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A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c550 and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd1 Nitrite Reductase. Isobel V. Pearson, 2003.In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc1 complex to the periplasmic nitrite reductase, cytochrome cd1 . The periplasmic protein cytochrome c550 has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells . Here, the hypothesis that cytochrome c550 and pseudoazurin are alternative electron carriers from the cytochrome bc1 complex to the nitrite reductase was tested by construction of mutants of P . denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c550 . The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium . Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c550 or both these proteins . We concluded that pseudoazurin and cytochrome c550 are the alternative electron mediator proteins between the cytochrome bc1 complex and the cytochrome cd1-type nitrite reductase . We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP . Reconstruction of Mycobacterial Dehalogenase Rv2579 by Cumulative Mutagenesis of Haloalkane Dehalogenase LinB. Yuji Nagata, 2003.The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579 . To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB . The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family . Genetic Engineering of a Highly Solvent-Tolerant Pseudomonas putida Strain for Biotransformation of Toluene to p-Hydroxybenzoate. María-Isabel Ramos-González, 2003.The solvent-tolerant strain Pseudomonas putida DOT-T1E has been engineered for biotransformation of toluene into 4-hydroxybenzoate (4-HBA) . P . putida DOT-T1E transforms toluene into 3-methylcatechol in a reaction catalyzed by toluene dioxygenase . The todC1C2 genes encode the  and ß subunits of the multicomponent enzyme toluene dioxygenase, which catalyzes the first step in the Tod pathway of toluene catabolism . A DOT-T1E todC mutant strain was constructed by homologous recombination and was shown to be unable to use toluene as a sole carbon source . The P . putida pobA gene, whose product is responsible for the hydroxylation of 4-HBA into 3,4-hydroxybenzoate, was cloned by complementation of a Pseudomonas mendocina pobA1 pobA2 double mutant . This pobA gene was knocked out in vitro and used to generate a double mutant, DOT-T1EtodCpobA, that was unable to use either toluene or 4-HBA as a carbon source . The tmo and pcu genes from P . mendocina KR1, which catalyze the transformation of toluene into 4-HBA through a combination of the toluene 4-monoxygenase pathway and oxidation of p-cresol into the hydroxylated carboxylic acid, were subcloned in mini-Tn5Tc and stably recruited in the chromosome of DOT-T1EtodCpobA . Expression of the tmo and pcu genes took place in a DOT-T1E background due to cross-activation of the tmo promoter by the two-component signal transduction system TodST . Several independent isolates that accumulated 4-HBA in the supernatant from toluene were analyzed . Differences were observed in these clones in the time required for detection of 4-HBA and in the amount of this compound accumulated in the supernatant . The fastest and most noticeable accumulation of 4-HBA (12 mM) was found with a clone designated DOT-T1E-24 .
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