Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.

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“…Pseudomonas spp. belonging to the rRNA group I and bearing pWW0-Km grow on toluene and m-xylene (18).…”

Section: Methodsmentioning

confidence: 99%

“…Alternatively, a single organism able to eliminate nitroaromatics may be produced by appropriate recruitment of catabolic functions, as described for the metabolism of haloaromatics (10,18,21,22).…”

mentioning

confidence: 99%

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene

et al. 1993

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“…Genes encoding the TOL meta-cleavage pathway are grouped into a single operon, the expression of which is positively regulated at the level of transcription by the xylS gene product, which is activated by benzoate effectors (1)(2)(3)(4). Stimulation of transcription from the Pm promoter requires a DNA sequence extending to about 80 bp 1 upstream of the transcription initiation point (5)(6)(7).…”

mentioning

confidence: 99%

Critical Nucleotides in the Upstream Region of the XylS-dependent TOL meta-Cleavage Pathway Operon Promoter as Deduced from Analysis of Mutants

González-Pérez

Ramos

Gallegos

et al. 1999

Journal of Biological Chemistry

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The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effectors, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. This promoter is unique in that in vivo transcription is mediated by RNApolymerase with different sigma factors. In vivo footprinting analysis shows that XylS interacted with nucleotides in the ؊40 to ؊70 region. In vivo and in vitro methylation of Pm shows extensive methylation of T at position ؊42 in the bottom strand, suggesting that it represents a key distortion point that may favor XylS/ RNA polymerase interactions. Methylation of T ؊42 was highest in cells bearing XylS and in the presence of an effector. Gs in the ؊47 to ؊61 region appeared to be more protected in cells harboring XylS in the presence than in the absence of the effector. Almost 100 mutants in the Pm region between ؊41 and ؊78 were generated; transcriptional analysis of these mutants defined the XylS target as two direct repeats with the sequence TGCAN 6 GGNCA. These motifs cover the ؊70 to ؊56 and the ؊49 to ؊35 regions. Single point mutations revealed that nucleotides located at ؊49 to ؊46 and at ؊59, ؊60, ؊62, and ؊70 are the most critical for appropriate XylS-Pm interactions.

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“…XylS1-pWW53 and XylSpWW0 differ in only 5 amino acids (Cys-4, Cys-53, Gly-90, Asp-137, and His-153 in XylS versus Arg-4, Gly-53, Asp-90, Glu-137, and Asn-153 in XylS1), but despite this minor difference the latter exhibited a much narrower effector profile than XylS-pWW0 (7). In fact, XylS1-pWW53 recognizes only 3MB and 3CB as effectors, whereas XylS-pWW0 recognizes in addition other alkyl-and chlorobenzoates with substituents at positions 2 or 4 (14,15). The difference in effector profile is consistent with the changes occurring at the N-terminal end of these regulators.…”

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confidence: 97%

Residues 137 and 153 at the N Terminus of the XylS Protein Influence the Effector Profile of This Transcriptional Regulator and the ς Factor Used by RNA Polymerase to Stimulate Transcription from Its Cognate Promoter

Ruíz¹,

Ramos

2002

Journal of Biological Chemistry

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The 321-residue XylS and XylS1 proteins, encoded by the pWW0 and pWW53 plasmids respectively, differ in only 5 residues at positions 4, 53, 90, 137, and 153. As a result, the effector profile of XylS is wider than that of XylS1, and XylS mediates higher levels of transcription from its cognate-regulatable promoter than does XylS1. We generated a series of XylS-pWW0 mutants and found that the single mutants Asp-137 3 Glu and His-153 3 Asn exhibited an activation pattern different from that of the wild-type regulator. In the double-mutant XylSD137E,H153N the effector profile for benzoates was similar to that of XylS1. This suggests that these two residues are crucial for effector recognition and regulator activation to stimulate transcription. XylS-dependent transcription from its cognate promoter is mediated by RNA polymerase with 32 38 . This suggests that a positive transcriptional regulator can choose the RNA polymerase complex that mediates transcription from a given promoter.

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Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria.

Cited by 171 publications

References 19 publications

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene

Critical Nucleotides in the Upstream Region of the XylS-dependent TOL meta-Cleavage Pathway Operon Promoter as Deduced from Analysis of Mutants

Residues 137 and 153 at the N Terminus of the XylS Protein Influence the Effector Profile of This Transcriptional Regulator and the ς Factor Used by RNA Polymerase to Stimulate Transcription from Its Cognate Promoter

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