Transcription initiation at multiple promoters of the pfl gene by E sigma 70-dependent transcription in vitro and heterologous expression in Pseudomonas putida in vivo

Sawers, R. Gary; Wagner, A.; Böck, August

doi:10.1128/jb.171.9.4930-4937.1989

Cited by 24 publications

(14 citation statements)

References 46 publications

(40 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fnr protein regulates the expression of a number of genes required for anaerobic growth in E. coli (reviewed by Spiro and Guest [41]). A sequence resembling the consensus sequence of the Ϫ10 region in 70 promoters, present in other Fnr-dependent promoters (37), was also identified at the right distance from 5b (Fig. 2C).…”

Section: Resultsmentioning

confidence: 92%

The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene

et al. 1995

View full text Add to dashboard Cite

Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H 2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO 2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P 5b , with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5-TTGAgccatgTCAA-3, centered at position ؊39.5. Expression of the P 5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum bv. viciae UPM791 gene bank by complementing an R. meliloti FixK ؊ mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN ؊ mutant of R. leguminosarum bv. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P 5b was reduced to ca. 50% of the wild-type level in the FnrN ؊ mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum bv. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.A few strains of Rhizobium leguminosarum bv. viciae have been found to synthesize, in symbiosis with peas, an H 2 uptake (hup) system that recycles hydrogen evolved during the nitrogen reduction process in root nodules (2). In R. leguminosarum bv. viciae UPM791, the genetic determinants for this H 2 uptake system are clustered in a 20-kb DNA region which has been isolated in cosmid pAL618 (21). About 15 kb of this region have been sequenced, and 17 genes closely linked and oriented in the same direction were identified (14,15,31,33). The first two genes of the cluster, hupS and hupL, encode the structural polypeptides of a dimeric (NiFe)-type hydrogenase (14) and belong to an operon that also includes four additional genes, hupCDEF (15). The remaining genes, named hupG, H, I, J, and K and hypA, B, F, C, D, and E, are located downstream of hupF (31, 33). These genes encode hydrogenase accessory proteins that are required for H 2 oxidation in pea bacteroids, but their specific molecular functions...

show abstract

Section: Resultsmentioning

confidence: 92%

The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene

et al. 1995

View full text Add to dashboard Cite

show abstract

“…Analysis of pfl gene expression in L. lactis MG1363. The expression of pfl is induced under anaerobiosis in E. coli (30). Physiological studies have shown that pyruvate is converted mainly to lactate when L. lactis is growing in glucose.…”

Section: Resultsmentioning

confidence: 99%

Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase

Arnau¹,

Jørgensen²,

Madsen³

et al. 1997

J Bacteriol

View full text Add to dashboard Cite

The Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized. The deduced amino acid sequence of the L. lactis PFL protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL. The genetic organization of the chromosomal pfl region in L. lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene. The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L. lactis. Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used. During growth with galactose, pfl showed the highest levels of expression. Constructed L. lactis pfl strains were unable to produce formate under anaerobic growth. Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp. lactis biovar diacetylactis pfl strain.Pyruvate has a key role in the metabolic network of Lactococcus lactis. In this industrially relevant organism, sugars are metabolized predominantly to generate energy and as a carbon source. The catabolism of sugars leads to pyruvate, whose further metabolism determines the nature of the fermentation (see reference 5 for a review). Lactate is the major end product responsible for acidification, while other minor metabolites, mainly diacetyl, are responsible for aroma in fermented milk products. Several genes involved in pyruvate metabolism have been studied for their role in the production of diacetyl (Fig.

show abstract

“…Previously, it has been shown that FNR-dependent genes of E. coli are transcribed and expressed in Pseudomonas species (28,46), but regulation by oxygen limitation in the heterologous host has not been demonstrated. Our present data show that the "strict aerobe" (38) P. aeruginosa correctly regulates an artificial FNR-dependent gene of E. coli in response to oxygen availability and that this regulator is defective in an anr mutant.…”

Section: Discussionmentioning

confidence: 99%

Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa

et al. 1991

View full text Add to dashboard Cite

The preferred way of life of Pseudomonas aeruginosa is aerobic respiration (38). When aeration is reduced or absent and nitrate is present, P. aeruginosa obtains energy from nitrate respiration (14,44). When neither oxygen nor nitrate is available, arginine can be used as the sole energy source for motility and growth (48,62). In this case, the arginine deiminase pathway serves to degrade arginine to ornithine and carbamoylphosphate, which allows regeneration of ATP from ADP without respiration (41,62). Anaerobic growth on arginine requires a rich medium and extended incubation; colonies take 4 to 5 days to form. Under these conditions, arginine is converted quantitatively to omithine and hence does not serve as a carbon source (62). Good aeration inhibits nitrate uptake (21) and prevents induction of the nitrate respiration enzymes (12) and of the arginine deiminase pathway in P. aeruginosa (34). Aerobic conditions favor the utilization of arginine as a carbon source via the succinylarginine pathway (13). The molecular mechanisms involved in the switch from aerobic to anaerobic metabolism in P. aeruginosa are unknown.In Escherichia coli and other enteric bacteria, thefnr gene is essential for the expression of fumarate and nitrate respiration and many other anaerobic processes (26,47,54,57,61). Thefnr-dependent genes and operons have a conserved symmetrical sequence, the FNR box, upstream of their promoters. Mutations in the FNR box interfere with anaerobic induction (8,16,24,54). It is generally accepted that under anaerobic conditions the FNR protein acts as a transcriptional activator by binding to the FNR box; however, a physical interaction between FNR and the FNR box has not been demonstrated (9, 54, 59).We are interested in the regulation of anaerobic metabolism in P. aeruginosa, especially in the induction of the arginine deiminase pathway by oxygen limitation. for this pathway are organized as an operon (arcDABC), which encodes a transmembrane protein having arginine/ ornithine antiporter activity (arcD), arginine deiminase (arcA), catabolic ornithine carbamoyltransferase (arcB), and carbamate kinase (arcC) (5, 6, 9a, 30, 31, 62). Most P. aeruginosa mutants that cannot grow anaerobically on arginine (Arc-phenotype) have a defect in the arcDABC operon (43, 62). Recently, we found a mutant whose Arc-phenotype is due to a mutation lying outside the arc operon (19). This mutation had originally been obtained by van Hartingsveldt et al. (63,64) and designated nirD. It causes inability of P. aeruginosa to grow anaerobically on nitrate (Nar-phenotype) and loss of dissimilatory nitrite reductase activity. Other genes required for nitrate respiration are unlinked to nirD (63,64). Here we show that a chromosomal fragment of P. aeruginosa complements the nirD mutant for anaerobic growth on nitrate and arginine and also restores anaerobic induction of nitrate reductase and arginine deiminase in the mutant. We therefore propose that nirD should be renamed anr (for anaerobic regulation of arginine deiminase and ni...

show abstract

Transcription initiation at multiple promoters of the pfl gene by E sigma 70-dependent transcription in vitro and heterologous expression in Pseudomonas putida in vivo

Cited by 24 publications

References 46 publications

The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene

The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene

Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase

Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa

Contact Info

Product

Resources

About