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Results show that secreted adenylate kinase mediates macrophage cell death by the production of a toxic pool of AMP, ADP, and ATP, whch endows on Pseudomonas aeruginosa the virulence capability. Data also show that adenylate kinase is detected in macrophages during Pseudomonas infection.

Secreted products of a nonmucoid Pseudomonas aeruginosa strain, strain 808, have been found to bring on two types of macrophage killing. One is external-ATP-dependent, P2Z-receptor-mediated necrosis and the other ATP-independent, caspase-mediated apoptosis. Cyotoxic factors active against macrophages were secreted during the exponential phase of growth in a complex medium. Two secreted redox proteins, azurin and a cytochrome c, induce mast cell apoptosis where there is no exogenous ATP. Researchers are interested in whether secretion of ATP-utilizing enzymes and associated cyotoxic agents is restricted to cystic fibrosis isolate mucoid strains. The nature of the pathway promoting macrophage cell death through the P2Z-receptor-independent pathway is of interest.

Research has been conducted on the nucleoside diphosphate kinase (Ndk). The localization of the Ndk secretion motif has been carried out.

Results reveal that in Pseudomonas aeruginosa both ATP and GTP are synthesized via succinyl coenzyme A synthetase using succinyl-CoA, inorganic phosphate, and an equimolar mixture of ADP and GDP. Data indicate that although the enzyme can synthesize other nucleotide phosphates, it preferentially synthesizes ATP and GTP.

Results show a 5.7 kb six-gene cluster, cbnRABorfXCD, is involved in the chlorocatechol degradation in Ralstonia eutropha NH9. Transcriptional activation of the gene cluster is mediated by 2-chloro-cis,-cis-muconate, an intermediate product of the chlorocatechol degradation pathway, and the process is regulated by the cbnR gene.

Results show that affinity purified proteins serine/threonine kinase and serine/threonine phosphoprotein phosphatase are 39 and 27 kDa proteins respectively with the former exhibiting autophosphorylation and the latter dependent on manganese for activity.

The genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX) were isolated from Pseudomonas aeruginosa 8830 to study polyphosphate metabolism and clarify the function of polyphosphate in the microorganism. In contrast to Escherichia coli, the ppk and ppx genes in P aeruginosa are transcribed divergently and no coregulation between their expression was shown by a study of the two promoters. A residual low PPK activity was detected in the mutant, representing a separate mechanism of polyP synthesis.

A study was conducted to test whether tftG encodes a dechlorinase enzyme that transforms 5-chlorohydroquinol (5-CHQ) to to hydroxyquinol. Escherichia coli crude cell extracts were incubated at room temperature in a sodium phosphate buffer solution while polymerase chain reaction was carried out to determine the tftG gene accommodating six histidine codons. Results indicated that 5-CHQ supports specific product formation with three enzymes and that HBQ reductase is involved in reductive steps.

A study was conducted to determine and describe the tftCD gene to characterize the upper portion of the aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) pathway in Burkholderia cepacia. Plasmid-preparative columns were utilized to perform a large-scale purification of cosmids while agarose plugs with intact chromosomal DNA were determined from overnight cultures. Results showed that the genes encoding the 2,4,5-T oxygenase, tftAB, were on replicon IV.

The enzymes responsible for benzoate catabolism in Pseudomonas putida are encoded by the catBCA operon which requires the transcription regulator CatR and an inducer. CatR has been shown to bind to two sites in the promoter region of the operon. A third binding region within the catB structural gene has been identified. This region is designated as the internal binding site and functions as a cis-acting repressor based on site-directed mutagenesis studies.

A study was conducted on the repression of the expression of the clcABD operon during growth with TCA cycle intermediates which occurs at the transcriptional level. The results indicate a repression of the expression from the clc promoter when the cells are grown on succinate, citrate or fumarate. Moreover, the formation of the clcA transcript is inhibited directly and specifically by the TCA cycle intermediate fumarate.

The analogous intermediate of the 3-chlorocatechol degradation in Pseudomonas putida, 2-chloromuconate, was found to be the inducer of the clcABD operon. The DNase footprints of ClcR with and without 2-chloromuconate were also determined. An extended region of the promoter from -79 to -25 was occupied in the absence of the inducer but the -35 region was unprotected. The footprint contracted at the proximal end of the promoter and the -35 region was contacted when 2-chloromuconate was added to the binding assays.

An in vitro transcription assay was utilized to examine the various components of the catBCA transcriptional activation mechanism in Pseudomonas putida. Point mutations of the catBCA promoter region indicated the correlation between the levels of in vitro and in vitro transcripts. An intact alpha subunit of CatR-RNA polymerase was also required by the regulatory CatR protein for the activation of catBCA and pheBA promoters.

Proteins from the membrane fraction of Pseudomonas aeruginosa were analyzed to determine the effects of guanosine triphosphatase (GTPase) activity on the nucleoside diphosphate kinase-pyruvate kinase (Ndk-Pk) complex. Analysis of the Pseudomonas aeruginosa 48-kDa protein that exhibited GTPase activity indicated the presence of an amino acid sequence that was similar to the Escherichia coli Ras-like protein. Furthermore, the association of the Ras-like protein of Pseudomonas aeruginosa with Ndk or Pk restricted its intrinsic GTPase activity.

The cloning and sequencing of the new gene called algK which is involved in the biosynthesis of alginate from Pseudomonas aeruginosa was identified. This gene which lies between alg44 and algE genes of the operon encodes a poorly expressed preprotein of approximately 53 kDa which is processed to form a mature polypeptide. AlgK is found to be periplasmic in location and is most likely anchored to the inner membrane.

Site-directed and random mutagenesis techniques in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa were employed to study the function of Ndk in the production of alginate by this organism. Six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91 and Asp-135) were found whose alteration caused the inability of Ndk to complement alginate production.

The enzyme hydroxyquinol 1,2-dioxygenase and maleylacetate reductase have been purified from Escherichia coli cells expressing the Burkholderia cepacia AC1100 tftH gene and tftE gene, respectively. The two enzymes catalyze the formation of 3-oxoadipate from hydroxyquinol. Genetic studies reveal that these enzymes bring about degradation of 2,4,5-trichlorophenoxyacetic acid. Hydroxyquinol 1,2-dioxygenase is a dimeric molecule of 68 kDa.

Interaction between the sigma factor AlgT and anti-sigma factor MucA mediates the conversion of the non mucoid form of Pseudomonas aeruginosa to the virulent mucoid form in cystic fibrosis patients. The algT gene encodes for sigma(super E), which mediates the conversion to mucoidy. The mucA gene suppresses conversion to mucoidy. Immunological assay shows that MucA interacts physically with AlgT. Thus inhibition of AlgT dependent transcription may be responsible for suppression of mucoidy by mucA.

An intracellular protease was found to cleave the 16-kDa form of nucleoside diphosphate kinase (Ndk) in Pseudomonas aeruginosa to a 12-kDa form that undergoes autophosphorylation with an efficiency almost similar to that of the 16-kDa form. The 12 kDa-form of Ndk synthesizes GTP in preference to other nucleoside triphosphates. The generation of the 12-kDa form of Ndk and its association with the cell membrane are linked to the growth stage of P. aeruginosa.

The Burkholderia cepacia AC1100 oxygenase (TftAB) converts 2,4,5-trichlorophenoxyacetic acid to 2,4,5-trichlorophenol and many other chlorinated aromatic phenoxyacetates to their phenolic derivatives. The rate of substrate consumption by TftAB depends upon the degree of substrate chlorination, chlorines position and phenoxy group. There is a mechanistic similarity between TftAB and TfdA of Alcaligenes eutrophus. Escherichia coli and Burkholderia cepacia expresses the promoter cloned upstream of the beta-galactosidase gene and show beta-galactosidase activity.

Chronic infection by Pseudomonas aeruginosa is the leading cause of death in cystic fibrosis patients. The algT (algU) gene encoding a putative alternative sigma factor sigma(super E) is one of the main regulators of alginate synthesis during the sustained chronic infection by alginate-producing Pseudomonas aeruginosa. The N-terminal sequence of the purified protein matches exactly that of the predicted DNA sequence, which, in the presence of the E. coli RNA polymerase core enzyme, is able to initiate transcription of an algT promoter.

Maleylacetate reductase from Pseudomonas sp. strain B13 is one of the enzymes of the modified ortho pathway that is involved in chlorocatechol degradation. Purification and characterization of the enzyme by high-performance liquid chromatography detected tfdF and tcbF genes on plasmids pJP4 of Alcaligenes eutrophus and pP51 of Pseudomonas strain P51. These genes were shown to have significant similarities with those on plasmid pAC27 of Pseudomonas putida AC866.

The newly discovered algH gene in Pseudomonas aeruginosa is involved in the production of nucleoside diphosphate kinase (Ndk). The algH gene is one of several genes that are activated under conditions which cause P. aeruginosa to convert from its nonmucoid form to the mucoid form. The mucoid form is covered by an alginate capsule that promotes adherence to cells of the respiratory tract.

LacZ-transcriptional fusion expression experiments assess the potential of the LysR-type regulatory proteins CatR, and ClcR, to cross-regulate catechol and 3-chlorocatechol pathways in Pseudomonas putida. CatR interacts with the clcABD promoter region while ClcR interacts with the catBC promoter region. Only CatR and not ClcR, can cross-activate, as explained by differences in the CatR and ClcR binding sites and their binding properties.

Sequencing of the 2.7-kb Sst-XbaI DNA fragment involved in the conversion of 2,4,5-T, the carbon source of Pseudomonas cepacia, to 2,4,5-trichlorophenol, indicates the presence of two open reading frames (ORFs) in the fragment. The ORFs are homologous to the BenA and BenB and XylX and XylX proteins and other biodegradative dioxygenase complexes in their amino acid sequences. Pulse-labeling experiments enable the identification of the directions of transcription of these genes and the number and sizes of the polypeptide synthesized. The enzymes encoded by the two ORFs, TftA1 and TftA2, exhibit a relaxed substrate specificity.

Levels of the complex-forming nucleoside diphosphate kinase (Ndk) and phosphorylated succinyl coenzyme A synthetase (Scs) in Pseudomonas aeruginosa are diminished in the algR2 mutant. Phosphorylation of Scs is low due to a decline in its function, but the presence of another kinase function that is sensitive to Tween 20 contributes the missing Ndk activity. AlgR2 exhibits an Scs-specific effect on tricarboxylic acid (TCA) cycle enzymes, but its capacity to grow on TCA substrates is minimized.

Gel filtration and amino acid sequencing of the regulator of LYSR family, ClcR, reveals that ClcR is a dimer with its active fractions having molecular mass of 74 plus or minus 12 kDa. DNAse I foot printing analysis of ClcR helps determine its binding sites. Comparison of the binding of ClcR and the chlorocatechol operon promoter CatR, of Pseudomonas putida ClcABD regulators to their promoters showed that unlike CatR, ClcR attaches to activation binding site and repression binding site (RBS) target regions without any effector. Further analysis indicates the presence of a second element between the ATG start site and ClcR-ABD RBS region.

Purification and characterization of phosophomannomutase (PMM) derived from Pseudomonas aeruginosa show that the PMM enzyme affects the biosynthesis of alginate and lipopolysaccharide by using both phosphomannose and phosphoglucose as substrates. PMM catalyzes the interconversion of mannose 1-phosphate, mannose 6-phosphate, glucose 1-phosphate and glucose 6-phosphate and the conversion of ribose 1-phosphate and 2-deoxyglucose 6-phosphate. The enzymes PMM and phosphoglucomutase do not affect the nonmucoid algC mutant strain 8858 and the LPS-rough algC mutant strain AK1012.

High-level expression of alginate lyase in the 8830 mucoid strain induces degradation of alginate and increased cell detachment of Pseudomonas aeruginosa cells. The degradation occurs when alginate lyase is induced in the early or late stages of cell growth. Alginate lyase expressed from a modulated promoter sequence, causes improved cell sloughing due to alginate degradation process.

The features of AlgR1-binding sites (ABS) are similar to those of eukaryotic enhancer elements and identical to some of the prokaryotic enhancer sequences. In addition to the two ABSs, algC-ABB1 and algC-ABS2, that are upstream of the algC gene, another ABS, algC-ABS3, located within the structural gene of algC, is essential for the activation of the algC promoter. Though the algC-ABS2 is not strongly active, it can completely activate the algC promoter in the presence of algC-ABS1. The number of copies, the orientation or the situation of the algC-ABS1 does not influence the activation of the algC promoter.

The activity of an alginate promoter of Pseudomonas aeruginosa was monitored by a reporter gene technology. Chromogenic and fluorogenic substrates from beta-galactosidase were used to demonstrate reporter activity. Fourier transform infrared spectroscopy, uronic acid accumulation and alginate- specific enzyme-linked immunosorbent assay measured the correlation between gene-up expression and alginate biosynthesis. The activation of the specific gene was demonstrated.

The effect of two types of inhibitors on the activities of AlgR1 and AlgR2, the two-component signal transduction system that regulates alginate gene expression in Pseudomonas aeruginosa, were studied. These compounds inhibit alginate gene expression by inhibiting the phosphorylation/dephosphorylation of AlgR2 and the DNA-binding activity of AlgR1. In addition, it was shown that one compound has the ability to significantly inhibit the kinase activities of other two-component kinases, CheA, NR(sub II) and KinA.

The Pseudomonas putida clcABD operon encodes a degradative pathway converting 3-chlorocatechol to maleylacetic acid. Sequence analysis of this operon identified a partial open reading frame similar to the N terminus of the LysR family of regulatory proteins. The complete clcR regulatory gene was isolated and characterized. The results showed that clcR could autoregulate its own product as well as the clcABD operon. Operon regulation involved ClcR binding to the clcR-clcA intergenic promoter region.

The mechanism by which CatR activates the catBC operon which encodes enzymes involved in benzoate degradation in Pseudomonas putida was examined. Gel shift studies demonstrated that the inducer, cis,cis-muconate, increases the binding affinity of CatR for the catBCpromoter region. In addition, cic,cis-muconate allows CatR to bind to a second site of the catBC promoter region, thereby activating catBC transcription. However, binding of CatR to this activation binding site required an intact repression binding site.

The transcriptional regulation of algC, a distantly located alginate biosynthetic gene in the Pseudomonas aeruginosa chromosome, was studied. Transcriptional activation of algC increases with the osmolarity of the medium and is dependent on the response regulator, AlgR1. The algC promoter has partial dependence on the rpoNgene product sigma-54. Moreover, it was shown that AlgR1 binds to two regions of the algC promoter, upstream and downstream of the mRNA start site. Thus, AlgR1 is involved in regulating and modulating alginate gene expression.

Alginate is a major virulence factor synthesized by Pseudomonas aeruginosa in the lung of cystic fibrosis patients. Biosynthesis of this exopolysaccharide is dependent on the expression of the gene algD which encodes an enzyme essential to alginate production. Expression of this algD gene is dependent on the regulatory genes alg1 and alg2. Characterization of alg2 suggests that it encodes a kinase that undergoes autophosphorylation in the presence of either ATP or GTP. The phosphate group it has acquired is then transferred to alg1 leading to alg1 activation and algD transcription promotion resulting in alginate synthesis.

Results show that Burkholderia cepacia, a major pathogen of cystic fibrosis, secretes two redox proteins, azurin homologue and cytochrome c(sub)551. Data indicate that the azurin homologue is cytotoxic towards macrophages and also induces apoptosis in them.

This article examines the mechanism of action of the bacteria for their ability to cause tumor regression. Research suggests that they may activate the immune system leading to cancer mitigation. However, an alternative hypothesis indicates that it may be the inhibition of angiogenesis or production of redox proteins which cause tumor regression instead.

In the industrialized world halogenated compounds play a major part in day-to-day life. Large amounts of these compounds used as refrigerants, fire retardants, paints and varnishes, solvents, herbicides, pesticides, and [...]