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A M Chakrabarty

Publications and source records attributed to A M Chakrabarty.

At least 19 recordsLinked to original sources
PubMed1995

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

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

Adenosine Triphosphate↗
PubMed1995

The algT (algU) gene of Pseudomonas aeruginosa, a key regulator involved in alginate biosynthesis, encodes an alternative sigma factor (sigma E).

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is the leading cause of mortality among cystic fibrosis (CF) patients. During the course of sustained infection, the production of an alginate capsule protects the bacteria and allows them to persist in the CF lung. One of the key regulators of alginate synthesis is the algT (algU) gene encoding a putative alternative sigma factor (sigma E). AlgT was hyperproduced and purified from Escherichia coli. The N-terminal sequence of the purified protein matched perfectly with that predicted from the DNA sequence. The purified protein, in the presence of E. coli RNA polymerase core enzyme, was able to initiate transcription of an algT promoter. Deletion of the -35 region of this promoter abolished this activity in vitro as well as in vivo. These data indicate that the algT gene encodes a sigma factor that is autoregulatory.

Alginates↗
PubMed1995

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

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

Alginates↗
PubMed1995

Differential DNA bending introduced by the Pseudomonas putida LysR-type regulator, CatR, at the plasmid-borne pheBA and chromosomal catBC promoters.

The plasmid-borne pheBA operon of Pseudomonas putida strain PaW85 allows growth of the host cells on phenol. The promoter of this operon is activated by the chromosomally encoded LysR-type regulator CatR, in the presence of the inducer cis,cis-muconate. cis,cis-muconate is an intermediate of catechol degradation by the chromosomally encoded ortho or beta-ketoadipate pathway. The catBC operon encodes two enzymes of the beta-ketoadipate pathway and also requires CatR and cis,cis-muconate for its expression. The promoters of the pheBA and catBC operons are highly homologous, and since both respond to CatR, it is likely that the pheBA promoter was recruited from the ancestral catBC promoter. Gel shift assays and DNase I footprinting have shown that the pheBA promoter has a higher binding affinity for CatR than the catBC promoter. Like the catBC promoter, the pheBA promoter forms two complexes (C1 and C2) with CatR in the absence of cis,cis-muconate, but only forms a single complex (C2) in the presence of cis,cis-muconate. Like the catBC promoter CatR repression binding site (RBS) and activation binding site (ABS) arrangement, the pheBA promoter demonstrates the presence of a 26 bp segment highly homologous to the RBS that is protected by CatR from DNase I digestion in the absence of the inducer. An additional 16 bp sequence, similar to the catBC promoter ABS, is protected only when the inducer cis-cis-muconate is present. The binding of CatR in absence of cis,cis-muconate bends the catBC and pheBA promoter regions to significantly different degrees, but CatR binding in the presence of cis,cis-muconate results in a similar degree of DNA bending. The evolutionary implications of the interactions of CatR with these two promoters are discussed.

Adipates↗
PubMed1995

Sequence analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 utilizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. PT88 is a chromosomal deletion mutant of B. cepacia AC1100 and is unable to grow on 2,4,5-T. The nucleotide sequence of a 5.5-kb chromosomal fragment from B. cepacia AC1100 which complemented PT88 for growth on 2,4,5-T was determined. The sequence revealed the presence of six open reading frames, designated ORF1 to ORF6. Five polypeptides were produced when this DNA region was under control of the T7 promoter in Escherichia coli; however, no polypeptide was produced from the fourth open reading frame, ORF4. Homology searches of protein sequence databases were performed to determine if the proteins involved in 2,4,5-T metabolism were similar to other biodegradative enzymes. In addition, complementation studies were used to determine which genes were essential for the metabolism of 2,4,5-T. The first gene of the cluster, ORF1, encoded a 37-kDa polypeptide which was essential for complementation of PT88 and showed significant homology to putative trans-chlorodienelactone isomerases. The next gene, ORF2, was necessary for complementation and encoded a 47-kDa protein which showed homology to glutathione reductases. ORF3 was not essential for complementation; however, both the 23-kDa protein encoded by ORF3 and the predicted amino acid sequence of ORF4 showed homology to glutathione S-transferases. ORF5, which encoded an 11-kDa polypeptide, was essential for growth on 2,4,5-T, but the amino acid sequence did not show homology to those of any known proteins. The last gene of the cluster, ORF6, was necessary for complementation of PT88, and the 32-kDa protein encoded by this gene showed homology to catechol and chlorocatechol-1,2-dioxygenases.

2,4,5-Trichlorophenoxyacetic Acid↗
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

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↗
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↗