Cloning of the glnA, ntrB and ntrC genes of Klebsiella pneumoniae and studies of their role in regulation of the nitrogen fixation (nif) gene cluster

Abstract

The glnA, ntrB and ntrC genes of Klebsiella pneumoniae have been cloned, on a 12 kb HindIII fragment, into the plasmid pACYC184. In a coupled in vitro transcription/translation system the resultant plasmid, pGE100, directed synthesis of five polypeptides (molecular weights 73, 53, 51, 39, 36 kd) from the cloned fragment. A number of plasmids were derived from pGE100 and studied by complementation analysis and in vitro transcription/translation in order to locate particular genes and identify their products. On the basis of the results presented here, together with previous genetic and physical characterisation of the glnA gene and its product in other enteric bacteria, we propose that the 53 kd polypeptide is the glnA gene product (glutamine synthetase monomer). Two polypeptides (36 kd and 51 kd) were synthesised from a 3 kb region previously defined as glnR. In E. coli and S. typhimurium this region comprises two genes ntrB and ntrC with products of 36 kd and 54 kd respectively. This analogy supports the idea that the 36 kd and 51 kd polypeptides are the products of the K. pneumoniae ntrB and ntrC genes respectively. Comparison of these assignments with the physical map of the region indicates a gene order glnA, ntrB, ntrC. Assessment of the Nif phenotype of a glnA-ntrC deletion strain carrying various clones from pGE100 demonstrated that glnA is not required for expression of the nif regulon and that of the three genes cloned, ntrC alone is sufficient for nif expression.

Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum

“…Consistent with this, the intracellular glutamine concentration increases rapidly after NH 4 ϩ treatments in both R. rubrum and A. brasilense (11,15,18). GS activity is reduced in the A. brasilense ntrBC mutants (40), and the products of ntrBC regulate GS synthesis in other nitrogen-fixing bacteria, such as K. pneumoniae (6), Rhizobium meliloti (4,34), and B. japonicum (22). In some of these organisms, two or three genes encoding GS have been found (4,22).…”

“…In E. coli and K. pneumoniae, ntrBC are linked to glnA (6,29). There was, however, no hybridization of this cloned region with the glnA probe from A. brasilense, and it has been reported that glnA is not linked to ntrBC in either A. brasilense (5,20) or R. capsulatus (17).…”

Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation

“…The R. meliloti 1021 gln locus cloned in plasmid pFB69 is analogous to the gInII loci of B. japonicum (9) and A. tumefaciens (57) (50), in contrast to the situation in enteric bacteria (2,16,21,24 The glnT locus appears to consist of a complex operon, based on maxicell analysis of polypeptides encoded by glnT and glnT::TnS plasmids. TnS mutations at the beginning of the locus (A and B, Fig.…”

“…E. coli and Rhizobium cells were inoculated into 5 ml of LB or TY medium and grown overnight to saturation. A 0.5-ml sample was reinoculated in 50 ml of minimal medium supplemented with 0.2% (NH4)2SO4 and 0.2% glutamine, and the culture was incubated for 24 h at 37°C (E. coli) or 28°C (R. meliloti). The cells were harvested and resuspended in 50 ml of minimal medium without an N source (derepressing conditions) and grown for 10 h. Crude extract preparations, GS enzyme assays, and sucrose gradient analyses were performed as described before (56).…”

The product of the Klebsiella pneumoniae nifX gene is a negative regulator of the nitrogen fixation (nif) regulon

“…Transcription of the nifLA operon is itself activated through the action of rpoN, ntrB, and ntrC gene products on NH4+ starvation (9,25).…”