Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols. (1/14)

The predominant bacterial pathway for nitrobenzene (NB) degradation uses an NB nitroreductase and hydroxylaminobenzene (HAB) mutase to form the ring-fission substrate ortho-aminophenol. We tested the hypothesis that constructed strains might accumulate the aminophenols from nitroacetophenones and other nitroaromatic compounds. We constructed a recombinant plasmid carrying NB nitroreductase (nbzA) and HAB mutase A (habA) genes, both from Pseudomonas pseudoalcaligenes JS45, and expressed the enzymes in Escherichia coli JS995. IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells of strain JS995 rapidly and stoichiometrically converted NB to 2-aminophenol, 2-nitroacetophenone (2NAP) to 2-amino-3-hydroxyacetophenone (2AHAP), and 3-nitroacetophenone (3NAP) to 3-amino-2-hydroxyacetophenone (3AHAP). We constructed another recombinant plasmid containing the nitroreductase gene (nfs1) from Enterobacter cloacae and habA from strain JS45 and expressed the enzymes in E. coli JS996. Strain JS996 converted NB to 2-aminophenol, 2-nitrotoluene to 2-amino-3-methylphenol, 3-nitrotoluene to 2-amino-4-methylphenol, 4-nitrobiphenyl ether to 4-amino-5-phenoxyphenol, and 1-nitronaphthalene to 2-amino-1-naphthol. In larger-scale biotransformations catalyzed by strain JS995, 75% of the 2NAP transformed was converted to 2AHAP, whereas 3AHAP was produced stoichiometrically from 3NAP. The final yields of the aminophenols after extraction and recovery were >64%. The biocatalytic synthesis of ortho-aminophenols from nitroacetophenones suggests that strain JS995 may be useful in the biocatalytic production of a variety of substituted ortho-aminophenols from the corresponding nitroaromatic compounds.  (+info)

Molecular breeding of 2,3-dihydroxybiphenyl 1,2-dioxygenase for enhanced resistance to 3-chlorocatechol. (2/14)

3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 1.13.11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.  (+info)

Bacterial degradation of cyanide and its metal complexes under alkaline conditions. (3/14)

A bacterial strain able to use cyanide as the sole nitrogen source under alkaline conditions has been isolated. The bacterium was classified as Pseudomonas pseudoalcaligenes by comparison of its 16S RNA gene sequence to those of existing strains and deposited in the Coleccion Espanola de Cultivos Tipo (Spanish Type Culture Collection) as strain CECT5344. Cyanide consumption is an assimilative process, since (i) bacterial growth was concomitant and proportional to cyanide degradation and (ii) the bacterium stoichiometrically converted cyanide into ammonium in the presence of l-methionine-d,l-sulfoximine, a glutamine synthetase inhibitor. The bacterium was able to grow in alkaline media, up to an initial pH of 11.5, and tolerated free cyanide in concentrations of up to 30 mM, which makes it a good candidate for the biological treatment of cyanide-contaminated residues. Both acetate and d,l-malate were suitable carbon sources for cyanotrophic growth, but no growth was detected in media with cyanide as the sole carbon source. In addition to cyanide, P. pseudoalcaligenes CECT5344 used other nitrogen sources, namely ammonium, nitrate, cyanate, cyanoacetamide, nitroferricyanide (nitroprusside), and a variety of cyanide-metal complexes. Cyanide and ammonium were assimilated simultaneously, whereas cyanide strongly inhibited nitrate and nitrite assimilation. Cyanase activity was induced during growth with cyanide or cyanate, but not with ammonium or nitrate as the nitrogen source. This result suggests that cyanate could be an intermediate in the cyanide degradation pathway, but alternative routes cannot be excluded.  (+info)

Epoxide formation on the aromatic B ring of flavanone by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes KF707. (4/14)

Prokaryotic dioxygenase is known to catalyze aromatic compounds into their corresponding cis-dihydrodiols without the formation of an epoxide intermediate. Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 showed novel monooxygenase activity by converting 2(R)- and 2(S)-flavanone to their corresponding epoxides (2-(7-oxabicyclo[4.1.0]hepta-2,4-dien-2-yl)-2, 3-dihydro-4H-chromen-4-one), whereby the epoxide bond was formed between C2' and C3' on the B ring of the flavanone. The enzyme also converted 6-hydroxyflavanone and 7-hydroxyflavanone, which do not contain a hydroxyl group on the B-ring, to their corresponding epoxides. In a previous report (S.-Y. Kim, J. Jung, Y. Lim, J.-H. Ahn, S.-I. Kim, and H.-G. Hur, Antonie Leeuwenhoek 84:261-268, 2003), however, we found that the same enzyme showed dioxygenase activity toward flavone, resulting in the production of flavone cis-2',3'-dihydrodiol. Extensive structural identification of the metabolites of flavanone by using high-pressure liquid chromatography, liquid chromatography/mass spectrometry, and nuclear magnetic resonance confirmed the presence of an epoxide functional group on the metabolites. Epoxide formation as the initial activation step of aromatic compounds by oxygenases has been reported to occur only by eukaryotic monooxygenases. To the best of our knowledge, biphenyl dioxygenase from P. pseudoalcaligenes KF707 is the first prokaryotic enzyme detected that can produce an epoxide derivative on the aromatic ring structure of flavanone.  (+info)

Pleiotropic costs of niche expansion in the RNA bacteriophage phi 6. (5/14)

Natural and experimental systems have failed to universally demonstrate a trade-off between generalism and specialism. When a trade-off does occur it is difficult to attribute its cause to antagonistic pleiotropy without dissecting the genetic basis of adaptation, and few previous experiments provide these genetic data. Here we investigate the evolution of expanded host range (generalism) in the RNA virus phi6, an experimental model system allowing adaptive mutations to be readily identified. We isolated 10 spontaneous host range mutants on each of three novel Pseudomonas hosts and determined whether these mutations imposed fitness costs on the standard laboratory host. Sequencing revealed that each mutant had one of nine nonsynonymous mutations in the phi6 gene P3, important in host attachment. Seven of these nine mutations were costly on the original host, confirming the existence of antagonistic pleiotropy. In addition to this genetically imposed cost, we identified an epigenetic cost of generalism that occurs when phage transition between host types. Our results confirm the existence in phi6 of two costs of generalism, genetic and environmental, but they also indicate that the cost is not always large. The possibility for cost-free niche expansion implies that varied ecological conditions may favor host shifts in RNA viruses.  (+info)

Steady-state kinetic characterization of evolved biphenyl dioxygenase, which acquired novel degradation ability for benzene and toluene. (6/14)

Biphenyl dioxygenase (Bph Dox) catalyzes initial oxygenation in the bacterial biphenyl degradation pathway. Bph Dox in Pseudomonas pseudoalcaligenes KF707 is a Rieske type three-component enzyme in which a large subunit (encoded by the bphA1 gene) plays an important role in the substrate specificity of Bph Dox. Steady-state kinetic assays using purified enzyme components demonstrated that KF707 Bph Dox had a kcat/Km of 33.1 x 10(3) (M(-1) s(-1)) for biphenyl. Evolved 1072 Bph Dox generated by the process of DNA shuffling (Suenaga, H. et al., J. Bacteriol., 184, 3682-3688 (2002)) exhibited enhanced degradation activity not only for biphenyl (kcat/Km of 62.2 x 10(3) [M(-1) s(-1)]) but also for benzene and toluene, compounds that are rarely attacked by KF707 Bph Dox. These results suggest that evolved 1072 Bph Dox acquires higher affinities and catalytic efficiencies for various substrates than the original KF707 enzyme.  (+info)

Cross-regulation of biphenyl- and salicylate-catabolic genes by two regulatory systems in Pseudomonas pseudoalcaligenes KF707. (7/14)

Pseudomonas pseudoalcaligenes KF707 grows on biphenyl and salicylate as sole sources of carbon. The biphenyl-catabolic (bph) genes are organized as bphR1A1A2(orf3)A3A4BCX0X1X2X3D, encoding the enzymes for conversion of biphenyl to acetyl coenzyme A. In this study, the salicylate-catabolic (sal) gene cluster encoding the enzymes for conversion of salicylate to acetyl coenzyme A were identified 6.6-kb downstream of the bph gene cluster along with a second regulatory gene, bphR2. Both the bph and sal genes were cross-regulated positively and/or negatively by the two regulatory proteins, BphR1 and BphR2, in the presence or absence of the effectors. The BphR2 binding sequence exhibits homology with the NahR binding sequences in various naphthalene-degrading bacteria. Based on previous studies and the present study we propose a new regulatory model for biphenyl and salicylate catabolism in strain KF707.  (+info)

Essential role of cytochrome bd-related oxidase in cyanide resistance of Pseudomonas pseudoalcaligenes CECT5344. (8/14)

Pseudomonas pseudoalcaligenes CECT5344 grows in minimal medium containing cyanide as the sole nitrogen source. Under these conditions, an O2-dependent respiration highly resistant to cyanide was detected in cell extracts. The structural genes for the cyanide-resistant terminal oxidase, cioA and cioB, are clustered and encode the integral membrane proteins that correspond to subunits I and II of classical cytochrome bd, although the presence of heme d in the membrane could not be detected by difference spectra. The cio operon from P. pseudoalcaligenes presents a singular organization, starting upstream of cioAB by the coding sequence of a putative ferredoxin-dependent sulfite or nitrite reductase and spanning downstream two additional open reading frames that encode uncharacterized gene products. PCR amplifications of RNA (reverse transcription-PCR) indicated the cyanide-dependent up-regulation and cotranscription along the operon. The targeted disruption of cioA eliminates both the expression of the cyanide-stimulated respiratory activity and the growth with cyanide as the nitrogen source, which suggests a critical role of this cytochrome bd-related oxidase in the metabolism of cyanide by P. pseudoalcaligenes CECT5344.  (+info)