Nucleoid-associated proteins (NAPs) are DNA-binding proteins critical for the organization and function of the bacterial chromosome. A subclass of NAPs, including Caulobacter crescentus GapR and Escherichia coli H-NS, preferentially bind AT-rich regions of the nucleoid, but phylogenetic groups that encode GapR rarely encode H-NS. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recent DNA-bound crystal structure of GapR (Guo et al, 2018), we show that although evolutionarily distant, GapR and H-NS possess two regions that are structurally and functionally conserved. These regions are involved in self-association and DNA-binding, even though the two proteins oligomerize and regulate transcription differently. Functional analysis of GapR and H-NS protein chimeras identified structural elements present in H-NS but absent in GapR that rationalize differences in transcriptional regulation. In addition, we identified a sequence element unique to GapR that enables assembly into its tetrameric state. Using fluid-atomic force microscopy, we showed that GapR is capable of bridging DNA molecules in vitro. Together, these results demonstrate that two distantly related NAPs utilize evolutionarily conserved structural elements to serve specialized cellular roles via distinct mechanisms. nucleoid structuring protein H-NS is remarkable due to its ability to silence AT-rich sequences (3-7).By spreading across promoter sequences, H-NS is thought to prevent RNA polymerase from accessing these regions (5). Further, H-NS filaments assemble at sites of Rho-dependent termination, where they bridge segments of the nucleoid and contribute to both the pausing and termination of transcription (8).Members of the H-NS family have been widely recognized in the β and γ subdivisions of Proteobacteria (9). Even though some NAPs in distantly related bacteria, such as Lsr2 in Mycobacterium tuberculosis and the Rok protein in Bacillus subtilis, display low sequence similarity to H-NS, they exhibit the same preference for DNA with high AT content and can functionally replace H-NS in Escherichia coli (10-12). In addition, Lsr2 bridges DNA as does H-NS (13). Regardless of whether Lsr2 and the Rok protein are H-NS orthologs or represent convergent evolution, NAPs that specifically recognize AT-rich sequences are more widely spread in bacteria than can be assumed by only considering proteins with clear sequence similarity to H-NS.GapR, a newly discovered NAP with binding preference for regions with high AT content, was identified in the asymmetrically dividing bacterium Caulobacter crescentus and was found to have a broad distribution in the α subdivision of Proteobacteria (14-17). Interestingly, over-occupation of high AT chromosome sites in C. crescentus due to high expression of either GapR or H-NS from E coli, causes morphological and division defects (16). However, association of GapR with these sites does not lead to downregulation of gene expression (16). Furthermore, gapR expressed in E. coli fails to restore t...
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