Summary auto-generated
This study characterizes the glnB gene and its protein product (PII protein) in Nostoc punctiforme, a filamentous nitrogen-fixing cyanobacterium. The researchers cloned the glnB gene using heterologous hybridization, confirmed its expression in N. punctiforme cells, and demonstrated that the purified PII protein could be phosphorylated in vitro by a Synechococcus kinase in an ATP and 2-oxoglutarate-dependent manner. However, modified forms of PII were not detected in N. punctiforme cell extracts under any growth condition tested, including nitrogen starvation, which contrasts with observations in other cyanobacteria. Critically, attempts to create a glnB null mutant through targeted gene replacement were unsuccessful unless a second glnB copy was provided in trans, suggesting the gene or its product is essential for growth under the tested conditions. The genomic rearrangements observed during gene replacement experiments prevented full characterization of glnB disruption effects, but the data collectively indicate PII plays an essential role in N. punctiforme physiology, though its specific function in this organism remains to be determined.
Key findings
- The N. punctiforme PII protein shares 98% amino acid identity with Calothrix PII and contains conserved phosphorylation sites, yet shows no detectable modification in vivo under various growth conditions.
- Purified N. punctiforme PII protein is phosphorylatable in vitro by Synechococcus 7942 kinase in an ATP and 2-oxoglutarate-dependent manner, demonstrating the protein is biochemically capable of modification.
- The glnB gene cannot be disrupted by targeted replacement unless a second copy is provided in trans, indicating the gene is essential for growth under standard laboratory conditions.
- N. punctiforme carries a single chromosomal copy of glnB with monocistronic transcription producing a 470 nucleotide message that increases in abundance following nitrogen limitation.
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Abstract
Bacterial PII proteins, encoded by glnB genes, are central signalling molecules in nitrogen regulatory pathways and are modulated by post- translational modification in response to the cellular nitrogen status. The glnB gene was cloned from the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme strain ATCC 29133 (PCC 73102) by heterologous hybridization to a Synechococcus sp. strain PCC 7942 gene fragment. Expression of the cloned gene was verified by hybridization to N. punctiforme total RNA and a single cross-reactive polypeptide was observed in immunoblots of N. punctiforme extracts probed with anti- Synechococcus 7942 PII antiserum. Modification of the purified N. punctiforme PII protein by a Synechococcus 7942 PII kinase was observed, but modified forms of PII were not detected in extracts of N. punctiforme from a variety of incubation conditions. The N. punctiforme glnB gene could not be disrupted by targeted gene replacement unless a second copy of glnB was provided in trans, suggesting that the gene or gene product is essential for growth under the conditions tested.