Structure and function of the Pseudomonas putida integration host factor

Abstract

Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P U promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.Integration host factor (IHF) is a sequence-specific DNAbinding and -bending heterodimeric protein that is abundant in the Escherichia coli cell. Its two subunits are coded by the ihfA gene (previously termed himA) and by the ihfB gene (previously termed hip or himD) (33). IHF belongs to the family of bacterial histone-like proteins that share amino acid sequence homology with HU proteins from a variety of bacteria (3,5,7,23). One member of the family, HU of Bacillus stearothermophilus, was crystallized (28, 35), and its three-dimensional structure serves as a model for the whole family. According to the model, the N-terminal half of the protein is involved in dimer formation, and the long arms, consisting of antiparallel double-stranded ␤ ribbons of the two subunits, wrap around the DNA. Recent solution structure studies of HU showed that its arms are highly flexible (32).IHF participates in a number of cellular processes such as site-specific recombination, transposition and inversion, phage DNA packaging, plasmid and phage DNA replication, and the positive and negative control of gene expression (for reviews, see references 6 and 7). Yang and Nash (37) proposed a model for IHF interaction with DNA in which the "arms" of the IHF protein contact DNA through the minor groove; the C termini provide for additional contacts made possible by the bending of the DNA. Although the two subunits of IHF are similar in structure, experimental evidence indicates that the two subunits contribute differently to the function of IHF (34, 38).IHF was found to participate in gene regulation in a number of gram-negative bacteria. A large number of these genes are transcribed by ...

Role of Integration Host Factor in the Transcriptional Activation of Flagellar Gene Expression in Caulobacter crescentus

“…These findings are analogous to those reported for an ihfA mutant of P. putida that exhibited reduced expression of the 54 -dependent xyl operon and consequently was unable to degrade benzyl alcohol (9). We propose that the motility defect in the C. crescentus ihfB mutant strain is attributable to markedly reduced levels of the 27-kDa flagellin (FljL).…”

LacZ-promoter fusions: the effect of growth

“…In contrast, an inverse relation between the level of expression and growth rate, as observed here, would be indicative of a growth-rate-independent rate of transcription (10). Such considerations may be helpful when discussing growth-rate-dependent expression (2,3,4,5,8) and, at least, show the importance of careful use of terminology. At any time, it should be kept in mind that the rate of transcription is a complex parameter containing many different contributions.…”

“…Four had already been described and characterized by experimental methods. Two of these ORFs encode the two subunits of the IHF protein of P. putida (5). Two others match the HupB and HupN protein sequences (2).…”

Transcription from Fusion Promoters Generated during Transposition of Transposon Tn 4652 Is Positively Affected by Integration Host Factor in Pseudomonas putida

“…The cleavage of pUT mini-Tn5 luxAB (11) by NotI enabled us to delete the luxAB genes from this plasmid and to clone the Ptac-ihfAB-lacI q cassette as NotI DNA fragment into mini-Tn5 to obtain plasmid pUTtetPF. Mating between E. coli S17-1 pir (33) carrying the plasmid pUTtetPF and the ihfA knockout mutant P. putida A8759 (6), and selection of kanamycin-tetracycline-resistant P. putida transconjugants containing chromosomal insertion of Ptac-ihfAB-lacI q cassette plus the lacI q gene was carried out as previously described (10). The presence of this cassette in the chromosome of P. putida RT31 was verified by PCR by using the primers specific to the 5Ј end of the ihfA and the 3Ј end of the ihfB.…”

“…The amplified DNA fragment was cloned into EcoRI and HindIII sites in pBluescript SK vector. The gene ihfB, originating from plasmid pHip (6), was cloned as a 600-bp XbaI-SmaI fragment downstream of the ihfA gene. Thereafter, the ihfA and ihfB genes were inserted as a XbaI-HindIII fragment into plasmid pMMB208 (34) containing Ptac promoter and lacI q gene.…”