Smith et al. – Page S1
Supplementary Materials
RESULTS
A supplementary assay for retrohoming
For purposes of clarity, we refer to the retrohoming assay of Cousineau et al. (1998) used in the body of the paper as assay A, and the supplementary assay of Guo et al. (2000), to be described below, as assay B. These two assays are compared in Supplementary Figure S1.
In assay B, used principally for verification of host effects, the donor plasmid expresses an unmarked intron that contains a phage T7 promoter (Fig. S1) (Guo et al. 2000). The recipient plasmid has a minimal Ll.LtrB target sequence (E1-E2, -30 to +15), followed by a promoterless tetracycline resistance gene (tet),and is drug sensitive (TetS). After integration of the intron into the target site, the T7 promoter in the intron drives the expression of the tet gene rendering the recombinant TetR. Homing products are then directly selected for TetR and the recipient PM2. The efficiency of assay B, in the 0.2%-100% range, as for assay A depends on whether the ltrA gene is in its native position in the intron, or whether it has been deleted from the intron and cloned downstream of exon 2 (Fig. S1, cf. crosses 4 and 5).
Assay B was performed as described (Guo et al. 2000; Karberg et al. 2001) and detailed below in Materials and Methods. Briefly, cotransformants with donor and recipient plasmids were induced for intron expression with IPTG, plated directly onto media containing tetracycline + ampicillin and incubated for 1 to 2 days.
Homing efficiencies are not strictly comparable between assays A and B, rather only within assays (Tables S1-S3). This is because in assay A, only individual retrohoming events occurring within 3 h of induction are scored as positive. In assay B, however, a single retrohoming event in a cell after one or two days of incubation may register as a positive, even if most intron recipients in that cell remain unoccupied, leading to a higher estimate of homing efficiencies relative to assay A (A. Beauregard and M.B., unpublished results). Furthermore, the intron is longer by up to four-fold in donors used in assay A compared with assay B. Nevertheless, in most cases there is good agreement on host effects between the two assays, and where not, the disparities can be explained by the difference in assay conditions, as discussed below.
Comparison of results with assays A and B
The most satisfying correspondence between assays A and B was the polymerase mutants. Retrohoming was reduced ~4-fold in the Pol III ts mutant at 37oC in both assays (Table S3). The Pol I 5'3' exo point mutant was also depressed about 4-fold in both assays and more dramatically in the deletion mutant, whereas homing was not affected in either assay in the Pol I Kklenow mutant, deficient in polymerase and 3'5' exo activities (Table S3), cf. crosses 1 and 6). Additionally, the two assays yielded similar results with respect to the repair polymerases (Table S3). Whereas retrohoming was reduced about 3-fold in the Pol II and Pol IV mutants in assay A and depressed slightly in assay B, the Pol V showed a 1.5- fold increase in both assays. In the Pol II/IV double mutant, homing was again reduced 3-fold in assay A but not in assay B. However, in the Pol II/V, IV/V and II/IV/V multiple mutants, homing was reduced ca. 10-fold in both assays. Furthermore, the in assays performed similarly in the ligase ts mutant, both showeding a four-fold reduction in homing at 37oC, and there was no change in either case in the RecG mutant (Table S1).
Some mutants did, however, give different results in assays A and B. The ribonucleases RNase I and E showed a 4- and 12-fold increase in homing levels, respectively in assay A, whereas in assay B, both showed only a ~2-fold increase (Table S2). This may reflect that the intron is susceptible to degradation at the RNA level, particularly with the lengthier intron used in assay A. Furthermore, the RNase E experiment was performed at 37oC in assay B, rather than 44oC, as was used for assay A, due to the nature of the assays. In assay A, plasmid DNA is extracted after a 3-h time frame of homing, and this DNA is retransformed into a non-ts host. However, with assay B, the experiment is performed in the ts host from beginning to end, so the cells do not maintain viability if kept at the non-permissive temperature for the time of the assay (24-48h).
In the RNase H mutants, for assay A, retrohoming was reduced >30-fold in the rnhA mutant, 2-fold in the rnhB mutant, and again >30-fold in the double mutant (Table S2). , cf. crosses 1). Again, the rnhA effect was less dramatic in assay B, where the RNase H1 mutant caused a 2-fold reduction with the lengthier intron donor (cross 4), but homing occurred at wild-type levels with the short intron donor (cross 5). However, the double mutant showed 3- to 5-fold effects with both introns (crosses 4 and 5). These results suggest not only that RNases H contributes to degradation of the intron RNA template, with RNaseH1 playing the greater role, but also that RNase H1 appears more important for longer templates, while either RNase H1 or RNase H2 appear to be capable of degrading shorter RNA templates. RNA degradation by other enzymes or RNA displacement during second-strand synthesis may contribute to the relaxed RNase H requirement for shorter RNA templates.
The RecJ, MutD and SbcD nucleases all showed about a 4-fold decrease in homing in assay A, but no effect in assay B (Table S1, cf. crosses 1 and 5). The remediative effects of RecJ, MutD and SbcCD may be more pronounced within the narrow time-window of assay A (3 h) than in the protracted assay B (24 to 48 h), in which the need for such nucleases may be suppressed by complementing activities. Likewise, a mutant in Exo III, the product of the xthA gene, showed a >8-fold elevation in retrohoming in assay A, but not assay B (Table S1, cf. cross 1 with 4 and 5). Again, the relatively extended time-frame of assay B may allow repair of degraded products, whereas this is precluded in the short time-window of assay A.
materials and methods
Plasmids used in this study
Plasmids for retrohoming assays are described relative to assay A, which was used for experiments in the main text (Figs. 2A, 2B, and S1A and S1C), and assay B, which is described appears in Supplementary Materials (Fig. S1B and C). Plasmids used for assay A were described previously (Cousineau et al. 1998). Briefly, the pLI1td+KR' twintron donor contains a kanR marker gene as well as a group I td intron for verification of the retrohoming pathway, cloned in loop IV of the Ll.LtrB intron in a pET11a (Novagen) backbone. The CamR pLHS1 plasmid carries the 271-bp HindIII fragment containing the Ll.LtrB homing site, as well as some flanking pET11a sequences in pSU18. Plasmids pSCLtrAtd+KR' and pRIA-HS used in cross 2 are similar to those used in cross 1, except that the replication origins were changed to those indicated in Figures 2B and S1C, to replicate in a polA host. Plasmid pSCLtrAtd+KR' was made by first, deleting the Col El origin from pLI1td+KR' by inverse PCR with oligonucleotides containing Bg1II ends, and then cloning in the pSC101 origin from pWSK29 on a BglII fragment. The pRIA-HS plasmid for cross 2 was made by linearizing pRIA vector with HindIII and cloning in the Ll.LtrB homing site on a 271-bp HindIII fragment to generate pRIA-LtrBHS. Plasmid pACD3-TpR8 for cross 3, containing a trimethoprim-resistant marker, is similar to pACD3-RAM (Zhong et al. 2003), but lacks the td intron insertion.
The donor plasmid pACD-LtrB for assay B that expresses the full-length intron (Fig. S1C, cross 4) and pACD2 (cross 5) that expresses the ΔORF intron with the IEP synthesized from downstream of the 3' exon were previously described (Guo et al. 2000; Karberg et al. 2001). Plasmids pACSD-LtrB and pACSD2, which were used for assays in CamR-resistant strains, are similar to pACD-LtrB and pACD2, respectively, but with a spectinomycin-resistant marker (spcR) in place of camR. To construct pACSD2 (cross 5), we amplified the spcR marker by PCR from pMN1343 (Cousineau et al. 1998) and cloned it between the blunted PflmI and Bsu36I sites of pACD2. Plasmid pACSD-LtrB (cross 4) was constructed by insertion of the ClaI/PshAI fragment of pACSD2 containing spcRat the same sites of pACD-LtrB. To construct pR1A-LtrB1 containing a Bhr replicon for replication in polA mutants, (cross 6), we cloned the blunted NotI/Bsu36I fragment of pBBR1MCS-2 (Kovach et al. 1995)between the PvuI and PshAI sites of pACD2, to generate pR1A. The blunted ClaI/PshAI fragment of pACD2 was then inserted at the blunted BsaWI site of pR1A, to generate pR1A-LtrB1. The recipient plasmids pBRR3-ltrB and pB101B-ltrB, containing a minimal Ll.LtrB intron target DNA sequence (-30 to +15), were described previously described(Zhong and Lambowitz 2003).
Homing assays
For the retrohoming assay used in the printed text (assay A), cotransformants with donor and recipient plasmids were grown with appropriate antibiotics (Cousineau et al. 1998). Different donor and recipient pairs are listed in Figures 2B and S1C. Antibiotics These were added at the following concentrations: ampicillin (100 g/ml), kanamycin (50 g/ml), chloramphenicol (25 g/ml), tetracycline (10 g/ml), or spectinomycin (50 g/ml), depending on the donor and recipient used (Fig. 2C2B and S1C). Overnight cultures were diluted 1/100 and grown to OD600 = 0.2, then induced with 2 mM IPTG for 3 h. Plasmid DNA was extracted from 2-ml aliquots and digested with SapI, PpuMI and AvaII, each of which cut the donor only, to enrich for homing products. When the TetR recipient was used, AvaII was omitted from this mix and when the SpcR recipient was used, SapI was omitted because of sites in those marker genes. When pLHStetpLHS1-rnhA and pLHStetpLHS1-rnhB were used in complementation assays, SapI and HpaI were used to cut the donor, but not the recipient. Additionally, when pSCLtrAtdKR' and pRIAHS were used in the polA hosts, Bgl II was used to cut the donor, but not the recipient. In all cases, the digested DNA was then electroporated into DH5 and plated onto selective media for PM2. For example selection was for CamR transformants (recipients, homing products and cotransformants of donor and recipient), CamRKanR transformants (homing products and cotransformants) or CamRKanRAmpR (to determine the cotransformant background). The CamRKanR colonies were patched to ensure that they were homing products, as well as to probe for the loss of the group I intron, to estimate retrohoming. Homing was determined by the number of CamRKanR colonies relative to the number of CamR colonies.
To test the dnaE mutant, we grew cotransformants of pACD3-TpR8 and pBRR3-ltrB in LB medium with ampicillin and chloramphenicol to OD598 = 0.1. The temperature was shifted to 37oC or 42oC, and cells were grown at the elevated temperature for an additional 1 h before IPTG induction. IPTG was added to 400 µM, to induce intron expression, for 3 h. Induction was stopped by placing cells on ice for 10 min. Cells were then harvested and plasmid DNA was isolated using a Qiagen miniprep kit. After restriction digestion with AgeI, which specifically cleaves the donor plasmid but not the recipient plasmid or the homing product, the plasmid DNA was transformed back into the DH5α cells and transformants were plated on Muller-Hinton medium containing ampicillin alone, or ampicillin plus trimethoprim, or ampicillin plus trimethoprim plus chloramphenicol. Mobility frequencies were calculated as the ratio of (AmpRTpR - AmpRTpRCamR)/AmpR colonies.
For assay B, described in Supplementary Materials, donor and recipient plasmids, listed in Figure S1C, were co-electroporated into the E. coli strain to be tested. The co-transformants were grown overnight in LB liquid medium with ampicillin (100 g/ml) and chloramphenicol (25 g/ml) or spectinomycin (100 g/ml). The culture was diluted 1/100 into fresh LB medium with ampicillin and chloramphenicol and grown at 37oC. When OD598 reached 0.2, IPTG was added to 100 M, to induce intron expression, and the culture was aerated at 37oC for 1 h. Cells were washed with fresh LB medium, diluted and spread onto LB plates with ampicillin or onto LB plates with ampicillin and tetracycline (25 g/ml). Plates were incubated for 1 to 2 days, and the ratio of AmpRTetR colonies to AmpR colonies was used to determine homing efficiency of the LtrB intron.
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Table S1. Homing of the Ll.LtrB intron in mutants defective in DNA processing functions
Mutant (mutation)a / Function / Homing efficiencybAssay A / Assay B
Cross 1 (RH)c / Cross 4 / Cross 5
DNA Exonucleases
RecJ (ΔrecJ)
SbcB (sbcB15)
RecB (recB21)
MutD (mutD5)
XthA (xthA1)
SbcC (sbcC201)
SbcD (sbcD300::kan)
RecD (recD1014)
RecF (recF143)
DNA Helicases
RecG (recG258 Tn10::kan)
RuvB (ruvB9)
UvrD (uvrDΔ291::tet)
RecQ (recQ1)
Rep (Δrep::kan)
DNA repair Functions
Lig (lig7 [ts]) / 5' - 3' ssd
3' - 5' ss
5' - 3' ss, 3' - 5' ss
3' - 5' dsd and ss
3' - 5' ds
ATP dependent exo
suppressors of recB+C
5' - 3' ss, 3' - 5' ss
5' - 3' ds
3' - 5' helicase
5' - 3' jxnd helicase
RNA/DNA hybrids
3' - 5' RNA/DNA
hybrids
3' - 5' DNA helicase
3' - 5' RNA/DNA
hybrids
Ligase mutant [37oC] / 0.18 + 0.05 (53 + 16)
2.58 + 0.89 (50 + 14)
2.19 + 0.56 (75 + 3)
0.27 + 0.17 (53 + 9)
8.63 + 2.69 (75 + 6)
0.51 + 0.17 (41 + 10)
0.28 + 0.14 (30 + 9)
0.92 + 0.31 (71 + 5)
0.69 + 0.16 (57 + 18)
1.08 + 0.34 (22 + 9)
1.46 + 0.28 (55 + 10)
1.17 + 0.31 (30 + 11)
0.70 + 0.13 (69 + 3)
1.61 + 1.0 (56 + 13)
0.26 + .08 (63) / ---
---
---
---
0.79+0.03
---
---
---
---
---
---
---
---
---
--- / 0.71+0.06
---
0.78+0.07
1.40 + 0.10
1.02+0.08
---
1.02 + 0.17
---
---
1.02+0.14
---
---
---
---
0.24+0.03
aFull genotype of parental strains, and sources of mutants, are listed in Supplementary Table S4.
bHoming efficiency was determined using assays A and B (Fig. 2A S1A and BC) and expressed relative to an isogenic "wild-type" host, in which homing efficiency was in the range indicated in Figure 2CS1C. Numbers reflect the mean + standard error for at least three independent experiments.
cRetrohoming (RH) frequencies reflect the percentage of homing products that lost the group I intron, verifying the retrohoming pathway. Loss of the td intron occurred in 22-75% of the homing products, reflecting the lower limit of mobility via an RNA intermediate. It is uncertain whether this range reflects differences in retrohoming, or slightly altered splicing efficiencies of the group I intron in the mutant hosts.
dss = single-stranded, ds = double-stranded, jxn = junction
Table S2. Homing of the Ll.LtrB intron in mutants defective in RNA processing functions
Mutant (mutation)/Plasmida / Function / Homing efficiencybAssay A / Assay B
Cross 1 (RH) / Cross 4 / Cross 5
General
RNase I (rna-19)
RNase E (rne-1) ts
StpAc (ΔstpA::tet)
H-NSc (Δhns:: cat)
Intron degradation
RNase H1 (rnhA339::cat)
RNase H2 (rnhB716::kan)
RNase H1 + H2 (rnhA, rnhB)
RNase H1/pLHS1d
RNase H1/pLHS1-rnhA
RNase H1/plHS1pLHS1-rnhB / RNA degradation
RNA processing and decay [44oC for assay A, 37oC for assay B]
RNA annealing
Regulator +
nucleoid
protein
Degrade RNA from
RNA/DNA hybrids
Complementationd
Complementationd
Complementationd / 4.19 + 2.09 (79 + 2)
11.68 + 4.09 (83 + 7)
0.58 + 0.11 (88 + 7)
1.06 + 0.3 (82 + 9)
0.03 + 0.01 (34 + 10)
0.49 + 0.16 (55 + 1)
0.03 + 0.02 (54 + 4)
0.05 + 0.01 (27 + 4)
0.48 + 0.18 (67 + 12)
0.06 + 0.02 (45 + 13) / 1.82 + 0.35
1.67 + 0.12
---
---
0.49 + 0.27
0.62 + 0.12
0.29 + 0.08
---
---
--- / ---
---
---
---
1.06+0.28
1.04+0.2
0.2+0.03
---
---
---
a, bAs footnotes a and b of Table S1.
cAlthough StpA action on RNA is more potent than that of its paralog H-NS, H-NS does have some RNA-binding capability (Cusick and Belfort 1998; Brescia et al. 2004) and was therefore included in this table.
dThe rnhA mutant was transformed with the recipient plasmid pLHS1 (Fig. 2AS1A), either with or without the rnhA and rnhB genes.
Smith et al. – Page S1