Project description:Acidovorax sp. strain JS42 is able to utilize 2-nitrotoluene (2NT) as its sole carbon, nitrogen, and energy source. We report here that strain JS42 is chemotactic to 2NT and that the response is increased when cells are grown on compounds such as 2NT that are known to induce the first step of 2NT degradation. Assays with JS42 mutants unable to oxidize 2NT showed that the first step of 2NT metabolism was required for the induced response, but not for a portion of the constitutive response, indicating that 2NT itself is an attractant. The 2NT metabolite nitrite was shown to be a strong attractant for strain JS42, and sufficient nitrite was produced during the taxis assay to account for a large part of the induced response. A mutant with an inactivated ntdY gene, which is located adjacent to the 2NT degradation genes and codes for a putative methyl-accepting chemotaxis protein, showed a defect in taxis toward 2NT that may involve a reduced response to nitrite. Responses of a mutant defective for the energy-taxis receptor, Aer, indicated that a functional aer gene is required for a substantial part of the wild-type induced response to 2NT. In summary, strain JS42 utilizes three types of taxis to sense and respond to 2NT: constitutive 2NT-specific chemotaxis to directly sense 2NT, metabolism-dependent nitrite-specific chemotaxis that may be mediated by NtdY, and energy taxis mediated by Aer.
Project description: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 beta-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.
Project description:Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the ? subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.
Project description:The bacterium Novosphingobium sp. THN1 (THN1) is capable of degrading microcystin-LR (MCLR). To get an insight into genes expression during MCLR degradation and the regulation of different carbon concentrations on MCLR degradation, we performed RNA-seq of THN1 during MCLR degradation under different carbon concentrations.
Project description:This study examines the transcriptomic response of biofilms of the PAH-degrading Sphingomonas sp. LH128 on solute stress when actively degrading and growing on the PAH compound. To address the effect of solute stress on bacterial physiology and transcriptomic response, NaCl was used as osmolyte. Both acute and chronic solute stress was invoked to assess differences in short-term and long-term responses.