Overproduction of the capsular polysaccharide alginate appears to confer a selective advantage for Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The regulators AlgB and AlgR, which are both required as positive activators in alginate overproduction, have homology with the regulator class of two-component environmental responsive proteins which coordinate gene expression through signal transduction mechanisms. Signal transduction in this class of proteins generally occurs via autophosphorylation of the sensor kinase protein and phosphotransfer from the sensor to a conserved aspartate residue, which is present in the amino terminus of the response regulator. Recently,kinB was identified downstream of algB and was shown to encode the cognate histidine protein kinase that efficiently phosphorylates AlgB. However, we show here that a null mutation inkinB in a mucoid cystic fibrosis isolate, P. aeruginosa FRD1, did not block alginate production. The role of the conserved aspartate residue in the phosphorylation of AlgB was examined. The predicted phosphorylation site of AlgB (D59) was mutated to asparagine (N), and a derivative of an AlgB lacking the entire amino-terminal phosphorylation domain (AlgBΔ1-145) was constructed. A hexahistidine tag was included at the amino terminus of the wild-type (H-AlgB), H-AlgBΔ1-145, and mutant (H-AlgB.59N) AlgB proteins. These derivatives were purified by Ni2+affinity chromatography and examined for in vitro phosphorylation by the purified sensor kinase protein, KinB. The results indicated that while KinB efficiently phosphorylated H-AlgB, no phosphorylation of H-AlgBΔ1-145 or H-AlgB.D59N was apparent. An allelic exchange system was developed to transfer mutantalgB alleles onto the chromosome of a P. aeruginosa algB mutant to examine the effect on alginate production. Despite the defect in AlgB phosphorylation,P. aeruginosa strains expressing AlgB.D59N or H-AlgBΔ1-145 remained mucoid. The roles of the conserved aspartate residues in the phosphorylation of AlgR were also examined. As seen with AlgB, mutations in the predicted phosphorylation site of AlgR (AlgR.D54N and AlgR.D85N) did not affect alginate production. These results indicate that in vivo phosphorylation of AlgB and AlgR are not required for their roles in alginate production. Thus, the mechanism by which these response regulators activate alginate genes in mucoid P. aeruginosa appears not to be mediated by conventional phosphorylation-dependent signal transduction.

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Industrial effluents from tanneries and electroplating industries from small-and large-scale sector industrial plants contain substantial amount of toxic heavy metal, which pollutes rivers and lakes, land, air and sea leading to imbalance of ecosystem and certain health issues to humans, animals as well as plants. The worldwide environmental regulations stipulate the reduction of heavy metals in the effluents to permissible levels before discharging into water bodies. Enzyme-mediated precipitation of heavy metals affords a novel eco-friendly method for remediation of toxic heavy metals from various industrial effluents like tannery, electroplating and dye industries. This chapter has paid attention to bacterial alkaline phosphatase (BAP) from Escherichia coli C90 and calf-intestinal alkaline phosphatase (CIAP), which catalyses phospho mono-and diesters and produces inorganic phosphate (Pi). The Pi thus generated precipitates the heavy metals as metal-phosphate complexes. The kinetic behaviour of both the enzymes with para-nitrophenyl phosphate, ascorbic acid 2-phosphate and α-naphthyl phosphate was investigated at various pH regimes from 8 to 11. The chapter also explains in detail the descriptive information on the capability of BAP-and CIAP-mediated precipitation of heavy metals, which is desirable and convenient method for the toxic heavy metals such as chromium, cadmium, nickel and cobalt.

Enzyme-mediated bioremediation is an eco-friendly process for removing hazardous toxic heavy metals from the environment. The potential use of mutant alkaline phosphatase H412R for bioprecipitation of heavy metals such as Co 2+ , Cd 2+ , Cr 6+ , Ni 2+ , Mn 2+ and Zn 2+ from single-ion solutions and electroplating effluents was analysed in the present study. Purified wild-type and H412R mutant alkaline phosphatase enzymes were incubated with an initial concentration of 100 ppm metal solutions for various time periods along with the substrate p-nitrophenol phosphate. Upon catalysis, the enzyme-substrate reaction liberates inorganic phosphate which in turn binds to heavy metals and precipitates them as metal-phosphates. The amount of metal ions precipitated as a result of formation of metal ion-phosphate complexes was determined by estimating the amount of free metal ions present in the solution using atomic absorption spectroscopy. Based on the results obtained, maximum bioprecipitation of metal ions, in general, was observed at 180 min of incubation period. The H412R mutant enzyme exhibited higher efficiency and precipitated 96% of Mn 2+ from electroplating effluent and 92% of Co 2+ from the metal ion solution. The pattern of precipitation of various metal ions was in the order Co 2+ > Cr 6+ > Ni 2+ > Cd 2+ > Mn 2+ > Zn 2+ for H412R mutant enzyme and Co 2+ > Cr 6+ > Zn 2+ > Ni 2+ > Cd 2+ > Mn 2+ for wild-type enzyme. The results emphasise the use of novel H412R, a mutated alkaline phosphatase enzyme in its catalytic site, as an efficient way of achieving bioremediation of heavy metals from real-time effluents.