A Novel AT-Rich DNA Recognition Mechanism for Bacterial Xenogeneic Silencer MvaT

Abstract

Bacterial xenogeneic silencing proteins selectively bind to and silence expression from many AT rich regions of the chromosome. They serve as master regulators of horizontally acquired DNA, including a large number of virulence genes. To date, three distinct families of xenogeneic silencers have been identified: H-NS of Proteobacteria, Lsr2 of the Actinomycetes, and MvaT of Pseudomonas sp. Although H-NS and Lsr2 family proteins are structurally different, they all recognize the AT-rich DNA minor groove through a common AT-hook-like motif, which is absent in the MvaT family. Thus, the DNA binding mechanism of MvaT has not been determined. Here, we report the characteristics of DNA sequences targeted by MvaT with protein binding microarrays, which indicates that MvaT prefers binding flexible DNA sequences with multiple TpA steps. We demonstrate that there are clear differences in sequence preferences between MvaT and the other two xenogeneic silencer families. We also determined the structure of the DNA-binding domain of MvaT in complex with a high affinity DNA dodecamer using solution NMR. This is the first experimental structure of a xenogeneic silencer in complex with DNA, which reveals that MvaT recognizes the AT-rich DNA both through base readout by an “AT-pincer” motif inserted into the minor groove and through shape readout by multiple lysine side chains interacting with the DNA sugar-phosphate backbone. Mutations of key MvaT residues for DNA binding confirm their importance with both in vitro and in vivo assays. This novel DNA binding mode enables MvaT to better tolerate GC-base pair interruptions in the binding site and less prefer A tract DNA when compared to H-NS and Lsr2. Comparison of MvaT with other bacterial xenogeneic silencers provides a clear picture that nature has evolved unique solutions for different bacterial genera to distinguish foreign from self DNA.

MvaT Family Proteins Encoded on IncP-7 Plasmid pCAR1 and the Host Chromosome Regulate the Host Transcriptome Cooperatively but Differently

“…Regarding possibility iv, the coupling ratio of TurB-Pmr was found to be lower than those of Pmr-Pmr and TurA-Pmr (9). Recently, Ding and colleagues identified the important residues for DNA binding in MvaT of Pseudomonas aeruginosa PAO1 (10). Considering that these residues are completely conserved in TurA but not in Pmr or TurB (see Fig.…”

“…Thus, we decided to use KT2440(pCAR1) derivatives expressing C-terminally His-tagged forms of Pmr, TurA, and TurB instead of the corresponding intact forms. It should be noted that the C-terminal His tags were not likely to affect DNA-binding ability or the sequence preference of the three proteins, because the C terminus of MvaT is not involved in DNA binding (10). Although the genome-wide Pmr-binding regions were determined previously (18), we again obtained the ChAP-chip data on Pmr-binding regions accompanied by those on TurA-and TurB-binding regions, because in this study we modified the method of ChAP-chip analysis to incorporate a different sonication system.…”

“…are recognized as members of the H-NS family because they can complement Escherichia coli H-NS-deficient phenotypes (5)(6)(7)(8)(9). H-NS and MvaT homologs share similar characteristics (e.g., dimerization, oligomerization, and DNA-binding mechanisms) (6)(7)(8), although the regions important for homo-oligomerization and DNA sequence preference are different (9,10). Pseudomonas putida KT2440 (11) possesses five genes encoding MvaT proteins, designated turA (PP_1366), turB (PP_3765), turC (PP_0017), turD (PP_3693), and turE (PP_2947) (12).…”

Cell cycle progression inCaulobacterrequires a nucleoid-associated protein with high AT sequence recognition

“…This result, in light of the correlation between GapR ChIP-seq enrichment and local AT richness, suggests that the association of GapR with DNA is specified by local AT content rather than by an explicit consensus binding sequence. Indeed, DNA-associated proteins with generic affinity for AT-rich sequences have been described in various prokaryotic and eukaryotic organisms, including mammals (26)(27)(28)(29)(30)(31)(32).…”

Silencing of cryptic prophages inCorynebacterium glutamicum

“…This mechanism relies on the function of small nucleoid-associated proteins (NAPs) to target and inhibit the expression of foreign DNA, which is recognizable by its typically higher AT content in comparison to the host genome (1,14). The major role of XS proteins is the binding of foreign DNA elements and the inhibition of transcription by a complex formation of AT-rich DNA stretches causing either the occlusion or trapping of the RNA polymerase (15,16). Currently known XS proteins belong to one of four classes, consisting of H-NS-type proteins found in several proteobacteria (12,17), Lsr2-like proteins of the actinomycetes (18), MvaT of Pseudomonas species (16) and Rok of Bacillus subtilis (19).…”