Abstract
Ammonium concentration and nitrogen source regulate promoter activity and use for the transcription of chiA, the major chitinase gene of Pseudoalteromonas sp. S91 and S91CX, an S91 transposon lacZ fusion mutant. The activity of chiA was quantified by β-galactosidase assay of S91CX cultures containing different ammonium concentrations (NH4+; 0, 9.5 or 191 mM) and with different nitrogen sources (N-acetylglucosamine (GlcNAc) or glutamate (glt)). S91 chiA expression was found to depend on both the NH4+ concentration and source of nitrogen in marine minimal medium (MMM). Pseudoalteromonas sp. S91 and S91CX can use either GlcNAc or glt as a sole source of carbon in MMM containing a standard concentration of 9.5 mM NH4+. Adding excess NH4+, 20 times the standard concentration, to MMM significantly reduced chiA activity below that found in the presence of either GlcNAc or glt. When no NH4+ was added to MMM, S91CX was also able to use either GlcNAc or glt as a source of nitrogen; under these conditions chiA activity was significantly increased. Under all conditions tested, GlcNAc induced chiA activity significantly more than glt. Regulation of bacterial chitinases by nitrogen has not been previously reported. Transcriptional start point analysis of S91 chiA, using 5′RACE (ligation-anchored PCR), showed that during growth in MMM supplemented with (1) maltose (solely a carbon source for S91), chiA transcription occurred from only one putative σ70-dependent promoter; (2) the chitin monomer GlcNAc, transcription initiated from two putative σ54-dependent promoters and (3) glt, transcription initiated from all three putative promoters.
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Accession codes
Abbreviations
- bp:
-
base pair
- GlcNAc:
-
N-acetylglucosamine
- glt:
-
glutamate
- LA-PCR:
-
ligation-anchored PCR
- MMM:
-
marine minimal medium
- MMM 20N:
-
marine minimal medium with 20 times the standard concentration of NH4Cl
- MMM NN:
-
marine minimal medium with no added NH4Cl
- sp. act:
-
β-galactosidase-specific activity
- TSP:
-
transcriptional start point
- TSPs:
-
transcriptional start points
- wt:
-
wild type
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Acknowledgements
This work was supported in part by Flinders University and the Australian Research Council. Marina Delpin was supported by an Australian Postgraduate Award.
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Delpin, M., Goodman, A. Nitrogen regulates chitinase gene expression in a marine bacterium. ISME J 3, 1064–1069 (2009). https://doi.org/10.1038/ismej.2009.49
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DOI: https://doi.org/10.1038/ismej.2009.49
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