Genetic Organization of Genes Encoding Phenol Hydroxylase, Benzoate 1,2-Dioxygenase Alpha Subunit and Its Regulatory Proteins in Acinetobacter calcoaceticus PHEA-2

Acinetobacter calcoaceticus PHEA-2 is a phenol-degrading bacterium isolated from the wastewater from an oil refinery. A 10-kb XhoI fragment consisting of nine complete Open Reading Frames (ORFs) and one partial ORF was screened from a lambda library of PHEA-2 by Southern hybridization. The sequence analyses revealed that ORF2–ORF7, designated mphKLMNOP, are homologous to dmpKLMNOP of Pseudomonas sp. CF600 and mopKLMNOP of Acinetobacter calcoaceticus NCIB8250, sharing 38%–72% and 58.5%–93.5% respectively. The products encoded by dmp and mop genes convert phenol to catechol. The mph-operon and downstream ORFs, ORF9 and ORF10, sharing high identities to benM and benA, which encode ben-operon regulatory protein and benzoate 1,2-dioxygenase alpha subunit respectively, are separated by ORF8, whose function is unknown. The organization of the mph and ben operons is different from that described previously. Received: 8 April 2002 / Accepted: 8 May 2002     

Single-culture aerobic granules with <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

Aerobic granules are cultivated by a single bacterial strain, Acinetobacter calcoaceticus, in a sequencing batch reactor (SBR). This strain presents as a good phenol reducer and an efficient auto coagulator in the presence of phenol, mediated by heat-sensitive adhesins proteins. Stable 2.3-mm granules were formed in the SBR following a 7-week cultivation. These granules exhibit excellent settling attributes and degrade phenol efficiently at concentrations of 250–2,000 mg l⁻¹. The corresponding phenol degradation rate reached 993.6 mg phenol g⁻¹ volatile suspended solids (VSS) day⁻¹ at 250 mg l⁻¹ phenol and 519.3 mg phenol g⁻¹ VSS day⁻¹ at 2,000 mg l⁻¹ phenol concentration. Meanwhile, free A. calcoaceticus cells were fully inhibited at phenol >1,500 mg l⁻¹. Denaturing gradient gel electrophoresis fingerprint profile demonstrated no genetic modification in the strain during aerobic granulation. The present single-strain granules showed long-term structural stability and performed high phenol degrading capacity and high phenol tolerance. The confocal laser scanning microscopic test revealed that live A. calcoaceticus cells principally distributed at 200–250 μm beneath the outer surface, with an extracellular polymeric substance layer covering them to defend phenol toxicity. Autoaggregation assay tests demonstrated the possibly significant role of secreted proteins on the formation of single-culture A. calcoaceticus granules.     

Diversity of Degradation Pathways of Some Aromatic Compounds by Phenotype and Genotype Testing in Acinetobacter Strains

Phenotype and genotype testing were combined in this study to identify the diversity of five Acinetobacter strains to degrade some low-molecular-weight aromatic compounds. Three groups were identified: Group one, A. calcoaceticus PHEA-2 and A. baumanii UMI-95, which had the ability to degrade catechol and protocatchuate, and could catabolize vanillic acid and phenol; Group two, Acinetobacter sp. MM1, which had the ability to degrade protocatchuate, but not catechol, just could catabolize vanillic acid; and Group three, A. lwoffi UMI-8, A. junii 85, which degraded neither catechol nor protocatchuate, not could catabolize vanillic acid or phenol. This revised version was published online in July 2006 with corrections to the Cover Date.     

Degradation of phenol via carboxylation to benzoate by a defined,obligate syntrophic consortium of anaerobic bacteria

Summary An obligate syntrophic culture was selected in mineral medium with phenol as the only carbon and energy source. The consortium consisted of a short and a long rod-shaped bacterium and of low numbers of Desulfovibrio cells, and grew only in syntrophy with methanogens, e. g. Methanospirillum hungatei. Under N₂/CO₂, phenol was degraded via benzoate to acetate, CH₄ and CO₂, while in the presence of H₂/CO₂ benzoate was formed, but not further degraded. When 4-hydroxybenzoate was fed to the mixed culture, it was decarboxylated to phenol prior to benzoate formation and subsequent ring cleavage. Isolation of pure cultures of the two rod-shaped bacteria failed. Microscopic observations during feeding of either 4-hydroxybenzoate, phenol or benzoate implied an obligate syntrophic interdependence of the two different rod-shaped bacteria and of the methanogen. The non-motile rods formed phenol from 4-hydroxybenzoate and benzoate from phenol, requiring an as yet unknown co-substrate or co-factor, probably cross-fed by the short, motile rod. The short, motile rodshaped bacterium grew only in syntrophy with methanogens and degraded benzoate to acetate, CO₂ and methane. Desulfovibrio sp., present in low numbers, apparently could not contribute to the degradation of phenol or 4-hydroxybenzoate.     

Anaerobic degradation of phenol in sewage sludge

Summary Anaerobic phenol degrading consortia were selected in sewage sludge and culture conditions were improved to allow maximum degradation rates of 0.9 g/l·d. Phenol had to be added in two portions of 0.45 g/l at intervals of 12 h to keep the fermentation at stable conditions. From U-¹⁴C-phenol little benzoate and acetate were formed as intermediates under a N₂:CO₂ gas phase. Final products were methane and CO₂. When methanogenesis was inhibited by BESA, less labeled methane and CO₂ were formed and labeled acetate remained undegraded. Turnover rates of phenol were significantly reduced in the presence of a H₂:CO₂ gas atmosphere and benzoate was formed from phenol and CO₂. Acetate did not accumulate remarkably. After the H₂:CO₂ was converted to methane or was exchanged by N₂:CO₂ the accumulated benzoate was further degraded to methane and CO₂. Elevated pools of acetate in sewage sludge led also to a reduction of the phenol degradation rates and presumably to an increased concentration of benzoate. In fresh sewage sludge benzoate degradation proceeds immediately, while the degradation of phenol starts only after a lag-phase of 3–10 days.     

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron‐reducing enrichment culture

Anaerobic benzene degradation was studied with a highly enriched iron‐reducing culture (BF) composed of mainly Peptococcaceae‐related Gram‐positive microorganisms. The proteomes of benzene‐, phenol‐ and benzoate‐grown cells of culture BF were compared by SDS‐PAGE. A specific benzene‐expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N‐terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI‐MS/MS‐based shotgun proteomic analysis revealed other specifically benzene‐expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate‐CoA ligase (BamY) of Geobacter metallireducens and 67% to 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (～17 kb) composed of carboxylase‐related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX).     

Effect of fluorinated analogues of phenol and hydroxybenzoates on the anaerobic transformation of phenol to benzoate

The effects of fluorinated analogues on the anaerobic transformation of phenol to benzoate were examined. At 250 M 2- or 3-fluorophenol, phenol transformation was delayed. 2-Fluorophenol had no apparent effect on subsequent degradation of benzoate, but benzoate accumulated in the presence of 250 M 3-fluorophenol. In contrast, 4-fluorophenol at 2 mM had no effect on either phenol transformation or benzoate degradation. Phenol and 2-, or 3-fluorophenol were transformed simultaneously, but phenol was transformed more rapidly than either fluorophenol. Thus, fluorinated analogues of phenol did not prevent anaerobic transformation of phenol to benzoate. 2-Fluorophenol was converted to 3-fluorobenzoate, and phenol enhanced the rate and extent of its transformation. 3-Fluorophenol was transformed to 2-fluorobenzoate to a limited extent (3%) when phenol was present. 4-Fluorophenol was not transformed regardless of the presence of phenol. 3-Fluoro-4-hydroxybenzoate, a potential fluorinated intermediate product of para-carboxylation, was transformed rapidly to 2-fluorophenol and 3-fluorobenzoate, irrespective of the presence of phenol, indicating that both dehydroxylation and decarboxylation occurred. Initially, 2-fluorophenol and 3-fluorobenzoate were rapidly formed in an approximate molar ratio of 2 : 1. Once 3-fluoro-4-hydroxybenzoate was completely removed, the 2-fluorophenol, initially formed, was converted to 3-fluorobenzoate at a slower rate. Thus, phenol enhanced transformation of the fluorinated analogues, and the products of transformation suggested para-carboxylation. 3-Fluoro-2-hydroxybenzoate was not transformed in either the presence or absence of phenol, indicating that ortho-carboxylation did not occur.Abbreviations 3F4HB 3-fluoro-4-hydroxybenzoate - 3F2HB 3-fluoro-2-hydroxybenzoate (3-fluorosalicylate) Contribution No. 692, Environmental Research Laboratory, U.S. EPA, Gulf Breeze, FL. 32561, USA     

Inducibility of benzoate oxidizing cell activities in Acinetobacter calcoaceticus strain Bs 5 by chlorobenzoates as influenced by the position of chlorine atoms and the inducer concentration

Summary Among four chlorobenzoates tested, only 3-chlorobenzoate and 4-chlorobenzoate were capable of inducing benzoate oxidizing cell activities in Acinetobacter calcoaceticus strain Bs 5, whereas 2-chlorobenzoate and 2,6-dichlorobenzoate were not. With the monochlorobenzoates, this inducing capability decreased with increasing proximity of the chlorine atom to the carboxyl group, i.e. in the order: 4-chlorobenzoate > 3-chlorobenzoate > 2-chlorobenzoate. It is therefore supposed that the induction of benzoate oxidizing cell activities is inhibited primarily be sterical influences of the chlorine substituents of the various chlorobenzoates.With decreasing concentration of 3-chlorobenzoate and 4-chlorobenzoate, the induction of benzoate oxidizing cell activities decreased. Below a critical concentration of 1 M, these activities were no longer detectable in the cells of Acinetobacter calcoaceticus, with the consequence that below this concentration limit, the degradation of 3-chlorobenzoate and 4-chlorobenzoate was no longer possible.     

Anaerobic aniline degradation via reductive deamination of 4-aminobenzoyl-CoA in Desulfobacterium anilini

The initial reactions involved in anaerobic aniline degradation by the sulfate-reducing Desulfobacterium anilini were studied. Experiments for substrate induction indicated the presence of a common pathway for aniline and 4-aminobenzoate, different from that for degradation of 2-aminobenzoate, 2-hydroxybenzoate, 4-hydroxybenzoate, or phenol. Degradation of aniline by dense cell suspensions depended on CO₂ whereas 4-aminobenzoate degradation did not. If acetyl-CoA oxidation was inhibited by cyanide, benzoate accumulated during degradation of aniline or 4-aminobenzoate, indicating an initial carboxylation of aniline to 4-aminobenzoate, and further degradation via benzoate of both substrates. Extracts of alinine or 4-aminobenzoategrown cells activated 4-aminobenzoate to 4-aminobenzoyl-CoA in the presence of CoA, ATP and Mg²⁺. 4-Aminobenzoyl-CoA-synthetase showed a K _m for 4-aminobenzoate lower than 10 M and an activity of 15.8 nmol · min^-1 · mg^-1. 4-Aminobenzoyl-CoA was reductively deaminated to benzoyl-CoA by cell extracts in the presence of low-potential electron donors such as titanium citrate or cobalt sepulchrate (2.1 nmol · min^-1 · mg^-1). Lower activities for the reductive deamination were measured with NADH or NADPH. Reductive deamination was also indicated by benzoate accumulation during 4-aminobenzoate degradation in cell suspensions under sulfate limitation. The results provide evidence that aniline is degraded via carboxylation to 4-aminobenzoate, which is activated to 4-aminobenzoyl-CoA and further metabolized by reductive deamination to benzoyl-CoA.     

Inhibition of benzene,toluene, phenol and benzoate in single and combination on Anammox activity: implication to the denitrification–Anammox synergy

A previous study demonstrated that denitrification synergized with Anammox could accelerate the anaerobic degradation of benzene. The inhibitory effects of benzene, toluene, phenol and benzoate in single and combination on Anammox activity were investigated by short-term batch tests. The results indicated that the inhibition of single compounds on Anammox could be well fitted with the extended non-competitive and Luong inhibition kinetic models. The inhibitions of the individual compound were in order as follows: benzene?>?toluene?>?phenol?>?benzoate. The joint inhibitions of bi-component mixtures of benzene with toluene, benzene with phenol and benzene with benzoate on Anammox activity were additive; the joint inhibition of a tri-component mixture (benzene, toluene and phenol) was partly additive; and the joint inhibition of a multicomponent mixture (benzene, toluene, phenol and benzoate) was synergistic. The effect of benzoate on the denitrification–Anammox synergy for benzene degradation was evaluated using a long-term test. Although the average rate of benzene degradation decreased by 13% with the addition of 10 mg L^?1 benzoate, the average rate of NO₃^? and NH₄⁺ increased by approximately 1- and 0.56-fold, respectively, suggesting that benzoate favors the stability of the denitrification–Anammox synergy. The carboxylation of benzene would be a more favorable pathway for the anaerobic degradation of benzene under denitrification synergized with Anammox.     

Biodegradation of mixture containing monohydroxybenzoate isomers by <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

A bacterial strain capable of utilizing a mixture containing 2-hydroxybenzoic acid (2-HBA), 3-hydroxybenzoic acid (3-HBA) and 4-hydroxybenzoic (4-HBA) acid was isolated through enrichment from a soil sample. Based on 16SrDNA sequencing, the microorganism was identified as Acinetobacter calcoaceticus. The sequence of biodegradation of the three isomers when provided as a mixture (0.025%, w/v each) was elucidated. The dihydroxylated metabolites formed from the degradation of 2-HBA, 3-HBA and 4-HBA were identified as catechol, gentisate and protocatechuate, respectively, using the cell-free supernatant and cell-free crude extracts. Monooxygenases and dioxygenases that were induced in the cells of Acinetobacter calcoaceticus in response to growth on mixture containing 2-HBA, 3-HBA and 4-HBA could be detected in cell-free extracts. These data revealed the pathways operating in Acinetobacter calcoaceticus for the sequential metabolism of monohydroxybenzoate isomers when presented as a mixture.     

The importance of phenol oxidase activity in lignin degradation by the white-rot fungus Sporotrichum pulverulentum

The lignin degradation abilities of wildtype, a phenol oxidase-less mutant and a phenol oxidase-positive revertant of Sporotrichum pulverulentum were compared to determine if phenol oxidase activity is necessary for lignin degradation by white-rot fungi. The phenol oxidase-less mutant was unable to degrade kraft lignin or wood. The phenol oxidase-positive revertant, however, regained the ability of the wildtype to degrade kraft lignin and all of the major components of wood. It was found that kraft lignin and lignin-related phenols decreased cellulase and xylanase production by the phenol oxidase-less mutant. Addition of highly purified laccase increased the production of endo-1,4--glucanase in the phenol oxidase-less mutant in the presence of vanillic acid and kraft lignin. After addition of laccase to kraft lignin agar plates, the phenol oxidase-less mutant could degrade kraft lignin.It is proposed that phenol oxidase function in regulating the production of both lignin-and polysaccharide-degrading enzymes by oxidation of lignin and lignin-related phenols when S. pulverulentum is growing on wood.Abbreviation WT wildtype Sporotrichum pulverulentum Research supported by a grant from Stiftelsen Nils and Dorthi Troëdssons forskningsfond     

Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.     

Microbial production of <Emphasis Type="Italic">cis</Emphasis>-1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylate by genetically modified <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid (DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after 24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC production rate was 0.356 g L⁻¹ h⁻¹. DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by nuclear magnetic resonance.