Summary
A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage λ47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated. A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively. Cells of Escherichia coli harbouring these plasmids expressed CMCase activity. The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis. pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter. All four cloned enzymes cleaved p-nitrophenyl-β-D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity. In common with other extracellular enzymes cloned in E. coli, all the CMCases were exported to the periplasmic space in the enteric bacterium. The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E. coli.
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Abbreviations
- SSC:
-
0.15 M NaCl, 0.015 M sodium citrate
- Smr :
-
resistance to streptomycin
- Kmr :
-
resistance to kanamycin
- Apr :
-
resistance to ampicillin
- Tcr :
-
resistance to tetracycline
- Cmr :
-
resistance to chloramphenicol
- CMCase:
-
carboxymethylcellulase
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Communicated by H. Hennecke
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Gilbert, H.J., Jenkins, G., Sullivan, D.A. et al. Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa . Mol Gen Genet 210, 551–556 (1987). https://doi.org/10.1007/BF00327211
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DOI: https://doi.org/10.1007/BF00327211