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S Shankar

Publications and source records attributed to S Shankar.

At least 19 recordsLinked to original sources
PubMed1996

Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis.

We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2 and algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P. aeruginosa. The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction events within the cell and in the production of GDP-mannose, an essential alginate precursor, clearly indicates the importance of Ndk in cellular processes as well as in alginate synthesis.

Amino Acid Sequence↗
PubMed1996

Sigma factor-anti-sigma factor interaction in alginate synthesis: inhibition of AlgT by MucA.

Conversion from the nonmucoid to the mucoid phenotype is a typical feature of Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients. One of the key genetic controls in this conversion to mucoidy is from the algT(U)-mucA-mucB(algN) locus, located at 67.5 min on the standard P. aeruginosa chromosomal map. The algT gene promotes conversion to mucoidy and encodes an alternative sigma factor (sigma E) which belongs to the ECF (for extracytoplasmic function) family. On the other hand, the mucA and mucB (algN) genes suppress conversion to mucoidy. Loss-of-function mutations in mucA have been postulated to be the cause of mucoidy in some P. aeruginosa strains isolated from cystic fibrosis patients. We expressed and purified the protein products from the mucA and mucB open reading frames. The purified MucA protein abolishes the in vitro transcription specified by AlgT and the ability of AlgT to compete with an Escherichia coli sigma factor, FliA, suggesting that inhibiting AlgT-dependent transcription could be the mechanism by which mucA suppresses mucoidy in vivo. Enzyme-linked immunosorbent assay and glycerol density gradient sedimentation experiments suggest that MucA physically interacts with AlgT.

Bacterial Proteins↗
PubMed1996

Two forms of the nucleoside diphosphate kinase of Pseudomonas aeruginosa 8830: altered specificity of nucleoside triphosphate synthesis by the cell membrane-associated form of the truncated enzyme.

Nucleoside diphosphate kinase (EC 2.7.4.6) (Ndk) is a ubiquitous enzyme functioning in the intracellular distribution of terminal phosphate bond energy among the various nucleotides used in synthetic and regulatory functions in cells. We have previously reported that in Pseudomonas aeruginosa, this important enzyme is transcriptionally regulated by the gene algR2 and posttranslationally regulated by a phosphoprotein phosphatase for the phosphorylated form of Ndk. We report here that an intracellular protease cleaves the 16-kDa form of Ndk to a 12-kDa form that undergoes autophosphorylation with an efficiency almost identical to that of the 16-kDa form. The 12-kDa form was found to be predominantly associated with the P. aeruginosa cell membrane fraction, whereas the 16-kDa form was predominantly cytoplasmic. In the membrane-associated state, the 12-kDa form of Ndk was found to synthesize GTP in preference to other nucleoside triphosphates. The specificity toward GTP synthesis could be abolished by the addition of Tween 20 or Triton X-100. The activity itself could be abolished by the addition of anti-Ndk antibody to the assay mixture. The formation of the 12-kDa form of Ndk and its association with the cell membrane were found to be related to the growth stage of P. aeruginosa, with less than 1% of the 12-kDa Ndk detectable in the membrane fraction at early log phase in comparison with the levels present at late stationary phase.

Cytoplasm↗
PubMed1995

Characterization of a phosphoprotein phosphatase for the phosphorylated form of nucleoside-diphosphate kinase from Pseudomonas aeruginosa.

Nucleoside-diphosphate kinase (ATP:nucleoside-diphosphate phosphotransferase, EC 2.7.4.6; NDP kinase) is an important enzyme for the maintenance of the correct cellular levels of nucleoside triphosphates (NTPs) and their deoxy derivatives (dNTPs) and is involved in the regulation of cellular development. The enzyme is under the dual control of algR2 and algH in Pseudomonas aeruginosa. We report here the purification and characterization of a protein that dephosphorylates the phosphorylated intermediate form of the P. aeruginosa NDP kinase (Ndk). Dephosphorylation of Ndk phosphate leads to loss of its enzymatic activity. The 10.1-kDa polypeptide shows 77% homology at the N terminus with the Spo0E phosphatase, identified as a negative regulator of sporulation in Bacillus subtilis and 66% with the human Bax protein, identified as an effector of programmed cell death. The phosphatase termed Npp showed varied specificity toward phosphorylated Ndks from different sources including human erythrocyte Ndk phosphate. Its activity toward other histidine phosphates such as CheA or the alpha-subunit of succinyl-CoA synthetase or phosphoesters such as p-nitrophenyl phosphate was quite limited. Npp was stable at room temperature up to 2 h and required Mg2+ for activity. The presence of a phosphatase capable of dephosphorylating the phosphorylated form of P. aeruginosa Ndk represents an interesting and efficient mode of post-translational modification of an enzyme crucial to cellular development.

Adenosine Triphosphate↗
PubMed1995

Regulation of nucleoside diphosphate kinase and an alternative kinase in Escherichia coli: role of the sspA and rnk genes in nucleoside triphosphate formation.

We have previously reported that two genes cloned from a cosmid library of Escherichia coli can restore mucoidy to an algR2 mutant of Pseudomonas aeruginosa. AlgR2 is a protein involved in the regulation of nucleoside diphosphate kinase (Ndk) as well as alginate synthesis in P. aeruginosa. One of the E. coli genes, rnk, encodes a 14.9 kDa protein with no homology to any other proteins. The other gene, sspA, encodes the stringent starvation protein, a regulatory protein involved in stationary-phase regulation and the stringent response of E. coli. While both rnk and sspA restored alginate production to the P. aeruginosa algR2 mutant, only rnk restored Ndk activity to the mutant. In this report, we have examined the effect of mutations in rnk and sspA on the levels of Ndk in E. coli. We find that a mutation in rnk drastically reduces the level of Ndk in E. coli. A mutation in sspA, however, affects the level of another nucleoside diphosphate kinase distinct from Ndk. The proteins can be easily distinguished from each other by their different affinities for nucleoside diphosphates (NDPs) and also by the differential effect of anti-Ndk antibodies on the reactions they catalyse. The ability of either of these two proteins to restore alginate synthesis in the algR2 mutant of P. aeruginosa demonstrates the importance of nucleoside triphosphate synthesis and energy metabolism for alginate synthesis. Additionally, a role for the stringent starvation protein (SspA) in the modulation of nucleoside triphosphate (NTP) levels in E. coli is also suggested from these experiments.

Adhesins, Bacterial↗
PubMed1995

The Escherichia coli genes sspA and rnk can functionally replace the Pseudomonas aeruginosa alginate regulatory gene algR2.

The algR2 (also known as algQ) gene of Pseudomonas aeruginosa has previously been identified as being necessary for alginate production at 37 degrees C. We have cloned two genes, from a cosmid library of Escherichia coli, which can restore mucoidy to an algR2 mutant of P. aeruginosa. The complementing regions of both cosmids were localized by subcloning restriction fragments. One of the E. coli genes identified here has not previously been described; we have named this gene rnk (regulator of nucleoside diphosphate kinase). It encodes a 14.9 kDa protein with no homology to any other protein. The other gene, sspA, is a regulator involved in stationary-phase regulation in E. coli. Either gene will restore mucoidy to an algR2-deficient strain of P. aeruginosa. AlgR2 has been shown to regulate at least two enzymes, succinyl-CoA synthetase (Scs) and nucleoside diphosphate kinase (Ndk), which form a complex in P. aeruginosa. When we examined the ability of the E. coli analogues to regulate Ndk, we found that rnk but not sspA was able to restore Ndk activity to the P. aeruginosa algR2 mutant. Furthermore, rnk was able to restore growth of the algR2 mutant in the presence of Tween 20, which inhibits other Ndk-like activities.

Alginates↗
PubMed1995

Substrate diversity and expression of the 2,4,5-trichlorophenoxyacetic acid oxygenase from Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid as a sole source of carbon and energy. The genes encoding the proteins involved in the first step (tftA and tftB [previously designated tftA1 and tftA2, respectively]) have been cloned and sequenced. The oxygenase, TftAB, is capable of converting not only 2,4,5-trichlorophenoxyacetic acid to 2,4,5-trichlorophenol but also a wide range of chlorinated aromatic phenoxyacetates to their corresponding phenolic derivatives, as shown by whole-cell and cell-free assays. The rate of substrate utilization by TftAB depends upon the extent of chlorination of the substrate, the positions of the chlorines, and the phenoxy group. These results indicate a mechanistic similarity between TftAB and the 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate-dependent dioxygenase, TfdA, from Alcaligenes eutrophus JMP134. The promoter of the oxygenase genes was localized by promoter-probe analysis, and the transcriptional start site was identified by primer extension. The beta-galactosidase activity of the construct containing the promoter region cloned upstream of the beta-galactosidase gene in the promoter-probe vector pKRZ-1 showed that this construct is constitutively expressed in Escherichia coli and in AC1100. The -35 and -10 regions of the oxygenase genes show significant sequence identity to typical Escherichia coli sigma 70 promoters.

Base Sequence↗
PubMed1995

Regulation of nucleoside diphosphate kinase and secretable virulence factors in Pseudomonas aeruginosa: roles of algR2 and algH.

Alginate is an important virulence factor for Pseudomonas aeruginosa during infection of the lungs of cystic fibrosis patients. The genes encoding enzymes for alginate production by P. aeruginosa are normally silent. They are activated in response to several environmental conditions, including high osmolarity, exposure to ethanol, or long-term growth under conditions of nutrient deprivation. Several genes which participate in the activation of alginate gene promoters have been identified; among these is the algR2 (algQ) gene. AlgR2 is an 18-kDa protein which has been shown to regulate the critical algD gene encoding GDP-mannose dehydrogenase as well as to regulate the levels of a tricarboxylic acid cycle enzyme, i.e., succinyl coenzyme A synthetase, and nucleoside diphosphate kinase (Ndk), an enzyme involved in nucleoside triphosphate synthesis. Succinyl coenzyme A synthetase and Ndk form a complex in P. aeruginosa. While algR2 is required for alginate synthesis at 37 degrees C, an algR2 insertion mutant was still able to make alginate slowly at 37 or at 30 degrees C. We used this observation to identify and clone a gene, termed algH. A strain with mutations in both algR2 and algH is unable to produce alginate at either 37 or 30 degrees C, and it is fully defective in Ndk production.

Alginates↗
PubMed1995

Polyamines exert regulatory control on mycobacterial transcription: a study using RNA polymerase from Mycobacterium phlei.

We have used purified RNA polymerase from Mycobacterium phlei to study the role of polyamines in mycobacterial transcription. Both initiation and elongation phases of the process were affected biphasically by polyamines. Interaction of polyamines with DNA template plays an important role in transcription modulation. Our studies emphasize that polyamines can exert a regulatory control on mycobacterial transcription.

DNA-Directed RNA Polymerases↗
PubMed1995

The 'Rastelli' operation--results at the Singapore General Hospital.

Reconstruction of the right ventricular outflow tract in patients with right ventricle-pulmonary artery discontinuity with a valved conduit is well established. The aim of this study was to review our results at the Singapore General Hospital. Between October 1989 and May 1993, 13 patients underwent the 'Rastelli' operation. The mean age at definitive repair was 68 months. Ten patients had pulmonary atresia with ventricular septal defect, two patients truncus arteriosus (one patient each with type 3 and type 2) and one patient had tetralogy of Fallot with absent pulmonary valve syndrome. In 12 patients cryo-preserved pulmonary artery homografts with a mean diameter of 21mm (19-23mm) were used, and a 11mm composite Hancock dacron valved conduit in one patient. There were no operative deaths. The mean duration of follow-up is 37 months (23-48 months). All patients were well and in NYHA class I on recent follow-up. On echocardiographic assessment 12 patients had trivial or no gradient across the right ventricular outflow tract, while one patient had a gradient of 25mmHg. The "Rastelli" operation can be undertaken with low mortality and morbidity and early results are encouraging.

Blood Vessel Prosthesis↗
PubMed1995

Cancer prevention behaviors among African-American adults: a survey of wards 7 and 8 in Washington, DC.

A telephone survey of knowledge, attitude, and health practices regarding cancer was undertaken in wards 7 and 8, Washington, DC in 1988. These wards have the highest cancer rates in the city and are predominantly African American. Of the 670 randomly selected persons over 18 years of age, 243 were males and 427 were females. Among females, 84% believed cigarette smoking causes cancer, and 48% thought alcohol causes cancer; 31% smoked cigarettes and 38% consumed alcoholic beverages. Among males, 91% and 52% thought cigarettes and alcohol causes cancer respectively; 41% smoked and 54% consumed alcoholic beverages. Only 6% of the males over age 40 practiced all eight recommended cancer prevention behaviors, while 2% of the females over age 40 practiced all preventive health behaviors. Cancer preventive behavior was examined in relation to socioeconomic status. This study indicates that preventive health behaviors were not associated with socioeconomic status. Data suggest that cancer prevention and control programs and services targeted to this Washington, DC population should be increased and intensified.

Adolescent↗
PubMed1994

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

Infection with mucoid, alginate-producing strains of Pseudomonas aeruginosa is the leading cause of mortality among patients with cystic fibrosis. Alginate production by P. aeruginosa is not constitutive but is triggered by stresses such as starvation. The algR2 (also termed algQ) gene has been previously identified as being necessary for mucoidy; an algR2 mutant strain is unable to produce alginate when grown at 37 degrees C. We show here that the levels of phosphorylated succinyl coenzyme A synthetase (Scs) and nucleoside diphosphate kinase (Ndk), which form a complex in P. aeruginosa, are reduced in the algR2 mutant. We were able to correlate the lower level of phosphorylated Scs with a decrease in Scs activity. Western blots (immunoblots) also showed a decreased level of Ndk in the algR2 mutant, but the presence of another kinase activity sensitive to Tween 20 provides the missing Ndk function. The effect of AlgR2 on tricarboxylic acid (TCA) cycle enzymes appears to be specific for Scs, since none of the other TCA cycle enzymes measured showed a significant decrease in activity. Furthermore, the ability of the algR2 mutant to grow on TCA cycle intermediates, but not glucose, is impaired. These data indicate that AlgR2 is responsible for maintaining proper operation of the TCA cycle and energy metabolism.

Alginates↗
PubMed1993

MchAI and MchAII, two class-II restriction endonucleases from Mycobacterium chelonei.

We have purified and characterized two class-II restriction endonucleases from the saprophyte Mycobacterium chelonei AMB 82. MchAI was an isoschizomer of NotI recognizing and cleaving at 5'-GC/GGCCGC. MchAI did not require Triton X-100 for activity and was fully active at concentrations over 10-200 mM KCl and MgCl2. MchAII was an isoschizomer of HaeIII recognizing and cleaving at 5'-GG/CC. MchAII was active in 10 mM Tris.HCl pH 8.0 containing MgCl2 at 10-200 mM, was strongly inhibited by NaCl at concentrations higher than 10 mM and was inactive at KCl concentrations lower than 250 mM.

Base Sequence↗
PubMed1992

Can consultants resuscitate?

Twenty-four of 53 hospital consultants responded to an offer to attend a resuscitation training course. Fourteen of them had never had resuscitation training. Their performance of basic life-support was assessed before and after training according to the Resuscitation Council UK recommendations. Their initial performance of basic life-support on a manikin was extremely poor. One hour of training and practice resulted in statistically significant improvements.

Cardiopulmonary Resuscitation↗