Fig 1.
RNA degradation causes chromosomal fragmentation.
(A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse If and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse If, 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.
Table 1.
Strains used in this study.
Fig 2.
(A) Representative radiogram showing RNase dose dependent chromosomal fragmentation in AB1157. Plugs were made with 0, 2, 10, 25, 50 or 100 μg RNase and lysed and electrophoresed under standard conditions. CZ, compression zone. (B) Quantification showing increase in chromosomal fragmentation in RNase dose-dependent manner. Data points are means of six independent assays ± SEM. (C) RNase-effect is not seen in the pre-lyzed cells. Plugs from AB1157 culture were made in the presence of proteinase K, but without any RNase. After overnight lysis and extensive washing, the plugs were incubated with 0, 2, 20 and 100 μg RNase or 100 U of EcoRI for 15 H at 37°C before PFGE. (D) Quantification of chromosomal fragmentation showing extreme sensitivity of chromosomes to EcoRI, but not RNase, when plugs were treated with the enzymes after lysis of cells. The experiment is done twice and a representative result is presented. (E) A representative radiogram showing kinetics of RiCF. Multiple plugs were made in the presence of RNase (50 μg/plug) and incubated at 62°C for 10, 30, 60, 180 or 900 minutes with lysis buffer in individual tubes. At the indicated times, one tube was removed, lysis buffer was replaced with ice-cold TE, and plugs were stored at 4°C until all plugs were ready for electrophoresis. (F) Quantification of kinetics of chromosomal fragmentation when plugs were made in the presence of RNase and lysed for 1, 5, 10, 30, 60, 180 or 900 minutes. Data points are means of three independent assays ± SEM. Arrow shows the value of fragmentation after 10 min lysis.
Fig 3.
Effect of the growth parameters on RiCF.
(A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.
Fig 4.
(A) Quantitative determination of RiCF in Δhns mutant. AB1157 and SRK254 were grown at 37°C to the same final OD, and plugs were made in the absence of proteinase K, but with or without RNase (50 μg/plug). After overnight incubation in the lysis buffer at 62°C, the plugs were electrophoresed under standard conditions. Data points are means of 6–10 independent assays± SEM. (B) Radiogram of a representative pulsed field gel from which data in (A) are calculated. (C) Comparison of RiCF in ΔhupA ΔhupB and ΔihfA ΔihfB double mutants. Experiment was done as described in (A), and values presented are means of 6–13 independent assays ± SEM. (D) Radiogram of a representative pulsed field gel from which data in (C) are calculated. (E) Effect of hns deletion on RiCF of ΔihfA ΔihfB mutant. Values presented are means of 7 independent assays ± SEM.
Fig 5.
Non-coding RNA and HU stabilize nucleoids.
(A) Comparison of spontaneous and RNase-induced fragmentation in Δnc1 Δnc5 ΔihfA ΔihfB, Δnc1 Δnc5 ΔhupA ΔhupB and Δnc1 Δnc5 Δhns mutants. AB1157, SRK254-12, SRK254-15 and SRK254-18 were grown at 37°C, and plugs were made in absence of proteinase K, both with or without RNase (50 μg/plug), and lysed under standard conditions. Data points are means of at least three independent assays± SEM. (B) Radiogram of a representative gel from which data in (A) are generated.
Fig 6.
Endonuclease I mediated RNA degradation causes RiCF.
(A) Schematics of a hypothetical scenario when RNase activates a DNase by degrading an inhibitory RNA. Activated DNase, in turn, attacks chromosomal DNA causing its fragmentation. (B) Quantitative determination of RiCF in ΔendA and ΔrnaA mutants. The strains were grown at 37°C to the same final OD, and plugs were made in the absence or presence of RNase (50 μg/plug). Data points are means of 4–8 independent assays ± SEM. (C) Radiogram of a representative pulsed field gel from which data in (B) are calculated. (D) Comparison of RiCF in Δrna mutants in two genetic backgrounds. AB1157, BW25113 and their Δrna mutants were grown and processed as described in (A). Values presented are means of 3–8 independent assays ± SEM. (E) Radiogram of a representative pulsed field gel from which data in (D) are derived.
Fig 7.
Endonuclease-I is critical for RiCF but not for spontaneous fragmentation.
(A) Comparison of spontaneous fragmentation and RiCF in AB1157, AB1157 Δnc1 Δnc5 ΔhupA ΔhupB and AB1157 Δnc1 Δnc5 ΔhupA ΔhupB ΔendA mutants. All strains were grown at 37°C to the final OD of 0.6, and plugs were made in the absence of proteinase K, both with or without RNase (50 μg/plug). Data points are means of at least three independent assays ± SEM. (B) Radiogram of a representative gel from which data in (A) are generated.
Fig 8.
RiCF is generated by activation of endonuclease I. When cells are embedded in agarose plugs without RNase (scenario A), disintegration of plasma membrane during lysis causes release of cytoplasmic RNA that inactivates periplasmic endonuclease I and saves the chromosomes from degradation. RNAse, when present in close proximity of the lysing cells (scenario B), degrades the released cytoplasmic RNA avoiding endonuclease I inhibition. Endonuclease I, in the presence of divalent cations, cuts the chromosomal DNA, but fails to degrade the chromosomes completely because of its prompt inactivation in lysis conditions.