CRISPR
            interference‐mediated gene regulation in
            Pseudomonas putida
            KT
            2440

ABSTRACT This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of β-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant. Complementation with the closely related regulators NagR (from Ralstonia sp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator.

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Effects of Mutations in the Pseudomonas putida miaA Gene: Regulation of the trpE andtrpGDC Operons in P. putida by Attenuation

Journal of Bacteriology ◽

10.1128/jb.183.10.3256-3260.2001 ◽

2001 ◽

Vol 183 (10) ◽

pp. 3256-3260 ◽

Cited By ~ 7

Author(s):

Igor Olekhnovich ◽

Gary N. Gussin

Keyword(s):

Escherichia Coli ◽

Gene Regulation ◽

Pseudomonas Putida ◽

Bacterial Species ◽

Nucleotide Sequences ◽

Leader Peptides ◽

Insertion Mutants

ABSTRACT Tn5 insertion mutants defective in regulation of thePseudomonas putida trpE and trpGDCoperons by tryptophan were found to contain insertions in the P. putida miaA gene, whose product (in Escherichia coli) modifies tRNATrp and is required for attenuation. Nucleotide sequences upstream of trpE andtrpG encode putative leader peptides similar in sequence to leader peptides found in other bacterial species, and the phenotypes of the mutants strongly suggest that transcription of these operons is regulated solely by attenuation.

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