Structural and functional analysis of the phosphoenolpyruvate carboxylase gene from the purple nonsulfur bacterium Rhodopseudomonas palustris No. 7

Inui, Masayuki; Dumay, Valérie; Zahn, Kenneth; Yamagata, Hisashi; Yukawa, Hideaki

doi:10.1128/jb.179.15.4942-4945.1997

Cited by 17 publications

(30 citation statements)

References 24 publications

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“…It is also possible that intracellular CA aids in the conversion of CO # to HCO − $ for fixation by phosphoenolpyruvate carboxylase . Both biochemical pathways are found in photosynthetic bacteria (Buchanan et al, 1967 ;Inui et al, 1997 ;Tabita, 1995) although carboxysomes are not found in PNSB and our results suggest that CA enzyme is not associated with RubisCo, because the main CA activity was found in the periplasmic fraction. Periplasmic localization and the presence of the enzyme only under anaerobic conditions suggests that CA participates in C i utilization in this bacterium.…”

Section: Localization and Expression Of Casupporting

confidence: 55%

A periplasmic, α-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake

et al. 2000

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Intact cells of the purple non-sulfur bacterium Rhodopseudomonas palustris growing anaerobically, but not aerobically, contain carbonic anhydrase (CA) activity. The native enzyme was purified S2000-fold to apparent homogeneity and found to be a dimer with an estimated molecular mass of 54 kDa and a subunit molecular mass of 27 kDa. The CA gene (acaP) was cloned and its sequence revealed that it was homologous to α-type CAs. The upstream region of acaP was fused to the lacZ gene and β-galactosidase activity was measured under different growth conditions. Acetazolamide inhibited purified CA with an IC 50 in the range of 10 N8 M, and in the culture media concentrations as low as 30 µM inhibited phototrophic growth under anaerobic, light conditions when bicarbonate was used. An acaP ::Kan r mutant strain was constructed by insertion of a kanamycin-resistance cassette and showed a growth pattern similar to wild-type cells grown in the presence of CA inhibitor. CO 2 gas supplied as an inorganic carbon source reversed the effect of mutation or acetazolamide. CA activity measurements, fusion and Western blot experiments confirmed that CA is expressed under different anaerobic conditions independently of bicarbonate or CO 2 and that there is no expression under aerobic conditions.

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Section: Localization and Expression Of Casupporting

confidence: 55%

A periplasmic, α-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake

et al. 2000

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“…Assays for GAPDH (Omumasaba et al, 2004), phosphoglycerate kinase (PGK) (Eikmanns, 1992), triosephosphate isomerase (TPI) (Eikmanns, 1992), PEPC (Inui et al, 1997) and LDH (Bunch et al, 1997) were performed as previously described. Malate dehydrogenase (MDH) activity was determined as described by Sridhar et al (2000), modified by using 0.1 M Tris/HCl (pH 8.0) and 0.15 mM NADH to obtain optimum enzyme activities.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions

et al. 2007

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A transcriptional profiling of the metabolism of Corynebacterium glutamicum under oxygen deprivation conditions is reported. It was observed that the glucose consumption rate per cell when C. glutamicum cells were incubated under oxygen deprivation conditions was higher than that achieved by cells incubated under aerobic growth conditions. Furthermore, DNA microarray and quantitative RT-PCR analyses revealed that the genes of several key enzymes of the glycolytic and organic acid production pathways, including gapA, pgk, tpi, ppc, ldhA and mdh, were significantly upregulated under oxygen deprivation conditions. The corresponding enzymic activities consistently correlated with the regulation patterns of the genetic expression observed at the transcriptional level. Studies of lacZ fusions with the gapA, ldhA and mdh genes indicated not only that these genes are strongly induced at the onset of the stationary phase under aerobic growth conditions, but also that high expression levels are maintained under oxygen deprivation conditions. These results indicate that the genetic expression of several key metabolic enzymes in C. glutamicum cells incubated under oxygen deprivation conditions is chiefly regulated at the transcriptional level. The physiological consequence of the observed increased transcription under oxygen deprivation conditions is an increased rate of carbon source consumption, which is accompanied by a concomitant increase in organic acid production.

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“…Under anaerobic conditions in the light, all species grow photoheterotrophically when supplied with various organic substrates or photoautotrophically with CO 2 as a sole carbon source. Under microaerobic to aerobic conditions in the dark, many representatives can grow chemoheterotrophically, and some grow chemoautotrophically (40).To develop a new CO 2 -fixing bioprocess, we have been performing biochemical and genetic analyses of intermediary metabolism, including CO 2 fixation, underlying the complex modes of growth in the PNSB, using Rhodopseudomonas palustris as a model microorganism (4,23,24). For this purpose, development of a versatile host-vector system would be helpful.…”

mentioning

confidence: 99%

“…To develop a new CO 2 -fixing bioprocess, we have been performing biochemical and genetic analyses of intermediary metabolism, including CO 2 fixation, underlying the complex modes of growth in the PNSB, using Rhodopseudomonas palustris as a model microorganism (4,23,24). For this purpose, development of a versatile host-vector system would be helpful.…”

mentioning

confidence: 99%

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Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

Inui

Roh

Zahn

et al. 2000

Appl Environ Microbiol

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A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of ␣-proteobacteria.Purple nonsulfur bacteria (PNSB) are an assemblage of phenotypically diverse species. Under anaerobic conditions in the light, all species grow photoheterotrophically when supplied with various organic substrates or photoautotrophically with CO 2 as a sole carbon source. Under microaerobic to aerobic conditions in the dark, many representatives can grow chemoheterotrophically, and some grow chemoautotrophically (40).To develop a new CO 2 -fixing bioprocess, we have been performing biochemical and genetic analyses of intermediary metabolism, including CO 2 fixation, underlying the complex modes of growth in the PNSB, using Rhodopseudomonas palustris as a model microorganism (4,23,24). For this purpose, development of a versatile host-vector system would be helpful.In R. palustris and other PNSB, broad-host-range vectors have been used to provide the tools for gene transfer. The most widely used vectors are derivatives of RK2 such as pRK415 (25) and pLAFR1 (14). Cloning vector pRK415 has been utilized for genetic analyses of several R. palustris genes (8, 17), and cosmid vector pLAFR1 has been used to make a library of R. palustris DNA (8). However, these plasmids were unstable in R. palustris under nonselective conditions (M. Inui, unpublished data), as also observed in R. sphaeroides (9) and in Rhodospirillum rubrum (42).Other vectors derived from the broad-host-range plasmid RSF1010 such as pDSK519 (25) can also replicate in PNSB, including R. palustris (Inui, unpublished data). But this vector was also unstable under nonselective conditions in R. palustris and R. sphaeroides (Inui, unpublish...

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Structural and functional analysis of the phosphoenolpyruvate carboxylase gene from the purple nonsulfur bacterium Rhodopseudomonas palustris No. 7

Cited by 17 publications

References 24 publications

A periplasmic, α-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake

A periplasmic, α-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake

Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

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