scholarly journals Binding of Escherichia coli integration host factor (IHF) to the origin segment of p15A plasmid.

1995 ◽  
Vol 42 (1) ◽  
pp. 103-108
Author(s):  
E Hiszczyńska-Sawicka ◽  
J Kur
Keyword(s):  
Escherichia Coli ◽  
Consensus Sequence ◽  
Integration Host Factor ◽  
Host Factor ◽  
Mobility Shift ◽  
Consensus Sequences ◽  
Report Data ◽  
Functional Dna ◽  
Gel Mobility Shift ◽  
Dna Binding And Bending

The integration host factor (IHF) is a sequence-specific, histone-like, multi-functional DNA-binding and -bending protein of Escherichia coli. Characterization and functional analysis of this protein has been carried out mainly in bacteriophage lambda and other mobile genetic elements. In this paper we report data concerning the binding of IHF protein to the plasmid orip15A region. IHF binds to the single site of the DNA fragment containing the orip15A, as shown by the gel mobility shift assays and footprinting experiment. On the basis of the ihf consensus sequences published, we have been able to identify one sequence of putative ihf site into the orip15A sequence with two mismatches in relation to the consensus sequence of Kur et al., 1989, Gene 81, 1-15. One ihf binding site was also found in the oriColE1 region sequence with three mismatches in relation to this consensus sequence.

scholarly journals An AT-Rich Tract Containing an Integration Host Factor-Binding Domain and Two UP-Like Elements Enhances Transcription from thepilEp1 Promoter of Neisseria gonorrhoeae

1998 ◽  
Vol 180 (8) ◽  
pp. 2152-2159 ◽  
Author(s):  
Janet A. M. Fyfe ◽  
John K. Davies
Keyword(s):  
Escherichia Coli ◽  
Neisseria Gonorrhoeae ◽  
Binding Site ◽  
Detailed Analysis ◽  
Consensus Sequence ◽  
Integration Host Factor ◽  
Host Factor ◽  
Rich Region ◽  
Factor Binding ◽  
Binding Core

ABSTRACT The pilE gene of Neisseria gonorrhoeae is transcribed from a ς70 promoter (pilEp 1) with an AT-rich tract extending 65 nucleotides upstream of the −35 box. Within this region is an integration host factor (IHF)-binding core consensus sequence. We have performed a detailed analysis to determine which upstream sequences are required for efficient transcription frompilEp 1 in N. gonorrhoeae. Deletion of sequences upstream of the AT-rich tract had no effect on the level of transcription. However, the IHF-binding core consensus sequence and the AT-rich sequence further upstream were both required for enhanced levels of transcription from this promoter in both N. gonorrhoeae and an Escherichia coli strain producing IHF. In addition, an UP-like element positioned between the −35 box and the IHF-binding site was required for maximal transcription. The AT-rich region upstream of the IHF-binding core consensus sequence can also act as an UP-like element when appropriately repositioned upstream of the −35 box.

scholarly journals Transcription from Fusion Promoters Generated during Transposition of Transposon Tn4652 Is Positively Affected by Integration Host Factor in Pseudomonas putida

2000 ◽  
Vol 182 (3) ◽  
pp. 589-598 ◽  
Author(s):  
Riho Teras ◽  
Rita Hõrak ◽  
Maia Kivisaar
Keyword(s):  
Pseudomonas Putida ◽  
Specific Binding ◽  
Stationary Growth Phase ◽  
Integration Host Factor ◽  
Host Factor ◽  
Mobile Dna ◽  
Mobility Shift ◽  
Transposase Gene ◽  
Gel Mobility Shift

ABSTRACT We have previously shown that both ends of the Tn3family transposon Tn4652 contain integration host factor (IHF) binding sites and that IHF positively regulates expression of the Tn4652 transposase gene tnpA inPseudomonas putida (R. Hõrak, and M. Kivisaar, J. Bacteriol. 180:2822–2829, 1998). Tn4652 can activate silent genes by creating fusion promoters during the transposition. The promoters are created as fusions between the −35 hexamer provided by the terminal inverted repeats of Tn4652 and the −10 hexamers in the target DNA. Two fusion promoters, PRA1 and PLA1, that contain sequences of the right and left termini of Tn4652, respectively, were chosen for the study of mechanisms of transcription activation. Gel mobility shift analysis using crude extracts fromP. putida cells allowed us to detect specific binding ofP. putida IHF to the ends of the transposon Tn4652. We found that the rate of transcription from the fusion promoter PRA1 is enhanced by IHF. Notably, the positive effect of IHF on transcription from the promoter PRA1 appeared only when cells of P. putida reached the stationary growth phase. We speculate that the intracellular concentration of IHF might be critical for the in vivo effect of IHF on transcription from the fusion promoters in P. putida. In the case of PLA1, the mechanism of transcription modulation by IHF is different than that observed for PRA1. Our results demonstrate that transcription of neighboring genes from outwardly directed promoters at the ends of a mobile DNA element could be influenced by the same factors that control transposition of the element.

scholarly journals Fis negatively affects binding of Tn4652 transposase by out-competing IHF from the left end of Tn4652

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1203-1214 ◽  
Author(s):  
Riho Teras ◽  
Julia Jakovleva ◽  
Maia Kivisaar
Keyword(s):  
Dnase I ◽  
Integration Host Factor ◽  
Mobile Dna ◽  
Mobility Shift ◽  
Transposase Gene ◽  
Transposition Activity ◽  
Global Regulators ◽  
Factor For Inversion Stimulation ◽  
Dna Elements ◽  
Gel Mobility Shift

Transposition activity in bacteria is generally maintained at a low level. The activity of mobile DNA elements can be controlled by bacterially encoded global regulators. Regulation of transposition of Tn4652 in Pseudomonas putida is one such example. Activation of transposition of Tn4652 in starving bacteria requires the stationary-phase sigma factor RpoS and integration host factor (IHF). IHF plays a dual role in Tn4652 translocation by activating transcription of the transposase gene tnpA of the transposon and facilitating TnpA binding to the inverted repeats of the transposon. Our previous results have indicated that besides IHF some other P. putida-encoded global regulator(s) might bind to the ends of Tn4652 and regulate transposition activity. In this study, employing a DNase I footprint assay we have identified a binding site of P. putida Fis (factor for inversion stimulation) centred 135 bp inside the left end of Tn4652. Our results of gel mobility shift and DNase I footprint studies revealed that Fis out-competes IHF from the left end of Tn4652, thereby abolishing the binding of TnpA. Thus, the results obtained in this study indicate that the transposition of Tn4652 is regulated by the cellular amount of P. putida global regulators Fis and IHF.

scholarly journals Characterization of Specific Nucleotide Substitutions in DtxR-Specific Operators of Corynebacterium diphtheriae That Dramatically Affect DtxR Binding, Operator Function, and Promoter Strength

2000 ◽  
Vol 182 (2) ◽  
pp. 432-438 ◽  
Author(s):  
John H. Lee ◽  
Randall K. Holmes
Keyword(s):  
Consensus Sequence ◽  
Corynebacterium Diphtheriae ◽  
Promoter Strength ◽  
Negative Effects ◽  
Nucleotide Substitutions ◽  
Mobility Shift ◽  
Diphtheria Toxin Repressor ◽  
Sense Strand ◽  
Gel Mobility Shift ◽  
The Cost

ABSTRACT The diphtheria toxin repressor (DtxR) of Corynebacterium diphtheriae uses Fe2+ as a corepressor. Holo-DtxR inhibits transcription from the iron-regulated promoters (IRPs) designated IRP1 through IRP5 as well as from the promoters for thetox and hmuO genes. DtxR binds to 19-bp operators with the consensus sequence 5′-TTAGGTTAGCCTAACCTAA-3′, a perfect 9-bp palindrome interrupted by a single C · G base pair. Among the seven known DtxR-specific operators, IRP3 exhibits the weakest binding to DtxR. The message (sense) strand of the IRP3 operator (5′-TTAGGTGAGACGCACCCAT-3′ [nonconsensus nucleotides underlined]) overlaps by 2 nucleotides at its 5′ end with the putative −10 sequence of the IRP3 promoter. The underlined C at position +7 from the center of the IRP3 operator [C(+7)] is unique, because T is conserved at that position in other DtxR-specific operators. The present study examined the effects of nucleotide substitutions at position +7 or −7 in the IRP3 operator. In gel mobility shift assays, only the change of C(+7) to the consensus nucleotide T caused a dramatic increase in the binding of DtxR, whereas other nucleotide substitutions for C(+7) or replacements for A(−7) had only small positive or negative effects on DtxR binding. All substitutions for C(+7) or A(−7) except for A(−7)C dramatically decreased IRP3 promoter strength. In contrast, the A(−7)C variant caused increased promoter strength at the cost of nearly eliminating repressibility by DtxR. The message (sense) strand of the IRP1 operator (5′-TTAGGTTAGCCAAACCTTT-3′) includes the −35 region of the IRP3 promoter. A T(+7)C variant of the IRP1 operator was also constructed, and it was shown to exhibit decreased binding to DtxR, decreased repressibility by DtxR, and increased promoter strength. The nucleotides at positions +7 and −7 in DtxR-specific operators are therefore important determinants of DtxR binding and repressibility of transcription by DtxR, and they also have significant effects on promoter activity for IRP3 and IRP1.

scholarly journals The Escherichia coli K-12 NarL and NarP Proteins Insulate the nrf Promoter from the Effects of Integration Host Factor

2006 ◽  
Vol 188 (21) ◽  
pp. 7449-7456 ◽  
Author(s):  
Douglas F. Browning ◽  
David J. Lee ◽  
Alan J. Wolfe ◽  
Jeffrey A. Cole ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Regulatory Region ◽  
Enteric Bacteria ◽  
Integration Host Factor ◽  
Host Factor ◽  
Receptor Protein ◽  
E Coli ◽  
Serovar Typhimurium ◽  
K 12

ABSTRACT The Escherichia coli K-12 nrf operon promoter can be activated fully by the FNR protein (regulator of fumarate and nitrate reduction) binding to a site centered at position −41.5. FNR-dependent transcription is suppressed by integration host factor (IHF) binding at position −54, and this suppression is counteracted by binding of the NarL or NarP response regulator at position −74.5. The E. coli acs gene is transcribed from a divergent promoter upstream from the nrf operon promoter. Transcription from the major acsP2 promoter is dependent on the cyclic AMP receptor protein and is modulated by IHF and Fis binding at multiple sites. We show that IHF binding to one of these sites, located at position −127 with respect to the nrf promoter, has a positive effect on nrf promoter activity. This activation is dependent on the face of the DNA helix, independent of IHF binding at other locations, and found only when NarL/NarP are not bound at position −74.5. Binding of NarL/NarP appears to insulate the nrf promoter from the effects of IHF. The acs-nrf regulatory region is conserved in other pathogenic E. coli strains and related enteric bacteria but differs in Salmonella enterica serovar Typhimurium.

scholarly journals Integration Host Factor Positively Regulates Virulence Gene Expression in Vibrio cholerae

2008 ◽  
Vol 190 (13) ◽  
pp. 4736-4748 ◽  
Author(s):  
Emily Stonehouse ◽  
Gabriela Kovacikova ◽  
Ronald K. Taylor ◽  
Karen Skorupski
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Virulence Gene ◽  
Toxin Production ◽  
Dnase I ◽  
Integration Host Factor ◽  
Host Factor ◽  
Mobility Shift ◽  
Virulence Gene Expression ◽  
Dnase I Footprinting

ABSTRACT Virulence gene expression in Vibrio cholerae is dependent upon a complex transcriptional cascade that is influenced by both specific and global regulators in response to environmental stimuli. Here, we report that the global regulator integration host factor (IHF) positively affects virulence gene expression in V. cholerae. Inactivation of ihfA and ihfB, the genes encoding the IHF subunits, decreased the expression levels of the two main virulence factors tcpA and ctx and prevented toxin-coregulated pilus and cholera toxin production. IHF was found to directly bind to and bend the tcpA promoter region at an IHF consensus site centered at position −162 by using gel mobility shift assays and DNase I footprinting experiments. Deletion or mutation of the tcpA IHF consensus site resulted in the loss of IHF binding and additionally disrupted the binding of the repressor H-NS. DNase I footprinting revealed that H-NS protection overlaps with both the IHF and the ToxT binding sites at the tcpA promoter. In addition, disruption of ihfA in an hns or toxT mutant background had no effect on tcpA expression. These results suggest that IHF may function at the tcpA promoter to alleviate H-NS repression.

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