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A M Chakrabarty

Publications and source records attributed to A M Chakrabarty.

At least 91 records · Page 5Linked to original sources
PubMed1993

Inhibitors of two-component signal transduction systems: inhibition of alginate gene activation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa strains infecting cystic fibrosis patients often produce copious amounts of the exopolysaccharide alginate. Expression of alginate genes in P. aeruginosa is regulated by several proteins including members of the two-component bacterial signal transduction systems. Two of these regulatory proteins are AlgR1, the DNA-binding response regulator that transcriptionally activates alginate gene expression, and AlgR2, the kinase that modifies AlgR1 via phosphorylation to enhance its activity. In this paper, we report the identification of compounds that inhibit alginate gene expression by inhibiting (i) the phosphorylation/dephosphorylation of AlgR2 and (ii) the DNA-binding activity of AlgR1. Compounds with these activities may have potential as components of therapy for eliminating P. aeruginosa infection from the cystic fibrosis lung. In addition, we describe the effect of these compounds on the autophosphorylation activity of other known two-component kinases and show the ability of one compound to significantly inhibit the kinase activities of CheA, NRII, and KinA.

Adenosine Triphosphate↗
PubMed1993

Nucleotide sequence and expression of the Pseudomonas aeruginosa algF gene controlling acetylation of alginate.

Colonization of the cystic fibrosis lung by Pseudomonas aeruginosa is greatly facilitated by the production of an exopolysaccharide called alginate. In this study we determined the nucleotide sequence of an alginate modification gene, algF, which controls the addition of acetyl groups to alginate. Expression of algF using a T7 promoter-expression system showed that algF codes for a 24.5 kDa polypeptide (predicted size 22,832 Da) that is processed to 19.5 kDa. The N-terminus of the processed polypeptide matched the predicted amino acid sequence of AlgF starting at Asp-29. An algF mutant failed to produce alginate owing to a polar effect on the downstream algA gene. Although the algA gene, provided in trans, restored synthesis of alginate, the alginate was non-acetylated. We show that a plasmid containing both the algF and algA gene complements the alginate acetylation defect of the algF mutant strain.

Acetylation↗
PubMed1993

Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa.

Reporter gene technology was employed to detect the activity of an alginate promoter of Pseudomonas aeruginosa when the organism was grown as a biofilm on a Teflon mesh substratum and as planktonic cells in liquid medium. Alginate biosynthetic activity was determined with a mucoid cell line derived from a cystic fibrosis isolate and containing an alginate algC promoter fused to a lacZ reporter gene. Reporter activity was demonstrated with chromogenic and fluorogenic substrates for beta-galactosidase. Expression of algC was shown to be upregulated in biofilm cells compared with planktonic cells in liquid medium. Gene up-expression correlated with alginate biosynthesis as measured by Fourier transform infrared spectroscopy, uronic acid accumulation, and alginate-specific enzyme-linked immunosorbent assay. The algC promoter was shown to have maximum activity in planktonic cultures during the late lag and early log phases of the cell growth cycle. During a time course experiment, biofilm algC activity exceeded planktonic activity except during the period immediately following inoculation into fresh medium. In continuous-culture experiments, conversion of lacZ substrate was demonstrated microscopically in individual cells by epifluorescence microscopy.

Alginates↗
PubMed1993

Enhancer-like activity of A1gR1-binding site in alginate gene activation: positional, orientational, and sequence specificity.

Significant activation of promoters of alginate genes such as algD or algC occurs in mucoid Pseudomonas aeruginosa during its proliferation in the lungs of cystic fibrosis patients. These promoters have been shown to be responsive to environmental signals such as high osmolarity. The signaling is mediated by a so-called two-component signal transduction system, in which a soluble protein, AlgR2, undergoes autophosphorylation and transfers the phosphate to a DNA-binding response regulator protein, AlgR1. The phosphorylated form of AlgR1 has a high affinity for binding at upstream sequences of both the algC and algD promoters. Two AlgR1-binding sites (ABS) have been reported upstream of the algC gene. One of the two ABSs (algC-ABS1, located at -94 to -81) is critical for the algC activation process, while the second ABS (algC-ABS2, located at +161 to +174) is only weakly active. We now report the presence of a third ABS within the structural gene of algC, and this ABS (algC-ABS3) is also important for algC promoter activation. algC-ABS1 can be replaced functionally by algC-ABS2, algD-ABS1, or algD-ABS2 and somewhat weakly by algD-ABS3. Introduction of a half-integral turn in the DNA helix between the algC site of transcription initiation and algC-ABS1 allowed only slight reduction of promoter activity, suggesting that the binding site could be appreciably functional even when present in the opposite face of the helix. Activation of the algC promoter is independent of the relative location (upstream or downstream of the mRNA start site), the number of copies, or the orientation of algC-ABS1, suggesting that it behaves like a eukaryotic enhancer element in promoting transcription from the algC promoter.

Alginates↗
PubMed1993

Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida.

The 3-chlorocatechol operon clcABD is central to the biodegradative pathway of 3-chlorobenzoate. The clcR regulatory gene, which activates the clcABD operon, was cloned from the region immediately upstream of the operon and was shown to complement an insertion mutation for growth on 3-chlorobenzoate. ClcR activated the clcA promoter, which controls expression of the clcABD operon, in trans by 14-fold in an in vivo promoter probe assay in Pseudomonas putida when cells were incubated with 15 mM 3-chlorobenzoic acid. Specific binding of ClcR to the clcR-clcA intergenic promoter region was observed in a gel shift assay. Nucleotide sequence analysis of the clcR gene predicts a polypeptide of 32.5 kDa, which was confirmed by using specific in vivo 35S labeling of the protein from a T7 promoter-controlled ATG fusion construct. ClcR shares high sequence identity with the LysR family of bacterial regulator proteins and has especially high homology to a subgroup of the family consisting of TcbR (57% amino acid sequence identity), TfdS, CatR, and CatM. ClcR was shown to autoregulate its own production in trans to 35% of unrepressed levels but partially relieved this autorepression under conditions that induced transcription at the clcA promoter. Several considerations indicate that the clcR-clcABD locus is most similar to the tcbR-tcbCDEF regulon.

Amino Acid Sequence↗
PubMed1992

AlgR2 is an ATP/GTP-dependent protein kinase involved in alginate synthesis by Pseudomonas aeruginosa.

The exopolysaccharide alginate is a major virulence factor in the pathogenicity of Pseudomonas aeruginosa infecting the lungs of cystic fibrosis (CF) patients. Alginate synthesis by P. aeruginosa is believed to occur in response to environmental signals present in the CF lung. Transcription of a critical alginate biosynthetic gene, algD, is triggered by environmental signals and is known to be controlled by regulatory proteins AlgR1, AlgR2, and AlgR3. AlgR1 is a member of the family of response regulators of the phosphorylation-dependent two-component bacterial signal transduction systems. In this report, we describe the characterization of AlgR2 as the kinase involved in phosphorylation of AlgR1. AlgR2, an 18-kDa soluble protein undergoes rapid autophosphorylation in the presence of either ATP or GTP and transfers the phosphate to AlgR1. AlgR2 retains high affinity for both ATP and GTP with an apparent Km of 137 and 249 nM, respectively, for phosphorylation by these two substrates. ADP and GDP exhibit competitive inhibition with an apparent Ki of 94 and 314 nM, respectively, during phosphorylation by ATP and 481 and 273 nM during phosphorylation by GTP. AlgR1 and AlgR2 can be isolated in the form of an 80-kDa complex that is capable of undergoing phosphorylation and intracomplex phosphotransfer in vitro. A 16-kDa AlgR2 analog, capable of autophosphorylation in the presence of ATP or GTP and transferring the phosphate moiety to AlgR1, has been characterized in Escherichia coli.

Adenosine Triphosphate↗
PubMed1992

Signal transduction in exopolysaccharide alginate synthesis: phosphorylation of the response regulator AlgR1 in Pseudomonas aeruginosa and Escherichia coli.

Synthesis of alginate by Pseudomonas aeruginosa correlates with its pathogenicity in the lungs of patients suffering from cystic fibrosis (CF). Alginate synthesis-encoding genes (alg) in P. aeruginosa are normally silent, but are specifically triggered in the CF lung environment. The promoter for the algD gene, located at the upstream end of the alg cluster, is activated by environmental factors such as high osmolarity, nutrient limitation and dehydration. Several regulatory proteins are known to control transcription from the algD promoter. Among these proteins is AlgR1 which is homologous to the phosphorylation-dependent response regulators of the two-component signal transduction system. In this paper, we report that AlgR2, an 18-kDa protein which in cooperation with AlgR1 regulates the algD promoter, undergoes phosphorylation in the presence of ATP. The phosphate group acquired by AlgR2 is then transferred to AlgR1. In addition, we show that AlgR1 can be phosphorylated by an AlgR2-analog in Escherichia coli. AlgR1 is isolated in a phosphorylatable 80-kDa complex in association with AlgR2 in P. aeruginosa and the AlgR2-analog in E. coli.

Alginates↗
PubMed1992

Characterization of guanosine diphospho-D-mannose dehydrogenase from Pseudomonas aeruginosa. Structural analysis by limited proteolysis.

Alginate is believed to be a major virulence factor in the pathogenicity of Pseudomonas aeruginosa in the lungs of patients suffering from cystic fibrosis. Guanosine diphospho-D-mannose dehydrogenase (GDPmannose dehydrogenase, EC 1.1.1.132) is a key enzyme in the alginate biosynthetic pathway which catalyzes the oxidation of guanosine diphospho-D-mannose (GDP-D-mannose) to GDP-D-mannuronic acid. In this paper, we report the structural analysis of GMD by limited proteolysis using three different proteases, trypsin, submaxillary Arg-C protease, and chymotrypsin. Treatment of GMD with these proteases indicated that the amino-terminal part of this enzyme may fold into a structural domain with an apparent molecular mass of 25-26 kDa. Multiple proteolytic cleavage sites existed at the carboxyl-terminal end of this domain, indicating that this segment may represent an exposed region of the protein. Initial proteolysis also generated a carboxyl-terminal fragment with an apparent molecular mass of 16-17 kDa which was further digested into smaller fragments by trypsin and chymotrypsin. The proteolytic cleavage sites were localized by partial amino-terminal sequencing of the peptide fragments. Arg-295 was identified as the initial cleavage site for trypsin and Tyr-278 for chymotrypsin. Catalytic activity of GMD was totally abolished by the initial cleavage. However, binding of the substrate, GDP-D-mannose, increased stability toward proteolysis and inhibited the loss of enzyme activity. GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.

Affinity Labels↗
PubMed1992

Alginate synthesis in Pseudomonas aeruginosa: environmental regulation of the algC promoter.

The exopolysaccharide alginate is a major virulence factor of Pseudomonas aeruginosa strains that infect the lungs of cystic fibrosis patients. The synthesis of alginate is almost uniquely associated with the pathogenicity of P. aeruginosa within the environment of the cystic fibrosis lung. The gene algC is one of the essential alginate biosynthetic genes and codes for the enzyme phosphomannomutase. In this report, we present data on the transcriptional regulation of algC expression. The activity of the algC promoter is modulated by the response regulator, AlgR1, a member of the two-component signal transduction protein family, which also regulates other alginate-specific promoters. In both mucoid (alginate-positive) and nonmucoid (alginate-negative) P. aeruginosa strains, transcriptional activation of algC increased with the osmolarity of the culture medium. This osmolarity-induced activation was found to be dependent on AlgR1. AlgR1 was found to interact directly with the algC promoter. Deletion mapping, in conjunction with mobility shift assays, showed that AlgR1 specifically bound with two regions of algC upstream DNA. A fragment spanning nucleotide positions -378 to -73 showed strong specific binding, while a fragment located between positions -73 and +187 interacted relatively weakly with AlgR1. Phosphorylation of the AlgR1 protein resulted in the stimulation of its in vitro ability to bind to the algC promoter region (a fragment spanning nucleotides -378 to -73). Transcription from the algC promoter, which has significant homology with the RNA polymerase sigma-54 (RpoN) recognition sequence, decreased in an rpoN mutant of P. aeruginosa.

Alginates↗
PubMed1992

Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida.

In Pseudomonas putida, the catBC operon encodes enzymes involved in benzoate degradation. Previous studies have determined that these enzymes are induced when P. putida is grown in the presence of benzoate. Induction of the enzymes of the catBC operon requires an intermediate of benzoate degradation, cis,cis-muconate, and a regulatory protein, CatR. It has been determined that CatR binds to a 27-bp region of the catBC promoter in the presence or absence of inducer. We have called this the repression binding site. In this study, we used a gel shift assay to demonstrate that the inducer, cis,cis-muconate, increases the affinity of CatR for the catBC promoter region by 20-fold. Furthermore, in the absence of cis,cis-muconate, CatR forms two complexes in the gel shift assay. The inducer cis,cis-muconate confers specificity primarily for the formation of complex 2. DNase I footprinting showed that an additional 27 bp of the catBC promoter region is protected by CatR in the presence of cis,cis-muconate. We have named this second binding site the activation binding site. Methylation interference footprinting determined that in the presence or absence of inducer, five G nucleotides of the catBC promoter region were necessary for CatR interaction with the repression binding site, while a single G residue was important for CatR interaction with the activation binding site in the presence of cis,cis-muconate. Using polymerase chain reaction-generated constructs, we found that the binding of CatR to the repression binding site is independent of the activation binding site. However, binding of CatR to the activation binding site required an intact repression binding site.

Bacterial Proteins↗
PubMed1991

Nucleotide sequence and expression of the algE gene involved in alginate biosynthesis by Pseudomonas aeruginosa.

Alginate (Alg), a random polymer of mannuronic acid and glucuronic acid residues, is synthesized and secreted by Pseudomonas aeruginosa primarily during its infection of the lungs of cystic fibrosis patients. The molecular biology and biochemistry of the enzymatic steps leading to the production of the Alg precursor GDP-mannuronic acid have been elucidated, but the mechanism of polymer formation and export of Alg are not understood. We report the nucleotide sequence of a 2.4-kb DNA fragment containing the algE gene, previously designated alg76, encoding the AlgE protein (Mr 54,361) that is believed to be involved in these late steps of Alg biosynthesis. Expression of algE appears to occur from its own promoter. The promoter region contains several direct and inverted repeat sequences and shares structural similarity with promoters of several other alg genes from P. aeruginosa. In addition, the AlgE protein was overproduced from the tac promoter in P. aeruginosa. N-terminal amino acid sequence analysis showed that the polypeptide contains a signal peptide which is cleaved to form the mature protein during AlgE export from the cell cytoplasm.

Alginates↗
PubMed1991

Characterization and regulation of the Pseudomonas aeruginosa algC gene encoding phosphomannomutase.

The nucleotide sequence of the Pseudomonas aeruginosa algC gene encoding phosphomannomutase (PMM; EC 5.4.2.8) was determined. The codon usage in algC in the wobble base position was 90.4% G+C, typical of Pseudomonas genes. The predicted amino acid sequence of phosphomannomutase (PMM) showed homology over a stretch of 112 amino acids in the carboxyl terminus with rabbit muscle phosphoglucomutase (PGM), an enzyme that catalyzes a reaction analogous to that catalyzed by PMM. In addition, a specific amino acid sequence within PMM showed homology with the catalytic site of PGM. DNA sequence analysis of a defective algC gene (algC') cloned from a mutant of P. aeruginosa that lacked PMM activity revealed one point mutation (a C to T transition) in the carboxyl terminus of PMM which resulted in an amino acid change from arginine 420 to cysteine 420. The mutation identified in the algC' gene was not within the regions of homology with PGM. The algC promoter showed significant homology with the promoters of two other P. aeruginosa genes involved in alginate synthesis, algD and algR1. Both the algD and algR1 promoters are activated by the product of the algR1 gene in P. aeruginosa. The upstream region of the algC gene contained a sequence identical to the algD upstream sequence that is known to be the binding site for the AlgR1 protein. Expression of algC was reduced 5.7-fold in an algR1 mutant of P. aeruginosa compared to its isogenic parent strain (lacking the algR1 mutation), suggesting that the algR1 gene product activates the transcription of the algC gene.

Amino Acid Sequence↗
PubMed1991

Purification of the regulatory protein AlgR1 and its binding in the far upstream region of the algD promoter in Pseudomonas aeruginosa.

A regulatory protein AlgR1, previously suggested to be a member of a two-component sensory transduction system because of its homology to OmpR and NtrC and its ability to allow activation of the algD promoter under conditions of high osmolarity, has been hyperproduced in Escherichia coli after deletion of the upstream region including part of the Shine-Dalgarno sequence of the algR1 gene and its subsequent cloning under the tac promoter. The AlgR1 protein is purified as a monomer, and the sequence of the nine N-terminal amino acids of the monomer matches with that predicted from the DNA sequence of the algR1 gene. The purified AlgR1 protein binds to two separate DNA fragments of the algD upstream region. DNase protection experiments identify these two DNA segments as 14-mer sequences centered at -382 and -458 regions, which contain a common CCGT-TCGTC sequence in them. While the presence of at least one AlgR1 binding site is important for the activation of the algD promoter, the presence of both of the binding sites in the upstream region leads to a higher level of activation.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa.

We report here the purification and characterization of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase, a bifunctional enzyme (PMI-GMP) which catalyzes both the phosphomannose isomerase (PMI) and guanosine 5'-diphospho-D-mannose pyrophosphorylase (GMP) reactions of the Pseudomonas aeruginosa alginate biosynthetic pathway. The PMI and GMP activities co-eluted in the same protein peak through successive fractionation on hydrophobic interaction, ion exchange, and gel filtration chromatography. The purified enzyme migrated as a 56,000 molecular weight protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native protein migrated as a monomer of 54,000 molecular weight upon gel filtration chromatography. The apparent Km for D-mannose 6-phosphate was 3.03 mM, and the Vmax was 830 nmol/min/mg of enzyme. For the GMP forward reaction, apparent Km values of 20.5 microM and 29.5 microM for D-mannose 1-phosphate and GTP, respectively, were obtained from double reciprocal plots. The GMP forward reaction Vmax (5,680 nmol/min/mg of enzyme) was comparable to the reverse reaction Vmax (5,170 nmol/min/mg of enzyme), and the apparent Km for GDP-D-mannose was determined to be 14.2 microM. Both reactions required Mg2+ activation, but the PMI reaction rate was 4-fold higher with Co2+ as the activator. PMI (but not GMP) activity was sensitive to dithiothreitol, indicating the involvement of disulfide bonds to form a protein structure capable of PMI activity. DNA sequencing of a cloned mutant algA gene from P. aeruginosa revealed that a point mutation at nucleotide 961 greatly decreased the levels of both PMI and GMP in a crude extract.

Alginates↗
PubMed1991

Cloning and characterization of a chromosomal DNA region required for growth on 2,4,5-T by Pseudomonas cepacia AC1100.

A series of spontaneous 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) nonmetabolizing mutants of Pseudomonas cepacia AC1100 were characterized to be defective in either 2,4,5-T uptake or conversion of this compound to 2,4,5-trichlorophenol (2,4,5-TCP). Two of these mutants, RHC22 and RHC23, were complemented for growth on 2,4,5-T using an AC1100 genomic library constructed in the cosmid vector pCP13. Recombinant cosmids isolated from the complemented mutants contained a 27.5-kb insert which frequently underwent various-sized deletions in Escherichia coli. Hybridization studies showed this DNA to be of chromosomal origin and totally deleted in RHC22, RHC23 and other similar mutants. Complementation analyses of RHC22 with a series of subcloned fragments and spontaneously deleted derivatives of the recombinant cosmid pRHC21 showed the 2,4,5-T (tft) genes to occur within an 8.9-kb region. Pseudomonas aeruginosa cells transformed with this DNA acquired the ability to convert 2,4,5-T to 2,4,5-TCP. The genetic determinant for this function was further localized within a 3.7-kb region. This DNA, in the absence of other sequences from the 8.9-kb tft gene region allowed RHC22 cells to metabolize 2,4,5-T, but at low rates which were insufficient to support growth. Copies of the insertions sequence element IS931 were identified either adjacent to or within this tft gene region in the genomes of two independent wild-type AC1100 isolates. Preliminary evidence suggests that these sequences either facilitate or are required for growth on 2,4,5-T and hence may be implicated in the genetic evolution of the 2,4,5-T metabolic pathway.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1991

Environmentally regulated algD promoter is responsive to the cAMP receptor protein in Escherichia coli.

The environmentally activated algD promoter of Pseudomonas aeruginosa has been shown to be influenced by DNA supercoiling. It is believed that protein-induced bending or looping is required for this activation. We studied the role of Escherichia coli cAMP-CRP on algD promoter activation in E. coli and show that a functional CRP is required for this activation. We also demonstrate that the algD promoter is sensitive to glucose repression both in E. coli and P. aeruginosa. Deletion of a putative consensus CRP binding sequence upstream of the algD promoter renders the promoter non-responsive to glucose repression. The involvement of cAMP-CRP complex in the activation of the algD promoter in E. coli has been demonstrated directly through binding of a 255 base pair DNA fragment containing the putative consensus CRP binding sequence. Other fragments, upstream or downstream but without any consensus CRP binding sequence, did not show any binding with CRP. A CRP-like analogue, similar to that in Xanthomonas campestris, but capable of activating genes without forming a complex with cAMP, is believed to allow glucose repression in P. aeruginosa.

Bacterial Proteins↗