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A M Chakrabarty

Publications and source records attributed to A M Chakrabarty.

At least 73 records · Page 4Linked to original sources
PubMed1995

Copper as a signal for alginate synthesis in Pseudomonas syringae pv. syringae.

Plant-associated pseudomonads are commonly exposed to copper bactericides, which are applied to reduce the disease incidence caused by these bacteria. Consequently, many of these bacteria have acquired resistance or tolerance to copper salts. We recently conducted a survey of 37 copper-resistant (Cur) Pseudomonas spp., including P. cepacia, P. fluorescens, P. syringae, and P. viridiflava, and found that a subset of the P. syringae strains showed a dramatic increase in exopolysaccharide (EPS) production on mannitol-glutamate medium containing CuSO4 at 250 micrograms/ml. A modified carbazole assay indicated that the EPS produced on copper-amended media contained high levels of uronic acids, suggesting that the EPS was primarily alginic acid. Uronic acids extracted from selected strains were further confirmed to be alginate by demonstrating their sensitivity to alginate lyase and by descending paper chromatography following acid hydrolysis. Subinhibitory levels of arsenate, cobalt, lithium, rubidium, molybdenum, and mercury did not induce EPS production, indicating that alginate biosynthesis is not induced in P. syringae cells exposed to these heavy metals. A 200-kb plasmid designated pPSR12 conferred a stably mucoid phenotype to several P. syringae recipients and also increased their resistance to cobalt and arsenate. A cosmid clone constructed from pPSR12 which conferred a stably mucoid phenotype to several P. syringae strains but not to Pseudomonas aeruginosa was obtained. Results obtained in this study indicate that some of the signals and regulatory genes for alginate production in P. syringae differ from those described for alginate production in P. aeruginosa.

Alginates↗
PubMed1995

Evidence that operons tcb, tfd, and clc encode maleylacetate reductase, the fourth enzyme of the modified ortho pathway.

The maleylacetate reductase from Pseudomonas sp. strain B13 functioning in the modified ortho pathway was purified and digested with trypsin. The polypeptides separated by high-performance liquid chromatography were sequenced. Alignments with the polypeptides predicted from the tfdF and tcbF genes located on plasmids pJP4 of the 2,4-dichlorophenoxyacetate-degrading Alcaligenes eutrophus JMP134 and pP51 of the 1,2,4-trichlorobenzene-degrading Pseudomonas sp. strain P51 as well as polypeptides predicted from the tftE gene located on the chromosome of the 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were obtained. In addition, the deduced protein sequence encoded by the nucleotide sequence downstream of clcD on plasmid pAC27 of the 3-chlorobenzoate-degrading Pseudomonas putida AC866 was tested for homology. Significant sequence similarities with the polypeptides encoded by the tfdF, tcbF, and tftE genes as well as the nucleotide sequence downstream of the clcD gene gave evidence that these genes might encode maleylacetate reductases. A NAD-binding motif in a beta alpha beta-element was detected.

2,4-Dichlorophenoxyacetic Acid↗
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↗
PubMed1994

Interaction of two LysR-type regulatory proteins CatR and ClcR with heterologous promoters: functional and evolutionary implications.

The soil bacteria Pseudomonas putida can use benzoate or 3-chlorobenzoate as a sole carbon source. Benzoate and 3-chlorobenzoate are converted into catechol and 3-chlorocatechol, respectively, which are in turn converted into tricarboxylic acid cycle intermediates. The catabolic pathways of both compounds proceed through similar intermediates, have similar genetic organization, and have homologous enzymes responsible for different catabolic steps. This has led to suggestions that the plasmid-borne 3-chlorocatechol degradation genes evolved from the chromosomal catechol degradation genes. Both catechol and 3-chlorocatechol pathways are positively regulated by the homologous regulatory proteins CatR and ClcR, respectively. These proteins belong to the LysR family of DNA binding proteins and bind to highly conserved target sequences. We examined the ability of CatR and ClcR to cross-regulate the two pathways. CatR was shown in vitro by DNase I footprinting and gel-shift assays to interact with the clcABD promoter region. Likewise, ClcR was shown to interact in vitro with the catBC promoter region. In in vivo experiments, CatR complemented a ClcR- P. putida strain harboring the clcABD operon for growth on 3-chlorobenzoate. However, ClcR was not capable of complementing a CatR- P. putida strain for growth on benzoate. These observations were confirmed by lacZ-transcriptional fusion expression experiments. Differences in the CatR and ClcR binding sites and their in vitro binding characteristics may explain the ability of CatR and not ClcR to cross-activate. These differences may provide insight about the evolution of regulatory systems in P. putida.

Bacterial Proteins↗
PubMed1994

Post-transcriptional regulation of the Pseudomonas aeruginosa algC gene.

The algC gene (encoding phosphomannomutase) of Pseudomonas aeruginosa, similarly to the algD gene, is environmentally regulated through transcriptional activation of its promoter. This gene, like algD, has a long (244 bp) 5' untranslated leader region (5' UTR). Using transcriptional and translational algC::lacZ fusions, we show that even though the transcript levels are similar, the beta-galactosidase-specific activities of the translational fusions are much higher than those of the transcriptional fusions during the entire growth phase. Both the 5' UTR and the ribosomal-binding site are shown to be important for efficient translation of the algC mRNA.

Carbohydrate Dehydrogenases↗
PubMed1994

Characterization of nucleoside-diphosphate kinase from Pseudomonas aeruginosa: complex formation with succinyl-CoA synthetase.

The enzyme nucleoside-diphosphate kinase (Ndk), responsible for the conversion of (deoxy)ribonucleoside diphosphates to their corresponding triphosphates, has been purified from Pseudomonas aeruginosa. The N-terminal 12 amino acid sequence of P. aeruginosa Ndk shows significant homology with that of Myxococcus xanthus and that of Escherichia coli. Ndk enzyme activity is also associated with succinyl-CoA synthetase activity in P. aeruginosa, whose alpha and beta subunits show extensive sequence homology with those of E. coli and Dictyostelium discoideum. The 33-kDa alpha subunit of succinyl-CoA synthetase of P. aeruginosa appears to undergo autophosphorylation in the presence of either ATP or GTP, although the presence of small amounts of Ndk activity may influence the level of such phosphorylation.

Adenosine Triphosphate↗
PubMed1994

Critical nucleotides in the interaction of a LysR-type regulator with its target promoter region. catBC promoter activation by CatR.

We have used site-directed mutagenesis to analyze the importance of nucleotides in the catBC promoter region for the binding of CatR, a member of the LysR family of DNA-binding proteins and for the in vivo activation of the catBC operon. The binding affinity of CatR for its binding site in the catBC promoter region was shown to increase about 2.2-fold in the presence of the inducer, cis,cis-muconate. Nucleotides were targeted for mutagenesis on the basis of previous footprinting data and sequence analysis of CatR binding sites. Critical nucleotides for CatR binding were found within an imperfect inverted repeat. Previous studies have indicated that the LysR family of DNA-binding proteins shares a consensus T-N11-A binding motif (Goethals, K., Van Montagu, M., and Holsters, M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1646-1650), but the CatR binding site sequence has located within the imperfect inverted repeat a G-N11-A instead of the predicted T. Mutagenesis of the G to a T resulted in an increase in both the binding of CatR to the catBC promoter and in the in vivo activation. Nucleotides within the -35 and -10 regions of the catBC promoter were found to be important for promoter activity but not for CatR binding.

Bacterial Proteins↗
PubMed1994

Identification of amino acid residues involved in the activity of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa.

Phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase (PMI-GMP), which is encoded by the algA gene, catalyzes two noncontiguous steps in the alginate biosynthetic pathway of Pseudomonas aeruginosa; the isomerization of D-fructose 6-phosphate to D-mannose 6-phosphate and the synthesis of GDP-D-mannose and PPi from GTP and D-mannose 1-phosphate. Amino acids that are required for the GMP enzyme activity were identified through site-directed mutagenesis of the algA gene. Mutation of Lys-175 to arginine, glutamine, or glutamate produced an enzyme whose Km for D-mannose 1-phosphate was 470-3,200-fold greater than that measured for the wild type enzyme. In addition, these mutant enzymes had a lower Vmax for the GMP activity as compared with the wild type PMI-GMP. These results indicate that Lys-175 is primarily involved in the binding of the substrate D-mannose 1-phosphate, although it is likely that other residues are required for the specificity of binding. Mutation of Arg-19 to glutamine, histidine, or leucine resulted in a 2-fold lower Vmax for the GMP enzyme activity and a 4-7-fold increase in the Km for GTP as compared with the wild type enzyme. Thus, it appears that Arg-19 functions in the binding of GTP. In addition, chymotryptic digestion of PMI-GMP showed that the carboxyl terminus is critical for PMI activity but not for GMP activity. Taken together, these results support the hypothesis that the bifunctional PMI-GMP protein is composed of two independent enzymatic domains.

Alginates↗
PubMed1994

Pseudomonas aeruginosa: genes and enzymes of alginate synthesis.

Alginate is an important virulence factor of Pseudomonas aeruginosa, a bacterium that colonizes the pulmonary tracts of cystic fibrosis patients. Alginate is also widely used in the food, pharmaceutical and chemical industries, and consequently there is considerable interest in the molecular biology and biochemistry of alginate synthesis. As well as its therapeutic potential, research on mucoid P. aeruginosa may provide a lead to an alternative source of alginate for industrial use.

Alginates↗
PubMed1994

Nucleotide sequence and functional analysis of the genes encoding 2,4,5-trichlorophenoxyacetic acid oxygenase in Pseudomonas cepacia AC1100.

Pseudomonas cepacia AC1100 is able to use the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as the sole source of carbon and energy. One of the early steps in this pathway is the conversion of 2,4,5-T to 2,4,5-trichlorophenol (2,4,5-TCP). 2,4,5-TCP accumulates in the culture medium when AC1100 is grown in the presence of 2,4,5-T. A DNA region from the AC1100 genome has been subcloned as a 2.7-kb SstI-XbaI DNA fragment, which on transfer to Pseudomonas aeruginosa PAO1 allows the conversion of 2,4,5-T to 2,4,5-TCP. We have determined the directions of transcription of these genes as well as the complete nucleotide sequences of the genes and the number and sizes of the polypeptides synthesized by pulse-labeling experiments. This 2.7-kb DNA fragment encodes two polypeptides with calculated molecular masses of 51 and 18 kDa. Proteins of similar sizes were seen in the T7 pulse-labeling experiment in Escherichia coli. We have designated the genes for these proteins tftA1 (which encodes the 51-kDa protein) and tftA2 (which encodes the 18-kDa protein). TftA1 and TftA2 have strong amino acid sequence homology to BenA and BenB from the benzoate 1,2-dioxygenase system of Acinetobacter calcoaceticus, as well as to XylX and XylY from the toluate 1,2-dioxygenase system of Pseudomonas putida. The Pseudomonas aeruginosa PAO1 strain containing the 2.7-kb SstI-XbaI fragment was able to convert not only 2,4,5-T to 2,4,5-TCP but also 2,4-dichlorophenoxyacetic acid to 2,4-dichlorophenol and phenoxyacetate to phenol.

Amino Acid Sequence↗
PubMed1994

Role of alginate lyase in cell detachment of Pseudomonas aeruginosa.

The exopolysaccharide alginate of Pseudomonas aeruginosa was shown to be important in determining the degree of cell detachment from an agar surface. Nonmucoid strain 8822 gave rise to 50-fold more sloughed cells than mucoid strains 8821 and 8830. Alginate anchors the bacteria to the agar surface, thereby influencing the extent of detachment. The role of the P. aeruginosa alginate lyase in the process of cell sloughing was investigated. Increased expression of the alginate lyase in mucoid strain 8830 led to alginate degradation and increased cell detachment. Similar effects were seen both when the alginate lyase was induced at the initial stage of cell inoculation and when it was induced at a later stage of growth. It appears that high-molecular-weight alginate polymers are required to efficiently retain the bacteria within the growth film. When expressed from a regulated promoter, the alginate lyase can induce enhanced sloughing of cells because of degradation of the alginate. This suggests a possible role for the lyase in the development of bacterial growth films.

Agar↗
PubMed1994

Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide.

The algC gene from Pseudomonas aeruginosa has been shown to encode phosphomannomutase (PMM), an essential enzyme for biosynthesis of alginate and lipopolysaccharide (LPS). This gene was overexpressed under control of the tac promoter, and the enzyme was purified and its substrate specificity and metal ion effects were characterized. The enzyme was determined to be a monomer with a molecular mass of 50 kDa. The enzyme catalyzed the interconversion of mannose 1-phosphate (M1P) and mannose 6-phosphate, as well as that of glucose 1-phosphate (G1P) and glucose 6-phosphate. The apparent Km values for M1P and G1P were 17 and 22 microM, respectively. On the basis of Kcat/Km ratio, the catalytic efficiency for G1P was about twofold higher than that for M1P. PMM also catalyzed the conversion of ribose 1-phosphate and 2-deoxyglucose 6-phosphate to their corresponding isomers, although activities were much lower. Purified PMM/phosphoglucomutase (PGM) required Mg2+ for maximum activity; Mn2+ was the only other divalent metal that showed some activation. The presence of other divalent metals in addition to Mg2+ in the reaction inhibited the enzymatic activity. PMM and PGM activities could not be detected in nonmucoid algC mutant strain 8858 and in LPS-rough algC mutant strain AK1012, while they were present in the wild-type strains as well as in algC-complemented mutant strains. This evidence suggests that AlgC functions as PMM and PGM in vivo, converting phosphomannose and phosphoglucose in the biosynthesis of both alginate and LPS.

Alginates↗
PubMed1994

Purification of the LysR family regulator, ClcR, and its interaction with the Pseudomonas putida clcABD chlorocatechol operon promoter.

Previous studies have shown that the clcABD operon is under the transcriptional control of the LysR-type activator ClcR. In this study, the conditions leading to its aggregation were avoided and ClcR was purified and confirmed by amino-terminal sequencing. Gel filtration indicated that ClcR exists as a dimer in solution. The DNase I footprint of ClcR was determined. The binding properties of ClcR and the catechol operon regulator, CatR, were compared.

Bacterial Proteins↗
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

Sequence of the alg8 and alg44 genes involved in the synthesis of alginate by Pseudomonas aeruginosa.

The alg8 and alg44 genes, which are required for alginate biosynthesis by Pseudomonas aeruginosa, are located in the alginate biosynthetic gene cluster between the algD and algE genes. The nucleotide sequence of these two genes is presented. Although the functions of the Alg8 and Alg44 proteins are not known, we believe that they may be involved in the polymerization of mannuronic acid residues to form alginate.

Alginates↗
PubMed1993

Sequence of the algL gene of Pseudomonas aeruginosa and purification of its alginate lyase product.

The alginate lyase-encoding gene (algL) of Pseudomonas aeruginosa was localized to a 1.7-kb EcoRI-XbaI fragment within the alginate biosynthetic gene cluster at 34 minutes on the chromosome. The nucleotide sequence of this DNA fragment revealed an ORF encoding a protein of M(r) 40,885 which is transcribed in the same orientation as the other alg genes within the biosynthetic gene cluster. The predicted protein has a potential N-terminal signal peptide which is consistent with its proposed periplasmic location. The AlgL protein was overproduced in Escherichia coli and purified. The purified protein was shown to have alginate lyase activity. In addition, an algL insertion mutant of the mucoid P. aeruginosa 8830 was constructed. This mutant (alm1) had a nonmucoid phenotype due to a polar effect on the transcription of an essential alg gene, algA. Thus, the algL gene is located within a region of the alginate biosynthetic gene cluster that appears to be non-essential for alginate production.

Alginates↗