Energy metabolism and alginate biosynthesis in Pseudomonas aeruginosa: role of the tricarboxylic acid cycle

Schlictman, David; Kavanaugh-Black, Andrew; Shankar, Sandeep; Chakrabarty, Ananda M.

doi:10.1128/jb.176.19.6023-6029.1994

Cited by 38 publications

(44 citation statements)

References 49 publications

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“…The 8830R2::Cm cell extract supernatant also demonstrated the transfer of the terminal phosphate from ATP to CDP, GDP, and UDP; however, Ndk activity was undetectable when anti-Pk antibodies were incorporated in the assay, suggesting that the phosphate transfer reaction in the extract was mostly accomplished by Pk. While it is known that Pk alone incubated with [␥- 32 (41) showing the amino acid residues which were altered in the present study. The new amino acid residue replacing that in the wild-type sequence is shown below the site of the mutation.…”

Section: Resultsmentioning

confidence: 69%

“…We have previously determined that an autophosphorylation assay using [␥- 32 P]ATP provides a sensitive means to assess Ndk levels and corresponds well to the measurement of Ndk levels by Western blotting (32,33). In this study, each of the 12 mutant ndk genes were hyperexpressed in 8830R2::Cm, which has very little chromosomally encoded Ndk, and the Ndk levels in supernatant extracts were examined by autophosphorylation and by Western blotting.…”

Section: Resultsmentioning

confidence: 99%

“…Ndk can transfer the terminal phosphate from ATP to any NDP, and the enzyme is known for its lack of specificity to nucleotide substrates. In this assay system, cellular fractions were incubated with [␥- 32 P]ATP and a mixture of nonradioactive UDP, CDP, and GDP, producing [␥-…”

Section: Resultsmentioning

confidence: 99%

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Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis

1996

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We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-143Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as critical to this process.Nucleoside diphosphate kinase (EC 2.7.4.6; Ndk) catalyzes the reversible transfer of the 5Ј-terminal phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs) via a ping-pong mechanism summarized as follows: N 1 TP ϩ N 2 DP ϭ N 1 DP ϩ N 2 TP (29). The transphosphorylation reaction involves a phosphohistidine intermediate, and Ndk is characterized as showing little substrate specificity, utilizing the ribose and deoxyribose forms of both purine and pyrimidine NDPs as substrates. Ndk plays a key role as a housekeeping enzyme that functions in the maintenance of nucleotide pools for DNA and RNA synthesis. The sequencing of ndk genes from a variety of sources has revealed that most encode proteins consisting of approximately 150 amino acids and that Ndks from prokaryotes to humans are extensively homologous (41). Crystal structure information also confirms the relatedness of Ndks, although eukaryotic Ndks (e.g., those from Dictyostelium discoideum and Drosophila melanogaster) are arranged as hexamers while a prokaryo...

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Section: Resultsmentioning

confidence: 69%

Section: Resultsmentioning

confidence: 99%

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Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis

1996

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“…Repression of Salmonella enterica serovar Typhimurium ndk by the DNA-binding and transcription regulatory protein, Fis [13] lowers GTP levels, thereby regulating pppGpp and ppGpp pool size. On the contrary, in Pseudomonas aeruginosa, AlgQ, which modulates the levels of alginate [14], upregulates ndk, thereby positively regulates the levels of GTP, ppGpp, and inorganic polyphosphate (polyp) [15,16]. Similarly, under osmotic and oxidative stress conditions, ndk is upregulated in order to restore cell homeostasis in Sinorhizobium meliloti, carrying a mutation in the outer membrane protein, TolC [17].…”

Section: Introductionmentioning

confidence: 99%

NUCLEOSIDE DIPHOSPHATE KINASE GENE IS EXPRESSED THROUGH MULTIPLE TRANSCRIPTS IN Mycobacterium smegmatis

KUMAR¹,

Arumugam²,

Anand³

et al. 2012

Int J of Micr Res

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Abstract-Nucleoside diphosphate kinase, NDK, plays a vital role in maintaining pools of nucleoside triphosphates and their respective deoxynucleoside triphosphates for the synthesis of RNA and DNA. Transcriptional regulation of ndk in mycoacteria remains unknown, although modulation of ndk expression under stress conditions involving DNA and, RNA synthesis arrest and cell division arrest had been studied in several bacterial systems. Therefore, in the present study, the start sites of transcription of ndk of Mycobacterium smegmatis (Msmndk) were identified and putative promoter regions were predicted. Using transcriptional fusions of the cloned putative promoter regions to mycobacterial codon-optimised reporter gene, gfpm 2+ , promoter activity was examined under active phase of growth, nutrient starvation and other stress conditions involving DNA replication inhibition and cell division arrest. Msmndk was found to be expressed through two transcripts, T1 and T2, arising from P1 and P2 promoters, respectively. Both the promoters belonged to C group of mycobacterial promoters, which do not possess consensus to any known canonical sigma factor recognition sequences. The levels of T2, but not of T1, were found to be low under the different stress conditions studied. The data documents modulation of ndk transcripts in mycobacteria.

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“…However, the Ndk-deficient mutant still makes small amounts of alginate. Since an alginate-negative algR2 mutant in P. aeruginosa is deficient in both Ndk and Succinyl-coenzyme A (CoA) synthetase (Scs) levels (23), an important question is whether Scs, like Ndk, plays a role in providing the GTP precursors for alginate synthesis.…”

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confidence: 99%

Succinyl Coenzyme A Synthetase of Pseudomonas aeruginosa with a Broad Specificity for Nucleoside Triphosphate (NTP) Synthesis Modulates Specificity for NTP Synthesis by the 12-Kilodalton Form of Nucleoside Diphosphate Kinase

2000

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Pseudomonas aeruginosa secretes copious amounts of an exopolysaccharide called alginate during infection in the lungs of cystic fibrosis patients. A mutation in the algR2 gene of mucoid P. aeruginosa is known to exhibit a nonmucoid (nonalginate-producing) phenotype and showed reduced activities of succinyl-coenzyme A (CoA) synthetase (Scs) and nucleoside diphosphate kinase (Ndk), implying coregulation of Ndk and Scs in alginate synthesis. We have cloned and characterized the sucCD operon encoding the ␣ and ␤ subunits of Scs from P. aeruginosa and have studied the role of Scs in generating GTP, an important precursor in alginate synthesis. We demonstrate that, in the presence of GDP, Scs synthesizes GTP using ATP as the phosphodonor and, in the presence of ADP, Scs synthesizes ATP using GTP as a phosphodonor. In the presence of inorganic orthophosphate, succinyl-CoA, and an equimolar amount of ADP and GDP, Scs synthesizes essentially an equimolar amount of ATP and GTP. Such a mechanism of GTP synthesis can be an alternate source for the synthesis of alginate as well as for the synthesis of other macromolecules requiring GTP such as RNA and protein. Scs from P. aeruginosa is also shown to exhibit a broad NDP kinase activity. In the presence of inorganic orthophosphate (P i ), succinyl-CoA, and either GDP, ADP, UDP or CDP, it synthesizes GTP, ATP, UTP, or CTP. Scs was previously shown to copurify with Ndk, presumably as a complex. In mucoid cells of P. aeruginosa, Ndk is also known to exist in two forms, a 16-kDa cytoplasmic form predominant in the log phase and a 12-kDa membrane-associated form predominant in the stationary phase. We have observed that the 16-kDa Ndk-Scs complex present in nonmucoid cells, synthesizes all three of the nucleoside triphosphates from a mixture of GDP, UDP, and CDP, whereas the 12-kDa Ndk-Scs complex specifically present in mucoid cell predominantly synthesizes GTP and UTP but not CTP. Such regulation may promote GTP synthesis in the stationary phase when the bulk of alginate is synthesized by mucoid P. aeruginosa.Our laboratory is interested in studying the regulation of biosynthesis of alginate in Pseudomonas aeruginosa, an opportunistic pathogen. A number of cystic fibrosis (CF) isolates have been studied for their enhanced synthesis and secretion of alginate (16). Alginate is a polymer of D-manuronic and its C-5 epimer L-guluronic acid. GDP-mannuronic acid, derived from GDP-mannose, is the building block of alginate, which requires mannose 1-phosphate and GTP as precursors (17). Thus, alginate synthesis demands a steady supply of GTP for every molecule of mannuronic acid incorporated into a molecule of alginate. Recently, Kamath et al. (9) have shown that the mucoid strains of P. aeruginosa retain an active form of elastase in their periplasm, and this periplasmic elastase acts on 16-kDa Ndk to generate a truncated 12-kDa form that is membrane associated. It has been demonstrated that the membrane-associated 12-kDa nucleoside diphosphate (NDP) kinase (Ndk), in complex with pyruvat...

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Energy metabolism and alginate biosynthesis in Pseudomonas aeruginosa: role of the tricarboxylic acid cycle

Cited by 38 publications

References 49 publications

Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis

Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis

NUCLEOSIDE DIPHOSPHATE KINASE GENE IS EXPRESSED THROUGH MULTIPLE TRANSCRIPTS IN Mycobacterium smegmatis

Succinyl Coenzyme A Synthetase of Pseudomonas aeruginosa with a Broad Specificity for Nucleoside Triphosphate (NTP) Synthesis Modulates Specificity for NTP Synthesis by the 12-Kilodalton Form of Nucleoside Diphosphate Kinase

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