Novel small multidrug resistance protein Tmt endows the Escherichia coli with triphenylmethane dyes bioremediation capability.

Dye contamination in printing and dyeing wastewater has long been a major concern due to its serious impact on both the environment and human health. In the quest for bioremediation of these hazardous dyes, biological resources such as biodegradation bacteria and enzymes have been investigated in severely polluted environments. In this context, the triphenylmethane transporter gene (tmt) was identified in six distinct clones from a metagenomic library of the printing and dyeing wastewater treatment system. Escherichia coli expressing tmt revealed 98.1% decolorization efficiency of triphenylmethane dye malachite green within 24 h under shaking culture condition. The tolerance to malachite green was improved over eightfold in the Tmt strain compared of the none-Tmt expressed strain. Similarly, the tolerance of Tmt strain to other triphenylmethane dyes like crystal violet and brilliant green, was improved by at least fourfold. Site-directed mutations, including A75G, A75S and V100G, were found to reinforce the tolerance of malachite green, and double mutations of these even further improve the tolerance. Therefore, the tmt has been demonstrated to be a specific efflux pump for triphenylmethane dyes, particularly the malachite green. By actively pumping out toxic triphenylmethane dyes, it significantly extends the cells tolerance in a triphenylmethane dye-rich environment, which may provide a promising strategy for bioremediation of triphenylmethane dye pollutants in the environments.

Pigmentiphaga kullae CHJ604 improved the growth of tobacco by degrading allelochemicals and xenobiotics in continuous cropping obstacles.

Plant autotoxicity is considered to be one of the important causes of continuous cropping obstacles in modern agriculture, which accumulates a lot of allelochemicals and xenobiotics and is difficult to solve effectively. To overcome tobacco continuous obstacles, a strain Pigmentiphaga kullae CHJ604 isolated from the environment can effectively degrade these compounds in this study. CHJ604 strain can degrade 11 types of autotoxicity allelochemicals and xenobiotics (1646.22 µg/kg) accumulated in the soil of ten-years continuous cropping of tobacco. The 11 allelochemicals and xenobiotics significantly reduced Germination Percentage (GP), Germination Index (GI), and Mean Germination Time (MGT) of tobacco seeds, and inhibited the development of leaves, stems, and roots. These negative disturbances can be eliminated by CHJ604 strain. The degradation pathways of 11 allelochemicals and xenobiotics were obtained by whole genome sequence and annotation of CHJ604 strain. The heterologous expression of a terephthalate 1,2-dioxygenase can catalyze 4-hydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 4-hydroxybenzaldehyde, and 4-hydroxy-3-methoxy-benzaldehyde, respectively. The phthalate 4,5-dioxygenase can catalyze phthalic acid, diisobutyl phthalate, and dibutyl phthalate. These two enzymes are conducive to the simultaneous degradation of multiple allelochemicals and xenobiotics by strain CHJ604. This study provides new insights into the biodegradation of autotoxicity allelochemicals and xenobiotics as it is the first to describe a degrading bacterium of 11 types of allelochemicals and xenobiotics and their great potential in improving tobacco continuous obstacles.

Dichlorodiphenyltrichloroethane inhibits soil ammonia oxidation by altering ammonia-oxidizing archaeal and bacterial communities.

Dichlorodiphenyltrichloroethane (DDT), a persistent organic pollutant, has known effects on natural microbes. However, its effects on soil ammonia-oxidizing microbes, significant contributors to soil ammoxidation, remain unexplored. To address this, we conducted a 30-day microcosm experiment to systematically study the effects of DDT contamination on soil ammonia oxidation and the communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Our findings revealed that DDT inhibited soil ammonia oxidation in the early stage (0-6 days), but it gradually recovered after 16 days. The copy numbers of amoA gene of AOA decreased in all DDT-treated groups from 2 to 10 days, while that of AOB decreased from 2 to 6 days but increased from 6 to 10 days. DDT influenced the diversity and community composition of AOA but had no significant effect on AOB. Further, the dominant AOA communities comprised uncultured_ammonia-oxidizing_crenarchaeote and Nitrososphaera sp. JG1: while the abundance of the latter significantly and negatively correlated with NH 4+-N (P ≤ 0.001), DDT (0.001 < P ≤ 0.01), and DDD (0.01 < P ≤ 0.05) and positively correlated with NO3--N (P ≤ 0.001), that of the former significantly and positively correlated with DDT (P ≤ 0.001), DDD (P ≤ 0.001), and NH 4+-N (0.01 < P ≤ 0.05) and negatively correlated with NO3--N (P ≤ 0.001). Among AOB, the dominant group was the unclassified Nitrosomonadales in Proteobacteria, which showed significant negative correlation with NH 4+-N (0.01 < P ≤ 0.05) and significant positive correlation with NO3--N (0.001 < P ≤ 0.01). Notably, among AOB, only Nitrosospira sp. III7 exhibited significant negative correlations with DDE (0.001 < P ≤ 0.01), DDT (0.01 < P ≤ 0.05), and DDD (0.01 < P ≤ 0.05). These results indicate that DDT and its metabolites affect soil AOA and AOB, consequently affecting soil ammonia oxidation.

The Complete Genome Sequence of a Gossypol-Degrading Bacterial Strain, Raoultella sp. YL01.

Cottonseed meal is an important source of plant protein for the meal fodder materials. But its usage in animal breeding industry is limited by a type of toxic phenol, gossypol, that has toxic effects on animal health. Microbial degradation is a promising way to lower down gossypol in cottonseed meal. However, the molecular mechanisms of bio-degradation of gossypol is still unclear. In this study we isolated a gossypol-degrading bacterial strain, YL01, and sequenced its complete genome via Oxford Nanopore sequencing method. There is a chromosome (5,737,005 bp) and a plasmid (136,446 bp) in YL01. 5489 protein coding genes in total were functionally annotated. 16S rRNA analysis showed that YL01 taxonomically belongs to the genus of Raoultella. YL01 is the first published complete genome sequence of microbes capable of gossypol degradation. Gene function annotation showed that 126 protein coding genes may involve in gossypol catabolism. Sequence similarity analysis showed that, as the only gossypol-degrading strain in the genus of Raoultella, YL01 uniquely holds 260 genes that are not possessed by other Raoultella strains. Our work gives a preliminary list for genes responsible for gossypol degradation but further investigations are needed to completely disclose this molecular processes.

The XylR/NtrC-type regulator CbsR positively regulates upstream pathway of chlorobenzene degradation in Pandoraea pnomenusa.

AIMS: Pandoraea pnomenusa MCB032 completely degrades chlorobenzene, whose metabolic pathway is encoded by cbs and clc gene clusters. The putative regulatory factors ClcR and CbsR are predicted to regulate the cbs and clc gene clusters. This research aims to understand the function of ClcR and CbsR. METHODS AND RESULTS: RT-PCR analyses demonstrated that the cbsFAaAbAcAdB operon that encodes catabolic pathways for the degradation of chlorobenzene to chlorocatechol is located on an operon. Moreover, the clcABCDE operon is involved in the 3-chlorocatechol pathway. Gene knockout and transcriptional analysis showed that the transcription of the cbsFAaAbAcAdB operon is positively regulated by CbsR, whereas the clcABCDE operon is activated by ClcR. Primer extension analysis was used to locate the transcription start sites of the cbsFAaAbAcAdB and cbsR operons. Electrophoretic mobility shift assay analyses showed that CbsR is bound to the sites in the promoter regions of cbsFAaAbAcAdB and cbsR operons. CONCLUSION: The XylR/NtrC-type regulator CbsR positively regulates the transcription of the cbsFAaAbAcAdB operon encoding the upstream pathway of chlorobenzene catabolism, while the LysR-type regulator ClcR activates the clcABCDE operon encoding the downstream pathway.

Microbial detoxification of 3,5-xylenol via a novel process with sequential methyl oxidation by Rhodococcus sp. CHJ602.

The compound 3,5-xylenol is an essential precursor used in pesticides and industrial intermediate in the disinfectants and preservatives industry. Its widespread application makes it an important source of pollution. Microbial bioremediation is more environmentally friendly than the physicochemical treatment process for removing alkylphenols from a polluted environment. However, the 3,5-xylenol-degrading bacteria is unavailable, and its degradation mechanism remains unclear. Here, a 3,5-xylenol-metabolizing bacterial strain, designated Rhodococcus sp. CHJ602, was isolated using 3,5-xylenol as the sole source of carbon and energy from a wastewater treatment factory. Results showed that strain CHJ602 maintained a high 3,5-xylenol-degrading performance under the conditions of 30.15 °C and pH 7.37. The pathway involved in 3,5-xylenol degradation by strain CHJ602 must be induced by 3,5-xylenol. Based on the identification of intermediate metabolites and enzyme activities, this bacterium could oxidize 3,5-xylenol by a novel metabolic pathway. One methyl oxidation converted 3,5-xylenol to 3-hydroxymethyl-5-methylphenol, 3-hydroxy-5-methyl benzaldehyde, and 3-hydroxy-5-methylbenzoate. After that, another methyl oxidation is converted to 5-hydroxyisophthalicate, which is metabolized by the protocatechuate pathway. It is catalyzed by a series of enzymes in strain CHJ602. In addition, toxicity bioassay result indicates that 3,5-xylenol is toxic to zebrafish and Rhodococcus sp. CHJ602 could eliminate 3,5-xylenol in water to protect zebrafish from its toxicity. The results provide insights into the bioremediation of wastewater contaminated 3,5-xylenol.

The auto-inhibition mechanism of transcription factor Ets-1 induced by phosphorylation on the intrinsically disordered region.

As the most abundant post-translation modifications (PTMs), the phosphorylation usually occurred on the intrinsically disordered regions (IDRs). The regulation on the structures and interactions of IDRs induced by phosphorylation is critical to the function performing. The eukaryotic transcription factor 1 (Ets-1) is a member of transcription factor family, which participates in many important biological processes. The DNA-binding ability of Ets-1 is auto-inhibited by a disordered serine-rich region (SRR) on the Ets-1. The inhibition ability of SRR is greatly enhanced by the phosphorylation of the serine on the SRR. Nevertheless, the molecular mechanisms of the phosphorylation regulation on the structure and activity of Ets-1 are still unclear and under debates. By using both of the molecular simulations and biochemical experiments, we studied the molecule mechanism of phosphorylation regulation on the auto-inhibition of the Ets-1. The reasons of stabilization of Ets-1 core by phosphorylation on SRR region were elucidated. More important, the free energy landscapes (FEL) show that both of the steric hindrance and allosteric regulation are responsible for the DNA-binding inhibitory induced by phosphorylation, but the steric effects contribute greater than the allosteric regulation. The phosphorylation not only enhances the electrostatic interactions to facilitate the steric impedance, but also promotes the formation of hydrophobic residue clusters, which provide major driven force for the allosteric regulation. The structural basis of auto-inhibition of Ets-1 induced by the phosphorylation revealed in this study would great help the developing of inhibitor for the cancer therapy.

The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli.

Escherichia coli has been developed as the most common host for recombinant protein expression. Unfortunately, there are still some proteins that are resistant to high levels of heterologous soluble expression in E. coli. Protein and peptide fusion tags are one of the most important methods for increasing target protein expression and seem to influence the expression efficiency and solubility as well. In this study, we identify a short 15-residue enhancing solubility peptide, the PCDS (protocatechuate 3,4-dioxygenase solubility) tag, which enhances heterologous protein expression in E. coli. This PCDS tag is a 45-bp long sequence encoding a peptide tag involved in the soluble expression of protocatechuate 3,4-dioxygenase, encoded by the pcaHG98 genes of Pseudomonas putida NCIMB 9866. The 45-bp sequence was also beneficial for pcaHG98 gene amplification. This tag was shown to be necessary for the heterologous soluble expression of PcaHG98 in E. coli. Purified His6-PcaHG98e04-PCDS exhibited an activity of 205.63±14.23U/mg against protocatechuate as a substrate, and this activity was not affected by a PCDS tag. This PCDS tag has been fused to the mammalian yellow fluorescent protein (YFP) to construct YFP-PCDS without its termination codons and YFPt-PCDS with. The total protein expressions of YFP-PCDS and YFPt-PCDS were significantly amplified up to 1.6-fold and 2-fold, respectively, compared to YFP alone. Accordingly, His6-YFP-PCDS and His6-YFPt-PCDS had 1.6-fold and 3-fold higher soluble protein yields, respectively, than His6-YFP expressed under the same conditions. His6-YFP, His6-YFP-PCDS, and His6-YFPt-PCDS also showed consistent fluorescence emission spectra, with a peak at 530nm over a scanning range from 400 to 700nm. These results indicated that the use of the PCDS tag is an effective way to improve heterologous protein expression in E. coli.

Molecular cloning and characterization of a novel xylanase from Microbacterium imperiale YD-01.

Xylaneses are very common xylanolytic enzymes, which are widely used in food, papermaking, and other industries. In this study, a xylanase-encoding gene xyn1923, which encodes a protein of 1352 amino acids, was identified through the whole genome analysis of Microbacterium imperiale YD-01. Bioinformatics analysis showed that Xyn1923 only had maximum similarity of 37% with the reported xylanase from Alkalihalobacillus halodurans C-125, indicating that Xyn1923 was a novel xylanase. The enzymatic properties of Xyn1923 were systematically analyzed after purification. The results showed that the specific activity of the enzyme was 10.582 ± 0.413 U/mg, while the optimum pH and temperature of the enzyme were 7.0 and 70°C, respectively. The enzyme is stable in the pH range of 6.0-9.0, and the enzyme activity could maintain more than 85% of the original activity after 16 hr incubation at pH 9.0. The enzyme activity is relatively stable in the range of 30-60°C, and its enzyme activity could maintain more than 89% of the original activity after treatment at 60°C for 30 min. Low concentrations (≤1 mM) of Co2+ , Ba2+ , Fe2+ , and Fe3+ metal ions exerted a stimulatory effect on the activity of Xyn1923. And in contrast, high concentrations (≥2 mM) of the above metal ions inhibit the activity of Xyn1923. Mg2+ , Ag+ , Cu2+ , Ca2+ , Mn2+ , and Pb2+ ions showed a negative effect on the activity of Xyn1923. Enzyme kinetic studies showed that Km and Vmax values for xylan were 7.842 ± 0.538 mg/ml and 15.208 ± 0.822 U/mg, respectively. Xyn1923 was found to be a weakly alkaline thermophilic xylanase through an enzymatic property analysis. PRACTICAL APPLICATIONS: Xylanases are widely used in food and feed, biofuels, papermaking, and other industries. However, their use is limited by poor performance under the conditions of pH and temperature. Therefore, the discovery of xylanases with the capability of working efficiently at alkaline pH and high temperature is the priority for its industrial applications. In this study, a novel xylanase-encoding gene xyn1923 from Microbacterium imperiale YD-01 was cloned and heterologously expressed in Escherichia coli. Enzymatic properties of this novel xylanase were investigated, indicating that the robust thermal stability and alkali resistance of Xyn1923 make it a potential candidate for the food and paper industries.

CpxR regulates the Rcs phosphorelay system in controlling the Ysc-Yop type III secretion system in Yersinia pseudotuberculosis.

The CpxRA two-component regulatory system and the Rcs phosphorelay system are both employed by the Enterobacteriaceae family to preserve bacterial envelope integrity and function when growing under stress. Although both systems regulate several overlapping physiological processes, evidence demonstrating a molecular connection between Cpx and Rcs signalling outputs is scarce. Here, we show that CpxR negatively regulates the transcription of the rcsB gene in the Rcs phosphorelay system in Yersinia pseudotuberculosis. Interestingly, transcription of rcsB is under the control of three promoters, which were all repressed by CpxR. Critically, synthetic activation of Cpx signalling through mislocalization of the NlpE lipoprotein to the inner membrane resulted in an active form of CpxR that repressed activity of rcsB promoters. On the other hand, a site-directed mutation of the phosphorylation site at residue 51 in CpxR generated an inactive non-phosphorylated variant that was unable to regulate output from these rcsB promoters. Importantly, CpxR-mediated inhibition of rcsB transcription in turn restricted activation of the Ysc-Yop type III secretion system (T3SS). Moreover, active CpxR blocks zinc-mediated activation of Rcs signalling and the subsequent activation of lcrF transcription. Our results demonstrate a novel regulatory cascade linking CpxR-RcsB-LcrF to control production of the Ysc-Yop T3SS.

The Complete Genome Sequence of a Bacterial Strain with High Alkalic Xylanase Activity Isolated from the Sludge Near a Papermill.

Many organisms secrete xylanase, an import group of proteins hydrolyzing xylan, and thus are able to use xylan as their carbon source. In this study, we sequenced the whole genome of a bacterial strain, YD01, which was isolated from the sludge near the sewage discharge outlet of a papermill and showed high alkalic xylanase activity. Its genome consists of a chromosome and two plasmids. Six rRNA genes, 46 tRNA genes, 3136 CDSs as well as 955 repetitive sequences were predicted. 3046 CDSs were functionally annotated. Phylogenetic analysis on 16S rRNA shows that YD01 is a new species in Microbacterium genus and is taxonomically close to M. jejuense THG-C31T and M. kyungheense THG-C26T. A comparative study on phylogenetic trees of 16S rRNA and xylanase genes suggests that xylanase genes in YD01 may originate from horizontal gene transfer instead of ancestral gene duplication.

Evaluation of carboxymethylpullulan-AlCl3 as a coagulant for water treatment: A case study with kaolin.

A reduction in the use of aluminum (Al)-based flocculants in the treatment of drinking water is considered essential for human health reasons. In this study, a novel composite flocculant, made of carboxymethylpullulan-AlCl3 , is evaluated in a lab-scale, jar test system for the flocculation of kaolin. The results showed that the coagulation efficiency of carboxymethylpullulan-AlCl3 was more effective in reducing turbidity than the solo use of carboxymethylpullulan or AlCl3 . The optimum treatment conditions assessed by a response surface methodology were obtained at pH 6.50, 13.03 mg/L carboxymethylpullulan, and 94.87 mg/L AlCl3 . Zeta potential measurements and photometric dispersion analysis demonstrated that AlCl3 had a more significant influence on charge neutralization than carboxymethylpullulan, whilst carboxymethylpullulan facilitated absorption and the development of particle bridges. Thus, the composite flocculant possessed both advantages that enhanced flocculation, and decreased the dosage of AlCl3 , thereby reducing the potential for secondary environment pollution. When 90 mg/L carboxymethylpullulan-AlCl3 was added to the model kaolin suspension characterized by a turbidity of 50 nephelometric turbidity units, the zeta potential and the maximum flocculating activity were determined as -2.28 mV and 98.0%, respectively. The results provide insight into the development of an environment-friendly composite flocculant prepared from water-dissolved polysaccharide and inorganic flocculants. PRACTITIONER POINTS: A novel composite flocculant CMP-AlCl3 was achieved by combining CMP and AlCl3 for water treatment. The coagulation efficiency of CMP-AlCl3 was more effective in reducing turbidity than the solo use of CMP or AlCl3 . The flocculation efficiency and mechanism were investigated by Zeta potential analysis, surface morphology, electron microscopy, and coagulation.

A low-voltage pulse electrolysis method for the degradation of anthraquinone and azo dyes in chloride medium by anodic oxidation on Ti/IrO2 -RuO2 -SnO2 electrodes.

Wastewater produced by the textile industry containing azo dyes and anthraquinone dyes is significant source of pollution to the environment and is toxic for aquatic life. To overcome the high-energy cost of traditional electrochemical oxidation, a custom-built power supply device for the degradation of anthraquinone and azo dyes by low voltage of 15.0-20.0 V pulsed discharge was investigated. Titanium coated with mixed oxide (Ti/IrO2 -RuO2 -SnO2 ) plates and pure titanium plates were used as the anode and cathode, respectively, for the generation of chlorine in the dye solution. For the anthraquinone dye Reactive Blue 19, 60.0% of the chemical oxygen demand (COD) and 22.0% of the total organic carbon (TOC) were removed using this system. A comparison of the direct current electrolysis and pulsed discharge revealed that using the pulsed discharge method reduced the energy cost by 68.6%. UV-visible, LC-MS, and GC-MS were used to identify the intermediate compounds formed during the degradation of Reactive Blue 19. The results indicate that in the process of oxidation by chlorine/hypochlorite, the chromophore group was first oxidized to -NH2 , followed by decolorization via chlorination of the aromatic rings. The results confirm that low-voltage pulse electrolysis can be used for the degradation of industrial dyes in waste effluents. PRACTITIONER POINTS: Low-voltage pulse electrolysis can be used for the degradation of industrial dyes and/or dyes in waste effluents. For anionic dye Reactive Blue 19, 60.0% of COD and 22.0% of TOC were removed using low-voltage (20.0 V) pulse electrolysis. The pulsed discharge method reduced the energy cost of this degradation process by 68.6% compared with direct current electrolysis. The intermediate compounds formed during the degradation of Reactive Blue 19 were confirmed by UV-visible spectroscopy, LC-MS, and GC-MS.

Complete Genome Sequence of a Chlorobenzene Degrader, Pandoraea pnomenusa MCB032.

Chlorobenzenes are ubiquitously distributed, highly persistent, and toxic environmental contaminants. Pandoraea pnomenusa MCB032 was isolated as a new dominant chlorobenzene-utilizing strain from a functionally stable bioreactor during the treatment of chlorobenzenes when strain Burkholderia sp. JS150 disappeared. In study, we report the complete genome sequence of strain MCB032 which consists of a circular chromosome and three plasmids, which are ~ 6 Mb in length with 5450 open reading frames-12 encoding rRNAs and 77 encoding tRNAs. We further identified 17 putative genes encoding the enzymes involved in the methyl-accepting chemotaxis proteins in sensing chemical gradients during chemotaxis. The annotated complete genome sequence of this strain will provide genetic insights into the degradation of chlorinated aromatic compounds. The information will empower the elucidation of chlorobenzene affinity hierarchy and species succession in the bioreactor.

PnpM, a LysR-Type Transcriptional Regulator Activates the Hydroquinone Pathway in para-Nitrophenol Degradation in Pseudomonas sp. Strain WBC-3.

A LysR-type transcriptional regulator (LTTR), PnpR, has previously been shown to activate the transcription of operons pnpA, pnpB, and pnpCDEFG for para-nitrophenol (PNP) degradation in Pseudomonas sp. strain WBC-3. Further preliminary evidence suggested the possible presence of an LTTR additional binding site in the promoter region of pnpCDEFG. In this study, an additional LTTR PnpM, which shows 44% homology to PnpR, was determined to activate the expression of pnpCDEFG. Interestingly, a pnpM-deleted WBC-3 strain was unable to grow on PNP but accumulating hydroquinone (HQ), which is the catabolic product from PNP degradation by PnpAB and the substrate for PnpCD. Through electrophoretic mobility shift assays (EMSAs) and promoter activity detection, only PnpR was involved in the activation of pnpA and pnpB, but both PnpR and PnpM were involved in the activation of pnpCDEFG. DNase I footprinting analysis suggested that PnpR and PnpM shared the same DNA-binding regions of 27 bp in the pnpCDEFG promoter. In the presence of PNP, the protection region increased to 39 bp by PnpR and to 38 bp by PnpM. Our data suggested that both PnpR and PnpM were involved in activating pnpCDEFG expression, in which PNP rather than the substrate hydroquinone for PnpCD is the inducer. Thus, during the PNP catabolism in Pseudomonas sp. strain WBC-3, pnpA and pnpB operons for the initial two reactions were controlled by PnpR, while the third operon (pnpCDEFG) for HQ degradation was activated by PnpM and PnpR. This study builds upon our previous findings and shows that two LTTRs PnpR and PnpM are involved in the transcriptional activation of these three catabolic operons. Specifically, our identification that an LTTR, PnpM, regulates pnpCDEFG expression provides new insights in an intriguing regulation system of PNP catabolism that is controlled by two regulators.

Constitutive Expression of a Nag-Like Dioxygenase Gene through an Internal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1.

UNLABELLED: The gene cluster encoding the 2-chloronitrobenzene (2CNB) catabolism pathway in Pseudomonas stutzeri ZWLR2-1 is a patchwork assembly of a Nag-like dioxygenase (dioxygenase belonging to the naphthalene dioxygenase NagAaAbAcAd family from Ralstonia sp. strain U2) gene cluster and a chlorocatechol catabolism cluster. However, the transcriptional regulator gene usually present in the Nag-like dioxygenase gene cluster is missing, leaving it unclear how this cluster is expressed. The pattern of expression of the 2CNB catabolism cluster was investigated here. The results demonstrate that the expression was constitutive and not induced by its substrate 2CNB or salicylate, the usual inducer of expression in the Nag-like dioxygenase family. Reverse transcription-PCR indicated the presence of at least one transcript containing all the structural genes for 2CNB degradation. Among the three promoters verified in the gene cluster, P1 served as the promoter for the entire catabolism operon, but the internal promoters P2 and P3 also enhanced the transcription of the genes downstream. The P3 promoter, which was not previously defined as a promoter sequence, was the strongest of these three promoters. It drove the expression of cnbAcAd encoding the dioxygenase that catalyzes the initial reaction in the 2CNB catabolism pathway. Bioinformatics and mutation analyses suggested that this P3 promoter evolved through the duplication of an 18-bp fragment and introduction of an extra 132-bp fragment. IMPORTANCE: The release of many synthetic compounds into the environment places selective pressure on bacteria to develop their ability to utilize these chemicals to grow. One of the problems that a bacterium must surmount is to evolve a regulatory device for expression of the corresponding catabolism genes. Considering that 2CNB is a xenobiotic that has existed only since the onset of synthetic chemistry, it may be a good example for studying the molecular mechanisms underlying rapid evolution in regulatory networks for the catabolism of synthetic compounds. The 2CNB utilizer Pseudomonas stutzeri ZWLR2-1 in this study has adapted itself to the new pollutant by evolving the always-inducible Nag-like dioxygenase into a constitutively expressed enzyme, and its expression has escaped the influence of salicylate. This may facilitate an understanding of how bacteria can rapidly adapt to the new synthetic compounds by evolving its expression system for key enzymes involved in the degradation of a xenobiotic.

HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866.

In addition to growing on p-cresol, Pseudomonas putida NCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of the para-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the gene hipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6 was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6 catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg(-1) and showed apparent Km values of 11.40 ± 3.05 µM for 4-hydroxyisophthalate with NADPH and 11.23 ± 2.43 µM with NADH and similar Km values for NADPH and NADH (64.31 ± 13.16 and 72.76 ± 12.06 µM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6 has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated that hipH is essential for 2,4-xylenol catabolism but not for p-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds.

Transcriptional activation of multiple operons involved in para-nitrophenol degradation by Pseudomonas sp. Strain WBC-3.

Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole carbon and energy source. The genes involved in PNP degradation are organized in the following three operons: pnpA, pnpB, and pnpCDEFG. How the expression of the genes is regulated is unknown. In this study, an LysR-type transcriptional regulator (LTTR) is identified to activate the expression of the genes in response to the specific inducer PNP. While the LTTR coding gene pnpR was found to be not physically linked to any of the three catabolic operons, it was shown to be essential for the growth of strain WBC-3 on PNP. Furthermore, PnpR positively regulated its own expression, which is different from the function of classical LTTRs. A regulatory binding site (RBS) with a 17-bp imperfect palindromic sequence (GTT-N11-AAC) was identified in all pnpA, pnpB, pnpC, and pnpR promoters. Through electrophoretic mobility shift assays and mutagenic analyses, this motif was proven to be necessary for PnpR binding. This consensus motif is centered at positions approximately -55 bp relative to the four transcriptional start sites (TSSs). RBS integrity was required for both high-affinity PnpR binding and transcriptional activation of pnpA, pnpB, and pnpR. However, this integrity was essential only for high-affinity PnpR binding to the promoter of pnpCDEFG and not for its activation. Intriguingly, unlike other LTTRs studied, no changes in lengths of the PnpR binding regions of the pnpA and pnpB promoters were observed after the addition of the inducer PNP in DNase I footprinting.

Involvement of the global regulator GlxR in 3-hydroxybenzoate and gentisate utilization by Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrially important producer of amino acids and organic acids, as well as an emerging model system for aromatic assimilation. An IclR-type regulator GenR has been characterized to activate the transcription of genDFM and genKH operons for 3-hydroxybenzoate and gentisate catabolism and represses its own expression. On the other hand, GlxR, a global regulator of the cyclic AMP (cAMP) receptor protein-fumarate nitrate reductase regulator (CRP-FNR) type, was also predicted to be involved in this pathway. In this study, electrophoretic mobility shift assays and footprinting analyses demonstrated that GlxR bound to three sites in the promoter regions of three gen operons. A combination of site-directed mutagenesis of the biding sites, promoter activity assay, and GlxR overexpression demonstrated that GlxR repressed their expression by binding these sites. One GlxR binding site (DFMx) was found to be located -13 to +8 bp upstream of the genDFM promoter, which was involved in negative regulation of genDFM transcription. The GlxR binding site R-KHx01 (located between positions -11 to +5) was upstream of the genKH promoter sequence and involved in negative regulation of its transcription. The binding site R-KHx02, at which GlxR binds to genR promoter to repress its expression, was found within a footprint extending from positions -71 to -91 bp. These results reveal that GlxR represses the transcription of all three gen operons and then contributes to the synchronization of their expression for 3-hydroxybenzoate and gentisate catabolism in collaboration with the specific regulator GenR.

The catabolism of 2,4-xylenol and p-cresol share the enzymes for the oxidation of para-methyl group in Pseudomonas putida NCIMB 9866.

Pseudomonas putida NCIMB 9866 utilizes p-cresol or 2,4-xylenol as a sole carbon and energy source. Enzymes catalyzing the oxidation of the para-methyl group of p-cresol have been studied in detail. However, those responsible for the oxidation of the para-methyl group in 2,4-xylenol catabolism are still not reported. In this study, real-time quantitative PCR analysis indicated pchC- and pchF-encoded p-cresol methylhydroxylase (PCMH) and pchA-encoded p-hydroxybenzaldehyde dehydrogenase (PHBDD) in p-cresol catabolism were also likely involved in the catabolism of 2,4-xylenol. Enzyme activity assays and intermediate identification indicated that the PCMH and PHBDD catalyzed the oxidations of 2,4-xylenol to 4-hydroxy-3-methylbenzaldehyde and 4-hydroxy-3-methylbenzaldehyde to 4-hydroxy-3-methylbenzoic acid, respectively. Furthermore, the PCMH-encoding gene pchF was found to be necessary for the catabolism of 2,4-xylenol, whereas the PHBDD-encoding gene pchA was not essential for the catabolism by gene knockout and complementation. Analyses of the maximum specific growth rate (µ m) and specific activity of the gene-knockout strain to different intermediates revealed the presence of other enzyme(s) with PHBDD activity in strain 9866. However, PHBDD played a major role in the catabolism of 2,4-xylenol in contrast to the other enzyme(s).