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Constitutive Expression of a Nag-Like Dioxygenase Gene Through An

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Constitutive Expression of a Nag-Like Dioxygenase Gene Through An crossmark Constitutive Expression of a Nag-Like Dioxygenase Gene through an Internal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1 Yi-Zhou Gao,a,c Hong Liu,a Hong-Jun Chao,a Ning-Yi Zhoua,b Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Chinaa; State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, Chinab; University of Chinese Academy of Sciences, Beijing, Chinac ABSTRACT The gene cluster encoding the 2-chloronitrobenzene (2CNB) catabolism pathway in Pseudomonas stutzeri ZWLR2-1 is a patch- Downloaded from work 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 usu- ally 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 in- ternal promoters P2 and P3 also enhanced the transcription of the genes downstream. The P3 promoter, which was not previ- http://aem.asm.org/ ously 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 sug- gested that this P3 promoter evolved through the duplication of an 18-bp fragment and introduction of an extra 132-bp frag- ment. 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 expres- on January 6, 2017 by guest sion of the corresponding catabolism genes. Considering that 2CNB is a xenobiotic that has existed only since the onset of syn- thetic chemistry, it may be a good example for studying the molecular mechanisms underlying rapid evolution in regulatory net- works 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. volutionary changes in a regulated gene promoter have been Nag dioxygenase is thought to be the progenitor of nitroarene Eheavily studied, particularly changes in promoter sequences in dioxygenases (collectively called Nag-like dioxygenase) described the deployment of the transcriptional factors (1). Horizontal gene so far (8), including nitrobenzene dioxygenase (9), 2-nitrotoluene transfer can significantly alter the composition of bacterial regu- dioxygenase (10), 3-nitrotoluene dioxygenase (11), and 2,4-dini- lons (2). The promoter sequence in a regulatory system can be trotoluene dioxygenase (12, 13). The synthetic nitro- and chloro- changed by point mutation (3) or insertion of a transposable ele- aromatic compound, 2-chloronitrobenzene (2CNB), has been in ment (4) in order to allow the organism to adapt to environmental the environment only for a short period of time. Pseudomonas change. With the rapid development of the chemical industry, stutzeri ZWLR2-1 utilizes 2CNB as a sole carbon and energy many synthetic compounds have been released into the environ- source (14). In this strain, a 2CNB catabolism cluster was formed ment in large quantities. This places selective pressure on micro- organisms to develop their abilities to utilize these chemicals to grow. In general, a bacterium that has evolved pathways to metab- olize toxic compounds as its sole source of carbon and energy Received 20 January 2016 Accepted 28 March 2016 must surmount three problems: (i) shift the specificities of key Accepted manuscript posted online 1 April 2016 Citation Gao Y-Z, Liu H, Chao H-J, Zhou N-Y. 2016. Constitutive expression of a enzymes to run a complete pathway producing central intermedi- Nag-like dioxygenase gene through an internal promoter in the 2- ate metabolites, (ii) acquire or evolve a regulatory device for ex- chloronitrobenzene catabolism gene cluster of Pseudomonas stutzeri ZWLR2-1. pression of the corresponding catabolism genes, and (iii) avoid the Appl Environ Microbiol 82:3461–3470. doi:10.1128/AEM.00197-16. stress caused by the substrate itself or by its degradation of inter- Editor: R. E. Parales, University of California—Davis mediates (5, 6). Address correspondence to Ning-Yi Zhou, [email protected]. In Ralstonia sp. strain U2, the catabolism of naphthalene is Copyright © 2016, American Society for Microbiology. All Rights Reserved. initiated by naphthalene dioxygenase NagAaAbAcAd (7). This June 2016 Volume 82 Number 12 Applied and Environmental Microbiology aem.asm.org 3461 Gao et al. TABLE 1 Bacterial strains and plasmids in this study Bacterial strain or plasmid Genotype or phenotypea Reference(s) or source Strains Pseudomonas stutzeri ZWLR2-1 2-Chloronitrobenzene utilizer, wild type 8, 14 Pseudomonas putida PaW340 Strr TrpϪ 44, 45 Escherichia coli DH5␣ supE44 lacU169 (␾80dlacZ⌬M15) recA1 endA1 hsdR17 thi-1 gyrA96 relA1 46 Plasmids pCM130 IncP and colE1 origin, promoter probe vector, Tcr 23 pCMgfp-lacZ pCM130 containing gfp and new MCS 31 pCM130-P1 pCM130 carrying sequence between positions 2579 and 3869 This study pCM130-P2 pCM130 carrying sequence between positions 8474 and 8969 This study pCM130-P3 pCM130 carrying sequence between positions 10420 and 10760 This study pCMgfp-P3lacZ pCMgfp-lacZ carrying sequence between positions 10420 and 10760 This study pCMgfp-P32lacZ pCMgfp-lacZ carrying sequence between positions 10552 and 10760 This study pCMgfp-P3M1lacZ GG¡AA mutation at position 10630 on foundation of pCMgfp-P3lacZ This study Downloaded from pCMgfp-P3M2lacZ AC¡GT mutation at position 10632 on foundation of pCMgfp-P3lacZ This study pCMgfp-P3M3lacZ TA¡CG mutation at position 10634 on foundation of pCMgfp-P3lacZ This study pCMgfp-P3M4lacZ AA¡GG mutation at position 10636 on foundation of pCMgfp-P3lacZ This study pCMgfp-P3M5lacZ TG¡CA mutation at position 10638 on foundation of pCMgfp-P3lacZ This study a Strr, streptomycin resistant; Tcr, ticarcillin resistant; MCS, multiple-cloning site; TrpϪ, tryptophan auxotroph. by a patchwork assembly of a Nag-like 2CNB dioxygenase (cata- strains were grown in lysogeny broth (LB) medium at 37°C. Pseudomonas http://aem.asm.org/ lyzing the conversion of 2CNB to 3-chlorocatechol) gene cluster putida PaW340 strains were grown in LB medium at 30°C with 50 ␮g/ml and a 3-chlorocatechol degradation cluster, in which several streptomycin. Pseudomonas stutzeri ZWLR2-1 was grown at 30°C in LB or transposable elements are also present (8), as shown in Fig. 2. M9 medium (14) supplemented with 0.5 mM 2CNB or 10 mM succinate. Among the 2CNB catabolism cluster, cnbA encodes 2CNB dioxy- For solid media, agar was added to a final concentration of 12 g/liter. genase (cnbAa encodes ferredoxin reductase, cnbAb encodes ferre- When required, antibiotics were used at the following concentrations: doxin, and cnbAc and cnbAd encode the ␣- and ␤-subunits of ampicillin, 100 mg/liter; kanamycin, 50 mg/liter; and tetracycline hydro- chloride, 20 mg/liter. Substrate 2CNB was purchased from Fluka Chem- oxygenase, respectively), catalyzing the conversion of 2CNB to ical Co. (Buchs, Switzerland), and 3-chlorocatechol was from TCI (To- 3-chlorocatechol with the release of nitrite. CnbC is 3-chlorocat- kyo, Japan); catechol and o-nitrophenyl ␤-D-galactopyranoside (ONPG) echol 1,2-dioxygenase catalyzing the ring cleavage of 3-chlorocat- were purchased from Sigma (St. Louis, MO). All primers used are listed in on January 6, 2017 by guest echol to 2-chloro-cis,cis-muconate. The 2CNB dioxygenase, to- Table 2. gether with other nitroarene dioxygenases, is thought to originate Plasmid construction. All DNA manipulation was performed accord- from the naphthalene dioxygenase NagAcAd in Ralstonia sp. ing to standard procedures (18). Restriction enzymes, DNA polymerases, strain U2, which is activated by the LysR transcriptional regulator and T4 DNA ligase were purchased from New England BioLabs (Ipswich, NagR, with salicylate as an inducer (9, 13, 15–17). It has been MA), and TaKaRa (Dalian, China). A NOVA SLIC kit purchased from speculated that horizontal gene transfer is involved in the rapid LCP Biomed Co. (Lianyungang, China) was used for ligation-indepen- evolution of 2CNB catabolism pathway in strain ZWLR2-1 (8). dent cloning. The plasmid DNA preparation, DNA purification kit, and However, the transcriptional regulator gene usually present in the RNA preparation kit were purchased
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