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J. Biol. Chem., Vol. 276, Issue 20, 16641-16648, May 18, 2001
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From the A protein mixture containing two major components
able to catalyze a The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF345628 (hupB of P. putida); AF345629 (hupN
of P. putida), and AF345630 (hupN of P. aeruginosa).
The Essential HupB and HupN Proteins of Pseudomonas
putida Provide Redundant and Nonspecific DNA-bending
Functions*
§,,
, and Division of Microbiology, Gesellschaft
für Biotechnologische Forschung (GBF), D-38124,
Braunschweig, Germany and ¶ Department of Microbial Biotechnology,
Centro Nacional de Biotecnología, Consejo Superior de
Investigaciones Científicas, (CSIC), Campus de Cantoblanco,
28049 Madrid, Spain
-recombination reaction requiring nonspecific DNA
bending was obtained by fractionation of a Pseudomonas
putida extract. N-terminal sequence analysis and genomic data
base searches identified the major component as an analogue of HupB of
Pseudomonas aeruginosa and Escherichia coli,
encoding one HU protein variant. The minor component of the
fraction, termed HupN, was divergent enough from HupB to predict a
separate DNA-bending competence. The determinants of the two proteins
were cloned and hyperexpressed, and the gene products were purified.
Their activities were examined in vitro in
-recombination assays and in vivo by complementation of
the Hbsu function of Bacillus subtilis. HupB and
HupN were equally efficient in all tests, suggesting that they are
independent and functionally redundant DNA bending proteins. This was
reflected in the maintenance of in vivo activity of the
54 Ps promoter of the toluene degradation
plasmid, TOL, which requires facilitated DNA bending, in
hupB or hupN strains. However,
hupB/hupN double mutants were not viable. It is suggested
that the requirement for protein-facilitated DNA bending is met in
P. putida by two independent proteins that ensure an
adequate supply of an essential cellular activity.
*
This work was supported by Contracts BIO4-CT97-2040 and
QLRT-99-00041 from the European Commission and by Grants
BIO98-0808 and DGCYT BMC2000-0548 from the Spanish Comisión
Interministerial de Ciencia y Tecnología (CICYT).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by the Fonds der Chemische Industrie.
§
Current address: Fresenius HemoCare Adsorber Technology GmbH,
Frankfurter Str. 6-8, 66606 St. Wendel, Germany.
**
To whom correspondence should be addressed: Dept. of Microbial
Biotechnology, Centro Nacional de Biotecnología-CSIC, Campus de
Cantoblanco, 28049 Madrid, Spain. Tel.: +34 91-585-4536; Fax: +34 91-585-4506; E-mail: vdlorenzo@cnb.uam.es.
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