A DNA-binding protein apparently silences foreign DNA in Salmonella and perhaps other bacteria, helping regulate what they incorporate into their genomes, researchers reported in the June 8 online edition of Science.
This protein and other molecules could help protect against the potentially harmful effects of horizontal gene transfer while leaving open the door for beneficial genes to eventually get expressed, according to the paper. By helping bacteria distinguish between self- and foreign DNA, they "might be viewed as a primitive immune system," study author Ferric Fang at University of Washington in Seattle told The Scientist.
The researchers investigated H-NS (histone-like nucleoid structuring protein), which belongs to a family of small Gram-negative bacterial proteins that bind to DNA with relatively low sequence specificity -- important to any potential role in suppressing foreign DNA, which can be varied in sequence. Prior experiments showed H-NS can behave as a...
Fang and his colleagues identified genes H-NS regulates by comparing transcript levels between wild-type and hns-null strains of Salmonella via cDNA microarray analysis. In hns-null mutants, they saw transcript levels of 178 open reading frames (ORFs) were reduced by more than three-fold, including ones for mobility and chemotaxis, while 409 transcripts were more abundant, such as genes for virulence.
Of the 409 transcripts H-NS normally represses, nearly 65% seem acquired from foreign sources, as they are not universally present in Salmonella's close relatives. They also possess significantly reduced levels of guanine and cytosine (GC). While the entire Salmonella genome's average GC content is 52.2%, transcripts displaying three-fold or greater repression by H-NS had on average 46.8% GC. Chromatin immunoprecipitation (ChIP)-on-chip assays showed 740 of 745 H-NS binding sites surveyed were within 1,000 nucleotides of regions with 49% or less average GC content.
To see whether H-NS targeted presumably foreign sequences lower in GC and richer in adenine and thymine (AT), the researchers recombined a Helicobacter pylori gene with its promoter into a non-essential region of the Salmonella chromosome with an average GC content greater than 50% and no demonstrable interaction with H-NS. This gene, hp0226, was 39.7% GC.
Reverse transcriptase quantitative PCR (Q-PCR) analysis of transcript levels revealed wild-type strains repressed hp0226 expression more than 15-fold compared with hns-null mutants. Chromatin precipitation/Q-PCR found H-NS associated significantly with hp0226 but not with adjacent gene stm1033, which was 52.5% GC. This suggests H-NS binding silences AT-rich sequences.
The genome-wide average of GC content can vary from 25% to 75% between bacterial genera. "The use of AT content to distinguish 'self' from 'foreign' DNA may help to explain why different bacterial species retain characteristic AT/GC ratios," Fang said.
The roles of H-NS have been varied and confusing for years, and these findings provide "a platform to rationally tie many of these varied processes together," Stanley Maloy at San Diego State University in California, who did not participate in this study, told The Scientist. After hns-expressing bacteria incorporate foreign DNA into their genomes, Fang suggested H-NS silences it until counteracted by other proteins such as SlyA or Ler. Investigators should analyze how other molecules counter H-NS, such as competition with H-NS, changing the structure of DNA to lower H-NS affinity for it, or physical interaction with H-NS, Linda Kenney at the University of Illinois at Chicago, also not a co-author, told The Scientist.
The primary sequence of H-NS primary sequence is poorly conserved outside of enteric bacteria and close relatives such as Vibrio bacteria, which include cholera. Future experiments should uncover the mechanisms underlying H-NS recognition of AT-rich sequences, to perhaps discover proteins in other bacteria with similar structures and roles, Fang said. "It will be interesting to determine whether AT-rich bacteria have another type of protein that recognizes and silences GC-rich DNA," he added.
H-NS has a higher affinity for curved DNA. Experiments should investigate growth conditions that might affect DNA curvature and see whether H-NS completely silences genes or just down-regulates them, Charles Dorman at Trinity College Dublin, who did not participate in this study, told The Scientist.