Evaluation of non-cyanobacterial genome sequences for occurrence of genes encoding proteins homologous to cyanophycin synthetase and cloning of an active cyanophycin synthetase from Acinetobacter sp. strain DSM 587

Krehenbrink, Martin; Oppermann-Sanio, Fred-Bernd; Steinbüchel, Alexander

doi:10.1007/s00203-001-0396-9

Cited by 114 publications

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“…Homologs of cyanophycinase are typically found in cyanobacteria where they are used to break down an intracellular granular C and N storage molecule, cyanophycin (Richter et al, 1999). Cyanophycin synthetase and/or cyanophycinase homologs have been detected in about 10% of heterotrophic genomes analyzed (Krehenbrink et al, 2002;Fueser and Steinbuechel, 2007). The acI SAGs analyzed here have the genes required to break down cyanophycin granules and take up the resulting dipeptides and amino acids as a source of energy and N. However, it is unclear whether this cyanophycinase can be secreted, as we were unable to identify an obvious secretion signal peptide.…”

Section: Resultsmentioning

confidence: 83%

Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage

et al. 2014

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Members of the acI lineage of Actinobacteria are the most abundant microorganisms in most freshwater lakes; however, our understanding of the keys to their success and their role in carbon and nutrient cycling in freshwater systems has been hampered by the lack of pure cultures and genomes. We obtained draft genome assemblies from 11 single cells representing three acI tribes (acI-A1, acI-A7, acI-B1) from four temperate lakes in the United States and Europe. Comparative analysis of acI SAGs and other available freshwater bacterial genomes showed that acI has more gene content directed toward carbohydrate acquisition as compared to Polynucleobacter and LD12 Alphaproteobacteria, which seem to specialize more on carboxylic acids. The acI genomes contain actinorhodopsin as well as some genes involved in anaplerotic carbon fixation indicating the capacity to supplement their known heterotrophic lifestyle. Genome-level differences between the acI-A and acI-B clades suggest specialization at the clade level for carbon substrate acquisition. Overall, the acI genomes appear to be highly streamlined versions of Actinobacteria that include some genes allowing it to take advantage of sunlight and N-rich organic compounds such as polyamines, di-and oligopeptides, branched-chain amino acids and cyanophycin. This work significantly expands the known metabolic potential of the cosmopolitan freshwater acI lineage and its ecological and genetic traits.

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Section: Resultsmentioning

confidence: 83%

Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage

et al. 2014

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“…CphA 6308 has been described as having a broad substrate range in vitro (2) and incorporates up to 10 mol% of lysine, replacing arginine in the side chain of CGP, when it is expressed in E. coli (30). In contrast, CGP isolated from the natural host Synechocystis sp.…”

Section: Discussionmentioning

confidence: 99%

“…After the cells were washed three times with PBS for 20 min each, they were postfixed in 1% (wt/vol) osmium tetroxide in 0.1 M PBS (pH 7.3) and washed once with PBS for 20 min. Then, the water was removed by using a graded water/ethanol series (30,50,70,90, and 96%, and absolute ethanol) in which each step lasted about 15 min. To obtain thin sections, the samples were embedded in Spurr resin without propylene oxide (46).…”

Section: Vol 74 2008 Cyanophycin In Transgenic Strains Of S Cerevimentioning

confidence: 99%

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Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Steinle

Oppermann‐Sanio

Reichelt

et al. 2008

Appl Environ Microbiol

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Cyanophycin [multi-L-arginyl-poly(L-aspartic acid) (CGP)] was, for the first time, produced in yeast. As yeasts are very important production organisms in biotechnology, it was determined if CGP can be produced in two different strains of Saccharomyces cerevisiae. The episomal vector systems pESC (with the galactoseinducible promoter GAL1) and pYEX-BX (with the copper ion-inducible promoter CUP1) were chosen to express the cyanophycin synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA 6308 ) in yeast. Expression experiments with transgenic yeasts revealed that the use of the CUP1 promoter is much more efficient for CGP production than the GAL1 promoter. As observed by electrophoresis of isolated CGP in sodium dodecyl sulfate-polyacrylamide gels, the yeast strains produced two different types of polymer: the water-soluble and the water-insoluble CGP were observed as major and minor forms of the polymer, respectively. A maximum CGP content of 6.9% (wt/wt) was detected in the cells. High-performance liquid chromatography analysis showed that the isolated polymers consisted mainly of the two amino acids aspartic acid and arginine and that, in addition, a minor amount (2 mol%) of lysine was present. Growth of transgenic yeasts in the presence of 15 mM lysine resulted in an incorporation of up to 10 mol% of lysine into CGP. Anti-CGP antibodies generated against CGP isolated from Escherichia coli TOP10 harboring cphA 6308 reacted with insoluble CGP but not with soluble CGP, if applied in Western or dot blots.Cyanophycin, also referred to as multi-L-arginyl-poly(L-aspartic acid) or cyanophycin granule polypeptide (CGP), is a nonribosomally synthesized polypeptide consisting of a poly-(aspartic acid) backbone with arginine residues linked to the ␤-carboxyl group of each aspartate by the ␣-amino group (45). CGP is a polydispersed polymer; the molecular size distribution of CGP varies with the producing host strains (1,15,18,20,30,37,52). Due to its branched structure, CGP is not degradable by a wide range of proteinases (45). Biosynthesis of CGP from aspartate and arginine requires only one enzyme, cyanophycin synthetase, which is encoded by cphA (51). CGP is insoluble at neutral pH and under physiological ionic strength, but it is soluble at low (Ͼ3) or high (Ͻ9) pH. Ziegler et al. were the first to observe a water-soluble form of CGP after the heterologous expression of cphA from Desulfitobacterium hafniense strain DSM 10664 in Escherichia coli (52). A detailed study of the solubility behavior of CGP isolated from recombinant E. coli in inorganic salts has been carried out by Füser and Steinbüchel (16). It was shown that the occurrence of the soluble form was not dependent on the origin of cphA or on the host.Recently, several putative applications for CGP and its derivatives have become available, indicating there is a need for its efficient biotechnological production (36,42,43). For the production of CGP at a technical scale, cyanobacteria were shown to be unsuitable due to their low cell...

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“…The detailed genomic examination led to the discovery of a gene annotated as cyanophycin synthetase (cphA) in all three symbiont genomes. This functionally interesting enzyme generates cyanophycin (multi-L-arginylpoly-L-aspartate), a storage biopolymer containing both nitrogen and carbon (Ziegler et al, 1998;Krehenbrink et al, 2002). So far, glycogen and sulfur were the only storage compounds observed in the deep-sea symbionts (Sorgo et al, 2002;Pflugfelder et al, 2005).…”

Section: Gene Functionsmentioning

confidence: 99%

Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics

et al. 2011

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The two closely related deep-sea tubeworms Riftia pachyptila and Tevnia jerichonana both rely exclusively on a single species of sulfide-oxidizing endosymbiotic bacteria for their nutrition. They do, however, thrive in markedly different geochemical conditions. A detailed proteogenomic comparison of the endosymbionts coupled with an in situ characterization of the geochemical environment was performed to investigate their roles and expression profiles in the two respective hosts. The metagenomes indicated that the endosymbionts are genotypically highly homogeneous. Gene sequences coding for enzymes of selected key metabolic functions were found to be 99.9% identical. On the proteomic level, the symbionts showed very consistent metabolic profiles, despite distinctly different geochemical conditions at the plume level of the respective hosts. Only a few minor variations were observed in the expression of symbiont enzymes involved in sulfur metabolism, carbon fixation and in the response to oxidative stress. Although these changes correspond to the prevailing environmental situation experienced by each host, our data strongly suggest that the two tubeworm species are able to effectively attenuate differences in habitat conditions, and thus to provide their symbionts with similar micro-environments.

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Evaluation of non-cyanobacterial genome sequences for occurrence of genes encoding proteins homologous to cyanophycin synthetase and cloning of an active cyanophycin synthetase from Acinetobacter sp. strain DSM 587

Cited by 114 publications

References 0 publications

Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage

Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics

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