Hypernegative supercoiling inhibits growth by causing RNA degradation

Abstract

Transcription-induced hypernegative supercoiling is a hallmark of Escherichia coli topoisomerase I (topA) mutants. However, its physiological significance has remained unclear. Temperature downshift of a mutant yielded transient growth arrest and a parallel increase in hypernegative supercoiling that was more severe with lower temperature. Both properties were alleviated by overexpression of RNase HI. While ribosomes in extracts showed normal activity when obtained during growth arrest, mRNA on ribosomes was reduced for fis and shorter for crp, polysomes were much less abundant relative to monosomes, and protein synthesis rate dropped, as did the ratio of large to small proteins. Altered processing and degradation of lacA and fis mRNA was also observed. These data are consistent with truncation of mRNA during growth arrest. These effects were not affected by a mutation in the gene encoding RNase E, indicating that this endonuclease is not involved in the abnormal mRNA processing. They were also unaffected by spectinomycin, an inhibitor of protein synthesis, which argued against induction of RNase activity. In vitro transcription revealed that R-loop formation is more extensive on hypernegatively supercoiled templates. These results allow us, for the first time, to present a model by which hypernegative supercoiling inhibits growth. In this model, the introduction of hypernegative supercoiling by gyrase facilitates degradation of nascent RNA; overproduction of RNase HI limits the accumulation of hypernegative supercoiling, thereby preventing extensive RNA degradation.

FIG. 1.

Correlation between hypernegative supercoiling and growth inhibition after temperature downshifts. Cells were grown at 37°C to log phase as indicated in Materials and Methods before being transferred to 28°C (A), 26°C (B), or 21°C (C). Plasmid DNA from the topA-null strains was extracted just before the temperature downshift (time zero) and at the indicated times after the downshift. DNA was loaded on an agarose gel with 7.5 μg of chloroquine/ml and probed to reveal hypernegatively supercoiled pMD306 (top panels). Note that at this chloroquine concentration, the more relaxed topoisomers migrate faster, except hypernegatively supercoiled DNA, which also migrates rapidly (indicated by [-]). The dot points to a signal reflecting a negative supercoiling-dependent structural transition that was previously observed for this plasmid following two-dimensional gel analysis (4, 31). On the top of the gels, “+” and “-” indicate, respectively, that RNase HI was overproduced (pEM001) or not overproduced (pEM003). The arrow on the growth curves (bottom) indicates the time when growth resumed following the downshifts. Symbols: ⧫, VU7 [gyrB(Ts) topA/pMD306 and pEM001]; ▪, VU8 [gyrB(Ts) topA/pMD306 and pEM003]; ▴, RFM445 [gyrB(Ts)].

FIG. 2.

Association of yhdG and fis mRNAs with ribosomes after a temperature downshift. Cells were grown at 37°C to log phase as indicated in Materials and Methods before being transferred to 28°C. After 20 min, cells were recovered for sucrose gradient fractionation of ribosomes and mRNA analysis as described in Materials and Methods. Top panels show the ribosome profiles (A₂₆₀) with the numbers pointing to the peaks corresponding to monosomes (1, one ribosome per RNA) and to polysomes (2, 3, and 4; two or more ribosomes per RNA). The numbers below the ribosome profiles correspond to the different fractions from which the RNA was extracted. The middle panels show the ethidium bromide-stained gels with 23S and 16S rRNA. These gels were used for Northern blot analysis as described in Materials and Methods with probes corresponding to yhdG (a) or to fis (b). The strains used were MA249 [gyrB(Ts)], MA251 [gyrB(Ts) topA], and IB34 [gyrB(Ts) topA/pSK760]. + RNase HI indicates that RNase HI was overproduced (pSK760).

FIG. 3.

Association of crp mRNAs with ribosomes after a temperature downshift. Cells were grown and recovered for sucrose gradient fractionation of ribosomes and mRNA analysis as described in the legend to Fig. 2. The top, middle, and bottom panels are as described in the legend to Fig. 2. The probe used for the Northern blot analysis corresponds to crp.

FIG. 4.

Protein synthesis after a temperature downshift. Cells were grown at 37°C to log phase before being transferred to 28°C. After 20 min, l-[³⁵S]cysteine was added, and aliquots were recovered at different times to monitor protein synthesis as described in Materials and Methods. Samples were analyzed by polyacrylamide gel electrophoresis as described in Materials and Methods. Shown in panel a is the result of one experiment. The two areas delimited by the brackets, respectively, labeled large and small were chosen to calculate the large/small ratios graphically represented in panel b (three independent experiments). Symbols: □, MA249 [gyrB(Ts)]; ▵, MA251 [gyrB(Ts) topA]; ○, IB34 [gyrB(Ts) topA/pSK760].

FIG. 5.

Expression and processing of fis mRNAs after a temperature downshift. Cells were grown at 37°C to log phase as indicated in Materials and Methods before being transferred to 28°C. After 20 min, rifampin at 250 μg/ml (final concentration) was added to the cells, and the RNA was extracted at the indicated time (for time zero the RNA was extracted immediately before the addition of rifampin). Portions (10 μg) of the RNA samples were used for Northern blot analysis with an oligonucleotide probe hybridizing to fis as described in Materials and Methods. The strains used were MA249 [gyrB(Ts)], MA251 [gyrB(Ts) topA], and IB34 [gyrB(Ts) topA/pSK760]. + RNase HI indicates that RNase HI was overproduced (pSK760).

FIG. 6.

Expression and processing of lac mRNAs after a temperature downshift. Cells were grown at 37°C to log phase as indicated in Materials and Methods before being transferred to 28°C. At 5 min after this transfer, IPTG was added to 1 mM to induce lac expression from the chromosomal Ptrc-lac fusion, and 30 min later aliquots of the cell cultures were obtained for RNA extraction. The strains used were PS66 [gyrB(Ts) topA φ(lacI^q-Ptrc-lac)/pEM001] and PS68 [gyrB(Ts) topA φ(lacI^q-Ptrc-lac)/pEM003]. + and -, respectively, indicate that RNase HI was overproduced (pEM001) or not overproduced (pEM003). Fifteen μg of RNA were used for Northern blot analysis with random prime-labeled probes hybridizing to lacZ (top panel) or lacA (bottom panel) as described in Materials and Methods.

FIG. 7.

Effects of rneΔ14 and spectinomycin on the processing of lacA mRNAs after a temperature downshift. Cells were grown at 37°C to log phase as indicated in Materials and Methods. Spectinomycin (final concentration, 400 μg/ml) was added to one-half of the culture and 5 min later the cells were transferred to 28°C. Five minutes after this transfer, IPTG was added to 1 mM to induce lac expression from the chromosomal Ptrc-lac fusion, and thirty minutes later aliquots of the cell cultures were taken for RNA extraction. The strains used are PS63 [gyrB(Ts) topA φ(lacI^q-Ptrc-lac)], PS64 [gyrB(Ts) φ(lacI^q-Ptrc-lac)], PS123 [gyrB(Ts) topA φ(lacI^q-Ptrc-lac rneΔ14], and PS126 [gyrB(Ts) φ(lacI^q-Ptrc-lac rneΔ14]. Spc is spectinomycin.

FIG. 8.

R-loop formation on hypernegatively supercoiled templates in vitro. In vitro transcription reactions were performed as described in Materials and Methods in the presence of both [³²P]UTP and [³²P]ATP. Samples were loaded on an agarose gel without chloroquine. The bottom panel is the autoradiography of the gel to reveal RNA in R-loops. [-] indicates hypernegatively supercoiled templates.