Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al

Cali, Brian M.; Micca, J L; Stewart, Valley

doi:10.1128/jb.171.5.2666-2672.1989

J Bacteriol

1989

DOI: 10.1128/jb.171.5.2666-2672.1989

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Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al

Brian M. Cali

J L Micca

Valley Stewart

Abstract: We isolated Mu d11734 insertion mutants of Klebsiella pneumoniae that were unable to assimilate nitrate or nitrite as the sole nitrogen source during aerobic growth (Nas-phenotype). The mutants were not altered in respiratory (anaerobic) nitrate and nitrite reduction or in general nitrogen control. The mutations were linked and thus defined a single locus (nas) containing genes required for nitrate assimilation. ,I-Galactosidase synthesis in nas+l¢(nas-lacZ) merodiploid strains was induced by nitrate or nitrit… Show more

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Cited by 40 publications

(42 citation statements)

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“…4). Nitrate induces transcription of nitrate assimilation genes in higher plants (6), fungi (6,24), and bacteria (4,10,28), and nitrite has been shown to be an alternative inducer in the fungus Aspergillus nidulans (13,36) and the bacterium Klebsiella pneumoniae (4,10). The present results revealed that nitrate and nitrite activate the nitrate assimilation genes in the cyanobacteria Synechococcus sp.…”

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confidence: 48%

“…It is transported into the cells by an active transport system and reduced to ammonium by the sequential action of nitrate reductase (NR) and nitrite reductase (NiR) prior to fixation into the amide nitrogen of glutamine (Gln). As in other microorganisms capable of nitrate assimilation (3,4,6,8,19,24), expression of the nitrate assimilation system is inhibited by ammonium (12). In the unicellular non-nitrogen-fixing cyanobacterium Synechococcus sp.…”

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confidence: 99%

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Positive regulation by nitrite of the nitrate assimilation operon in the cyanobacteria Synechococcus sp. strain PCC 7942 and Plectonema boryanum

et al. 1996

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In the absence of fixation of ammonium to glutamine, nitrate and nitrite activated transcription of the nitrate assimilation (nirA-nrtABCD-narB) operon of Synechococcus sp. strain PCC 7942. In a nitrate reductasedeficient mutant, only nitrite activated transcription, indicating that nitrite is the actual activator of the operon. Nitrate and nitrite were also found to activate the transcription of a nitrate assimilation operon in the filamentous nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum.Nitrate is a major source of nitrogen for cyanobacteria (11,12). It is transported into the cells by an active transport system and reduced to ammonium by the sequential action of nitrate reductase (NR) and nitrite reductase (NiR) prior to fixation into the amide nitrogen of glutamine (Gln). As in other microorganisms capable of nitrate assimilation (3,4,6,8,19,24), expression of the nitrate assimilation system is inhibited by ammonium (12). In the unicellular non-nitrogen-fixing cyanobacterium Synechococcus sp. strain PCC 7942, the genes encoding the nitrate transporter (nrtABCD) (25-27), NR (narB) (2, 17), and NiR (nirA) (20, 32) form the nirA-nrtABCDnarB operon (32), and transcription of the operon is repressed by ammonium (32). Ammonium inhibits transcription through its fixation into Gln, but Gln is not the direct regulator of transcription (32). We have proposed that cyanate, a metabolite of Gln via carbamoylphosphate, acts as the metabolic signal for the ammonium-promoted repression of the nirA operon (34).Transcription of the nirA operon of Synechococcus sp. strain PCC 7942 is induced simply by removal of ammonium from the medium or by inhibition of ammonium fixation with L-methionine-DL-sulfoximine (MSX), showing no requirement for nitrate (32). Induction of the NiR gene is not dependent on nitrate in a filamentous, nonheterocystous nitrogen-fixing cyanobacterium, Plectonema boryanum IAM-M101, either (31). Thus, nitrate has seemingly no specific role in the transcription of the nitrate assimilation genes in the two strains of cyanobacteria. However, during studies of the expression of nitrate assimilation genes in MSX-treated cells, in which there is no negative feedback by the ammonium generated internally by reduction of nitrate and nitrite, we found that nitrate and nitrite do activate the transcription of the nitrate assimilation operons of Synechococcus sp. strain PCC 7942 and P. boryanum. By use of an NR-deficient mutant of strain PCC 7942 (⌬narB::kan) (33), the positive effect of nitrate was shown to be due to nitrite generated by reduction of nitrate.Cells of the cyanobacterial strains were grown photoautotrophically at 30ЊC under CO 2 -sufficient conditions as described previously (34). The basal medium used was a nitrogen-free medium obtained by modification of BG11 medium (30) as previously described (34). Ammonium-containing medium was prepared by adding 3.75 mM (NH 4 ) 2 SO 4 to the basal medium. Transcription of the nirA operon was induced by treatment of ammonium-grown cells with M...

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supporting

confidence: 48%

mentioning

confidence: 99%

Positive regulation by nitrite of the nitrate assimilation operon in the cyanobacteria Synechococcus sp. strain PCC 7942 and Plectonema boryanum

et al. 1996

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“…Further characterization of this translational coupling identified a secondary structure which prevents independent translation of nifA, making it dependent on full translation of nifL. (6) and the nifLA duplication-bearing strains SE1004 and SE1005 (as VJSK014Rif, but pIZ287::nifLA and pIZ288::nifLA, respectively; for details of construction, see below).…”

mentioning

confidence: 99%

Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae

1996

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The nifLA operon of Klebsiella pneumoniae codes for the two antagonistic regulatory proteins which control expression of all other nitrogen fixation genes. NifA is a transcriptional activator, and NifL inhibits NifA. The importance of a correct NifL-NifA stoichiometry for efficient regulation of nitrogen fixation genes has been investigated by constructing a strain with an altered nifL-nifA gene dosage ratio, resulting from the integration of an extra copy of nifA. Results showed that a balanced synthesis of both gene products is essential for correct regulation. Effects of mutations provoking translation termination of nifL upstream or downstream of its natural stop codon, combined with overproduction of both proteins when the genes are transcribed and translated from signals of the 10 gene of the phage T7, showed that, in addition to the previously reported transcriptional polarity, there is translational coupling between nifL and nifA. In spite of the apparently efficient ribosome binding site of nifA, its rate of independent translation is very low. This is due to a secondary structure masking the Shine-Dalgarno sequence of nifA, which could be melted by ribosomes translating nifL. Mutational analysis confirmed the functional significance of the secondary structure in preventing independent translation of nifA. Translational coupling between the two cistrons is proposed as an efficient mechanism to prevent production of an excess of NifA, which would affect the normal regulation of nitrogen fixation genes.

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“…The significance of this constitutivity remains unknown. Cali et al showed clearly that the K. pneumoniae nas-lac fusions were regulated by the NTR system at the level of gene expression (2). Macaluso et al (5) showed that NAS formation in K. aerogenes similarly required the NTR system (but not the nitrogen assimilation control system) for nitrogen regulation.…”

mentioning

confidence: 99%

“…Cali et al have recently considered the genetic regulation of NAS in Klebsiella pneumoniae (oxytoca) M5al (2). They overcame the instability of NAS in the assay mixture by isolating fusions of the lacZ gene to nas promoters and assaying P-galactosidase instead of NAS.…”

mentioning

confidence: 99%

Regulation of assimilatory nitrate reductase formation in Klebsiella aerogenes W70

Bender

Friedrich

1990

J Bacteriol

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Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al

Cited by 40 publications

References 43 publications

Positive regulation by nitrite of the nitrate assimilation operon in the cyanobacteria Synechococcus sp. strain PCC 7942 and Plectonema boryanum

Positive regulation by nitrite of the nitrate assimilation operon in the cyanobacteria Synechococcus sp. strain PCC 7942 and Plectonema boryanum

Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae

Regulation of assimilatory nitrate reductase formation in Klebsiella aerogenes W70

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