The essential HupB and HupN proteins of Pseudomonas putida provide redundant and nonspecific DNA-bending functions.
A protein mixture containing two major components able to catalyze a beta-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 beta-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 final sigma54 Ps promoter of the toluene degradation plasmid, TOL, which requires facilitated DNA bending, in DeltahupB or DeltahupN 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.
Division of Microbiology, Gesellschaft für Biotechnologische Forschung (GBF), D-38124, Braunschweig, Germany.
F
F
Bartels
S
S
Fernández
A
A
Holtel
K N
KN
Timmis
V
V
de Lorenzo
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; C087380:HupN protein, Bradyrhizobium japonicum; D008565:Membrane Proteins; D011994:Recombinant Proteins; D008741:Methyl Methanesulfonate