PubMed Health⌕ Search

Biomedical subjects

A M Chakrabarty

Publications and source records attributed to A M Chakrabarty.

At least 109 records · Page 6Linked to original sources
PubMed1991

Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting.

CatR, a LysR family protein, positively regulates the Pseudomonas putida catBC operon, which is required for growth on benzoate as a sole carbon source. Transcriptional studies show that the catR and catBC promoters are divergent and overlapping by 2 bp. A beta-galactosidase promoter probe vector was constructed to analyze expression from the catR and catBC promoters under induced and uninduced conditions. As predicted, the catBC promoter is expressed only under induced conditions, while the catR promoter is constitutive. CatR has been shown to specifically bind the catRBC promoter region, and this property was used to devise a purification protocol for CatR. Linear M13 DNA containing the catRBC control region was covalently bound to cyanogen bromide-activated Sepharose in order to construct a DNA affinity column. Crude extracts containing hyperproduced CatR protein were then incubated with the affinity resin under binding conditions, and the CatR protein was eluted with 1 M NaCl. CatR was also purified by heparin-agarose chromatography. This highly purified protein was used for gel retardation and hydroxyl-radical footprinting studies. From this analysis, it was shown that CatR binds upstream of the catBC promoter within the transcribed region of catR.

Bacterial Proteins↗
PubMed1990

Repeated sequences including RS1100 from Pseudomonas cepacia AC1100 function as IS elements.

Several lines of evidence were obtained that the previously identified, repeated sequence RS1100 of Pseudomonas cepacia strain AC1100 undergoes transposition events. DNA sequences flanking the chlorohydroxy hydroquinone (CHQ) degradative genes of this organism were examined from sources, including several independently isolated cosmid clones from an AC1100 genomic library and genomic DNAs of two independently maintained wild-type AC1100 isolates. Hybridization and restriction endonuclease mapping studies revealed these sequences to be similar except for their numbers and distributions of RS1100 copies. A recombinant plasmid containing the immediate chq gene region and excluding any copies of RS1100 was conjugated into AC1100 mutant RHA5 which was shown to have undergone a deletion of its corresponding DNA. Hybridization and restriction mapping analyses of several reisolated plasmids revealed the presence of RS1100 sequences at different positions within either the vector or insert portions. One such plasmid contained tandem copies of RS1100 with an intervening DNA sequence also of AC1100 origin. Similar experiments involving introduction of the promoter probe plasmid pKT240 into wild-type AC1100 cells resulted in the acquisition of high-concentration streptomycin resistance by a number of recipients. The reisolated plasmids in most cases also conferred streptomycin resistance to Escherichia coli transformants and in each case were found to contain insertions close to the upstream portion of the aphC structural gene. These insertions alternatively contained RS1100 sequences for a newly identified 3400 bp repeated sequence from AC1100. Based on these results, RS1100 has been redesignated as insertion sequence IS931 and the 3400 bp repeated sequence has been designated as IS932.

DNA Transposable Elements↗
PubMed1990

Enhanced removal of Exxon Valdez spilled oil from Alaskan gravel by a microbial surfactant.

Remediation efforts for the oil spill from the Exxon Valdez tanker in Alaska have focused on the use of pressurized water at high temperature to remove oil from the beaches. We have tested a biological surfactant from Pseudomonas aeruginosa for its ability to remove oil from contaminated Alaskan gravel samples under various conditions, including concentration of the surfactant, time of contact, temperature of the wash, and presence or absence of xanthan gum. The results demonstrate the ability of the microbial surfactant to release oil to a significantly greater extent (2 to 3 times) than water alone, particularly at temperatures of 30 degrees C and above.

Accidents↗
PubMed1990

AlgR3, a protein resembling eukaryotic histone H1, regulates alginate synthesis in Pseudomonas aeruginosa.

A regulatory mutation (alg52) in a Pseudomonas aeruginosa alginate-negative mutant (strain 8882) is complemented efficiently by the gene algR2 and somewhat inefficiently by a second gene termed algR3. algR3 and algR2 are located on a 4.4-kilobase-pair HindIII-BamHI fragment, which has been completely sequenced. algR2 has previously been characterized. Introduction of kanamycin-resistance cassettes and deletion-subcloning experiments involving various open reading frames in the HindIII-BamHI fragment have localized the algR3 gene, which encodes a 340-amino acid polypeptide. This highly basic regulatory protein contains 17% lysine and 36% alanine. The predicted amino acid sequence shows no significant similarity with any bacterial proteins and yet is highly similar to the sea urchin Lytechinus pictus histone H1 subtype of protein. Promoter localization by reverse transcriptase mapping of the algR3 gene shows the presence of Escherichia coli sigma 70 recognition sequences, and coupled transcription/translation experiments in E. coli demonstrate the presence of a 39-kDa polypeptide encoded by the cloned algR3 gene.

Alginates↗
PubMed1990

Pulmonary dehydration and infection in cystic fibrosis: evidence that ethanol activates alginate gene expression and induction of mucoidy in Pseudomonas aeruginosa.

Mortality among cystic fibrosis (CF) patients is most commonly attributed to pulmonary infection by mucoid, alginate-producing Pseudomonas aeruginosa. The initial infecting P. aeruginosa are typically non-mucoid; however, upon continued exposure to the CF lung environment, they become highly mucoid. The CF lung is an osmotically high environment because of the presence of substantial concentrations of electrolytes and dehydrated mucus. In this report we demonstrate that ethanol (a commonly used dehydrating agent) activates transcription from a critical alginate promoter, algD, and show that prolonged exposure to ethanol allows switching to the mucoid form. This activation appears to be dependent on DNA gyrase. Analysis of alginate gene activation, and the subsequent reversal of the activation process by bacterial DNA gyrase inhibitors, should aid the development of treatment strategies for CF patients infected with this organism.

Alginates↗
PubMed1990

Distribution of alginate gene sequences in the Pseudomonas rRNA homology group I-Azomonas-Azotobacter lineage of superfamily B procaryotes.

Chromosomal DNA from group I Pseudomonas species, Azotobacter vinelandii, Azomonas macrocytogens, Xanthomonas campestris, Serpens flexibilis, and three enteric bacteria was screened for sequences homologous to four Pseudomonas aeruginosa alginate (alg) genes (algA, pmm, algD, and algR1). All the group I Pseudomonas species tested (including alginate producers and nonproducers) contained sequences homologous to all the P. aeruginosa alg genes used as probes, with the exception of P. stutzeri, which lacked algD. Azotobacter vinelandii also contained sequences homologous to all the alg gene probes tested, while Azomonas macrocytogenes DNA showed homology to all but algD. X. campestris contained sequences homologous to pmm and algR1 but not to algA or algD. The helical bacterium S. flexibilis showed homology to the algR1 gene, suggesting that an environmentally responsive regulatory gene similar to algR1 exists in S. flexibilis. Escherichia coli showed homology to the algD and algR1 genes, while Salmonella typhimurium and Klebsiella pneumoniae failed to show homology with any of the P. aeruginosa alg genes. Since all the organisms tested are superfamily B procaryotes, these results suggest that within superfamily B, the alginate genes are distributed throughout the Pseudomonas group I-Azotobacter-Azomonas lineage, while only some alg genes have been retained in the Pseudomonas group V (Xanthomonas) and enteric lineages.

Alginates↗
PubMed1990

Degradation of the chlorinated phenoxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by pure and mixed bacterial cultures.

Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compounds provided as single substrates. These combined cell suspensions, however, poorly degraded mixtures of the two compounds provided at the same concentrations. Growth and viability studies revealed that such mixtures of 2,4-D and 2,4,5-T were toxic to AC1100 alone and to combinations of AC1100 and JMP134. High-pressure liquid chromatography analyses of culture supernatants of AC1100 incubated with 2,4-D and 2,4,5-T revealed the accumulation of chlorohydroquinone as an apparent dead-end catabolite of 2,4-D and the subsequent accumulation of both 2,4-dichlorophenol and 2,4,5-trichlorophenol. JMP134 cells incubated in the same medium did not catabolize 2,4,5-T and were also inhibited in initiating 2,4-D catabolism. A new derivative of strain AC1100 was constructed by the transfer into this organism of the 2,4-D-degradative plasmid pJP4 from strain JMP134. This new strain, designated RHJ1, was shown to efficiently degrade mixtures of 2,4-D and 2,4,5-T through the simultaneous metabolism of these compounds.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1990

Nucleotide sequencing and characterization of Pseudomonas putida catR: a positive regulator of the catBC operon is a member of the LysR family.

Pseudomonas putida utilizes the catBC operon for growth on benzoate as a sole carbon source. This operon is positively regulated by the CatR protein, which is encoded from a gene divergently oriented from the catBC operon. The catR gene encodes a 32.2-kilodalton polypeptide that binds to the catBC promoter region in the presence or absence of the inducer cis-cis-muconate, as shown by gel retardation studies. However, the inducer is required for transcriptional activation of the catBC operon. The catR promoter has been localized to a 385-base-pair fragment by using the broad-host-range promoter-probe vector pKT240. This fragment also contains the catBC promoter whose -35 site is separated by only 36 nucleotides from the predicted CatR translational start. Dot blot analysis suggests that CatR binding to this dual promoter-control region, in addition to inducing the catBC operon, may also regulate its own expression. Data from a computer homology search using the predicted amino acid sequence of CatR, deduced from the DNA sequence, showed CatR to be a member of a large class of procaryotic regulatory proteins designated the LysR family. Striking homology was seen between CatR and a putative regulatory protein, TfdS.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of a regulatory region controlling alginate synthesis in Pseudomonas aeruginosa: characterization of the algR2 gene.

Alginate (Alg), an exopolysaccharide with strong gelling properties, is produced by Pseudomonas aeruginosa primarily during its infection of the cystic fibrosis (CF) lung. The alg genes are normally not expressed in other environments. The promoter for a critical Alg biosynthetic gene, algD, encoding GDP-mannose dehydrogenase, is activated only under conditions reminiscent of the CF lung (i.e., under high osmolarity), and at least two regulatory genes, algR1 and algR2, have been implicated in this activation process. The physical mapping of a 4.4-kb region harboring algR2 has been accomplished and the complete nucleotide sequence of this fragment, including that of algR2, is presented. The cloning and complementation experiments also demonstrate the presence, on this fragment, of regulatory gene(s) different from algR1 and algR2. The expression of the algR2 gene allows a high level of activation of the algD promoter in Escherichia coli, in the presence of algR1 in a high osmotic environment, suggesting that the AlgR2 and AlgR1 proteins act cooperatively to activate the algD promoter. Hyperexpression of the algR2 gene from the tac promoter also allows the conversion of nonmucoid cells of strain 8822, a spontaneous revertant of the mucoid CF isolate strain 8821, back to mucoidy, but not that of the clinical isolate, strain PAO1.

Alcohol Oxidoreductases↗
PubMed1989

Control of alginate synthesis in Pseudomonas aeruginosa: regulation of the algR1 gene.

The triggering of mucoidy (formation of the exopolysaccharide alginate) in Pseudomonas aeruginosa is accomplished primarily in Cystic Fibrosis lung environment through activation of the promoter of a gene algD, which encodes GDP-mannose dehydrogenase, by the product of a regulatory gene algR1. Both algD and algR1 promoter regions have significant homology, including the presence of sequences recognized by the RNA polymerase sigma 54. We demonstrate that the algR1 promoter is partly constitutive and its activation, similar to that of algD, is dependent on high osmolarity as well as the presence of its own gene product and is repressed by high concentrations of AlgR1. The RpoN sigma factor also plays a critical role in the transcription of both algD and algR1 genes.

Alginates↗
PubMed1989

Purification and characterization of guanosine diphospho-D-mannose dehydrogenase. A key enzyme in the biosynthesis of alginate by Pseudomonas aeruginosa.

Alginate-producing Pseudomonas aeruginosa are usually associated with the cystic fibrosis lung environment and contribute to the high mortality rates observed among these patients. The present paper describes the purification and enzymatic properties of guanosine diphospho-D-mannose dehydrogenase (EC 1.1.1.132), a key enzyme in alginate biosynthesis by mucoid P. aeruginosa. The enzyme was overproduced using a plasmid vector containing algD (the gene encoding this enzyme) under control of the tac promoter. It was purified from cell-free lysates by lowering the pH to 5.0, heating the extract to 57.5 degrees C for 10 min, and discarding the protein pellet. The enzyme was selectively precipitated from the supernatant fraction with 45% acetone, resuspended in a 100 mM triethanolamine acetate buffer, pH 7.6, and ultimately purified by Bio-Sil TSK-400 gel filtration chromatography. The subunit molecular weight (Mr 48,000) as well as the N-terminal amino acid sequence corresponded to those predicted from the DNA sequence of algD. The native protein migrated as a hexamer of 290,000 molecular weight upon Bio-Gel A-1.5m gel filtration chromatography. Kinetic analysis demonstrated an apparent Km of 14.9 microM for the substrate GDP-D-mannose and 185 microM for the cofactor NAD+. GDP-D-mannuronic acid was identified as the enzyme reaction product. Several compounds (including GMP, ATP, GDP-D-glucose, and maltose) were found to inhibit enzymatic activity. GMP, the most potent of these inhibitors, exhibited competitive inhibition with an apparent Ki of 22.7 microM. Enzyme activity was also sensitive to the sulfhydryl group modifying agents iodoacetamide and p-hydroxymercuribenzoate. The addition of excess dithiothreitol restored enzyme activity, suggesting a possible involvement of cysteine residues in enzymatic activity.

Alginates↗
PubMed1989

Identification of nucleotides critical for activity of the Pseudomonas putida catBC promoter.

Pseudomonas putida utilizes the catBC operon, which encodes cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1) and muconolactone isomerase (MI; EC 5.3.3.4), for growth on benzoate as a sole carbon source. This operon is positively regulated, and the promoter is located 64 bp upstream of the catB translational start site. Using site-specific mutagenesis, we identified nucleotides that influenced the induction of this promoter. Promoter activity was monitored with the promoter probe vector pKT240. Transcription of mRNA from mutant promoters was determined by primer extension mapping. Comparison of the initiation start site of mutant promoters with that of the wild-type promoter identified a single functional promoter.

Bacterial Proteins↗
PubMed1989

Characterization and nucleotide sequence determination of a repeat element isolated from a 2,4,5-T degrading strain of Pseudomonas cepacia.

Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T- strain PT88 by a ColE1::Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) was identified which was repeated several times on the wild-type chromosome and was also present on AC1100 plasmid DNA. Various chromosomal fragments containing this sequence were cloned and their nucleotide sequence was determined. Examination of RS1100 revealed the presence of 38-39-bp terminal inverted repeats immediately flanked by 8-bp direct repeats. The translated sequence of the single large open reading frame of RS1100 showed structural similarity to the phage Mu transposase and other DNA-binding proteins. Thus the AC1100 repeated sequence has several structural features in common with insertion sequence elements. Three copies of RS1100 were mapped near 2,4,5-t genes encoding degradation of 5-chloro-1,2,4-trihydroxybenzene, an intermediate in 2,4,5-T degradation. Neither RS1100 nor the 2,4,5-t genes hybridized to DNA isolated from Pseudomonas strains, including P. cepacia, suggesting that both gene fragments may be of foreign origin recruited in strain AC1100. The origin of these two DNA segments as well as the role played by RS1100 in the recruitment of 2,4,5-t genes in AC1100 are presently under investigation.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1989

The algR gene, which regulates mucoidy in Pseudomonas aeruginosa, belongs to a class of environmentally responsive genes.

The Pseudomonas aeruginosa capsule, composed of polysaccharide alginate, is an important Pseudomonas virulence factor encountered primarily in cystic fibrosis. The regulatory algR gene positively controls transcription of a key alginate biosynthetic gene, algD. The algR gene was subcloned and sequenced by creating a set of nested deletions in M13 bacteriophage. DNA sequence analysis of algR revealed the homology of its gene product with a recently recognized class of environmentally responsive bacterial regulatory genes, including ompR, phoB, sfrA, ntrC, spoOA, dctD, and virG; these transcriptional activators control cellular reactions to osmotic pressure, phosphate limitations, or specific chemical compounds present in the medium or released from wounded host tissue. These findings indicate that novel conditions in lungs affected by cystic fibrosis may be participating in the control of mucoidy.

Alginates↗
PubMed1989

High osmolarity is a signal for enhanced algD transcription in mucoid and nonmucoid Pseudomonas aeruginosa strains.

Chronic lung infection with mucoid, alginate-producing strains of Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF) patients. Transcriptional activation of the P. aeruginosa algD gene, which encodes GDPmannose dehydrogenase, is essential for alginate synthesis. Activation of algD is dependent on the product of the algR gene. Sequence homology between the P. aeruginosa algR gene and the Escherichia coli ompR gene, which regulates the cellular response to changes in osmolarity of the growth medium, together with the abnormally high levels of Na+ and Cl- in respiratory tract fluid in CF patients suggested that high osmolarity in the lung of the CF patient might be a signal contributing to the induction of alginate synthesis (mucoidy) in infecting P. aeruginosa. In both mucoid and nonmucoid P. aeruginosa strains (containing a functional algR gene), transcriptional activation of algD increased as the osmolarity of the culture medium increased. The increased activation of algD at high osmolarity was not in itself sufficient to induce alginate synthesis in nonmucoid strains, however, suggesting that other environmental factors are involved in full activation of the alginate genes. The targets of AlgR and OmpR, the algD promoter and the ompC and ompF promoters, respectively, were found to have appreciable sequence homology in the -60 to -110 regions. In E. coli, OmpR was capable of activating the algD promoter nearly as well as AlgR, but in both cases, activation occurred only under conditions of high osmolarity.

Alginates↗
PubMed1988

Cloning, physical mapping and expression of chromosomal genes specifying degradation of the herbicide 2,4,5-T by Pseudomonas cepacia AC1100.

A genomic library of total DNA of Pseudomonas cepacia AC1100 was constructed on a broad-host-range cosmid vector pCP13 in Escherichia coli AC80. A 25-kb segment was isolated from the library which complemented a Tn5-generated, 2,4,5-trichlorophenoxyacetic acid-negative (2,4,5-T-) mutant, P. cepacia PT88. This mutation was partially characterized and appeared to be lacking functional enzyme required for metabolism of an intermediate of the 2,4,5-T pathway, recently identified as 5-chloro-1,2,4-trihydroxybenzene [Chapman et al., Abstr. Soc. Environ. Toxicol. Chem. USA 8 (1987) 127]. A simple colorimetric assay was developed to detect the presence of this active enzyme in intact cells and was used to determine the expression of complementing genes. Subcloning experiments showed that a 4-kb BamHI-PstI fragment and a 290-bp PstI-EcoRI fragment, separated by 1.3-kb, were required for complementation. Both fragments are identified to be chromosomal in origin. Hybridization studies using the subcloned fragments revealed that in addition to a Tn5 insertion, mutant PT88 contained an extensive chromosomal deletion accounting for its 2,4,5-T- phenotype. The cloned fragments did not show homology to plasmid DNAs carrying degradative genes for toluene, naphthalene and 3-chlorobenzoate.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1988

Transcriptional regulation, nucleotide sequence, and localization of the promoter of the catBC operon in Pseudomonas putida.

The catB and catC genes encode cis,cis-muconate lactonizing enzyme I (EC 5.5.1.1) and muconolactone isomerase (EC 5.3.3.4), respectively. These enzymes are required for the dissimilation of benzoate to beta-ketoadipate by Pseudomonas putida and are under coordinate transcriptional regulation. By deletion analysis and the use of pKT240 as a promoter probe vector, we located a single promoter region for the catBC operon upstream of catB. RNA-DNA hybridization studies, together with reverse transcriptase mapping, demonstrated that this promoter must be activated in the presence of an inducer molecule for effective transcription of the operon. In addition, the transcription initiation site was located 64 base pairs upstream of the catB initiation codon, and sequences upstream of -43 were required for promoter function. The catBC promoter was compared with other positively regulated procaryotic promoters to identify possible consensus sequences.

Amino Acid Sequence↗