title: Investigations on three genes in Ralstonia eutropha H16 encoding putative cyanophycin metabolizing enzymes.
abstract: The genome sequence of the facultative chemolithoautotrophic bacterium Ralstonia eutropha H16 exhibited two coding sequences with high homologies to cyanophycin synthetases (CphA) as well as one gene coding for a putative cyanophycinase (CphB). To investigate whether or not the genes cphA H16 (H16_A0774), cphA'H16 (H16_A0775) and cphB H16 (H16_B1013) encode active cyanophycin (CGP) metabolism proteins, several functional analyses were performed. Extensive in silico analysis revealed that all characteristic motifs are conserved within CphAH16, whereas CphA'H16 misses a large part of the so-called J-loop present in other active cyanophycin synthetases. Although transcription of both genes was demonstrated by RT-PCR, and heterologously expressed cphA genes led to light-scattering inclusions in recombinant cells of Escherichia coli, no CGP could be isolated from the cells or detected by HPLC analysis. For all enzyme assay experiments carried out, significant enzyme activities were determined for CphA and CphA' in recombinant E. coli cells if crude cell extracts were applied. Homologous expression of cphA genes in cells of R. eutropha H16∆phaC1 did not result in the formation of light-scattering inclusions, and no CGP could be isolated from the cells or detected by HPLC analysis. No transcription of cphB encoding a putative cyanophycinase could be detected by RT-PCR analysis and no overexpression was achieved in several strains of E. coli. Furthermore, no enzyme activity was detected by using CGP overlay agar plates.
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Adames
authors.forename: Karina
authors.initials: K
authors.lastname: Euting
authors.forename: Anna
authors.initials: A
authors.lastname: Bröker
authors.forename: Alexander
authors.initials: A
authors.lastname: Steinbüchel
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D019295:Computational Biology; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; C024250:cyanophycin; D010447:Peptide Hydrolases; C120585:cyanophycinase; D010453:Peptide Synthases; C113257:cyanophycin synthase, bacteria