Cloning of a creatinase gene from Pseudomonas putida in Escherichia coli by using an indicator plate

Chang, Ming; Chang, Chun Chin; Chang, Jinq Chyi

doi:10.1128/aem.58.10.3437-3440.1992

Cited by 13 publications

(5 citation statements)

References 10 publications

(4 reference statements)

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“…Minicells were isolated from E. coli P678-54 containing the appropriate plasmid, and proteins were labelled with [ 35 S] methionine by methods described previously (Doagan & Kehoe, 1984; Chang et al, 1992a). The labelled plasmid-encoded proteins were separated by SDS-PAGE (Laemmli, 1970) and identified by direct autoradiography of the dried gel, using X-ray film.…”

Section: Methodsmentioning

confidence: 99%

Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2

Chuang

Chiou

et al. 1997

Microbiology

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The structural gene encoding the extracellular lipase of Aeromonas hydrophila MCC-2 was cloned and found to be expressed in Escherichia coli using its own promoter. When the cloned gene (lip) was expressed in E. coli minicells, an 80 kDa protein was identified. Subcellular fractionation of E. coli carrying the lip gene indicated that the Lip protein was mainly associated with the membrane fraction. Nucleotide sequence analysis revealed that the gene is 2253 bp long, coding for a 79.9 kDa protein with an estimated pl of 1036. The deduced protein contains two putative signal peptide cleavage sites; one is a typical signal peptidase cleavage site and the other bears a strong resemblance to known lipoprotein leader sequences. Radioactivity from [3H]palmitate was incorporated into the Lip protein when expressed in E. coli.The deduced protein contains a sequence of VHFLGHSLGA which is very well conserved among lipases. It shows 67% and 65% overall identity to the amino acid sequences of lipase from A. hydrophila strains H3 and JMP636, respectively, but shows little homology to those of other lipases. The Lip protein was purified to homogeneity from both A. hydrophila and recombinant E. coli. In hydrolysis of p-nitrophenyl esters and triacylglycerols, using purified enzyme, the optimum chain lengths for the acyl moiety on the substrate were C,, to C,, for ester hydrolysis and C, to C,,, for triacylglycerol hydrolysis.

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Section: Methodsmentioning

confidence: 99%

Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2

Chuang

Chiou

et al. 1997

Microbiology

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“…The metabolism of creatine by P. putida creatinase to the intermediate sarcosine has been observed by multiple groups and is supported by the strong transcriptional induction of the predicted sarcosine metabolic genes, orthologous to P. aeruginosa's soxBDAG, 1 h postcreatine exposure in P. putida KT2440 (Fig. 5, Table 1) [5][6][7][8][9][45][46][47]. Genes involved in the subsequent steps of sarcosine metabolism, including glyA1 encoding the serine hydroxymethyltransferase and tdcG-I encoding the l-serine dehydratase, are amongst the next most highly transcribed genes in the presence of creatine (Fig.…”

Section: Discussionmentioning

confidence: 91%

“…CahR binds with specificity to the promoter region of creA (Fig. 3), from which we conclude direct transcriptional induction of creA, which encodes a creatinase with the ability to efficiently cleave creatine into sarcosine and urea [5][6][7][8][9][10][45][46][47]. Transcription of creA occurs quickly in WT P. putida, detectable by the GFP reporter within 1 h after exposure to creatine, and with rapid increase over the first 5 h post-exposure (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…putida to grow on creatine as the sole nitrogen source. CahR binds with specificity to the promoter region of creA (), from which we conclude direct transcriptional induction of creA , which encodes a creatinase with the ability to efficiently cleave creatine into sarcosine and urea [5–10, 45–47]. Transcription of creA occurs quickly in WT P.…”

Section: Discussionmentioning

confidence: 99%

“…aeruginosa ’s soxBDAG , 1 h post-creatine exposure in P. putida KT2440 (, ) [5–9, 45–47]. Genes involved in the subsequent steps of sarcosine metabolism, including glyA1 encoding the serine hydroxymethyltransferase and tdcG-I encoding the l -serine dehydratase, are amongst the next most highly transcribed genes in the presence of creatine (, ), providing a more complete picture of creatine metabolism in P.…”

Section: Discussionmentioning

confidence: 99%

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Creatine utilization as a sole nitrogen source in Pseudomonas putida KT2440 is transcriptionally regulated by CahR

et al. 2022

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Glutamine amidotransferase-1 domain-containing AraC-family transcriptional regulators (GATRs) are present in the genomes of many bacteria, including all Pseudomonas species. The involvement of several characterized GATRs in amine-containing compound metabolism has been determined, but the full scope of GATR ligands and regulatory networks are still unknown. Here, we characterize Pseudomonas putida ’s detection of the animal-derived amine compound creatine, a compound particularly enriched in muscle and ciliated cells by a creatine-specific GATR, PP_3665, here named CahR (Creatine amidohydrolase Regulator). cahR is necessary for transcription of the gene encoding creatinase (PP_3667/creA) in the presence of creatine and is critical for P. putida’s ability to utilize creatine as a sole source of nitrogen. The CahR/creatine regulon is small, and an electrophoretic mobility shift assay demonstrates strong and specific CahR binding only at the creA promoter, supporting the conclusion that much of the regulon is dependent on downstream metabolites. Phylogenetic analysis of creA orthologues associated with cahR orthologues highlights a strain distribution and organization supporting probable horizontal gene transfer, particularly evident within the genus Acinetobacter . This study identifies and characterizes the GATR that transcriptionally controls P. putida ’s metabolism of creatine, broadening the scope of known GATR ligands and suggesting GATR diversification during evolution of metabolism for aliphatic nitrogen compounds.

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Creatine metabolism and the consequences of creatine depletion in muscle

Wyss

Wallimann

1994

Cellular Bioenergetics: Role of Coupled Creatine Kinases

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Currently, considerable research activities are focussing on biochemical, physiological and pathological aspects of the creatine kinase (CK) -phosphorylcreatine (PCr) -creatine (Cr) system (for reviews see [1,2]), but only little effort is directed towards a thorough investigation of Cr metabolism as a whole. However, a detailed knowledge of Cr metabolism is essential for a deeper understanding of bioenergetics in general and, for example, of the effects of muscular dystrophies, atrophies, CK deficiencies (e.g. in transgenic animals) or Cr analogues on the energy metabolism of the tissues involved. Therefore, the present article provides a short overview on the reactions and enzymes involved in Cr biosynthesis and degradation, on the organization and regulation of Cr metabolism within the body, as well as on the metabolic consequences of 3-guanidinopropionate (GPA) feeding which is known to induce a Cr deficiency in muscle. In addition, the phenotype of muscles depleted of Cr and PCr by GPA feeding is put into context with recent investigations on the muscle phenotype of 'gene knockout' mice deficient in the cytosolic muscle-type M-CK. (Mol Cell Biochem 133/134: 51-66, 1994).

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Cloning of a creatinase gene from Pseudomonas putida in Escherichia coli by using an indicator plate

Cited by 13 publications

References 10 publications

Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2

Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2

Creatine utilization as a sole nitrogen source in Pseudomonas putida KT2440 is transcriptionally regulated by CahR

Creatine metabolism and the consequences of creatine depletion in muscle

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