Characterization and Expression Analysis of Extradiol and Intradiol Dioxygenase of Phenol Degradation Haloalkaliphilic Bacterial Isolates

Haloaklophilic bacteria have a potential advantage as a bioremediation organism for high pH oil polluted and industrial wastewater. In the current study, Haloalkaliphilic isolates were obtained from Hamralake, Wadi EL-Natrun, Egypt. The phenotypic features, biochemical characters, and 16S rRNA sequence comparison were used to identify the bacterial isolates including Halomonas HA1 and Marinobacter HA2. These strains showed high requirement of NaCl for growth specially HA1 strain that essentially required NaCl for its growth. The isolates are capable of degrading phenol at optimal pH values between 8 and 9 with the ability to grow in levels of pH up to 11, like what was seen in Halomonas HA1 strain. Both isolates represent two different mechanistic pathways for phenol degradation. Halomonas HA1 exploits the 1,2 phenol meta cleavage pathway while Marinobacter HA2 using the 2,3 ortho cleavage pathway indicated by universal primer sets for 1,2 and 2,3 CTD genes. Phenol degradation showed a comparable pattern between both isolates, while Marinobacter HA2 isolate can eliminate the added phenol within an incubation period of 72 h, The Halomonas HA1isolate required 96 h to degrade 84% of the same amount of phenol. The phylogenetic analysis of the amino acid sequence of 1,2 CTD (catechol dioxygenase) of Halomonas HA1showed an evolutionary relationship between 1,2 dioxygenases of both Halomonadaceae and Pseudomonadaceae while 2,3 CTD of Marinobacter HA2 shared the main domains of the closely related species. Semi-quantitative RT PCR analysis proved the constitutive expression pattern of both dioxygenase genes.

Structure–Activity Relationships Delineate How the Maize Pathogen Cochliobolus heterostrophus Uses Aromatic Compounds as Signals and Metabolites

The necrotrophic maize pathogen Cochliobolus heterostrophus senses plant-derived phenolic compounds, which promote nuclear retention of the redox-sensitive transcription factor ChAP1 and alter gene expression. The intradiol dioxygenase gene CCHD1 is strongly upregulated by coumaric and caffeic acids. Plant phenolics are potential nutrients but some of them are damaging compounds that need to be detoxified. Using coumaric acid as an inducer (16 to 160 μM), we demonstrated the rapid and simultaneous upregulation of most of the β-ketoadipate pathway genes in C. heterostrophus. A cchd1 deletion mutant provided genetic evidence that protocatechuic acid is an intermediate in catabolism of a wide range of aromatic acids. Aromatics catabolism was slowed for compounds showing toxicity, and this was strongly correlated with nuclear retention of GFP-ChAP1. The activity of a structure series of compounds showed complementary requirements for upregulation of CCHD1 and for ChAP1 nuclear retention. Thus, there is an inverse correlation between the ability to metabolize a compound and the stress response (ChAP1 nuclear retention) that it causes. The ability to metabolize phenolics and to respond to them as signals should be an advantage to plant pathogens and may explain the presence of at least two response pathways detecting these compounds.

Elucidation of the 4-Hydroxyacetophenone Catabolic Pathway in Pseudomonas fluorescens ACB

ABSTRACT The catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB is known to proceed through the intermediate formation of hydroquinone. Here, we provide evidence that hydroquinone is further degraded through 4-hydroxymuconic semialdehyde and maleylacetate to β-ketoadipate. The P. fluorescens ACB genes involved in 4-hydroxyacetophenone utilization were cloned and characterized. Sequence analysis of a 15-kb DNA fragment showed the presence of 14 open reading frames containing a gene cluster (hapCDEFGHIBA) of which at least four encoded enzymes are involved in 4-hydroxyacetophenone degradation: 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenyl acetate hydrolase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF). In between hapF and hapB, three genes encoding a putative intradiol dioxygenase (hapG), a protein of the Yci1 family (hapH), and a [2Fe-2S] ferredoxin (hapI) were found. Downstream of the hap genes, five open reading frames are situated encoding three putative regulatory proteins (orf10, orf12, and orf13) and two proteins possibly involved in a membrane efflux pump (orf11 and orf14). Upstream of hapE, two genes (hapC and hapD) were present that showed weak similarity with several iron(II)-dependent extradiol dioxygenases. Based on these findings and additional biochemical evidence, it is proposed that the hapC and hapD gene products are involved in the ring cleavage of hydroquinone.

Positive Selection for Mutations Affecting Bioconversion of Aromatic Compounds in Agrobacterium tumefaciens: Analysis of Spontaneous Mutations in the Protocatechuate 3,4-Dioxygenase Gene

ABSTRACT A positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain ofAgrobacterium tumefaciens A348. The nucleotide sequence of the A348 pcaHGB genes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and β-carboxy-cis,cis-muconate cycloisomerase (PcaB) for the first two steps in catabolism of the diphenolic protocatechuate, was determined. An omega element was introduced into the pcaB gene of A348, creating strain ADO2077. In the presence of phenolic compounds that can serve as carbon sources, growth of ADO2077 is inhibited due to accumulation of the tricarboxylate intermediate. The toxic effect, previously described forAcinetobacter sp., affords a powerful selection for suppressor mutations in genes required for upstream catabolic steps. By monitoring loss of the marker in pcaB, it was possible to determine that the formation of deletions was minimal compared to results obtained with Acinetobacter sp. Thus, the tricarboxylic acid trick in and of itself does not appear to select for large deletion mutations. The power of the selection was demonstrated by targeting the pcaHG genes of A. tumefaciensfor spontaneous mutation. Sixteen strains carrying putative second-site mutations in pcaH or -G were subjected to sequence analysis. All single-site events, their mutations revealed no particular bias toward multibase deletions or unusual patterns: five (−1) frameshifts, one (+1) frameshift, one tandem duplication of 88 bp, one deletion of 92 bp, one nonsense mutation, and seven missense mutations. PcaHG is considered to be the prototypical ferric intradiol dioxygenase. The missense mutations served to corroborate the significance of active site amino acid residues deduced from crystal structures of PcaHG from Pseudomonas putida andAcinetobacter sp. as well as of residues in other parts of the enzyme.

A Functional 4-Hydroxysalicylate/Hydroxyquinol Degradative Pathway Gene Cluster Is Linked to the Initial Dibenzo-p-Dioxin Pathway Genes inSphingomonas sp. Strain RW1

ABSTRACT The bacterium Sphingomonas sp. strain RW1 is able to use dibenzo-p-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy. We have determined and analyzed the nucleic acid sequence of a 9,997-bpHindIII fragment downstream of cistronsdxnA1A2, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways. This fragment contains 10 colinear open reading frames (ORFs), apparently organized in one compact operon. The enzymatic activities of some proteins encoded by these genes were analyzed in the strain RW1 and, after hyperexpression, in Escherichia coli. The first three ORFs of the locus, designateddxnC, ORF2, and fdx3, specify a protein with a low homology to bacterial siderophore receptors, a polypeptide representing no significant homology to known proteins, and a putative ferredoxin, respectively.dxnD encodes a 69-kDa phenol monooxygenase-like protein with activity for the turnover of 4-hydroxysalicylate, anddxnE codes for a 37-kDa protein whose sequence and activity are similar to those of known maleylacetate reductases. The following gene, dxnF, encodes a 33-kDa intradiol dioxygenase which efficiently cleaves hydroxyquinol, yielding maleylacetate, the ketoform of 3-hydroxy-cis,cis-muconate. The heteromeric protein encoded by dxnGH is a 3-oxoadipate succinyl coenzyme A (succinyl-CoA) transferase, whereas dxnI specifies a protein exhibiting marked homology to acetyl-CoA acetyltransferases (thiolases). The last ORF of the sequenced fragment codes for a putative transposase. DxnD, DxnF, DxnE, DxnGH, and DxnI (the activities of most of them have also been detected in strain RW1) thus form a complete 4-hydroxysalicylate/hydroxyquinol degradative pathway. A route for the mineralization of the growth substrates 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin inSphingomonas sp. strain RW1 thus suggests itself.

Crystallization and preliminary crystallographic analysis of the hydroxyquinol 1,2-dioxygenase from Nocardioides simplex 3E: a novel dioxygenase involved in the biodegradation of polychlorinated aromatic compounds

Hydroxyquinol 1,2-dioxygenase (HQ1,2O) from Nocardioides simplex 3E, an enzyme involved in the aerobic biodegradation of a large class of chloroaromatic compounds such as 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T), has been crystallized. HQ1,2O, which specifically catalyzes the intradiol cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), an intermediate in the degradation of a variety of aromatic pollutants, to maleylacetate, has been recently purified to homogeneity. The enzyme is an homodimer composed of two identical subunits in a α2-type quaternary structure, has a molecular weight of about 65 kDa and contains a catalytically essential Fe(III) ion. Crystals of HQ1,2O obtained using 2% PEG 400 and 2 M ammonium sulfate at pH 7.5 as precipitants belong to the orthorhombic space group P212121, with unit-cell parameters a = 81.15 (6), b = 86.79 (7), c = 114.93 (8). Assuming one dimer per asymmetric unit, the Vm value is 2.51 Å3 Da−1. A complete native data set to 1.8 Å resolution has been collected on a laboratory source. This is the first intradiol dioxygenase which specifically catalyzes the cleavage of hydroxyquinol to give diffraction-quality crystals.

Characterization of the Maleylacetate Reductase MacA of Rhodococcus opacus 1CP and Evidence for the Presence of an Isofunctional Enzyme

ABSTRACT Maleylacetate reductases (EC 1.3.1.32 ) have been shown to contribute not only to the bacterial catabolism of some usual aromatic compounds like quinol or resorcinol but also to the degradation of aromatic compounds carrying unusual substituents, such as halogen atoms or nitro groups. Genes coding for maleylacetate reductases so far have been analyzed mainly in chloroaromatic compound-utilizing proteobacteria, in which they were found to belong to specialized gene clusters for the turnover of chlorocatechols or 5-chlorohydroxyquinol. We have now cloned the gene macA, which codes for one of apparently (at least) two maleylacetate reductases in the gram-positive, chlorophenol-degrading strain Rhodococcus opacus 1CP. Sequencing of macA showed the gene product to be relatively distantly related to its proteobacterial counterparts (ca. 42 to 44% identical positions). Nevertheless, like the known enzymes from proteobacteria, the cloned Rhodococcusmaleylacetate reductase was able to convert 2-chloromaleylacetate, an intermediate in the degradation of dichloroaromatic compounds, relatively fast and with reductive dehalogenation to maleylacetate. Among the genes ca. 3 kb up- and downstream of macA, none was found to code for an intradiol dioxygenase, a cycloisomerase, or a dienelactone hydrolase. Instead, the only gene which is likely to be cotranscribed with macA encodes a protein of the short-chain dehydrogenase/reductase family. Thus, the R. opacus maleylacetate reductase genemacA clearly is not part of a specialized chlorocatechol gene cluster.