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Bacterial Transcription Terminator and Helpers Silence Foreign DNA, May Contribute to Bacterial 'Immunity'

NEW YORK (GenomeWeb News) – A team of researchers has found three proteins that contribute to something akin to bacterial “immunity” in Escherichia coli, halting transcription before foreign DNA is transcribed.
Researchers from Columbia University, New York University, and Rutgers, the State University of New Jersey, made the find while investigating the role of a transcription termination factor called Rho in E. coli. Their results, which appeared online today in Science, suggest that in the absence of Rho, the expression of both intergenic regions and foreign genes — acquired from viruses or horizontal gene transfer — increases.
“That was the big surprise, of course,” co-senior author Max Gottesman, a microbiologist and biochemist at Columbia University, told GenomeWeb Daily News.
Some 90 percent of the E. coli genome consists of protein-coding sequence. And this tightly packed genetic information necessitates precise transcriptional control. About half of E. coli’s transcriptional units have a hairpin structure that seems to kick off the polymerase elongation complex. But the RNA-DNA helicase Rho factor is thought to be responsible for terminating transcription for the other half of the microbe’s transcriptional units.
In an effort to better understand Rho’s role, Gottesman and his colleagues used an antibiotic called bicyclomycin, or BCM, to specifically inhibit the protein. The team directly compared gene expression before and after Rho is turned off and between different E. coli strains, Gottesman explained, using the Affymetrix E. coli Genome 2.0 array.
The researchers found that when they inhibited Rho in the laboratory strain K-12 MG1655, the expression of genes acquired by recent horizontal transfer from other species or from viruses that infect bacteria, called bacteriophages, increased dramatically.
This is crucial, Gottesman explained. Cryptic prophages have strong promoters, Rho terminators, and downstream lethal genes. “It’s very important to prevent the read-through into these lethal genes,” he said.
They noted that some 14 percent to 18 percent of the K-12 strain’s genome differs from other E. coli strains. And most of these tend to cluster in blocks or “K islands” that also tend to contain defective prophages, transposons, and insertion sequences. For example, K-12 and an enterohemorrhagic strain called O157:H7 (EDL933) shared more than 3,600 nearly identical genes. But the K-12 strain’s genome housed nearly 650 genes that were absent in EDL933, while EDL933 contained 1,769 unique genes.
The expression of about half of the genes that were unique to one strain or the other increased dramatically — three-fold or more — following Rho inhibition. In contrast, about a quarter of genes shared by the two strains showed similar up-regulation in the absence of Rho. Inhibiting Rho also ramped up the expression of nearly three-quarters of the non-coding regions between genes measured.
“The first major role of Rho is to adjust the levels of transcription to the translational needs of the bacteria,” New York University biochemist Evgeny Nudler, a senior author on the paper, said in a statement. “Rho is needed for survival because it appears to keep potentially toxic genes at bay.”
As such, the system may contribute to a sort of bacterial immunity against invaders. “Rho-dependent termination may represent a separate ‘immunity’ system that protects bacterial cells from the harmful activity of certain foreign genes,” the authors wrote. “The existence of such different defensive tools against new acquisitions to the genome underscores the importance of this phenomenon for bacterial evolution.”
Next, the team investigated whether the changes they saw in gene expression spilled over into the proteome. Using a combination of difference gel electrophoresis and mass spectrometry, the researchers assessed how BCM treatment affected protein content in the K-12 MG1655 cells. Somewhat unexpectedly, the protein content did not shift as dramatically as expected based on the transcriptional changes observed. Just 101 of 3,341 spots analyzed had increased two-fold or more following Rho inhibition, while eight decreased.
While Gottesman admits that it’s possible that the team “missed our window” for catching increased protein content, he said it is very possible that this effect is genuine. “It could be that there are regulatory mechanisms to keep protein levels low,” he said. If so, such regulatory mechanisms could rein in translation even when transcription is ramped up in certain regions of the genome.
Even so, some proteins did light up, Gottesman said, including the prophage protein RecE, which may contribute to toxicity in the absence of Rho.
Indeed, such toxicity does seem to be dependent on foreign DNA: when the researchers knocked out foreign DNA — such as horizontally transferred genes and cryptic prophages — E. coli cells grew better in the presence of BCM. And in the absence of this foreign DNA, the cells no longer needed two Rho co-factors called NusA and NusG.

This realization not only sheds light on the transcription process, Gottesman said, but also gives researchers the opportunity to better understand the roles of NusA and NusB, since they now know that they can knock out the two co-factors if cells lack cryptic prophages or other foreign genes.

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