Silencing by H-NS potentiated the evolution of Salmonella.

The bacterial H-NS protein silences expression from sequences with higher AT-content than the host genome and is believed to buffer the fitness consequences associated with foreign gene acquisition. Loss of H-NS results in severe growth defects in Salmonella, but the underlying reasons were unclear. An experimental evolution approach was employed to determine which secondary mutations could compensate for the loss of H-NS in Salmonella. Six independently derived S. Typhimurium hns mutant strains were serially passaged for 300 generations prior to whole genome sequencing. Growth rates of all lineages dramatically improved during the course of the experiment. Each of the hns mutant lineages acquired missense mutations in the gene encoding the H-NS paralog StpA encoding a poorly understood H-NS paralog, while 5 of the mutant lineages acquired deletions in the genes encoding the Salmonella Pathogenicity Island-1 (SPI-1) Type 3 secretion system critical to invoke inflammation. We further demonstrate that SPI-1 misregulation is a primary contributor to the decreased fitness in Salmonella hns mutants. Three of the lineages acquired additional loss of function mutations in the PhoPQ virulence regulatory system. Similarly passaged wild type Salmonella lineages did not acquire these mutations. The stpA missense mutations arose in the oligomerization domain and generated proteins that could compensate for the loss of H-NS to varying degrees. StpA variants most able to functionally substitute for H-NS displayed altered DNA binding and oligomerization properties that resembled those of H-NS. These findings indicate that H-NS was central to the evolution of the Salmonellae by buffering the negative fitness consequences caused by the secretion system that is the defining characteristic of the species.

A biomechanical mechanism for initiating DNA packaging.

The bacterial chromosome is under varying levels of mechanical stress due to a high degree of crowding and dynamic protein-DNA interactions experienced within the nucleoid. DNA tension is difficult to measure in cells and its functional significance remains unclear although in vitro experiments have implicated a range of biomechanical phenomena. Using single-molecule tools, we have uncovered a novel protein-DNA interaction that responds to fluctuations in mechanical tension by condensing DNA. We combined tethered particle motion (TPM) and optical tweezers experiments to probe the effects of tension on DNA in the presence of the Hha/H-NS complex. The nucleoid structuring protein H-NS is a key regulator of DNA condensation and gene expression in enterobacteria and its activity in vivo is affected by the accessory factor Hha. We find that tension, induced by optical tweezers, causes the rapid compaction of DNA in the presence of the Hha/H-NS complex, but not in the presence of H-NS alone. Our results imply that H-NS requires Hha to condense bacterial DNA and that this condensation could be triggered by the level of mechanical tension experienced along different regions of the chromosome.

Empirical neuroenchantment: from reading minds to thinking critically.

While most experts agree on the limitations of neuroimaging, the unversed public-and indeed many a scholar-often valorizes brain imaging without heeding its shortcomings. Here we test the boundaries of this phenomenon, which we term neuroenchantment. How much are individuals ready to believe when encountering improbable information through the guise of neuroscience? We introduced participants to a crudely-built mock brain scanner, explaining that the machine would measure neural activity, analyze the data, and then infer the content of complex thoughts. Using a classic magic trick, we crafted an illusion whereby the imaging technology seemed to decipher the internal thoughts of participants. We found that most students-even undergraduates with advanced standing in neuroscience and psychology, who have been taught the shortcomings of neuroimaging-deemed such unlikely technology highly plausible. Our findings highlight the influence neuro-hype wields over critical thinking.

Structural insights into the regulation of foreign genes in Salmonella by the Hha/H-NS complex.

BACKGROUND: Hha facilitates H-NS-mediated silencing of foreign genes in bacteria. RESULTS: Two Hha monomers bind opposing faces of the H-NS N-terminal dimerization domain. CONCLUSION: Hha binds the dimerization domain of H-NS and may contact DNA via positively charged surface residues. SIGNIFICANCE: The structure of Hha and H-NS in complex provides a mechanistic model of how Hha may affect gene regulation. The bacterial nucleoid-associated proteins Hha and H-NS jointly repress horizontally acquired genes in Salmonella, including essential virulence loci encoded within Salmonella pathogenicity islands. Hha is known to interact with the N-terminal dimerization domain of H-NS; however, the manner in which this interaction enhances transcriptional silencing is not understood. To further understand this process, we solved the x-ray crystal structure of Hha in complex with the N-terminal dimerization domain of H-NS (H-NS(1-46)) to 3.2 Å resolution. Two monomers of Hha bind to symmetrical sites on either side of the H-NS(1-46) dimer. Disruption of the Hha/H-NS interaction by the H-NS site-specific mutation I11A results in increased expression of the Hha/H-NS co-regulated gene hilA without affecting the expression levels of proV, a target gene repressed by H-NS in an Hha-independent fashion. Examination of the structure revealed a cluster of conserved basic amino acids that protrude from the surface of Hha on the opposite side of the Hha/H-NS(1-46) interface. Hha mutants with a diminished positively charged surface maintain the ability to interact with H-NS but can no longer regulate hilA. Increased expression of the hilA locus did not correspond to significant depletion of H-NS at the promoter region in chromatin immunoprecipitation assays. However, in vitro, we find Hha improves H-NS binding to target DNA fragments. Taken together, our results show for the first time how Hha and H-NS interact to direct transcriptional repression and reveal that a positively charged surface of Hha enhances the silencing activity of H-NS nucleoprotein filaments.

Silencing of foreign DNA in bacteria.

Xenogeneic silencing proteins facilitate horizontal gene transfer by silencing expression of AT-rich sequences. By virtue of their activity these proteins serve as master regulators of a variety of important functions including motility, drug resistance, and virulence. Three families of silencers have been identified to date: the H-NS like proteins of Gram-negative bacteria, the MvaT like proteins of Pseudomonacae, and the Lsr2 proteins of Actinobacteria. Structural and biochemical characterization of these proteins have revealed that they share surprising commonalities in mechanism and function despite extensive divergence in both sequence and structure. Here we discuss the mechanisms that underlie the ability of these proteins to selectively target AT-rich DNA and the contradictory data regarding the mode by which H-NS forms nucleoprotein complexes.

The 5.5 protein of phage T7 inhibits H-NS through interactions with the central oligomerization domain.

The 5.5 protein (T7p32) of coliphage T7 (5.5(T7)) was shown to bind and inhibit gene silencing by the nucleoid-associated protein H-NS, but the mechanism by which it acts was not understood. The 5.5(T7) protein is insoluble when expressed in Escherichia coli, but we find that 5.5(T7) can be isolated in a soluble form when coexpressed with a truncated version of H-NS followed by subsequent disruption of the complex during anion-exchange chromatography. Association studies reveal that 5.5(T7) binds a region of H-NS (residues 60 to 80) recently found to contain a distinct domain necessary for higher-order H-NS oligomerization. Accordingly, we find that purified 5.5(T7) can disrupt higher-order H-NS-DNA complexes in vitro but does not abolish DNA binding by H-NS per se. Homologues of the 5.5(T7) protein are found exclusively among members of the Autographivirinae that infect enteric bacteria, and despite fairly low sequence conservation, the H-NS binding properties of these proteins are largely conserved. Unexpectedly, we find that the 5.5(T7) protein copurifies with heterogeneous low-molecular-weight RNA, likely tRNA, through several chromatography steps and that this interaction does not require the DNA binding domain of H-NS. The 5.5 proteins utilize a previously undescribed mechanism of H-NS antagonism that further highlights the critical importance that higher-order oligomerization plays in H-NS-mediated gene repression.