-1). Protein content of the same fraction in CPS generally decreased with increasing light intensity. The images from atomic force microscopy showed that light intensity significantly affected the molecular weight of EPS, as well as the topographical structures, namely, long chains only found in RPS at 80 mu E m(-2) s(-1) and CPS at 4 mu E m(-2) s(-1). Short chains and irregular lumps occurred under all test light intensities. The above results strongly suggested that light intensity not only affects the EPS yield but also its partition pattern, component, and spatial structure. Therefore, light intensity also likely influences BSCs gluing cohesion.
... The junctional pores that seemed to be inclined with respect to the axis of the filament (Figure 3D) generate a helical flow of mucilage surrounding the filament (Figure 9A). It has been suggested that the helical flow of mucilage was directed by the rows of oscillin fibrils [21], which were visualized by a cryoEM technique in P. uncinatum. Unfortunately, our current electron microscopic procedure poorly preserves the surface structures, such as the S-layer and oscillin fibrils, which were not visible inFigure 3. We are not sure if inclined junctional pores (this is still a hypothesis, but they look inclined inFigure 3D) alone can produce a helical flow of mucilage sufficient for driving the rotation and movement of a filament. ...
Motile filamentous cyanobacteria, such as Oscillatoria, Phormidium and Arthrospira, are ubiquitous in terrestrial and aquatic environments. As noted by Nägeli in 1860, many of them form complex three-dimensional or two-dimensional structures, such as biofilm, weed-like thalli, bundles of filaments and spirals, which we call supracellular structures. In all of these structures, individual filaments incessantly move back and forth. The structures are, therefore, macroscopic, dynamic structures that are continuously changing their microscopic arrangement of filaments. In the present study, we analyzed quantitatively the movement of individual filaments of Phormidium sp. KS grown on agar plates. Junctional pores, which have been proposed to drive cell movement by mucilage/slime secretion, were found to align on both sides of each septum. The velocity of movement was highest just after the reversal of direction and, then, attenuated exponentially to a final value before the next reversal of direction. This kinetics is compatible with the "slime gun" model. A higher agar concentration restricts the movement more severely and, thus, resulted in more spiral formation. The spiral is a robust form compatible with non-homogeneous movements of different parts of a long filament. We propose a model of spiral formation based on the microscopic movement of filaments.
... This suggests that, within the time of experimental runs, a major part of Ca 2C flux is not a result of calcite precipitation, but due to binding to the biofilm by other means, i.e., organic complexes, pH-dependent deprotonation and/or primary production of carboxylated polymers ( One explanation for the differences between both strains might be as follows: Tychonema SAG 2388 shows a polysaccharide sheath and gliding motility, while Synechococcus RCK4 has a thin sheath only, and is nonmotile. The high Ca 2C removal by Tychonema therefore might be related either to Ca 2C binding to sheath exopolymers and/or Oscillin, a Ca 2C -binding glycoprotein involved in gliding motility (Hoiczyk 2000;Hoiczyk and Baumeister 1997). ...
Effect of variable PCO2 on Ca2+ removal and potential calcification of cyanobacterial biofilms - an experimental microsensor study
... Cyanobacteria secrete various proteins into the culture medium. Extracellular proteases, nucleases, structural components of the outer membrane, and other proteins are among them[1][2][3][4][5]. Previously, we detected seven proteins in the Synechocystis sp. ...
The effects of various stresses (osmotic, salt, low-temperature, high-temperature, and high-light stress) on the amount of mRNA of eight genes encoding the secreted proteins of Synechocystis sp. PCC 6803 were studied. Osmotic stress (0.5 M sorbitol) reduced the amount of all mRNAs, with the exception of slr0924. Supposedly, this gene encodes Tic22, a polypeptide involved in the formation of the transport system for proteins crossing the internal thylakoid membrane on the way to the lumen. Salt stress (0.5 M NaCl) inhibited the expression of all genes for secreted proteins almost completely. Low temperature (20C) did not affect the expression of the sll1891 gene of an unknown function and the slr0924 gene. The high temperature (44C) suppressed the expression of all genes tested. A detailed study of the expression of the sll1694 (pilA1) gene, which encodes the main structural protein of cyanobacterial pili, pilin PilA1, demonstrated that virtually all stresses suppressed its expression. Thus, various stresses were shown to suppress the expression of most genes encoding Synechocystis secreted proteins.
... (a nonflagellated marine cyanobacterial species), has been shown to be glycosylated and to contain N-glycosylation sites (Brahamsha 1996). Oscillin is another motility-associated protein found on the cell surface and which has a glycosylated structure, containing xylose, glucose, rhamnose, fucose, arabinose, and galactose (Hoiczyk and Baumeister 1997). More recently, a glycoprotein has been identified in the toxic cyanobacterium Microcystis aeruginosa (Zilliges et al. 2008). ...
Arabinogalactan proteins (AGP) are a diverse family of proteoglycans associated with the cell surfaces of plants. AGP have been implicated in a wide variety of plant cell processes, including signaling in symbioses. This study investigates the existence of putative AGP in free-living cyanobacterial cultures of the nitrogen-fixing, filamentous cyanobacteria Nostoc punctiforme and Nostoc sp. strain LBG1 and at the symbiotic interface in the symbioses between Nostoc spp. and two host plants, the angiosperm Gunnera manicata (in which the cyanobacterium is intracellular) and the liverwort Blasia pusilla (in which the cyanobacterium is extracellular). Enzyme-linked immunosorbent assay, immunoblotting, and immunofluorescence analyses demonstrated that three AGP glycan epitopes (recognized by monoclonal antibodies LM14, MAC207, and LM2) are present in free-living Nostoc cyanobacterial species. The same three AGP glycan epitopes are present at the Gunnera-Nostoc symbiotic interface and the LM2 epitope is detected during the establishment of the Blasia-Nostoc symbiosis. Bioinformatic analysis of the N. punctiforme genome identified five putative AGP core proteins that are representative of AGP classes found in plants. These results suggest a possible involvement of AGP in cyanobacterial-plant symbioses and are also suggestive of a cyanobacterial origin of AGP.
... The outer surface of gliding cyanobacteria in Oscillatoriaceae consists of a parallel, helically arranged protein array (S-layer).100,101 The S-layer protein, oscillin, required for gliding motility in Phormidium102 is conserved in A. platensis (NIES39_A01430, 42% identity). NIES39_A01430 consists of 19 haemolysin-like Ca2+-binding domains. ...
A filamentous non-N2-fixing cyanobacterium, Arthrospira (Spirulina) platensis, is an important organism for industrial applications and as a food supply. Almost the complete genome of A. platensis NIES-39 was determined in this study. The genome structure of A. platensis is estimated to be a single, circular chromosome of 6.8 Mb, based on optical mapping. Annotation of this 6.7 Mb sequence
yielded 6630 protein-coding genes as well as two sets of rRNA genes and 40 tRNA genes. Of the protein-coding genes, 78% are
similar to those of other organisms; the remaining 22% are currently unknown. A total 612 kb of the genome comprise group
II introns, insertion sequences and some repetitive elements. Group I introns are located in a protein-coding region. Abundant
restriction-modification systems were determined. Unique features in the gene composition were noted, particularly in a large
number of genes for adenylate cyclase and haemolysin-like Ca2+-binding proteins and in chemotaxis proteins. Filament-specific genes were highlighted by comparative genomic analysis.
... In relation to motility in the cyanobacterium , Phormidium uncinatum, a type I system is probably involved in the secretion of oscillin, a Ca 2 binding RTX glycoprotein that has an important role in the locomotion of this organism. Oscillin forms an array of parallel ¢brils on top of the S-layer and acts as a platform for the assembly and attachment of carbohydrate ¢brils that guide the rotation of the bacterium [100]. Based on homology searches of the non-redundant protein database, type I systems can be predicted to be present in several species including the fresh water cyanobacterium Synechocystis sp., the soil bacterium Sphingomonas sp., the hydrogenobacterium Aquifex aeolicus and the spirochete Treponema pallidum [101] . ...
... Limited aspects of motility have been described for cyanobacteria (14,15). Filamentous gliding cyanobacteria, major components of microbial mats and biofilms, require contact with a solid surface and, in certain species, the extracellular glycoprotein, oscillin, for motility (16). An outer membrane protein with features similar to that of oscillin is required for swimming of the marine cyanobacterium Synechococcus WH8102 (17). ...
To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an "inverted motility response" (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.
Filamentous cyanobacteria from the LPP-group (Lyngbya sp., Plectonema sp., Phormidium sp.), diatoms and heterotrophic bacteria are the dominant microorganisms of North Sea microbial mats (Gerdes et al., 1986). Cultivated on solid or liquid media the cyanobacteria produce motile trichomes. They employ motility to optimize their positions in their microhabitat using a number of external factors. Light is the major factor in directing their search for a suitable survival and growth niche. Organic substances and their concentrations may be another trigger. However, chemotaxis is essentially very poorly described in cyanobacteria. A large potential area for research has therefore been left untouched (Kangatharalingam et al., 1991).
In filamentous cyanobacteria, the mechanism of gliding motility is undefined but posited to be driven by a polysaccharide secretion system known as the junctional pore complex (JPC). Recent evidence implies that the JPC is a modified type IV pilus-like structure encoded for in part by genes in the hps locus. To test this hypothesis, we conducted genetic, cytological, and comparative genomics studies on hps and pil genes in Nostoc punctiforme, a species in which motility is restricted to transiently differentiated filaments called hormogonia. Inactivation of most hps and pil genes abolished motility and abolished or drastically reduced secretion of hormogonium polysaccharide, and the subcellular localization of several Pil proteins in motile hormogonia corresponds to the site of the junctional pore complex. The non-motile ΔhpsE-G strain, which lacks three glycosyltransferases that synthesize hormogonium polysaccharide, could be complemented to motility by the addition of medium conditioned by wild-type hormogonia. Based on this result, we speculate that secretion of hormogonium polysaccharide facilitates, but does not provide the motive force for gliding. Both the Hps and Pil homologs characterized in this study are almost universally conserved among filamentous cyanobacteria, with the Hps homologs rarely found in unicellular strains. These results support the theory that Hps and Pil proteins compose the JPC, a type IV pilus-like nanomotor which drives motility and polysaccharide secretion in filamentous cyanobacteria.
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Microcystine sind die wohl bekanntesten cyanobakteriellen Toxine. Sie werden im Wesentlichen durch die im Süßwasser weltweit verbreitete, koloniebildende Gattung Microcystis synthetisiert. Die biologische Funktion dieser Peptide ist jedoch ungeklärt. In dieser Studie wurde die Fragestellung erstmals über einen globalen Ansatz auf molekularer Ebene analysiert. Die proteomischen Analysen zwischen M. aeruginosa PCC 7806/ Wildtyp und einigen Microcystin-freien Mutanten deuten auf eine physiologische Rolle der Microcystine. Microcystine beeinflussen die Abundanz zahlreicher Proteine. Prominentester Vertreter ist RubisCO – Schlüsselenzym des Calvin Zyklus. RubisCO und andere im 2D selektierte Proteine konnten außerdem als mögliche zelluläre Bindepartner des Microcystins identifiziert werden. Möglicherweise bindet MC an bestimmte Cysteinreste dieser Proteine. Mit dem Knockout der mcy-Gene geht außerdem eine Überexpression eines Morphotyp-spezifischen Proteins einher, das MrpC genannt wurde. Dieses Protein vermittelt möglicherweise Zell-Zell-Interaktionen in Microcystis.
Many ABC transporters, composed of appropriate membrane and ABC components, are concerned with import or export of relatively small molecules. In contrast, ABC transporters in bacteria required for secretion of RTX toxins and related proteins have been the exception, seemingly embracing a number of different concepts in order to account for translocation of protein substrates, in some cases with sizes over 400 kDa. In all probability, such substrates, secreted by the socalled type 1 pathway, directly access the interior of the transporter from the cytoplasm, by-passing the bilayer. This chapter reconciles the implications of such mammoth transport substrates, or allocrite with a transport mechanism, which still probably shares many of the same features fundamental to other ABC proteins. It reviews the best-studied examples of the type 1 system, which are in Gram-negative bacteria, where two membranes have to be negotiated. In this case, at least one further auxiliary protein in the outer membrane is required to provide the exit to the external medium. This chapter considers the three major examples of this kind of ABC transporter which have been studied in the most detail, HlyB (HlyA toxin transport), PrtD (protease transport), and HasD (transport of a hemebinding protein, HasA).
With an impressive and unparalleled evolutionary history spanning over two billion years, cyanobacteria have evolved to thrive in a diverse range of habitats and numerically dominate vast regions, such as the open oceans. Impacts of this microbial lineage on our planet have been far-reaching: ancestors of extant cyanobacteria had a pivotal role not only in the establishment of oxygen as a major component of the atmosphere, but in the rise of embryophytic algae and land plants. The evolution of innovative cellular structure and function in response to abiotic and biotic selection pressures has resulted in a striking diversity in cyanobacteria. Given the fact that the relationship between form and function is complex, and that cellular structures are often multifunctional and dynamic, this review uses a comparative approach in understanding how biological functions or strategies impact cellular architecture. Notably, differences existing in cellular architecture among genera, and even between strains of the same genus, often reflect evolutionary innovations that have permitted a group to flourish in a particular environment. This review also addresses how a conserved cellular feature, namely the compartmentalization of key functions, has promoted metabolic flexibility and survival. This compartmentalization involves structures such as the internal membranes, heterocysts and carboxysomes, and has permitted the functioning and integration of diverse, and at times, incompatible, processes in a single cell. With heightened interest in utilizing photosynthetic organisms to address global challenges in food and energy resources, research on this remarkable lineage will undoubtedly continue to inform, as well as inspire.
Hemispheroidal domes (microbialites) produced by natural populations of filamentous cyanobacteria belonging to four distinct Phormidium species, and one probable new species of Schizothrix, were collected alive from 0 to 25 in depth habitats in the lagoon of Tikehau atoll (Tuamotu, French Polynesia). This study establishes the biochemical controls on in situ carbonate precipitation processes ("organomineralization" processes) occurring merely in the alveolate network of non-coalescent microfibrils that characterizes the degraded parts of the microbialite domes. Comparison is made between amino-acid and monosaccharide composition of purified cyanobacterially produced organic matter and that of intramineral (soluble and insoluble) organic matrices associated with carbonate precipitates. The results emphasize the importance of dicarboxylic (aspartic and glutamic) acids, released by the decay of cyanobacterial sheaths, in CaCO3 formation and demonstrates that the in situ precipitation of ultra-fine micrites is a highly selective process regarding the available external organic matter. This diagenetic process is thought to result from incipient hydrolysis of cyanobacterial S-layer proteins attached to extracellular polysaccharide fibrils composing the sheath. Taxonomic affinity of cyanobacterial populations responsible for microbialite construction is one of the major factors allowing biochemical discrimination of in situ precipitated carbonates, indicating that specific mucilages or their degradational products are guiding forces for the calcification processes. Another possible source for the formation of carbonate-associated organic matrices is derived from metabolites (e.g., mucus) released in water by lagoon-dwelling benthic organisms.
Filamentous cyanobacteria are capable of gliding motility, but the mechanism of motility is not well defined. Here we present a detailed characterization of the hmp locus from Nostoc punctiforme, which encodes chemotaxis-like proteins. Deletions of hmpB, C, D, and E abolished differentiation of hormogonia under standard growth conditions, but, upon addition of a symbiotic partner exudate, the mutant strains differentiated hormogonium-like filaments that lacked motility and failed to secrete hormogonium specific polysaccharide. The hmp locus is expressed as two transcripts, one originating 5' of hmpA and encompassing the entire hmp locus, and the other 5' of hmpB and encompassing hmpBCDE. The CheA-like HmpE donates phosphate to its own C-terminal receiver domain, and to the CheY-like HmpB, but not to the PatA family CheY-like HmpA. A GFP-tagged variant of each hmp-locus protein localized to a ring adjacent to the septum on each end of the rod-shaped cell. Immunofluorescence demonstrated that PilA localizes to a ring at the junction between cells. The phenotype of the deletion strains, and the localization of the Hmp proteins and the putative PilA protein to rings at the cell junctions are consistent with the hypothesis that these proteins are part of the junctional pore complex observed in a number of filamentous cyanobacteria.
Many filamentous cyanobacteria are capable of gliding motility by an undefined mechanism. Within the heterocyst-forming clades, some strains, such as the Nostoc spp. and Fisherella spp., are motile only as specialized filaments termed hormogonia. Here we report on the phenotype of inactivation of a methyl-accepting chemotaxis-like protein in Nostoc punctiforme, designated HmpD. The gene hmpD was found to be essential for hormogonium development, motility, and polysaccharide secretion. Comparative global transcriptional profiling of the ΔhmpD strain demonstrated that HmpD has a profound effect on the transcriptional program of hormogonium development, influencing approximately half of the genes differentially transcribed during differentiation. Utilizing this transcriptomic data, we identified a gene locus, designated here as hps, that appears to encode for a novel polysaccharide secretion system. Transcripts for the genes in the hps locus are up-regulated in two steps, with the second step dependent on HmpD. Deletion of hpsA, hpsBCD, or hpsEFG resulted in the complete loss of motility and polysaccharide secretion, similar to deletion of hmpD. Genes in the hps locus are highly conserved in the filamentous cyanobacteria, but generally absent in unicellular strains, implying a common mechanism of motility unique to the filamentous cyanobacteria.
Single component equilibrium and uptake of methane and nitrogen in barium exchanged ETS-4, Ba-ETS-4, have been measured in a constant volume apparatus over a wide range of temperatures and pressures followed by binary measurements in a differential adsorption bed at some selected conditions. Similar binary data in two strontium exchanged samples have also been included for which unary data were earlier reported from our laboratory.(12) Adsorbent particles used in these measurements were obtained by pressure-binding very fine crystals of Ba-ETS-4 synthesized in this laboratory, thus giving rise to a bidispersed pore structure with the controlling resistance to diffusion in the micropores. The effect of dehydration temperature on equilibrium and kinetics of aforementioned gases in Ba-ETS-4 has also been investigated. The uncoupled kinetic selectivity of nitrogen over methane in Ba-ETS-4 measured in this study far exceeds the selectivity reported for methane−nitrogen separation in other adsorbents in the literature. Kinetic selectivity including the coupling of equilibrium isotherm and uptake has also been calculated from binary measurements and is found to be equally encouraging. The impact of isotherm models on the concentration dependence of micropore diffusivity has been analyzed on the basis of chemical potential gradient as the driving force for diffusion. Binary equilibrium and kinetic models based on parameters independently established from unary experiments have been proposed that are able to explain the transport mechanism and capture the essential features of measured binary data.
The Saprospira sp. strain SS98-5 cells form colonies on a nutrient-rich agar medium, but are motile by gliding under low-nutrient condition and then numerous microtubule-like fibril structures are found intracellularly. The fibril structures are composed of a proteinous subunit SCFP; this gene was cloned previously. Here, the organization of ORFs adjacent to the SS98-5 SCFP gene and its transcription were investigated, and a SCFP gene homolog was cloned from Saprospira sp. SS03-4, a relative strain of SS98-5. The SCFP gene was also encoded within the SS03-4 chromosome, and grouped into a phage tail sheath protein family, suggesting its bacteriophage genome origin. Four ORFs adjacent to the SCFP gene were identified and their organization was in common with several prokaryotes. The SCFP mRNA was detected by reverse transcriptase polymerase chain reaction from gliding and non-gliding cells, implying that the SCFP gene was transcribed independent of existence or absence of the fibril structures.
A synthesis of available data on the morphological diversity of polykrikoid dinoflagellates allowed us to formulate a hypothesis of relationships that help explain character evolution within the group. Phylogenetic analyses of new SSU rDNA sequences from Pheopolykrikos beauchampii Chatton, Polykrikos kofoidii Chatton, and Polykrikos lebourae Herdman helped refine this hypothetical framework. Our results demonstrated that “pseudocolonies” in dinoflagellates evolved convergently at least three times independently from different Gymnodinium-like ancestors: once in haplozoans; once in Ph. beauchampii; and at least once within a lineage containing Ph. hartmannii, P. kofoidii, and P. lebourae. The Gymnodiniales sensu stricto was strongly supported by the data, and the type species for the genus, namely Gymnodinium fuscum (Ehrenb.) F. Stein, formed the nearest sister lineage to a well-supported Polykrikos clade. The best synapomorphy for the Polykrikos clade was the presence of two nuclei irrespective of zooid number. Two unidentified Gymnodinium species formed the nearest sister clade to Ph. beauchampii, which has four nuclei and four zooids per pseudocolony. The chain-forming dinoflagellate G. catenatum L. W. Graham branched closely to the clade containing all members of Polykrikos and Pheopolykrikos, suggesting that an ancestral capacity toward chain formation existed before the evolution of pseudocolonies in this group. Our results also clarified the phylogenetic significance of nematocysts, ocelloids, and photosynthesis in reconstructing the evolution of polykrikoids and warnowiids. The molecular phylogenies exposed taxonomic problems associated with Polykrikos, Pheopolykrikos, and Gymnodinium, and suggested that a revision for some of these genera is warranted.
The cyanobacteria, like all organisms. sense and respond to changes in their environment. This chapter focuses on one aspect.
an analysis of the signal transduction pathways that permit the appropriate adaptive response to be set in train. The mechanisms
by which prokaryotes respond to their environments largely represent variations and combinations of common themes. The cyanobacteria
are no exception. but in many cases there are modifications to the basic signal transduction pathways that are related to
the photoautotrophic lifestyle. This is particularly true with respect to the need for integration of the intracellular signals.
and the possibility that the thylakoids represent the site for this central process is examined. Whilst transcriptional mechanisms
are an important component of the signal transduction processes. signalling elements may operate at any level of the genotype
to phenotype pathway and the importance of the post-translational modification of proteins. particularly by phosphorylation,
is recognised. The potential to understand cyanobacterial signalling mechanisms has been significantly enhanced by genomic
sequence information, and where possible this has been utilised to confirm or extend the present empirical knowledge.
Based on carbohydrate histochemistry, including the use of lectins, and TEM, the study describes the distribution of terminal sugars in different structures of the demosponge Chondrilla nucula. The results of the general and specific carbohydrate histochemical approaches confirmed the presence of acidic and neutral glycoconjugates in the cells, and, with declining amounts from the ectosome to the mesohyl, in the extracellular matrix (ECM). AB-PAS staining indicated acidic complex carbohydrates particularly in the exopinacoderm, and more neutral ones in the cells and the ECM of the mesohyl. The PO-lectins applied demonstrated a general spectrum of free saccharide residues (α-d-mannose, α-/β-d-N-acetylglucosamine, α-d-N-acetylgalactosamine, α-d-galactose, β-d-galactose) in both sponge parts; α-l-fucose was only distinct in the ectosome. Sialic acids [siaα(2,3)-galactose, siaα(2,6)-N-acetylgalactosamine] were dominant in the very thin exopinacoderm, indicating O-linked high molecular weight glycoproteins. In this way a glycophysiologically ‘rigid’ outer mucus cover is developed as protection against mechanical hazards. Some of the free sugars (α-d-mannose, N-acetylglucosamine, N-acetylgalactosamine β-d-galactose, α-l-fucose) are known to prevent the adherence of different bacteria and fungi to cellular surfaces. Thus a high concentration of such sugars, may impede massive attacks of micro-inhabitants on mobile sponge cells, pinacocytes, and the exopinacoderm layer.
Many diverse bacteria move in the absence of any visible locomotory organelle by amechanism
termed gliding. Gliding motility requires contact with asolid substrate and occurs in the direction
parallel to the long axis of the bacterial cell. Recent research indicates that bacteria use at least two
different mechanisms to glide. One mechanism, termed social motility, is based on the extension and retraction
of pili. The other mechanism, termed adventurous motility, involves the secretion of slime from aspecialized
organelle called the junctional pore complex. This work discusses the possible role of the junctional pore
complex in gliding motility of cyanobacteria, myxobacteria, and the alphaproteobacterium Gluconacetobacter.
The cell walls of a number of filamentous, gliding cyanobacteria of the genus Oscillatoria were examined by transmission electron microscopy of ultrathin sections, of freeze-etched replicas, and of whole cells crushed between glass slides and negatively stained. All three techniques revealed the presence of a highly ordered array of parallel fibrils, seen in transverse sections to be situated between the peptidoglycan and the outer membrane. Approximately 200 individual fibrils, each 25 to 30 nm in width, form a parallel, helical array that completely surrounds each cyanobacterial filament, running at an angle of 25 to 30 degrees to its long axis. This highly regular arrangement of the fibrillar layer may imply some underlying symmetry responsible for its organization. A possible source of such symmetry would be the peptidoglycan, and some form of interaction between this layer and the fibrils might provide the necessary scaffolding for the fibrillar array. In crushed, negatively stained samples of fresh cells, individual fibrils were seen outside the filament, released from the cell wall. These released fibrils were of the same width as those observed in situ but were in short lengths, mostly of 100 to 200 nm, and were invariably bent, sometimes even into U shapes, implying great flexibility. Negative staining of released fibrils showed no evidence that they were hollow tubes but did give some indication of a substructure, implying that they were composed of many subunits. The function of this fibrillar array is unknown, although its position in the cell wall, as well as the correspondence between the angle of the fibrils with respect to the long axis of the filament and the rotation of the filament during gliding, may imply an involvement in gliding motility.
We have recently shown that phototactic movement in the unicellular cyanobacterium Synechocystis sp. PCC6803 requires type IV pilins. To elucidate further type IV pilus-dependent motility, we inactivated key genes implicated in pilus biogenesis and function. Wild-type Synechocystis sp. PCC6803 cells have two morphologically distinct pilus types (thick and thin pili). Of these, the thick pilus morphotype, absent in a mutant disrupted for the pilin-encoding pilA1 gene, appears to be required for motility. The thin pilus morphotype does not appear to be altered in the pilA1 mutant, raising the possibility that thin pili have a function distinct from that of motility. Mutants disrupted for pilA2, which encodes a second pilin-like protein, are still motile and exhibit no difference in morphology or density of cell-surface pili. In contrast, inactivation of pilD (encoding the leader peptidase) or pilC (encoding a protein required for pilus assembly) abolishes cell motility, and both pilus morphotypes are absent. Thus, the PilA1 polypeptide is required for the biogenesis of the thick pilus morphotype which, in turn, is necessary for motility (hence we refer to them as type IV pili). Furthermore, PilA2 is critical neither for motility nor for pilus biogenesis. Two genes encoding proteins with similarity to PilT, which is considered to be a component of the motor essential for type IV pilus-dependent movement, were also inactivated. A pilT1 mutant is (i) non-motile, (ii) hyperpiliated and (iii) accumulates higher than normal levels of the pilA1 transcript. In contrast, pilT2 mutants are motile, but are negatively phototactic under conditions in which wild-type cells are positively phototactic.
Cyanobacteria are a morphologically diverse group of phototrophic prokaryotes that are capable of a peculiar type of motility characterized as gliding. Gliding motility requires contact with a solid surface and occurs in a direction parallel to the long axis of the cell or filament. Although the mechanistic basis for gliding motility in cyanobacteria has not been established, recent ultrastructural work has helped to identify characteristic structural features that may play a role in this type of locomotion. Among these features are the distinct cell surfaces formed by specifically arranged protein fibrils and organelle-like structures, which may be involved in the secretion of mucilage during locomotion. The possible role of these ultrastructural features, as well as consequences for understanding the molecular basis of gliding motility in cyanobacteria, are the topic of this review.
We investigated the spectrum of secreted proteins in the cyanobacterium Synechocystis, and identified these proteins by amino-terminal sequencing. In total, seven sequences have been determined that corresponded to the proteins Sll0044, Sll1694, Sll1891, Slr0924, Slr0841, Slr0168, and Slr1855. The protein Sll1694 of 18 kDa that formed one of two major bands on SDS-PAGE was identified as cyanobacterial pilin, PilA. The amino-terminal sequence of another protein that formed a second major band was blocked. The analysis of the data revealed that five of seven proteins had distinct putative leader sequences for secretion.
We generated random Tn5 mutations in Synechocystis sp. PCC 6803 in search for genes involved in the signal transduction cascade for the cyanobacterial gliding motility. One of the non-gliding Tn5 mutants, S1-105, had an insertional inactivation in the slr1044 gene encoding a putative methyl-accepting chemotaxis protein. Interposon mutation on the slr1044 (named ctr1) in the bacterium also eliminated gliding motility. In the interposon mutant, the expression of pilA1 was 5-fold decreased compared with that of wild-type and thick pili, that are believed to be the motor for gliding, could not be observed by an electron microscope. Therefore, we suggest that the Ctr1 protein functions as a transducer that regulates the expression of pilA1, and thus is required for the biogenesis of thick pili.
The mechanisms responsible for bacterial gliding motility have been a mystery for almost 200 years. Gliding bacteria move actively over surfaces by a process that does not involve flagella. Gliding bacteria are phylogenetically diverse and are abundant in many environments. Recent results indicate that more than one mechanism is needed to explain all forms of bacterial gliding motility. Myxococcus xanthus "social gliding motility" and Synechocystis gliding are similar to bacterial "twitching motility" and rely on type IV pilus extension and retraction for cell movement. In contrast, gliding of filamentous cyanobacteria, mycoplasmas, members of the Cytophaga-Flavobacterium group, and "adventurous gliding" of M. xanthus do not appear to involve pili. The mechanisms of movement employed by these bacteria are still a matter of speculation. Genetic, biochemical, ultrastructural, and behavioral studies are providing insight into the machineries employed by these diverse bacteria that enable them to glide over surfaces.
The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
Nodulation and host-specific recognition of legumes such as peas and Vicia spp. are encoded by the nodulation (nod) genes of Rhizobium leguminosarum biovar viciae. One of these genes, nodO, has been shown to encode an exported protein that contains a multiple tandem repeat of a nine amino acid domain. This domain was found to be homologous to repeated sequences in a group of bacterial exported proteins that includes haemolysin, cyclolysin, leukotoxin and two proteases. These proteins are secreted by a mechanism that does not involve an N-terminal signal peptide. The NodO protein is present in the growth medium of Rhizobium bacteria induced for nod gene expression, and partial protein sequencing of the purified protein showed that there is no N-terminal cleavage of the exported protein. It has been suggested that the internally repeated domain of haemolysin may be involved in Ca2(+)-mediated binding to erythrocytes and we show that the NodO protein can bind 45Ca2+. It is proposed that the NodO protein may interact directly with plant root cells in a Ca2(+)-dependent way, thereby mediating an early stage in the recognition that occurs between Rhizobium and its host legume.
The cell walls of four gliding filamentous Oscillatoriaceae species comprising three different genera were studied by freeze substitution, freeze fracturing, and negative staining. In all species, the multilayered gram-negative cell wall is covered with a complex external double layer. The first layer is a tetragonal crystalline S-layer anchored on the outer membrane. The second array is formed by parallel, helically arranged surface fibrils with diameters of 8 to 12 nm. These fibrils have a serrated appearance in cross sections. In all cases, the orientation of the surface fibrils correlates with the sense of revolution of the filaments during gliding, i.e., clockwise in both Phormidium strains and counterclockwise in Oscillatoria princeps and Lyngbya aeruginosa. The lack of longitudinal corrugations or contractions of the surface fibrils and the identical appearances of motile and nonmotile filaments suggest that this structure plays a passive screw thread role in gliding. It is hypothesized that the necessary propulsive force is generated by shear forces between the surface fibrils and the continuing flow of secreted extracellular slime. Furthermore, the so-called junctional pores seem to be the extrusion sites of the slime. In motile cells, these pores exhibit a different staining behavior than that seen in nonmotile ones. In the former, the channels of the pores are filled with electron-dense material, whereas in the latter, the channels appear comparatively empty, highly contrasting the peptidoglycan. Finally, the presence of regular surface structures in other gliding prokaryotes is considered an indication that comparable structures are general features of the cell walls of gliding microbes.
The three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa, a zinc metalloprotease, has been solved to a resolution of 1.64 A by multiple isomorphous replacement and non-crystallographic symmetry averaging between different crystal forms. The molecule is elongated with overall dimensions of 90 x 35 x 25 A; it has two distinct structural domains. The N-terminal domain is the proteolytic domain; it has an overall tertiary fold and active site zinc ligation similar to that of astacin, a metalloprotease isolated from a European freshwater crayfish. The C-terminal domain consists of a 21-strand beta sandwich. Within this domain is a novel 'parallel beta roll' structure in which successive beta strands are wound in a right-handed spiral, and in which Ca2+ ions are bound within the turns between strands by a repeated GGXGXD sequence motif, a motif that is found in a diverse group of proteins secreted by Gram-negative bacteria.
Active motility is one of the fundamental characteristics of many microorganisms and involves the interplay of a number of cellular functions that enable an organism to move in a coordinated fashion. Therefore, cell motility is a very complex and broad topic in research, one that involves problems associated with motor design, steering and control, and energy supply and distribution. As early as the Precambrian era, organisms in aquatic habitats developed two basic types of motility: flagellar and gliding motility. The first one is the subject of other chapters in this volume; the second one is the topic of this chapter.
The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
Motility of the marine filamentous cyanobacterium Spirulina subsalsa is both Ca2+ and Na+ dependent, and replacement of Na+ by mannitol arrests it. The data presented suggest that Ca2+ interacts with sites on the surface of the cell membrane. The inhibitory effect of dicyclohexylcarbodiimide (DCCD) hints at the possibility that the role of Ca2+ may be associated with a membrane bound Ca-ATPase. Motility is pH dependent, being nil at pH < 6.5 and > 10.0, with an optimum at 8.5. Norepinephrine abolishes most of the inhibitory effect of low pH on motility. Ca2+ has an “all-or-none” effect on motility that is triggered at 5 mM. Acetylcholine lowers the threshold of Ca2+ necessary for triggering motility.
A new hydrophobic glass-fiber support is presented, which is well suited to the electrophoretic transfer of proteins from polyacrylamide gels and subsequent protein-chemical analysis. Modified glass-fiber sheets are easily prepared by chemical reaction of the surface with poly(methyl-3,3,3-trifluoropropylsiloxane) in trifluoroacetic acid. The modification is stable during electroblotting, amino acid sequence analysis and hydrolysis. The siliconized glass fiber exhibits a high protein-binding capacity, allows the application of well-established staining procedures, and does not interfere with the analytical methods of modern protein chemistry at the low picomole level. Samples separated by electrophoresis and immobilized on hydrophobic supports fail to exhibit any detectable contamination in amino acid sequence analysis hence allowing the high performance of the available protein-chemical methods to be exploited.
The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA. We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats. The resulting mutant proteins were perfectly stable in E. coli and were secreted with the same efficiency as the wild-type HlyA. HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca2+ for activity, as compared to the wild-type haemolysin. Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca2+ concentrations. The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca2+ concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca2+. These data indicate that the repeat domain of haemolysin is responsible for Ca2+-dependent binding of haemolysin to the erythrocyte membrane. A model for the possible functional role of Ca2+ in haemolysis is presented.
Recent discoveries of ultrastructures which might be involved in the gliding movements of blue-green algae have been reviewed, and in the light of these discoveries the role of mucilage secretion in movement has been reconsidered. The formation and behaviour of mucilage rings in filaments ofAnabaena cylindrica is described. The behaviour of the mucilage rings indicates that each cell has an autonomous gliding mechanism which is capable of immediate reversal, and that the gliding mechanism is probably located over the whole surface, rather than at the ends, of the cells. It follows that if mucilage secretion is the cause of movement it must take place over the whole surface of the cell: but if the ends of the cell are the sites of mucilage secretion, as seems likely, then gliding movement must be performed by some other process.
A rather remarkable clumping phenomenon is described which takes place in dense suspensions ofAnabaena. It results from the gliding movements of randomly orientated filaments made mutually adhesive by the mucilage which surrounds them.
A sensitive silver stain for detecting bacterial lipopolysaccharides in polyacrylamide gels is developed by modifying the silver-staining method used for proteins (cf. R. C. Switzer III, C. R. Merril, and S. Shifrin, Anal. Biochem.98, 231–237 (1979). Lipopolysaccharides are analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by visualization with either the modified silver stain or periodic acid-Schiff stain. The lipopolysaccharides are stained dark brown by the silver stain. The silver stain is 500 times more sensitive than the periodic acid-Schiff stain and can detect less than 5 ng of rough type lipopolysaccharides. Analyses of 5μg of smooth-type lipopolysaccharides from Salmonella typhimurium and Escherichia coli O111: B4 show each to have 30–40 components of different molecular weights. The use of a lipopolysaccharide having a known structure and variable numbers of repeating units in the O side chain, such as one of the two lipopolysaccharides mentioned above, as molecular weight markers is proposed for the estimation of the molecular weights of other lipopolysaccharides or their components. The lipopolysaccharides can also be stained grayish green, but become grayish blue with a heavy sample load, using a silver-based color-staining method (D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis2, 135–141 (1981)).
A glycoprotein responsible for the antiphagocytic properties of Campylobacter fetus has been identified by comparing cells of a wild-type strain with those of a mutant lacking this substance. The antiphagocytic component is demonstrable through electron microscopy as a discrete, negatively charged structure on the periphery of the cell. CLT is readily removed from the cell by mild extraction procedures and contributes to the inagglutinability in O antiserum normally displayed by C. fetus. Cells possessing this antigen are refractory to ingestion by macrophages except in the presence of specific antiserum. In its absence maximum phagocytosis occurs without a requirement for opsonins. It is concluded that the antiphagocytic component comprises a critical virulence factor of the bacterium.
Calcium ion induces in the myxobacterium Stigmatella aurantiaca the ability to glide on solid surfaces and to become cohesive (D. F. Gilmore and D. White, J. Bacteriol. 161:113-117, 1985; B. J. Womack, D. F. Gilmore, and D. White, J. Bacteriol. 171:6093-6096, 1989). The addition of calcium ion to the growth medium resulted in the formation of extracellular fibrils, the appearance in the membrane fractions of a 30-kDa protein, and the accumulation in a low-speed centrifugal pellet of 10 polypeptides that cross-reacted with affinity-purified antibody to one of the polypeptides. One of the polypeptides, a 55-kDa protein, was present in the membrane fraction of control cells not incubated with calcium ion and was apparently translocated to the extracellular matrix during incubation in medium containing calcium ion. The 55-kDa protein was immunologically related to a 65-kDa protein located on the fibrils of another myxobacterium, Myxococcus xanthus.
A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.
Publisher Summary Deglycosylation of glycoproteins is necessary for a number of studies such as in the structural determination of polypeptide chains, establishment of structure and function relationships of carbohydrates, 1-5 and their biosynthesis in glycoproteins. In all these studies it is important that the conditions used for chemical deglycosylation do not cause an impairment of the physicochemical integrity of the polypeptide chain. Two chemical reagents in the anhydrous form, trifluoromethanesulfonic acid (TFMS) and hydrogen fluoride (HF), have been employed for the deglycosylation of glycoproteins. TFMS by the modified procedure described herein is definitely superior to HF. It is much more potent, sensitive, convenient to use than HF because it does not require any special handling. To prevent any secondary reactions such as the alkylation of the polypeptide chains during deprotection of the protecting group, the use of anisole was made as a scavenger. This chapter discusses the chemical deglycosylation procedure by TFMS and HF in detail.
A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described. Tricine, used as the trailing ion, allows a resolution of small proteins at lower acrylamide concentrations than in glycine-SDS-PAGE systems. A superior resolution of proteins, especially in the range between 5 and 20 kDa, is achieved without the necessity to use urea. Proteins above 30 kDa are already destacked within the sample gel. Thus a smooth passage of these proteins from sample to separating gel is warranted and overloading effects are reduced. This is of special importance when large amounts of protein are to be loaded onto preparative gels. The omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.
A new hydrophobic glass-fiber support is presented, which is well suited to the electrophoretic transfer of proteins from polyacrylamide gels and subsequent protein-chemical analysis. Modified glass-fiber sheets are easily prepared by chemical reaction of the surface with poly(methyl-3,3,3-trifluoropropylsiloxane) in trifluoroacetic acid. The modification is stable during electroblotting, amino acid sequence analysis and hydrolysis. The siliconized glass fiber exhibits a high protein-binding capacity, allows the application of well-established staining procedures, and does not interfere with the analytical methods of modern protein chemistry at the low picomole level. Samples separated by electrophoresis and immobilized on hydrophobic supports fail to exhibit any detectable contamination in amino acid sequence analysis hence allowing the high performance of the available protein-chemical methods to be exploited.
An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.
Porin from Escherichia coli outer membranes has been analysed by high angle diffuse X-ray diffraction, and by attenuated total reflection infrared spectroscopy. These methods demonstrate independently that the majority of the polypeptide backbone is arranged in anti-parallel beta-pleated sheet structure. The average length of the beta-strands, which are oriented nearly normal to the membrane plane, is estimated to be 10-12 residues, independent of the method used. Although the details of strand arrangement (beta-barrels or stacked sheets) are not as yet known, porin represents the first transmembrane protein for which beta-structure has been established unequivocally.
An improved PAS method for the detection of glycoproteins after electrophoresis on acrylamide or urea-acrylamide gels is described. Stronger oxidizing conditions and a more controlled washing for the removal of periodic acid resulted in both increased staining intensity and band resolution. The method will not stain protein and it appears that a 2–3 μg sample of bound or precipitable carbohydrate would easily be detected. Mucopolysaccharides were not detectable by this method.
A new simple method of detecting calcium binding proteins in a protein mixture is described. A sample which might include calcium binding proteins was subjected to SDS-polyacrylamide gel electrophoresis and then electrophoretically transferred to a nitrocellulose membrane. The membrane was then incubated with ⁴⁵ Ca to detect calcium binding proteins as radioactive bands by autoradiography.
Purified troponin-C, calmodulin, myosin DTNB light chain, and parvalbumin were clearly identified by this method. In the whole homogenate of chicken skeletal muscle, myosin DTNB light chain, troponin-C, and 55K calcium binding protein were found to be radioactive. In the frog skeletal muscle, small molecular weight proteins of approximately 13–15K and 70K protein appeared to be the calcium binding proteins. In the case of the carp skeletal muscle, small molecular weight proteins including parvalbumin and two proteins of about 80K seemed to bind calcium ion. Two high molecular weight calcium binding proteins were present in the scallop striated muscle.
The procedure described can be completed within 24 h and can detect as little as 2 μg of calcium binding protein in the starting sample. Under appropriate conditions it was possible to detect only high affinity calcium binding proteins.
The unicellular cyanobacterium Aphanothece halophytica (PCC 7418) is motile, and spontaneous nonmotile (mot) mutants accumulate when the organism is subcultured. Analysis of mot mutants suggests that a glycoprotein in the cell wall is involved in the motility mechanism. Proteins from the wall fraction of the wild type and five mot clones were analyzed by gradient sodium dodecyl sulfate-acrylamide gel electrophoresis. Four clones were similar to the wild type, and one clone, mot-3, was missing a high-molecular-weight protein (approximately 200,000) and had at least one new polypeptide (160,000). The high-molecular-weight protein stained with periodic acid-Schiff reagent, suggesting that it was a glycoprotein. The absence of the protein in mot-3 did not affect the mechanical strength of the wall, since both mot-3 and wild-type cells were broken at the same rate by controlled cavitation. Several other cyanobacteria were also screened for the presence of glycoproteins. All motile strains have such proteins, although none had an apparent molecular weight as high as that in Aphanothece sp. Some motile strains, such as Oscillatoria limnetica and Phormidium sp., showed very large amounts of glycoproteins; whereas some nonmotile strains, such as Synechococcus sp. (UTEX 625) and Microcystis sp. (PCC 7820), showed no high-molecular-weight glycoproteins.
Certain marine unicellular cyanobacteria of the genus Synechococcus exhibit a unique and mysterious form of motility characterized by the ability to swim in liquid in the absence of flagella. An abundant cell-surface-associated polypeptide that is required for swimming motility by Synechococcus sp. strain WH8102 has been identified, and the gene encoding it, swmA, has been cloned and sequenced. The predicted SwmA protein contains a number of Ca2+-binding motifs as well as several potential N-glycosylation sites. Insertional inactivation of swmA in Synechococcus sp. strain WH8102 results in a loss of the ability to translocate, although the mutant strain, Swm-1, generates torque. This suggests that SwmA functions in the generation of thrust.
The sequence determination of the entire genome of the Synechocystis sp. strain PCC6803 was completed. The total length of the genome finally confirmed was 3,573,470 bp, including the previously reported sequence of 1,003,450 bp from map position 64% to 92% of the genome. The entire sequence was assembled from the sequences of the physical map-based contigs of cosmid clones and of lambda clones and long PCR products which were used for gap-filling. The accuracy of the sequence was guaranteed by analysis of both strands of DNA through the entire genome. The authenticity of the assembled sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA using the assembled sequence data. To predict the potential protein-coding regions, analysis of open reading frames (ORFs), analysis by the GeneMark program and similarity search to databases were performed. As a result, a total of 3,168 potential protein genes were assigned on the genome, in which 145 (4.6%) were identical to reported genes and 1,257 (39.6%) and 340 (10.8%) showed similarity to reported and hypothetical genes, respectively. The remaining 1,426 (45.0%) had no apparent similarity to any genes in databases. Among the potential protein genes assigned, 128 were related to the genes participating in photosynthetic reactions. The sum of the sequences coding for potential protein genes occupies 87% of the genome length. By adding rRNA and tRNA genes, therefore, the genome has a very compact arrangement of protein- and RNA-coding regions. A notable feature on the gene organization of the genome was that 99 ORFs, which showed similarity to transposase genes and could be classified into 6 groups, were found spread all over the genome, and at least 26 of them appeared to remain intact. The result implies that rearrangement of the genome occurred frequently during and after establishment of this species.
Die Glykosylierungsstellen und die Strukturen der Oligosaccharide des Membranglykoproteins Dipeptidylpeptidase IV (CD26)
- A Economou
- W D O Hamilton
- A W B Johnston
- K Feichtinger
Economou, A., Hamilton, W.D.O., Johnston, A.W.B., and Feichtinger, K. (1994) Die Glykosylierungsstellen und die Strukturen der Oligosaccharide des Membranglykoproteins Dipeptidylpeptidase IV (CD26). PhD thesis, Ludwig-Maxi-milians-Universitä t, Munich.
Detection of glycoproteins with PAS-method
- R A Kapitany
- E J Zebrowski
Kapitany, R.A., and Zebrowski, E.J. (1973) Detection of
glycoproteins with PAS-method. Anal Biochem 56: 361-369.
Die Glykosylierungsstellen und die Strukturen der Oligosaccharide des Membranglykoproteins Dipeptidylpeptidase IV (CD26)
- K Feichtinger
Feichtinger, K. (1994) Die Glykosylierungsstellen und die
Strukturen der Oligosaccharide des Membranglykoproteins
Dipeptidylpeptidase IV (CD26). PhD thesis, Ludwig-Maximilians-Universitä t, Munich.