Identification of multiple repressor recognition sites in the hut system of Pseudomonas putida.

Abstract:

:The hutC gene in Pseudomonas putida encodes a repressor protein that negatively regulates the expression of all hut genes. We have overexpressed this cloned hutC gene in Escherichia coli to identify P. putida hut regions that could specifically bind the repressor. Ten restriction fragments, some of which were partially overlapping and spanned the coding portions of the P. putida hut region, were labeled and tested for their ability to recognize repressor in a filter binding assay. This procedure identified three binding sites, thus supporting previous indications that there were multiple operons. A 1.0-kilobase-pair SalI restriction fragment contained the operator region for the hutUHIG operon, whereas a 1.9-kilobase-pair SmaI fragment contained the hutF operator. A 2.9-kilobase-pair XhoI segment appeared to contain the third operator, corresponding to a separate and perhaps little used control region for hutG expression only. The addition of urocanate, the normal inducer, caused dissociation of all operator-repressor complexes, whereas N-formylglutamate, capable of specifically inducing expression of the hutG gene, inhibited binding only of repressor to fragments containing that gene. Formylglutamate did not affect the action of urocanate on the repressor-hutUHIG operator complex, indicating that it binds to a site separate from urocanate on the repressor. DNA footprinting and gel retardation analyses were used to locate more precisely the operator for the hutUHIG operon. A roughly 40-base-pair portion was identified which contained a 16-base-pair region of dyad symmetry located near the transcription initiation site for this operon.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Hu L,Allison SL,Phillips AT

doi

10.1128/jb.171.8.4189-4195.1989

subject

Has Abstract

pub_date

1989-08-01 00:00:00

pages

4189-95

issue

8

eissn

0021-9193

issn

1098-5530

journal_volume

171

pub_type

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