Host Factor Titration by Chromosomal R-loops as a Mechanism for Runaway Plasmid Replication in Transcription Termination-defective Mutants of Escherichia coli

Compromised Factor-Dependent Transcription Termination in a nusA Mutant of Escherichia coli: Spectrum of Termination Efficiencies Generated by Perturbations of Rho, NusG, NusA, and H-NS Family Proteins

“…As described earlier (29,47), the dominant-negative H-NS⌬64 variant (that comprises the N-terminal segment of 63 amino acids, from the 137-aminoacid native protein) restores transcriptional polarity in the rho(A243E) or nusG(G146D) mutants. In the present study, we obtained suppressors of the hns effect on rho and nusG, by selection for mutants in these backgrounds in which polarity relief had been reestablished at trp and lac loci bearing polar mutations in the first genes of each of the two operons (trpE9777 [frameshift] and lacZ U118 [amber], respectively).…”

“…The strains of E. coli used in the present study are listed in Table 1. Phage NK1324, used for Tn10dCm Genotype b a Strains MC4100, RS445, GJ5147, GJ6504, GJ6509, and GJ6511 have been described in the accompanying study (47), and strain GJ3119 was described by Harinarayanan and Gowrishankar (29). Strains GJ6520 and GJ6524 were constructed by S. Aisha (unpublished); all other strains were constructed in this study.…”

“…Spectinomycin (Sp) supplementation was at 50 g/ml. Several plasmids were included (with salient features in parentheses): pACYC184 (p15A vector, Cm r Tet r [12]), pLG339 and pLG-H-NS⌬64 (pSC101 replicon vector and its derivative encoding H-NS⌬64, respectively, Kan r [56]), pCP20 (pSC101-based Ts replicon encoding Flp recombinase, Amp r [18]), pHYD2509 (pSC101 replicon encoding H-NS⌬64, Amp r [47]), pHYD2546 (p15A replicon carrying ydgT, Cm r [47]), and two plasmids that are IPTG dependent for replication (29), pHYD751 (nusG ϩ , Amp r ) and pHYD1201 (rho ϩ , Amp r ). The following three plasmids with nusA ϩ were used: pHYD2554 (Amp r , carrying the 10-kb EcoRI-HindIII fragment between kilobase coordinates 3310.06 and 3320.08 of the E. coli genome [45] cloned in a ColE1-based replicon, and obtained from Manjula Reddy); pHYD2557 (Cm r , carrying a 2.3-kb PCRamplified region between genomic nucleotide coordinates 3314061 and 3316393 [45] cloned in a pSC101-based Ts replicon and obtained from Ranjan Sen); and pHYD2556 (Sp r , carrying the minimal nusA ϩ open reading frame with its native ribosome-binding site between genomic nucleotide coordinates 3314061and 3315548 [45] cloned downstream of the ara regulatory region in a pSC101-based replicon and obtained from Ranjan Sen).…”

“…The mechanism is absolutely dependent on the essential Rho protein that binds the nascent transcript and is then believed to signal RNA polymerase to terminate transcription, but there is no consensus as yet on its details (reviewed in references 1,3,14,40,41,43, and 48; see also references 21 and 31). As with their effects on transcription elongation, NusA and NusG have been reported to function antagonistically for Rho-dependent transcription termination as well, with NusG increasing its efficiency (7,8,11,29,35,38,52) and NusA decreasing it (8,27,(32)(33)(34); indeed, it has been reported that mutations in rho can suppress the inviability associated with the loss of NusA (58). On the other hand, Gottesman and coworkers (9,(52)(53)(54) have suggested that both NusA and NusG act cooperatively to promote Rho-dependent termination.…”

“…In the present study, we identified a recessive substitution mutation in nusA that confers relief of transcriptional polarity (much like missense mutations in rho or nusG) but has no apparent effect on transcription elongation or antitermination in vivo. The nusA(R258C) mutation (in the KH1 domain) was synthetically lethal with rho or nusG mutations, and the lethality could be suppressed by certain perturbations of the H-NS family of nucleoid proteins that have earlier been shown (29,47) to restore the efficiency of tran-scription termination in rho and nusG mutants (for reviews of the H-NS family proteins, see references 19, 20, 22, and 36). Our data lend support to the findings of Cardinale et al (9) that NusA promotes factor-dependent transcription termination.…”