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A M Chakrabarty

Publications and source records attributed to A M Chakrabarty.

At least 127 records · Page 7Linked to original sources
PubMed1987

Pseudomonas aeruginosa infection in cystic fibrosis: nucleotide sequence and transcriptional regulation of the algD gene.

Pulmonary infection by mucoid, alginate producing, Pseudomonas aeruginosa is a major complication in patients suffering from cystic fibrosis (CF). To analyze the mechanisms leading to the emergence of mucoid P. aeruginosa in CF lungs, control of the algD gene coding for GDPmannose dehydrogenase was studied. Transcriptional activation of algD was shown to be necessary for alginate production. Sequencing of algD and its promoter revealed multiple direct repeats upstream of the transcription start and throughout the promoter region. Using the algD-xy1E transcriptional fusion the algD promoter was demonstrated to be under positive control by the algR gene. This gene has previously been shown to undergo antibiotic promoted chromosomal amplification resulting in the emergence of the mucoid phenotype. These findings provide a basis for better understanding the control of mucoidy in P. aeruginosa.

Alginates↗
PubMed1987

Cloning and complete nucleotide sequence determination of the catB gene encoding cis,cis-muconate lactonizing enzyme.

The enzyme, cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1), has been proposed to play a key role in the beta-ketoadipate pathway of benzoate degradation. A 10.2-kb EcoRI fragment isolated from a Pseudomonas putida genomic library complemented a mutant deficient in this enzyme. The MLEI coding gene, catB, was localized to a 1.6-kb fragment which was sequenced by the dideoxy chain termination method. MLEI was purified 25-fold from crude extracts of benzoate-grown P. putida PRS2015 harboring the cloned catB gene. Purified MLEI was greater than 95% homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit Mr was 40,000 which was in close agreement with the nucleotide sequence data. N-terminal sequence analysis of purified MLEI protein agreed with the N terminus predicted by the nucleotide sequence. Comparison of the nucleotide and amino acid sequences for catB with the corresponding sequences of the clcB gene (K.L. Ngai, B.F., D.K. Chatterjee, L.N. Ornston, and A.M.C., unpublished), whose gene product catalyzes the analogous reaction in 3-chlorobenzoate degradation, showed significant homology. These results suggest that catB and clcB have diverged from a common ancestral gene.

Amino Acid Sequence↗
PubMed1987

A set of cassettes and improved vectors for genetic and biochemical characterization of Pseudomonas genes.

A set of broad-host-range vectors allowing direct selection of recombinant DNA molecules to facilitate subcloning and expression analyses of Pseudomonas genes was constructed using Bg/II lacZ alpha cassette. Controlled expression vectors pVDtac39 and pVDtac24 were shown to be useful for determination of enzymatic activities encoded by the cloned DNA fragments and Mr determination of the corresponding polypeptides. A set of Pseudomonas putida xylE gene cassettes truncated at the 5' end was constructed for translational (protein) fusion studies. A protein fusion of the Pseudomonas aeruginosa algD gene, coding for GDPmannose dehydrogenase, and the truncated xylE gene cassette was used to verify the putative coding region and translational signals predicted from the algD nucleotide sequence.

Cloning, Molecular↗
PubMed1987

Microbial degradation of synthetic recalcitrant compounds.

Synthetic compounds, particularly highly chlorinated aromatics, comprise the bulk of the environmental pollutants that somehow must be removed from the environment. Microbial degradation of such compounds is usually very slow, making them highly persistent in nature. Some synthetic compounds, with a lower degree of chlorination are, however, biodegradable; biochemical, genetic, and molecular studies demonstrate the evolution of new plasmid-encoded enzymatic activities specifically designed for the chlorinated substrates. Nucleotide sequences of many of the genes encoding such enzymatic activities demonstrate considerable homology either near the active sites or throughout the molecules with the chromosomal genes encoding enzymes catalyzing analogous reactions. In some cases, unique repeated sequences, reminiscent of prokaryotic insertion sequence elements, are present at or near the newly evolved genes. This suggests gene duplication and divergence as well as recombinational events mediated by transposable type elements as key ingredients in the evolution of new degradative functions. An understanding of such evolutionary processes is an essential feature for the development of genetically-improved bacteria capable of utilizing and thereby removing highly chlorinated environmental pollutants from our environment.

Journal Article↗
PubMed1987

Organization and nucleotide sequence determination of a gene cluster involved in 3-chlorocatechol degradation.

Three critical enzymes catechol oxygenase II (chlorocatechol dioxygenase), muconate cycloisomerase II, and dienelactone hydrolase, are involved in the degradation of chlorocatechols, which are obligatory intermediates in the catabolism of chlorinated aromatic compounds. The organization and complete nucleotide sequence of the genes for these enzymes have been determined on a 4.2-kilobase-pair (kbp) Bgl II fragment cloned from the plasmid pAC27, based on the agreement of open reading frame lengths with apparent mobilities of polypeptides expressed in Escherichia coli maxicells, predicted N-terminal amino acid sequences with those of the purified proteins, and predicted total amino acid compositions with those of the purified proteins. The 4.2-kbp fragment contains the three genes and ribosome binding sites for those genes but no promoter. When placed downstream of the tac promoter in the broad-host-range plasmid pMMB22, this fragment directs the synthesis of all three enzymes in both E. coli and Pseudomonas putida only on induction with isopropyl beta-D-thiogalactopyranoside, suggesting that the gene cluster is regulated as a single unit under the control of a single promoter. Endogenous transcription initiation of the gene cluster on pAC27, however, occurs from a site present within a 386-bp Bgl II fragment upstream of the 4.2-kbp fragment, and sequences 5' to that site are similar to the sequences of other positively controlled Pseudomonas promoters occurring on the TOL and NAH plasmids.

Amino Acid Sequence↗
PubMed1987

Characterization of the Pseudomonas aeruginosa alginate (alg) gene region II.

Pseudomonas aeruginosa region II alginate genes are involved in the biosynthesis of the uronic acid containing exopolysaccharide, alginic acid. We have subcloned and overexpressed various DNA fragments contained within region II in an attempt to further characterize and more precisely localize the genes involved in alginate production. Overexpression of the genes controlling alginate biosynthesis within region II was accomplished by placing various cloned restriction fragments under the transcriptional control of the hybrid trp-lac (tac) promoter, and plasmid encoded proteins were examined in a maxicell expression system. We correlated various region II plasmid constructions with the ability to complement specific alginate negative (alg) mutants and code for polypeptides. Several proteins suspected of being involved in alginate production were encoded by sequences within region II. The results of this study further reveal that the transcriptional orientation of the alg loci within region II appears to be in the direction from argF to pmi. The specific activities of phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), two enzymes involved in the formation of the alginate precursor GDP-mannuronic acid, were measured in region II alg mutants and in cells overexpressing cloned segments from region II. Based on these enzyme measurements, we conclude that the remaining region II alg genes do not encode either PMM or GMP. These results support the suggestion that the remaining alg genes in region II are likely to be involved in post GDP-mannuronic acid processing events such as mannuronic acid transport, polymerization, secretion, epimerization and acetylation.

Alginates↗
PubMed1987

Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa.

Transcriptional regulation of alginate biosynthesis by Pseudomonas aeruginosa was studied. A DNA region complementing the alg-5 mutation within the alginate gene cluster was found by RNA-DNA dot blot and Northern hybridization to be transcriptionally activated in mucoid P. aeruginosa. This region was subcloned as a 3.2-kilobase BglII-ClaI DNA fragment on the broad-host-range controlled transcription vector pMMB24, and gene products were analyzed by expression from the tac promoter. A 48-kilodalton polypeptide was detected in extracts of P. aeruginosa and 35S-labeled Escherichia coli maxicells. By using the same expression system, GDPmannose dehydrogenase activity was detected in both P. aeruginosa and E. coli. Thus, gene algD coding for this enzyme was found to be present in the transcriptionally active DNA area. Insertion of the xylE gene within the BglII-ClaI fragment disrupted the induction of the 48-kilodalton polypeptide, GDPmannose dehydrogenase activity, and alg-5 complementing ability. With the algD-xylE transcription fusion, activation of algD gene expression was shown to occur in mucoid P. aeruginosa of different origins. In addition, regulation of the algD promoter activity was demonstrated to be mediated by a diffusible factor.

Base Sequence↗
PubMed1987

Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13.

The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.

Amino Acid Sequence↗
PubMed1987

Alginate biosynthetic enzymes in mucoid and nonmucoid Pseudomonas aeruginosa: overproduction of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase by overexpression of the phosphomannose isomerase (pmi) gene.

The specific activities of phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP), and GDP-mannose dehydrogenase (GMD) were compared in a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa and in two spontaneous nonmucoid revertants. In both revertants some or all of the alginate biosynthetic enzymes we examined appeared to be repressed, indicating that the loss of the mucoid phenotype may be a result of decreased formation of sugar-nucleotide precursors. The introduction and overexpression of the cloned P. aeruginosa phosphomannose isomerase (pmi) gene in both mucoid and nonmucoid strains led not only to the appearance of PMI levels in cell extracts several times higher than those present in the wild-type mucoid strain, but also in higher PMM and GMP specific activities. In extracts of both strains, however, the specific activity of GMD did not change as a result of pmi overexpression. In contrast, the introduction of the cloned Escherichia coli manA (pmi) gene in P. aeruginosa caused an increase in only PMI and PMM activities, having no effect on the level of GMP. This suggests that an increase in PMI activity alone does not induce high GMP activity in P. aeruginosa. The heterologous overexpression of the P. aeruginosa pmi gene in the E. coli manA mutant CD1 led to the appearance in cell extracts of not only PMI activity but also GMP activity, both of which are normally undetectable in extracts of CD1. We discuss the implications of these results and propose a mechanism by which overexpression of the P. aeruginosa pmi gene can cause an elevation in both PMM and GMP activities.

Alginates↗
PubMed1986

Nucleotide sequence analysis of the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa and comparison with the corresponding Escherichia coli gene manA.

Phosphomannose isomerase (PMI) has been proposed to catalyze the first step of the alginic acid biosynthetic pathway in Pseudomonas aeruginosa. The nucleotide sequence of the P. aeruginosa pmi gene contained on a 2.0-kb BamHI-SstI DNA fragment has been determined. The gene was defined by the start and stop codons and by in vitro disruption of an open reading frame of 1440 bp corresponding to a polypeptide product with a predicted Mr of 52 860. This polypeptide displayed an apparent Mr of approx. 56 000 upon electrophoresis of a maxicell extract on sodium dodecyl sulfate-polyacrylamide gels. The codon utilization of the pmi gene was distinct in the wobble base preference and influenced by the high G + C content (66 mol%) of the P. aeruginosa DNA. Computer assisted matching analysis failed to demonstrate any significant homology at the nucleotide level between the P. aeruginosa pmi and Escherichia coli manA (pmi) genes. However, sequences homologous to the P. aeruginosa pmi gene were found in other Pseudomonas species, such as P. putida and P. mendocina, and in Azotobacter vinelandii, all capable of producing alginic acid.

Amino Acid Sequence↗
PubMed1986

Gene amplification induces mucoid phenotype in rec-2 Pseudomonas aeruginosa exposed to kanamycin.

Gene amplification in the chromosome of rec-2 Pseudomonas aeruginosa PAO2003 upon growth on kanamycin-supplemented media led to a stable mucoid phenotype. The chromosomal region controlling alginate biosynthesis was shown to be amplified four to six times as a direct tandem repeat of at least 16.8 kilobase pairs. This amplification was deduced from Southern DNA-DNA hybridization patterns of the chromosomal DNA digested with restriction endonucleases BglII and EcoRI and probed with a cloned DNA segment complementing the alg-22 mutation. The part of the amplified unit carrying the novel DNA joint was cloned. The EcoRI junction fragment was further subcloned and used to probe chromosomes of parental strain PAO2003 and mucoid variant VD2003M. As predicted, the EcoRI junction fragment hybridized to the two chromosomal fragments required to produce the novel junction. Though the mucoid phenotype caused by gene amplification was stable, nonmucoid revertants were obtained at a low frequency on tetracycline-containing media. Southern hybridization of chromosomal DNA from a nonmucoid revertant revealed a reduction in the copy number of amplified DNA. These results suggest a direct relationship between amplification of this chromosomal segment and the induction of mucoidy.

Alginates↗
PubMed1986

Overproduction and assay of Pseudomonas aeruginosa phosphomannose isomerase.

Phosphomannose isomerase activity was undetectable in extracts of mucoid (alginate-producing) Pseudomonas aeruginosa. When a P. aeruginosa gene previously shown to complement an alginate-negative mutant was overexpressed under the control of the tac promoter in the broad-host-range controlled-expression vector pMMB22, phosphomannose isomerase activity could be measured in extracts of P. aeruginosa and in a manA (phosphomannose isomerase-negative) mutant of Escherichia coli. P. aeruginosa extracts containing induced levels of enzyme were shown to interconvert fructose 6-phosphate and mannose 6-phosphate. A 56,000-dalton polypeptide was visualized on sodium dodecyl sulfate-polyacrylamide gels after induction in both hosts. When RNA-DNA dot- blot hybridization analysis was used, transcription of algA, the gene coding for P. aeruginosa phosphomannose isomerase, was not measurable from the chromosomes of either mucoid or nonmucoid P. aeruginosa. However, a high level of algA transcription was detected after expression of algA under tac promoter control in pMMB22.

Alginates↗
PubMed1985

Genes specifying degradation of 3-chlorobenzoic acid in plasmids pAC27 and pJP4.

All of the structural genes for 3-chlorobenzoate degradation are clustered in a 4.2-kilobase (kb) region of plasmid pAC25 (or pAC27) in Pseudomonas putida. An approximate 10-kb DNA segment containing three structural genes for chlorocatechol metabolism present on plasmid pJP4 in Alcaligenes eutrophus shows homology with the above 4.2-kb region of pAC27. In spite of the detectable sequence homology in the structural genes present on both plasmids, the regulation of their expression seems quite different; unlike pAC27, structural rearrangements are prerequisite for efficient expression of the 3-chlorobenzoate genes on plasmid pJP4. Structural features such as stem-loop structures present on plasmid pJP4 are most likely the starting materials for such rearrangements.

Alcaligenes↗
PubMed1985

Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa.

The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate.

Alginates↗
PubMed1985

Fructose 1,6-bisphosphate aldolase activity is essential for synthesis of alginate from glucose by Pseudomonas aeruginosa.

We have isolated a mutant of Pseudomonas aeruginosa deficient in fructose 1,6-bisphosphate aldolase activity. This mutant, similar to the mutants deficient in any of the Entner-Doudoroff pathway enzymes, does not allow appreciable alginate formation from glucose and gluconate, but allows alginate synthesis from mannitol and fructose. This suggests that glucose and gluconate must be converted to fructose 1,6-bisphosphate via the Entner-Doudoroff pathway enzymes and fructose 1,6-bisphosphate aldolase.

Alginates↗
PubMed1985

Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa.

A 10-kilobase DNA fragment previously shown to contain the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa was used to construct a pBR325-based hybrid that can be propagated in P. aeruginosa only by the formation of a chromosomal-plasmid cointegrate. This plasmid, designated pAD4008, was inserted into the P. aeruginosa chromosome by recombination at a site of homology between the cloned P. aeruginosa DNA and the chromosome. Mobilization of pAD4008 into P. aeruginosa PAO and 8830 and selection for the stable acquisition of tetracycline resistance resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the phosphomannose isomerase gene region of the chromosomes. Chromosomal DNA from the tetracycline-resistant transformants was used to clone the drug resistance determinant with Bg/II or XbaI, thereby allowing the "walking" of the P. aeruginosa chromosome in the vicinity of the pmi gene. Analysis of overlapping tetracycline-resistant clones indicated the presence of sequences homologous to the DNA insert of plasmid pAD2, a recombinant clone of P. aeruginosa origin previously shown to complement several alginate-negative mutants. Restriction mapping, subcloning, and complementation analysis of a 30-kilobase DNA region demonstrated the tight clustering of several genetic loci involved in alginate biosynthesis. Furthermore, the tetracycline resistance determinant in PAO strain transformed by pAD4008 was mapped on the chromosome by plasmid FP2-mediated conjugation and was found to be located near 45 min.

Alginates↗