Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli

Abstract

Transcription of the bacterial genome by the RNA polymerase must terminate at specific points. Transcription can be terminated by Rho factor, an essential protein in enterobacteria. We used the antibiotic bicyclomycin, which inhibits Rho, to assess its role on a genome-wide scale. Rho is revealed as a global regulator of gene expression that matches Escherichia coli transcription to translational needs. We also found that genes in E. coli that are most repressed by Rho are prophages and other horizontally acquired portions of the genome. Elimination of these foreign DNA elements increases resistance to bicyclomycin. Although rho remains essential, such reduced-genome bacteria no longer require Rho cofactors NusA and NusG. Deletion of the cryptic rac prophage in wild-type E. coli increases bicyclomycin resistance and permits deletion of nusG. Thus, Rho termination, supported by NusA and NusG, is required to suppress the toxic activity of foreign genes.

Fig. 1. Genomic response of divergent E. coli strains to Rho inhibition

(A) Hierarchical cluster analysis of a concentration gradient of BCM (doses of 10, 25, and 100 μg/ml) in E. coli K-12 strain MG1655 showing only genes orthologous between K-12 and enterohemorrhagic E. coli. Arrays (columns) are shown in biological duplicates, normalized so that average of each gene on the untreated control arrays is equal to 1 and expression in treated cultures is displayed as a ratio of treated to untreated. Yellow blocks represent upregulation by BCM and blue represents downregulation. (B) Response to BCM of genes present in K-12 E. coli but absent from enterohemorrhagic E. coli, displayed as in (A). (C) Hierarchical cluster analysis of the response of orthologous genes in enterohemorrhagic E. coli O157:H7 strain EDL933. (D) Response to BCM treatment of genes present in enterohemorrhagic E. coli but absent from K-12. (E) Expression of ORFs in response to BCM displayed as a scatterplot of probe intensity in the control array (X-axis) and BCM-treated array (Y-axis) from a representative pair of arrays. The diagonal line represents equal probe hybridization intensity between both arrays; points above the diagonal are genes upregulated by treatment with BCM and points below the diagonal are downregulated. The red lines located at 100 intensity units represent the threshold below which probe-level analysis is 90% likely to call the probe absent. Therefore, probes in the upper left quadrant are ORFs whose expression was induced de novo. Grey points are orthologous genes and violet points are K-12-specific genes. (F) Scatterplot of probe intensity for intergenic (IG) regions of MG1655 after treatment by BCM.

Fig. 2. Proteomic response to Rho inhibition as detected by difference gel electrophoresis (DIGE)

Two-dimensional electrophoresis gel of protein extracted from BCM-treated and control cultures. Control protein is pseudocolored green and BCM-treated protein as red. Differentially-expressed proteins (indicated by name) were identified by mass spectrometry.

Fig. 3. Reduced-genome E. coli is resistant to Rho inhibition and deletion of elongation factors NusA and NusG

(A) Hierarchical cluster analysis of ORF gene expression in strain MDS42, BCM-treated MDS42, MDS42 ΔnusA, and MDS42 ΔnusG. Probe intensity is normalized to the untreated MDS42 strain. (B) Efficiency of colony formation assay of the indicated strains. Cultures at dilutions of 10⁻², 10⁻⁴, and 10⁻⁶ were spotted onto a control plate or a plate containing BCM at 25 μg/ml. int-kilR and int-ydaE are fragments of the rac prophage that were deleted.

Figure 4. Effect of BCM on the leftward operons of rac and λ phages

(A) Map of the leftward operon of the rac prophage. Gray arrows: genes; open-headed arrows: PCR primers; brackets: deletions of intR-ydaE and intR-kil; bent arrow: the operon’s promoter, (P_RM). (B) Map of the homologous operon of λ phage. Dashed lines show proposed transcripts produced. (C) RT-PCR using primer pairs indicated on the map in panel A shows that BCM treatment yields an elongated transcript. RT: reverse transcriptase. (D) Average β-galactosidase activity (lacZ expression) from the phage strain shown in panel B. The standard error is less than 5%.