Cloning, Characterization and Analysis of cat and ben Genes from the Phenol Degrading Halophilic Bacterium Halomonas organivorans (original) (raw)
Related papers
Research Square (Research Square), 2021
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.
springer, 2022
Haloalkophilic bacteria have a potential advantage as a bioremediation organism of high oil-polluted and industrial wastewater. In the current study, Haloalkaliphilic isolates were obtained from Hamralake, Wadi EL-Natrun, Egypt. The phenotype script, biochemical characters, and sequence analysis of bacterial-16S rRNA were used to identify the bacterial isolates; Halomonas HA1 and Marinobacter HA2. These strains required high concentrations of NaCl to ensure bacterial growth, especially Halomonas HA1 strain. Notably, both isolates can degrade phenol at optimal pH values, between 8 and 9, with the ability to grow in pH levels up to 11, like what was seen in the Halomonas HA1 strain. Moreover, both isolates represent two different mechanistic pathways for phenol degradation. Halomonas HA1 exploits the 1,2 phenol meta-cleavage pathway, while Marinobacter HA2 uses the 2,3 ortho-cleavage pathway as indicated by universal primers for 1,2 and 2,3 CTD genes. Interestingly, Marinobacter HA2 isolate eliminated the added phenol within an incubation period of 72 h, while the Halomonas HA1 isolate invested 96 h in degrading 84% of the same amount of phenol. Phylogenetic analysis of these 1,2 CTD (catechol dioxygenase) sequences clearly showed an evolutionary relationship between 1,2 dioxygenases of both Halomonadaceae and Pseudomonadaceae. In comparison, 2,3 CTD of Marinobacter HA2 shared the main domains of the closely related species. Furthermore, semi-quantitative RT-PCR analysis proved the constitutive expression pattern of both dioxygenase genes. These findings provide new isolates of Halomonas sp. and Marinobacter sp. that can degrade phenol at high salt and pH conditions via two independent mechanisms.
The structures of bacterial genes for the degradation of chlorocatechols and 2,4-dichlorophenoxyacetate were studied and were deduced to have been formed by some genetic recombination events including transposition. In vivo and in vitro analysis of chlorocatechol degradative operon cbnR-ABCD of a 3-chlorobenzoate degradative bacterium Ralstonia eutropha NH9 showed the transcriptional activation of the cbnA promoter by the LysR-type regulator CbnR upon recognition of the inducer molecules, (chloro)muconates produced from (chloro)catechols. Further in vivo analysis of LysR-type transcriptional regulators of (chloro)aromatic degradative operons indicated evolutionary divergence of these regulators in terms of the inducer recognition specificity and suggested specialization of some regulators of chlorocatechol operons for recognition of (chloro)muconates.
Phenol degradation by halophilic bacteria isolated from hypersaline environments
Biodegradation, 2013
Phenol is a toxic aromatic compound used or produced in many industries and as a result a common component of industrial wastewaters. Phenol containing waste streams are frequently hypersaline and therefore require halophilic microorganisms for efficient biotreatment without dilution. In this study three halophilic bacteria isolated from different saline environments and identified as Halomonas organivorans, Arhodomonas aquaeolei and Modicisalibacter tunisiensis were shown to be able to grow on phenol in hypersaline media containing 100 g/L of total salts at a concentration of 3 mM (280 mg/L), well above the concentration found in most waste streams. Genes encoding the aromatic dioxygenase enzymes catechol 1,2 dioxygenase and protocatechuate 3,4-dioxygenase were present in all strains as determined by PCR amplification using primers specific for highly conserved regions of the genes. The gene for protocatechuate 3,4-dioxygenase was cloned from the isolated H. organivorans and the translated protein was evaluated by comparative protein sequence analysis with protocatechuate 3,4-dioxygenase proteins from other microorganisms. Although the analysis revealed a wide range of sequence divergence among the protocatechuate 3,4-dioxygenase family, all of the conserved domain amino acid structures identified for this enzyme family are identical or conservatively substituted in the H. organivorans enzyme.
Journal of Bioscience and Bioengineering, 1999
For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol l,Z-dioxygenase (C120) and catechol 2,fdioxygenase (C230) genes. The C120-targeting primer set (Cl20 primers) was designed based on the homologous regions of 11 Cl20 genes listed in the GenBank, while the CZSO-targeting one (C230 primers) was designed based on those of 17 known C230 genes. Ollgonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the Cl20 and/or C230 genes used for the primer and probe design. Various authentic bacterial strains carrying neither Cl20 nor C230 genes were used as negative controls. PCR with the Cl20 primers ampliied DNA fragments of the expected sizes from 5 of the 6 known ClZO-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 ampliied fragments on Southern hybridization with the C12Op. The C230 primers amplified DNA fragments of the expected size from all the 11 tested C230-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol-and/or benzoatedegradlng bacteria newly isolated from a varlety of environments. The Cl20 and/or C230 primers amplified DNA fragments of the expected sizes from 69 of the 106 wild-type strains tested, while the C12Op and/or C23Op detected positive signals in the amplified fragments from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways.
Gene, 1994
Pseudomonas sp. strain HV3 degrades aromatics and chloroaromatics. It harbours a mega-plasmid, designated pSKY4, from which the gene cmpE, encoding a catechol 2,3-dioxygenase (C230) catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde, was cloned and sequenced. The deduced amino acid (aa) sequence shows the highest homology, 52%, to the deduced aa sequences of xylE1 and dmpB. The deduced 307-aa sequence of cmpE contains the extradiol ring-cleavage signature in the same position as other 307-aa C230-encoding genes.
Current Microbiology, 2009
The novel carbazole (CAR)-degrading bacterium Lysobacter sp. strain OC7 has been isolated from seawater and can also utilize naphthalene and phenanthrene as its sole carbon and energy source. The CAR-degradative gene cluster was isolated and encoded five complete open reading frames (ORFs) and two truncated ORFs. Among them, four ORFs showed 40–50% similarity with previously reported CAR-degradative genes. Ferredoxin (carAc) and ferredoxin reductase (carAd) genes, which are necessary for the CAR 1,9a-dioxygenase system, were not found in this car gene cluster. The car OC7 gene transcripts were strongly detected when CAR was provided. However, these transcripts were also detected when naphthalene was provided. The resting cell reaction with Escherichia coli revealed that CarAaOC7 can use CarAc and CarAd of Pseudomonas resinovorans CA10 as ferredoxin and ferredoxin reductase, respectively, and converted CAR to 2′-aminobiphenyl-2,3-diol. In 13 marine CAR-degrading isolates, only Caulobacter sp. strain OC6 hybridized with the car OC7 gene cluster probe. This is the first report showing CAR-degradative genes from the genus Lysobacter.
Indian Journal of Biotechnology, 2005
Catechol 1,2-dioxygenase (C12O) was purified to electrophoretic homogeneity from Acinetobacter sp. DS002. The pure enzyme appears to be a homodimer with a molecular mass of 66 kDa. The apparent K m and V max for intradiol cleavage of catechol were 1.58 lM and 2 units per mg of protein respectively. Unlike other C12Os reported in the literature, the catechol 1,2-dioxygenase of Acinetobacter showed neither intradiol nor extradiol cleavage activity when substituted catechols were used as substrates. However, it has shown mild intradiol cleavage activity when benzenetriol was used as substrate. As determined by two dimensional electrophoresis (2DE) followed MALDI-TOF/TOF analyses and gel permeation chromatography, no isoforms of C12O was observed in Acinetobacter sp. DS002. Further, the C12O was seen only in cultures grown in benzoate and it was completely absent in succinate grown cultures. Based on the sequence information obtained from MS/MS data, degenerate primers were designed to amplify catA gene from the genomic DNA of Acinetobacter sp. DS002. The sequence of the PCR amplicon and deduced amino acid sequence showed 97% similarity with a catA gene of Acinetobacter baumannii AYE (YP_001713609).
Archives of Microbiology, 2006
Phenol-and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1, 2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species-and biotype-speciWc phylogenetic grouping. LmPHs and C23Os clustered similarly in P. Xuorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. Xuorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the Wrst pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.