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Integration host factor (IHF) is a DNA–bending protein that recognizes its cognate sites through indirect readout. Previous studies have shown that binding of wild-type (WT)-IHF is disrupted by a T to A mutation at the center position of a conserved TTR motif in its binding site, and that substitution of βGlu44 with Ala prevented IHF from discriminating between A and T at this position. We have determined the crystal structures and relative binding affinities for all combinations of WT-IHF and IHF-βGlu44Ala bound to the WT and mutant DNAs. Comparison of these structures reveals that DNA twist plays a major role in DNA recognition by IHF, and that this geometric parameter is dependent on the dinucleotide step and not on the bound IHF variant. [Copyright &y& Elsevier]

The 41 kDa heat-modifiable OmpA-like protein from Bacteroides fragilis ATCC 25285 was purified and tryptic peptide sequences were used to design degenerate oligonucleotide primers which were then used in amplification and identification of the omp A gene. The omp A gene was cloned and sequenced (GenBank Accession Number AY028459). Sequence analysis revealed an open reading frame of 1188 bases. The deduced amino acid sequence (which contained the experimentally determined peptide sequences) has 395 amino acids; the mature protein consists of 376 amino acids, has a predicted MW of 41 448 and a theoretical pI of 7.95. It is preceded by 19 amino acids that correspond to elements typical of a signal peptide sequence, including the Lys-Lys sequence near the N-terminus followed by a hydrophobic region (LLALAGV). The C-terminus of B. fragilis OmpA is homologous to the highly characteristic OmpA signature domain (75% similarity with the OmpA consensus domain). Three-dimensional modeling of B. fragilis OmpA predicts an eight-stranded β-barrel; the predicted loops are in agreement with those predicted as hydrophilic maxima in hydrophobicity plots. B. fragilis OmpA was cloned into pET-27b(+) and expressed in E. coli BL21(DE3)pLysS. The recombinant B. fragilis OmpA was recovered from inclusion bodies, solubilized in 8 M urea and refolded in 4 mM Zwittergent 3-14. Recombinant, refolded B. fragilis OmpA was active in a liposome assay (swelling rate for arabinose was 0.495/5.6μg protein) and preliminary analysis indicated that channels of three sizes were formed in a black lipid bilayer assay. This is the first description of an ompA homolog in Bacteroides. [Copyright &y& Elsevier]

Highlights: [•] We mapped packaging cleavage sites for phage 80α and pathogenicity island SaPI1. [•] Packaging directed by 80α TerS initiates within the 80α terS coding sequence. [•] SaPI1 TerS-specific packaging initiates in an intergenic region. [•] The SaPI1 pac site determinants were mapped using a plasmid-based assay. [•] Deletion of the pac site from SaPI1 eliminates high frequency transduction. [ABSTRACT FROM AUTHOR]