Identification of basepairs within Tn5 termini that are critical sfor H-NS binding to the transpososome and regulation of Tn5 transposition

In the current work we have further examined determinants for H-NS binding to the Tn5 transpososome through both mutational studies on Tn5 termini (or 'transposon ends') and protein-protein cross-linking analysis. We identify mutations in two different segments of the transposon ends that abrogate H-NS binding and characterize the affinity of H-NS for wild type transposon ends in the context of the transpososome. We also show that H-NS forms cross-links with the Tn5 transposase protein specifically in the transpososome, an observation consistent with the two proteins occupying overlapping binding sites in the transposon ends. Finally, we make use of the end mutations to test the idea that H-NS exerts its impact on Tn5 transposition in vivo by binding directly to the transpososome. Consistent with this possibility, we show that two different end mutations reduce the sensitivity of the Tn5 system to H-NS regulation.H-NS typically regulates cellular functions through its potent transcriptional repressor function. Work presented here provides support for an alternative mechanism of H-NS-based regulation, and adds to our understanding of how bacterial transposition can be regulated.Most bacteria harbor a variety of different types of transposons [1]. While transposons can compromise genome stability through the various types of DNA rearrangements they promote, they can also confer a selective advantage to their hosts. This can come about through transposons acquiring genes that encode resistance to antibiotics and other environmental toxins, or through transposon insertion events that alter the expression of key host genes. In order for transposons and their hosts to coexist, transposition levels must be tightly regulated [2]. There are several examples where host proteins have been co-opted to down-regulate transposition. For instance, Dam methylase of E. coli methylates GATC sequences found in both the promoters controlling the expression of some transposase genes and