Regulation of assimilatory nitrate reductase formation in Klebsiella aerogenes W70

Bender, R.; Friedrich, B.

doi:10.1128/jb.172.12.7256-7259.1990

Cited by 32 publications

(21 citation statements)

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“…Although nasR and narK3-nirBD-nasBA are genomically distant and the leader sequence of the metabolic operon has no clearly identifiable stem-loop secondary structure motif (see below), we nonetheless observed the typical nitrate-induced regulatory patterns (Fig. 2) (30). In organisms such as Klebsiella oxytoca, a stem-loop structure forms in the leader region of the metabolic operon.…”

Section: Resultsmentioning

confidence: 76%

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment, Stem Colonization, and Virulence

Dalsing

Allen

2013

Journal of Bacteriology

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Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium's gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum's sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production.

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Section: Resultsmentioning

confidence: 76%

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment, Stem Colonization, and Virulence

Dalsing

Allen

2013

Journal of Bacteriology

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“…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%

“…Transcription of the nirA operon was induced by treatment of ammonium-grown cells with MSX, an inhibitor of glutamine synthetase, or by transfer of the ammonium-grown cells to ammonium-free media. MSX was added to cyanobacterial cultures in the mid-logarithmic phase of growth with or without simultaneous addition of KNO 3 or NaNO 2 . For transfer of the cells to ammonium-free media, the ammoniumgrown cells were collected by centrifugation at 5,000 ϫ g for 5 min at 25ЊC, washed twice with the basal medium, and inoculated into the basal medium with or without KNO 3 .…”

mentioning

confidence: 99%

“…MSX was added to cyanobacterial cultures in the mid-logarithmic phase of growth with or without simultaneous addition of KNO 3 or NaNO 2 . For transfer of the cells to ammonium-free media, the ammoniumgrown cells were collected by centrifugation at 5,000 ϫ g for 5 min at 25ЊC, washed twice with the basal medium, and inoculated into the basal medium with or without KNO 3 . The final concentrations of MSX, KNO 3 , and NaNO 2 were 0.1, 15, and 5 mM, respectively.…”

mentioning

confidence: 99%

“…For transfer of the cells to ammonium-free media, the ammoniumgrown cells were collected by centrifugation at 5,000 ϫ g for 5 min at 25ЊC, washed twice with the basal medium, and inoculated into the basal medium with or without KNO 3 . The final concentrations of MSX, KNO 3 , and NaNO 2 were 0.1, 15, and 5 mM, respectively. Total RNA was extracted and purified from cyanobacterial cells by the method of Aiba et al (1) and used for Northern (RNA) and dot hybridization analyses.…”

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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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Enzyme level N and O isotope effects of assimilatory and dissimilatory nitrate reduction

Treibergs

Granger

2016

Limnology & Oceanography

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To provide mechanistic constraints to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate ( NO3−), 15N/14N and 18O/16O, in the environment, we measured the enzymatic NO3− N and O isotope effects (15ε and 18ε) during its reduction by NO3− reductase enzymes, including (1) a prokaryotic respiratory NO3− reductase, Nar, from the heterotrophic denitrifier Paracoccus denitrificans, (2) eukaryotic assimilatory NO3− reductases, eukNR, from Pichia angusta and from Arabidopsis thaliana, and (3) a prokaryotic periplasmic NO3− reductase, Nap, from the photoheterotroph Rhodobacter sphaeroides. Enzymatic Nar and eukNR assays with artificial viologen electron donors yielded identical 18ε and 15ε of ∼28‰, regardless of [ NO3−] or assay temperature, suggesting analogous kinetic mechanisms with viologen reductants. Nar assays fuelled with the physiological reductant hydroquinone (HQ) also yielded 18ε ≈ 15ε, but variable amplitudes from 21‰ to 33.0‰ in association with [ NO3−], suggesting analogous substrate sensitivity in vivo. Nap assays fuelled by viologen revealed 18ε:15ε of 0.50, where 18ε ≈ 19‰ and 15ε ≈ 38‰, indicating a distinct catalytic mechanism than Nar and eukNR. Nap isotope effects measured in vivo showed a similar 18ε:15ε of 0.57, but reduced 18ε ≈ 11‰ and 15ε ≈ 19‰. Together, the results confirm identical enzymatic 18ε and 15ε during NO3− assimilation and denitrification, reinforcing the reliability of this benchmark to identify NO3− consumption in the environment. However, the amplitude of enzymatic isotope effects is apt to vary in vivo. The distinctive signature of Nap is of interest for deciphering catalytic mechanisms but may be negligible in most environments given its physiological role.

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Regulation of assimilatory nitrate reductase formation in Klebsiella aerogenes W70

Cited by 32 publications

References 8 publications

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment, Stem Colonization, and Virulence

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment, Stem Colonization, and Virulence

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

Enzyme level N and O isotope effects of assimilatory and dissimilatory nitrate reduction

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