Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 108 colony-forming units/μg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species.
We report the draft genome sequence of Variovorax boronicumulans strain HAB-30, which was isolated from a phenol-degrading chemostat culture. This strain contains genes encoding a multicomponent type of phenol hydroxylase, with degradation pathways for catechol and other aromatic compounds. The genome sequence will be useful for understanding the metabolic pathways involved in phenol degradation.
Hydrogenophaga sp. strain PBC is an effective degrader of 4-aminobenzenesulfonate isolated from textile wastewater. Here we present the assembly and annotation of its genome, which may provide further insights into its metabolic potential. This is the first announcement of the draft genome sequence of a strain from the genus Hydrogenophaga.
The gene coding for the oxygenase component, sadA, of 4-aminobenzenesulfonate (4-ABS) 3,4-dioxygenase in Hydrogenophaga sp. PBC was previously identified via transposon mutagenesis. Expression of wild-type sadA in trans restored the ability of the sadA mutant to grow on 4-ABS. The inclusion of sadB and sadD, coding for a putative glutamine-synthetase-like protein and a plant-type ferredoxin, respectively, further improved the efficiency of 4-ABS degradation. Transcription analysis using the gfp promoter probe plasmid showed that sadABD was expressed during growth on 4-ABS and 4-sulfocatechol. Heterologous expression of sadABD in Escherichia coli led to the biotransformation of 4-ABS to a metabolite which shared a similar retention time and UV/vis profile with 4-sulfocatechol. The putative reductase gene sadC was isolated via degenerate PCR and expression of sadC and sadABD in E. coli led to maximal 4-ABS biotransformation. In E. coli, the deletion of sadB completely eliminated dioxygenase activity while the deletion of sadC or sadD led to a decrease in dioxygenase activity. Phylogenetic analysis of SadB showed that it is closely related to the glutamine-synthetase-like proteins involved in the aniline degradation pathway. This is the first discovery, to our knowledge, of the functional genetic components for 4-ABS aromatic ring hydroxylation in the bacterial domain.
A co-culture consisting of Hydrogenophaga sp. PBC and Ralstonia sp. PBA, isolated from textile wastewater treatment plant could tolerate up to 100 mM 4-aminobenzenesulfonate (4-ABS) and utilize it as sole carbon, nitrogen and sulfur source under aerobic condition. The biodegradation of 4-ABS resulted in the release of nitrogen and sulfur in the form of ammonium and sulfate respectively. Ninety-eight percent removal of chemical oxygen demand attributed to 20 mM of 4-ABS in cell-free supernatant could be achieved after 118 h. Effective biodegradation of 4-ABS occurred at pH ranging from 6 to 8. During batch culture with 4-ABS as sole carbon and nitrogen source, the ratio of strain PBA to PBC was dynamic and a critical concentration of strain PBA has to be reached in order to enable effective biodegradation of 4-ABS. Haldane inhibition model was used to fit the degradation rate at different initial concentrations and the parameters μ(max), K(s) and K(i) were determined to be 0.13 h⁻¹, 1.3 mM and 42 mM respectively. HPLC analyses revealed traced accumulation of 4-sulfocatechol and at least four unidentified metabolites during biodegradation. This is the first study to report on the characterization of 4-ABS-degrading bacterial consortium that was isolated from textile wastewater treatment plant.
We report the draft genome sequence of Variovorax boronicumulans strain c24, which was isolated from a soil-inoculated chemostat culture amended with phenol as a sole carbon and energy source. The genome data will provide insights into phenol and other xenobiotic compound degradation mechanisms for bioremediation applications.
We improved upon the previously reported draft genome of Hydrogenophaga intermedia strain PBC, a 4-aminobenzenesulfonate-degrading bacterium, by supplementing the assembly with Nanopore long reads which enabled the reconstruction of the genome as a single contig. From the complete genome, major genes responsible for the catabolism of 4-aminobenzenesulfonate in strain PBC are clustered in two distinct genomic regions. Although the catabolic genes for 4-sulfocatechol, the deaminated product of 4-aminobenzenesulfonate, are only found in H. intermedia, the sad operon responsible for the first deamination step of 4-aminobenzenesulfonate is conserved in various Hydrogenophaga strains. The absence of pabB gene in the complete genome of H. intermedia PBC is consistent with its p-aminobenzoic acid (pABA) auxotrophy but surprisingly comparative genomics analysis of 14 Hydrogenophaga genomes indicate that pABA auxotrophy is not an uncommon feature among members of this genus. Of even more interest, several Hydrogenophaga strains do not possess the genomic potential for hydrogen oxidation, calling for a revision to the taxonomic description of Hydrogenophaga as "hydrogen eating bacteria."
Genes involved in the 4-aminobenzenesulfonate (4-ABS) degradation pathway of Hydrogenophaga sp. PBC were identified using transposon mutagenesis. The screening of 10,000 mutants for incomplete 4-ABS biotransformation identified four mutants with single transposon insertion. Genes with insertions that impaired the ability to utilize 4-ABS for growth included (1) 4-sulfocatechol 1,2-dioxygenase β-subunit (pcaH2) and 3-sulfomuconate cycloisomerase involved in the modified β-ketoadipate pathway; (2) 4-aminobenzenesulfonate 3,4-dioxygenase component (sadA) involved in aromatic ring hydroxylation; and (3) transposase gene homolog with a putative cis-diol dehydrogenase gene located downstream. The pcaH2 mutant strain accumulated brown metabolite during growth on 4-ABS which was identified as 4-sulfocatechol through thin layer chromatography and HPLC analyses. Supplementation of wild-type sadA gene in trans restored the 4-ABS degradation ability of the sadA mutant, thus supporting the annotation of its disrupted gene.
Glutathione S-transferases (GSTs) are a family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide. The enzyme also displayed dehalogenation function against dichloroacetate, permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.
A glutathione S-transferase (GST) with a potential dehalogenation function against various organochlorine substrates was identified from a polychlorobiphenyl (PCB)-degrading organism, Acidovorax sp. KKS102. A homolog of the gene BphK (biphenyl upper pathway K), named BphK-KKS, was cloned, purified and biochemically characterized. Bioinformatic analysis indicated several conserved amino acids that participated in the catalytic activity of the enzyme, and site-directed mutagenesis of these conserved amino acids revealed their importance in the enzyme's catalytic activity. The wild-type and mutant (C10F, K107T and A180P) recombinant proteins displayed wider substrate specificity. The wild-type recombinant GST reacted towards 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid, hydrogen peroxide and cumene hydroperoxide. The mutated recombinant proteins, however, showed significant variation in specific activities towards the substrates. A combination of a molecular docking study and a chloride ion detection assay showed potential interaction with and a dechlorination function against 2-, 3- and 4-chlorobenzoates (metabolites generated during PCB biodegradation) in addition to some organochlorine pesticides (dichlorodiphenyltrichloroethane, endosulfan and permethrin). It was demonstrated that the behavior of the dechlorinating activities varied among the wild-type and mutant recombinant proteins. Kinetic studies (using CDNB and glutathione) showed that the kinetic parameters Km, Vmax, Kcat and Km/Kcat were all affected by the mutations. While C10F and A180P mutants displayed an increase in GST activity and the dechlorination function of the enzyme, the K107T mutant displayed variable results, suggesting a functional role of Lys107 in determining substrate specificity of the enzyme. These results demonstrated that the enzyme should be valuable in the bioremediation of metabolites generated during PCB biodegradation.