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. 2016 May 31;82(12):3461-3470.
doi: 10.1128/AEM.00197-16. Print 2016 Jun 15.

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

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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 et al. Appl Environ Microbiol. .
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Abstract

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.

Figures

FIG 1
FIG 1
Real-time PCR for quantification of the expression of 2CNB catabolism genes under different conditions. In the 2CNB cluster, cnbC codes for 3-chlorocatechol 1,2-dioxygenase, and cnbAc codes for the large subunit of 2CNB dioxygenase. The data are derived from three replicates, and the error bars indicate standard deviations.
FIG 2
FIG 2
Transcription analysis of 2CNB-metabolic-pathway-encoding gene. (A) Catabolism pathway of 2CNB degradation in strain ZWLR2-1. (B) Genes involved in the 2CNB metabolic pathway (8). The gray boxes represent genes encoding 2CNB dioxygenase. The lines below the gene cluster show the location of the PCR fragments in panel C. (C) Electrophoretic analysis of 2CNB catabolism gene transcription by reverse transcription-PCR. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 show amplified products PP1, PP2, PP3, PP4, PP5, PP6, PP7, PP8, PP9, PP10, PP11, and PP12, respectively, using the corresponding primer pair sets listed in Table 2. Lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 show corresponding negative controls that do not have reverse transcriptase. Lane 25 shows a molecular size marker.
FIG 3
FIG 3
Expression pattern of cnb cluster and location of promoters. (A) Absolute quantification using real-time PCR to analyze 2CNB metabolic gene expression in Pseudomonas stutzeri ZWLR2-1. (B) Promoter activity assays of DNA fragments P1, P2, and P3 from 2CNB gene cluster in surrogate host Pseudomonas putida PaW340, using xylE as the reporter gene. The data are derived from at least three replicates, and the error bars indicate standard deviations. (C) Location of the coding genes and three promoter sequences on 2CNB metabolic gene cluster. The gray boxes represent genes encoding 2CNB dioxygenase. The transcription start sites are indicated with bent arrows.
FIG 4
FIG 4
Comparison of homologous sequence of strain JS42 and strain ZWLR2-1. (A) Sketch map of relationship between P3 DNA fragment from strain ZWLR2-1 and homologous sequence in Acidovorax sp. JS42. (B) DNA sequence of P3 fragment. Nucleotides 10419 to 10500 from the cnb cluster in strain ZWLR2-1 is 100% identical to the 132-bp fragment from plasmid pAOVO02 in strain JS42. Nucleotides 10551 to 10759 are almost identical to the sequence spanning ntdAb and ntdAc on the ntd cluster of strain JS42 chromosomal DNA, except a cytosine (C at nucleotide 10733, italics) in the cnb cluster instead of a thymine (T) in the ntd cluster, and a duplication of an 18-bp sequence in the ntd cluster to form a direct repeat in the cnb cluster, where the −10 and −35 regions of the P3 promoter are located. The transcriptional start site is marked +1, and the −10 and −35 regions are underlined. Start codon of the cnbAc gene at nucleotide 10743 is indicated with a bent arrow. LDR, left direct repeat; RDR, right direct repeat.
FIG 5
FIG 5
Mutation analysis of promoter activity of the newly formed −35 region at the junction of LDR and RDR. (A) Schematic diagram of mutant construction in the −35 region of the P3 promoter. (B) β-Galactosidase activities of promoter P3 and its mutants P3M1 to P3M5. The data are derived from at least 3 independent measurements, and the error bars indicate standard deviations.

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Grant support

This work, including the efforts of Hong Liu, was funded by National Natural Science Foundation of China (NSFC) (31170118) and the National Key Basic Research Program of China (973 Program, grant 2012CB721003).

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