Strain WBC-3 Research Articles

Protein 1619 of Pseudomonas putida WBC-3 participates in para-nitrophenol degradation by converting p-benzoquinone to hydroquinone

It is necessary to develop appropriate approaches to eliminate para-nitrophenol (PNP) in our environment, because the pollutant is highly toxic and also able to persist in the environment. Previously, Pseudomonas sp. strain WBC-3 isolated from polluted soil was found to be able to use PNP as the sole carbon and nitrogen source, but not very efficiently. In this study, WBC-3 was shown to belong to Pseudomonas putida through de novo genome sequencing. To enhance its efficiency of PNP utilization, a mutant strain (PM1-33) with a significantly increased PNP degradation rate was obtained. Although no increase in the expression levels of known PNP catabolizing genes/proteins were observed between WBC-3 and PM1-33, the expression level of protein 1619 significantly increased in PM1-33. Deleting GM1619 in WBC-3 and PM1-33 caused decreased PNP degradation rates in both strains and eliminated the difference in PNP degradation between the two strains. Functional prediction using AlphaFold2 showed that protein1619 might bind to p-benzoquinone (BQ). Consequently, protein 1619 was biochemically characterized, confirming its ability to convert BQ into hydroquinone (HQ). Thus, a new protein involved in PNP degradation was identified, thereby adding new knowledge to bacterial PNP degradation pathways.

Development of a novel promoter engineering-based strategy for creating an efficient para-nitrophenol-mineralizing bacterium

A toxic and persistent pollutant para-nitrophenol (PNP) enters into the environment through improper industrial waste treatment and agricultural usage of chemical pesticides, leading to a potential risk to humans. Although a variety of PNP-degrading bacteria have been isolated, their application in bioremediation has been precluded due to unknown biosafety, poor PNP-mineralizing capacity, and lack of genome editing tools. In this study, a novel promoter engineering-based strategy is developed for creating efficient PNP-mineralizing bacteria. Initially, a complete PNP biodegradation pathway from Pseudomonas sp. strain WBC-3 was introduced into the genome of a biosafety and soil-dwelling bacterium Pseudomonas putida KT2440. Subsequently, five strong promoters were identified from P. putida KT2440 by transcriptome analysis and strength characterization, and each of the five promoters was independently inserted into upstream of the pnp operon in the KT2440 genome. Consequently, a P8 promoter-substituted mutant strain showed the highest PNP degradation rate and strong tolerance against high concentrations of PNP. Furthermore, when using P8 promoter to regulate the transcription of all PNP degradation genes pnpABCDEF, the complete and efficient PNP mineralization was demonstrated by stable isotope 13C-labeled PNP transformation assay. Additionally, the finally constructed KTU-P8pnp can be monitored using integrated GFP on chromosome. This strategy of a combination of pathway construction and promoter engineering should open new avenues for creating efficient degraders for bioremediation.

Single point mutation in the transcriptional regulator PnpR renders Pseudomonas sp. strain WBC-3 capable of utilizing 2-chloro-4-nitrophenol

Pseudomonas sp. strain WBC-3 mineralizes para-nitrophenol (PNP) as a growth substrate. The genes responsible for PNP degradation constitute 3 operons: pnpA, pnpB, and pnpCDEFG. As a chlorinated PNP, 2-chloro-4-nitrophenol (2C4NP) serves as a substrate for the PNP 4-monooxygenase PnpA; however, strain WBC-3 is unable to utilize 2C4NP. The LysR-type transcriptional regulator (LTTR) PnpR activates the expression of the 3 catabolic operons in response to PNP. Furthermore, an additional LTTR PnpM responds to 2C4NP other than PNP to initiate the expression of pnpCDEFG operon. Using a long-term directed evolution selection, spontaneous mutants of strain WBC-3 that grew on 2C4NP were obtained. The 2C4NP+ strains had single mutation in codon 6 of the gene encoding PnpR, with substitution of cysteine for serine. The variant PnpRS6C drove the PNP-independent expression of pnpA and pnpB operons in an almost constitutive manner but allowed higher levels of induction upon addition of inducers. However, expression of pnpCDEFG operon was still PNP-dependent together with active PnpRS6C. When the pnpRS6C allele was introduced into a pnpR-deleted WBC-3 mutant, the corresponding strain acquired the ability to grow on 2C4NP but was still able to utilize PNP for growth.

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.

The crystal structures of native hydroquinone 1,2-dioxygenase from Sphingomonas sp. TTNP3 and of substrate and inhibitor complexes

The crystal structure of hydroquinone 1,2-dioxygenase, a Fe(II) ring cleaving dioxygenase from Sphingomonas sp. strain TTNP3, which oxidizes a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes, has been solved by Molecular Replacement, using the coordinates of PnpCD from Pseudomonas sp. strain WBC-3. The enzyme is a heterotetramer, constituted of two subunits α and two β of 19 and 38kDa, respectively. Both the two subunits fold as a cupin, but that of the small α subunit lacks a competent metal binding pocket. Two tetramers are present in the asymmetric unit. Each of the four β subunits in the asymmetric unit binds one Fe(II) ion. The iron ion in each β subunit is coordinated to three protein residues, His258, Glu264, and His305 and a water molecule. The crystal structures of the complexes with the substrate methylhydroquinone, obtained under anaerobic conditions, and with the inhibitors 4-hydroxybenzoate and 4-nitrophenol were also solved.The structures of the native enzyme and of the complexes present significant differences in the active site region compared to PnpCD, the other hydroquinone 1,2-dioxygenase of known structure, and in particular they show a different coordination at the metal center.

Simultaneous biodegradation of three mononitrophenol isomers by a tailor-made microbial consortium immobilized in sequential batch reactors.

Nitroaromatic compounds are readily spread in the environment and pose great potential toxicity concerns. Here, we report the simultaneous degradation of three isomers of mononitrophenol in a single system by employing a consortium of three bacteria, both in flasks and lab-scale sequential batch reactors. The results demonstrate that simultaneous biodegradation of three mononitrophenol isomers can be achieved by a tailor-made microbial consortium immobilized in sequential batch reactors, providing a pilot study for a novel approach for the bioremediation of mixed pollutants, especially isomers present in wastewater.

Effects of bioaugmentation in para-nitrophenol-contaminated soil on the abundance and community structure of ammonia-oxidizing bacteria and archaea.

Pseudomonas sp. strain WBC-3 mineralizes the priority pollutant para-nitrophenol (PNP) and releases nitrite (NO2 (-)), which is probably involved in the nitrification. In this study, the rate of PNP removal in soil bioaugmented with strain WBC-3 was more accelerated with more NO2 (-) accumulation than in uninoculated soils. Strain WBC-3 survived well and remained stable throughout the entire period. Real-time polymerase chain reaction (real-time PCR) indicated a higher abundance of ammonia-oxidizing bacteria (AOB) than ammonia-oxidizing archaea (AOA), suggesting that AOB played a greater role in nitrification in the original sampled soil. Real-time PCR and multivariate analysis based on the denaturing gradient gel electrophoresis showed that PNP contamination did not significantly alter the abundance and community structure of ammonia oxidizers except for inhibiting the AOB abundance. Bioaugmentation of PNP-contaminated soil showed a significant effect on AOB populations and community structure as well as AOA populations. In addition, ammonium (NH4 (+)) variation was found to be the primary factor affecting the AOB community structure, as determined by the correlation between the community structures of ammonia oxidizers and environmental factors. It is here proposed that the balance between archaeal and bacterial ammonia oxidation could be influenced significantly by the variation in NH4 (+) levels as caused by bioaugmentation of contaminated soil by a pollutant containing nitrogen.

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.

A unique fluorescence response of hexaphenylsilole to methyl parathion hydrolase: a new signal generating system for the enzyme label.

Methyl parathion hydrolase (MPH), an enzyme that catalyses the turnover of methyl parathion (MP) to p-nitrophenol (pNP), can be utilized as an enzyme label. In this paper, a unique fluorescence response of 1,1-bis[4-(diethylaminomethyl)phenyl]-2,3,4,5-tetraphenylsilole (A2HPS) to MPH whose gene was obtained from Pseudomonas sp. strain WBC-3 is described. In the absence of MP, A2HPS could only give a small fluorescence response to the enzyme (I/I0 = 1.1). The detection must be performed under low pH conditions, and the influence of BSA and hemoglobin (Hb) was high; upon addition of the enzyme's substrate, 1 × 10-5μg mL-1 or 2.85 × 10-13 M of the MPH could be reported by A2HPS with a higher I/I0 of 1.7. The detection limit was 105 times more sensitive than that given by a spectrophotometric method. In addition, the assay could be performed at a near neutral pH which was more biocompatible, and little influence was observed from BSA and Hb. The light-on response to the MPH was due to the different quenching effect of the MP and pNP on A2HPS and the improvement in the detection selectivity was due to combining the enzyme reaction with the detection. The findings of this work suggested that A2HPS and MP could form a new reporter system for the MPH enzyme label.

Bioaugmentation of a methyl parathion contaminated soil with Pseudomonas sp. strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes methyl parathion (MP) and para-nitrophenol as the sole source of carbon, nitrogen and energy. In this study, strain WBC-3 was inoculated into lab-scale MP-contaminated soil for bioaugmentation. Accelerated removal of MP was achieved in bioaugmentation treatment compared to non-bioaugmentation treatment, with complete removal of 0.536 mg g−1 dry soil in bioaugmentation treatment within 15 days and without accumulation of toxic intermediates. The analysis of denaturing gradient gel electrophoresis and real-time PCR showed that strain WBC-3 existed stably during the entire bioaugmentation period. Simultaneously, redundancy analysis for evaluating the relationships between the environmental factors and microbial community structure indicated that the indigenous bacterial community structure was significantly influenced by strain WBC-3 inoculation (P = 0.002).

Crystal structure of the γ-hydroxymuconic semialdehyde dehydrogenase from Pseudomonas sp. strainWBC-3, a key enzyme involved in para-Nitrophenol degradation

Backgroundpara-Nitrophenol (PNP) is a highly toxic compound with threats to mammalian health. The pnpE-encoded γ-hydroxymuconic semialdehyde dehydrogenase catalyzes the reduction of γ-hydroxymuconic semialdehyde to maleylacetate in Pseudomonas sp. strain WBC-3, playing a key role in the catabolism of PNP to Krebs cycle intermediates. However, the catalyzing mechanism by PnpE has not been well understood.ResultsHere we report the crystal structures of the apo and NAD bound PnpE. In the PnpE-NAD complex structure, NAD is situated in a cleft of PnpE. The cofactor binding site is composed of two pockets. The adenosine and the first ribose group of NAD bind in one pocket and the nicotinamide ring in the other.ConclusionsSix amino acids have interactions with the cofactor. They are C281, E247, Q210, W148, I146 and K172. Highly conserved residues C281 and E247 were identified to be critical for its catalytic activity. In addition, flexible docking studies of the enzyme-substrate system were performed to predict the interactions between PnpE and its substrate γ-hydroxymuconic semialdehyde. Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified. The importance of these residues for catalytic activity was confirmed by the relevant site-directed mutagenesis and their biochemical characterization.

Bioaugmentation with a consortium of bacterial nitrophenol-degraders for remediation of soil contaminated with three nitrophenol isomers

A consortium consisting of para-nitrophenol utilizer Pseudomonas sp. strain WBC-3, meta-nitrophenol utilizer Cupriavidus necator JMP134 and ortho-nitrophenol utilizer Alcaligenes sp. strain NyZ215 was inoculated into soil contaminated with three nitrophenol isomers for bioaugmentation. Accelerated removal of all nitrophenols was achieved in inoculated soils compared to un-inoculated soils, with complete removal of nitrophenols in inoculated soils occurring between 2 and 16 days. Real-time polymerase chain reaction (PCR) targeting nitrophenol-degradation functional genes indicated that the three strains survived and were stable over the course of the incubation period. The abundance of total indigenous bacteria (measured by 16S rRNA gene real-time PCR) was slightly negatively impacted by the nitrophenol contamination. Denaturing gradient gel electrophoresis profiles of total and group-specific indigenous community suggested a dynamic change in species richness occurred during the bioaugmentation process. Furthermore, Pareto–Lorenz curves and Community organization parameters indicated that the bioaugmentation process had little impact on species evenness within the microbial community.

Para-Nitrophenol 4-monooxygenase and hydroxyquinol 1,2-dioxygenase catalyze sequential transformation of 4-nitrocatechol in Pseudomonas sp. strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen and energy. PnpA (PNP 4-monooxygenase) and PnpB (para-benzoquinone reductase) were shown to be involved in the initial steps of PNP catabolism via hydroquinone. We demonstrated here that PnpA also catalyzed monooxygenation of 4-nitrocatechol (4-NC) to hydroxyquinol, probably via hydroxyquinone. It was the first time that a single-component PNP monooxygenase has been shown to catalyze this conversion. PnpG encoded by a gene located in the PNP degradation cluster was purified as a His-tagged protein and identified as a hydroxyquinol dioxygenase catalyzing a ring-cleavage reaction of hydroxyquinol. Although all the genes necessary for 4-NC metabolism seemed to be present in the PNP degradation cluster in strain WBC-3, it was unable to grow on 4-NC as a sole source of carbon, nitrogen and energy. This was apparently due to the substrate's inability to trigger the expression of genes involved in degradation. Nevertheless, strain WBC-3 could completely degrade both PNP and 4-NC when PNP was used as the inducer, demonstrating its potential in bioremediation of the environment polluted by both 4-NC and PNP.

Construction of an engineered strain free of antibiotic resistance gene markers for simultaneous mineralization of methyl parathion and ortho-nitrophenol

Pseudomonas sp. strain NyZ402, a native soil organism that grows on para-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of methyl parathion (MP) and ortho-nitrophenol (ONP) by integrating mph (methyl parathion hydrolase gene) from Pseudomonas sp. strain WBC-3 and onpAB (ONP 2-monooxygenase and ONP o-benzoquinone reductase genes) from Alcaligenes sp. strain NyZ215 into the genome of strain NyZ402. Methyl parathion hydrolase (MPH), ONP 2-monooxygenase (OnpA) and o-benzoquinone reductase (OnpB) were constitutively expressed in the engineered strain NyZ-MO. Strain NyZ-MO was free of exogenous antibiotic resistance gene markers and the introduced genes were genetically stable. Degradation experiments showed that strain NyZ-MO could utilize MP or ONP as the sole carbon and energy source, and mineralize 0.1 mM MP-0.1 mM ONP simultaneously. This method may serve as a useful strategy for the construction of engineered strains in the degradation of multiple environmental pollutants.

Characterization of a para-nitrophenol catabolic cluster in Pseudomonas sp. strain NyZ402 and construction of an engineered strain capable of simultaneously mineralizing both para- and ortho-nitrophenols

Pseudomonas sp. strain NyZ402 was isolated for its ability to grow on para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy, and was shown to degrade PNP via an oxidization pathway. This strain was also capable of growing on hydroquinone or catechol. A 15, 818 bp DNA fragment extending from a 800-bp DNA fragment of hydroxyquinol 1,2-dioxygenase gene (pnpG) was obtained by genome walking. Sequence analysis indicated that the PNP catabolic gene cluster (pnpABCDEFG) in this fragment shared significant similarities with a recently reported gene cluster responsible for PNP degradation from Pseudomonas sp. strain WBC-3. PnpA is PNP 4-monooxygenase converting PNP to hydroquinone via benzoquinone in the presence of NADPH, and genetic analysis indicated that pnpA plays a key role in PNP degradation. pnpA1 present in the upstream of the cluster (absent in the cluster from strain WBC-3) encodes a protein sharing as high as 55% identity with PnpA, but was not involved in PNP degradation by either in vitro or in vivo analyses. Furthermore, an engineered strain capable of growing on PNP and ortho-nitrophenol (ONP) was constructed by introducing onpAB (encoding ONP monooxygenase and ortho-benzoquinone reductase which catalyzed the transformation of ONP to catechol) from Alcaligenes sp. strain NyZ215 into strain NyZ402.

Identification and Characterization of Catabolicpara-Nitrophenol 4-Monooxygenase andpara-Benzoquinone Reductase fromPseudomonassp. Strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N. M. Kamerbeek, A. H. Westphal, S. A. Boeren, D. B. Janssen, M. W. Fraaije, and W. J. van Berkel, J. Bacteriol. 190:5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked.

A transposable class I composite transposon carryingmph(methyl parathion hydrolase) fromPseudomonassp. strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes methyl parathion (O,O-dimethyl O-p-nitrophenol phosphorothioate) or para-nitrophenol as the sole source of carbon, nitrogen and energy. A gene encoding methyl parathion hydrolase (MPH) had been characterized previously and found to be located on a typical class I composite transposon that comprised IS6100 (Tnmph). In this study, the transposability of this transposon was confirmed by transposition assays in two distinct mating-out systems. Tnmph was demonstrated to transpose efficiently in a random manner in Pseudomonas putida PaW340 by Southern blot and in Ralstonia sp. U2 by sequence analysis of the Tnmph insertion sites, both exhibiting MPH activity. The linkage of the mph-like gene with IS6100, together with the transposability of Tnmph, as well as its capability to transpose in other phylogenetically divergent bacterial species, suggest that Tnmph may contribute to the wide distribution of mph-like genes and the adaptation of bacteria to organophosphorus compounds.

Metabolism-independent chemotaxis of Pseudomonas sp. strain WBC-3 toward aromatic compounds

Pseudomonas sp. strain WBC-3 utilized methyl parathion or para-nitrophenol (PNP) as the sole source of carbon, nitrogen, and energy, and methyl parathion hydrolase had been previously characterized. Its chemotactic behaviors to aromatics were investigated. The results indicated that strain WBC-3 was attracted to multiple aromatic compounds, including metabolizable or transformable substrates PNP, 4-nitrocatechol, and hydroquinone. Disruption of PNP catabolic genes had no effect on its chemotactic behaviors with the same substrates, indicating that the chemotactic response in this strain was metabolism-independent. Furthermore, it was shown that strain WBC-3 had a constitutive beta-ketoadipate chemotaxis system that responded to a broad range of aromatic compounds, which was different from the inducible beta-ketoadipate chemotaxis described in other Pseudomonas strains.

Plasmid-borne catabolism of methyl parathion and p-nitrophenol in Pseudomonas sp. strain WBC-3

Pseudomonas sp. strain WBC-3 utilises methyl parathion (MP) or p-nitrophenol (PNP) as the sole source of carbon, nitrogen, and energy. A plasmid designated pZWL0 of ≈70 kb in this strain was found to be responsible for MP and PNP degradation. This was based on the fact that the plasmid-cured strains showed PNP −MP − phenotype and the PNP +MP + phenotype could be conjugally transferred. We have also cloned a 3.4-kb HindIII fragment which exhibited methyl parathion hydrolase activity, which revealed a methyl parathion hydrolase ( mph) gene whose DNA sequence is 99.5% identical to the recently identified mpd gene from Plesiomonas sp. M6 [C. Zhongli, L. Shunpeng, F. Guoping, Isolation of methyl parathion-degrading strain M6 and cloning of the methyl parathion hydrolase gene, Appl. Environ. Microbiol. 67 (2001) 4922–4925]. The mph gene was functionally expressed in Escherichia coli and the relative activities of the enzyme against different substrates were determined. The sequence alignment and phylogenetic analysis suggested that MPH and MPD evolved independently from other well-studied organophosphate hydrolases and may be originated from class B β-lactamase family. Subsequently obtained a 6.5-kb KpnI and BamHI fragment containing the above HindIII fragment revealed that the mph gene was physically located in a typical transposon.