Section 3. Synthetic combinations of various. termination defective rho, nusg and. nusa mutants: Spectrum of efficiency of

Transcription

1 Section 3 Synthetic combinations of various termination defective rho, nusg and nusa mutants: Spectrum of efficiency of Rho-dependent transcription termination

2 Introduction As described in Sections 1 and 2, the attempt to identify suppressors for H-NS 64 effect in the parental rho-a243e and nusg strains yielded suppressor mutations in rho and nusa that were themselves associated with defective transcription termination. Each of these newly identified mutations, namely rho-r102s,a243e, rho-q32r and nusa- R258C, exhibited transcription termination defects as single alleles which were suppressed by H-NS 64 to varying extents (not suppressed in rho-r102s,a243e but suppressed in rho-q32r and nusa-r258c). This led to the studies on synthetic phenotypes of the various mutant combinations in the absence and presence of H- NS 64. The contents of this chapter describe the studies with synthetic combinations of rho-a243e (mentioned as rho in the text); rho-q32r; rho-r102s,a243e; nusg- G146D (mentioned as nusg in text) and nusa-r258c (mentioned as nusa in the text) for their properties of polarity relief and ColE-plasmid lethality both in the absence and presence of H-NS 64. Effect of the RNA polymerase mutation rpob8 on the phenotypes of some of these double synthetic combinations was also studied. 3.3.A Results 3.3.A.1 Synthetic combinations with nusa 3.3.A.1.1 Construction and characterization of rho nusa and nusg nusa combinations The rho-a243e and nusg-g146d mutants have been shown to be defective for transcription termination thus exhibiting polarity relief (Harinarayanan and Gowrishankar, 2003). The findings that the suppressor selection for polarity relief in the 192

3 parental nusg-g146d derivative expressing H-NSΔ64 yielded nusa-r258c mutation that was itself associated with defective transcription termination, led to the examination of synthetic phenotypes, if any, of the various mutant combinations in the absence and presence of H-NSΔ64. It is important to note that since nusa mutant was obtained in combination with nusg in the presence of H-NS 64, it was already established that this combination was viable at least in the presence of H-NS 64. To study the behavior of the nusa mutation in combination with mutations in rho or nusg in the absence or presence of H-NS 64, nusa mutation was introduced into rho/nusg mutant strains carrying the cognate rho + or nusg + gene on an IPTG dependent replicon (phyd1201 or phyd751, respectively) by P1 transductions and the synthetic phenotypes of viability and transcriptional polarity relief were then determined by growth on media not supplemented with IPTG. This was achieved as follows. Using P1 lysate prepared on GJ7399 (nusa-r258c mutation approximately 86% linked to trub::cm), transduction was done into rho and nusg strains GJ6509/pHYD1201 and GJ6511/pHYD751 respectively. Selection was done for Cm R transductants on LB medium supplemented with Amp and IPTG. It was expected that approximately 86% of the transductants would inherit the nusa locus from the donor. Coming together of two transcription termination defective mutations was expected to cause accentuated defect in transcription termination and thus the double mutants obtained were screened for slow growth or lethality in the absence of cognate rho + /nusg + plasmids. As expected, 95% of the Cm R transductants in the rho derivative GJ6509/pHYD1201 (nineteen out of twenty) failed to subculture on LB medium without IPTG suggesting synthetic lethality. However, all the twenty transductants in 193

4 the nusg derivative GJ6511/pHYD751 background exhibited slow growth in the absence of IPTG. The results suggested lethality of rho nusa and slow growth phenotype for nusg nusa synthetic combinations. To confirm these observations, a more sensitive experiment was undertaken using an Efficiency of Plating (EOP) approach. Efficiency of Plating is defined as the the ratio of colony counts originating from single cells on a test medium versus a control medium and is a measure of cell viability on the former. Therefore, two slow growing and one fast growing transductant from the GJ6509/pHYD1201 transduction and two slow growing transductants from GJ6511/pHYD751 transduction were grown overnight in LB medium containing IPTG and appropriate dilution was used for plating on LB plates with and without IPTG to determine the EOP. (It was assumed that the slow growing and fast growing categories of transductants would represent those carrying the nusa mutation and nusa +, respectively.) The ratio of the number of colonies obtained on IPTG to +IPTG plates would determine the efficiency with which the double mutant colonies come up in the absence of the cognate rho + /nusg + plasmid and thus their viability (see Methods section 2.19). A ratio of 10 2 i.e or below, suggests synthetic lethality. The two slow growing transductants obtained using P1 on GJ7399 into the rho derivative GJ6509/pHYD1201, showed an EOP of 0.01 on plates without IPTG following 20 hr of incubation at 37 C or 30 C. One such transductant was designated GJ7417/pHYD1201 (Figure 3.23), while an EOP of 1 was obtained for the fast growing transductant, now designated GJ7418/pHYD1201. When incubated longer than 20 hr at 37 C, colonies subsequently appeared for GJ7417/pHYD1201 on the IPTG plate also that failed to subsequently subculture, suggesting lethality. 194

5 The two slow growing transductants from the transduction using P1 on GJ7399 into the nusg derivative GJ6511/pHYD751 gave an EOP of 1 following 20 hr of incubation at 37 C or 30 C however, these colonies failed to subculture. One such transductant was numbered GJ7419/pHYD751 (Figure 3.23). The difference in EOP results for both GJ7417 and GJ7419 were more clear at 30 C than at 37 C. Similar results were obtained in EOP done on tryptophan supplemented glucose-minimal A medium with GJ7417/pHYD1201 and GJ7419/pHYD751 following approximately 30 hr of incubation at 37 C. The rho nusa and nusg nusa strains GJ7417 and GJ7419 respectively, contain the trub::cm marker which is approximately 86% linked to nusa. Therefore these synthetic lethal combinations of rho nusa and nusg nusa were reconstructed to make the double mutant combinations devoid of any antibiotic marker for future use. This was done as follows. P1 lysate on argg::kan strain was used to transduce GJ6509/pHYD1201 and GJ6511/pHYD751 and one such Kan R Arg transductant in the two strains was designated as GJ10539/pHYD1201 and GJ10540/pHYD751 respectively. Using P1 lysate prepared on the original nusa suppressor strain GJ7387, transduction was done into GJ10539/pHYD1201 and GJ10540/pHYD751 selecting for Arg + prototrophs (since nusa is approximately 92% linked to argg). The Arg + transductants thus obtained were screened for synthetic lethalilty in the absence of IPTG. One such lethal transductant in GJ6509/pHYD1201 background was designated GJ10553/pHYD1201 (rho nusa) and one in GJ6511/pHYD751 background was designated GJ10554/pHYD751 (nusg nusa). 195

6 Figure 3.23 Synthetic lethal phenotypes of rho and nusg mutants. Mutants designated as rho without the suffix -Q32R carry the rho-4 (that is, rho-a243e) mutation. All strains also carry, as appropriate, the nusg + - or rho + -bearing plasmids (phyd751 and phyd1201, respectively) that are IPTGdependent for replication, so that the plasmids are retained only in growth media supplemented with IPTG. Derivatives additionally expressing H-NSΔ64 from plasmid plg-h-nsδ64 are marked. Cultures of the phyd1201-bearing derivatives of strains GJ6509 (rho), GJ7417 (rho nusa) and GJ10581 (rho-q32r nusa), and of the phyd751-bearing derivatives of strains GJ7419 (nusg nusa), GJ10591 (rho nusg) and GJ10557 (rho-q32r nusg) (including plg-h-nsδ64- carrying derivatives as indicated), were each plated at a suitable dilution on LB medium without and with IPTG supplementation and incubated for 20 h, and on glucose-minimal A with Anth supplementation and incubated for 40 h. For the nusg nusa derivative without H-NSΔ64, small colonies were obtained on LB without IPTG but they failed to grow upon subculturing, indicative of synthetic lethality. 196

7 It was therefore concluded from the studies performed above that the double combinations of rho nusa and nusg nusa are synthetic lethal in the absence of H- NS A.1.2 Rescue of rho nusa and nusg nusa synthetic lethality by H-NS 64: Polarity relief and pacyc184 lethality phenotypes As stated earlier, the nusa-r258c mutant was isolated along with nusg in the presence of H-NS 64 as a polarity-relieved suppressor. Therefore, although this combination of nusg nusa had been shown above to be synthetic lethal, it should be viable in the presence of H-NS 64, which explains the way it was obtained in the suppressor selection approach. However, the behavior of rho nusa in the presence of H- NS 64 was unknown. Therefore, the effect of H-NS 64 on the double synthetic lethal combinations for viability, polarity relief and pacyc184 lethality was studied as follows. Plasmid plg-h-nsδ64 was transformed into GJ7417/pHYD1201 (rho nusa) and GJ7419/pHYD751 (nusg nusa) and the lethality phenotype assessed using Efficiency of Plating (EOP) (see Methods section 2.19) approach on LB in the presence and absence of IPTG. The results indicated that H-NSΔ64 was able to rescue the lethality in the absence of IPTG as evidenced by an EOP of 1 for both GJ7417/pLG-H- NS 64 and GJ7419/pLG-H-NS 64 on LB-Kan medium without IPTG (Figure 3.23) and subsequent success in subculturing of these colonies on the same medium. These newly constructed viable double mutant combinations of rho nusa and nusg nusa expressing H-NS 64 (GJ7417/pLG-H-NS 64 and GJ7419/pLG-H-NS 64) were also tested for polarity relief on melibiose and anthranilate media. Furthermore, since polarity relief phenotype is correlated with lethality upon transformation with 197

8 ColE1-family plasmids like pacyc184, the viability of these mutants following transformation with pacyc184 was also assessed. It was observed that both GJ7417/pLG-H-NS 64 and GJ7419/pLG-H-NS 64 were Mel + Anth + (Figure 3.23) and exhibited delayed lethality with pacyc184 following 40 hr of incubation at 37 C. These results suggested that a strain doubly defective for both Rho and NusA or for both NusG and NusA exhibits an accentuated transcription termination defect and hence is inviable. However, the rescue of the synthetic lethality by H-NSΔ64 suggests that H-NS 64 helps restore transcription termination efficiency to a level that enables the cell to survive. How this is achieved, was a subject of further investigation. 3.3.A.1.3 rho nusa and nusg nusa synthetic lethality: Is rne-δcth a suppressor? As mentioned in section 3.2.A.20, rne-δcth, a variant of the rne gene that encodes a 493 amino acid truncated protein of the 1061 amino acid long RNase E protein, had been found to suppress rho, nusg (Dr. K. Anupama s work) and nusa (this study, section 3.2.A.20.1) phenotypes of lethality with ColE1-family plasmid pacyc184. Therefore, it was asked if it would also suppress the rho nusa and nusg nusa synthetic lethalities. This was tested as follows. The rne-δcth::kan allele (rne-493 ::Kan) was introduced using P1 transduction into rho nusa and nusg nusa strains namely, GJ7417/pHYD1201 and GJ7419/pHYD751, expressing cognate rho + and nusg + genes from IPTG dependent replicons. The Kan R strains thus constructed were designated as GJ7452/pHYD1201 and GJ7454/pHYD751 respectively. Lethality was tested by plating the overnight cultures raised in LB medium containing Amp and IPTG on LB medium in the presence and absence of IPTG. It was expected that rescue of rho nusa or nusg nusa synthetic 198

9 lethality by rne::kan would result in the formation of viable colonies in the absence of IPTG giving an EOP=1. It was however observed that upon depletion of rho + and nusg + plasmid on medium lacking IPTG, strains GJ7452 and GJ7454 did not give viable colonies, suggesting therefore that rne-δcth::kan did not rescue rho nusa and nusg nusa synthetic lethality. It was therefore concluded that although rne-δcth suppresses pacyc184 lethality in rho, nusg and nusa, it failed to suppress rho nusa and nusg nusa synthetic lethality. 3.3.A.1.4 rho nusa and nusg nusa synthetic lethality : Rescue by rpob8 rpob8 has been reported as a mutant slow moving RNA polymerase. Studies performed in section 3.2.A.19.3 have identified rpob8 suppression of rho and nusa phenotypes of defective transcription termination and pacyc184 lethality defects to varying extents (polarity relief suppressed in nusa but not in rho, pacyc184 lethality suppressed in both). It was therefore plausible that rpob8 would also suppress synthetic lethality which appears as a consequence of accentuated defect in transcription termination. Therefore, effect of rpob8 mutation in suppression of rho nusa and nusg nusa synthetic lethalities was tested as follows. P1 lysate prepared on strain with rpob8 mutation approximately 50% linked to btub::tn10 was used to transduce GJ10553/pHYD1201 and GJ10554/pHYD751 (rho nusa and nusg nusa strains respectively devoid of any chromosomal antibiotic markers and expressing the cognate rho + and nusg + gene from an IPTG dependent plasmid). Selection was made for Tet R transductants which were then screened for Rif R phenotype (since mutations in rpob are known to confer rifampicin resistance) (Jin and Gross, 1988). Rif R transductants were obtained at a frequency of 50% (ten out of twenty 199

10 tested) in GJ10553/pHYD1201 and 60% in GJ10554/pHYD751 (twelve out of twenty tested). The Rif R transductants in GJ10553/pHYD1201 and GJ10554/pHYD751 were numbered GJ10621/pHYD1201 and GJ10762/pHYD751 respectively while the Rif S transductants from the same cross were designated GJ10620/pHYD1201 and GJ10763/pHYD751 respectively. GJ10621/pHYD1201, GJ10620/pHYD1201, GJ10762/pHYD751 and GJ10763/pHYD751 were then used for the assessment of viability by determining the Efficiency of Plating (EOP) (see Methods section 2.19) on LB medium in the presence and absence of IPTG using appropriate dilution of overnight cultures grown in LB medium containing Amp and IPTG. An EOP of 1 was obtained for the rpob8 derivative of rho nusa i.e. GJ10621 which subsequently got subcultured on the same medium while the rpob + derivative GJ10621 gave an EOP of less than 0.01 suggestive of lethality. On the other hand both rpob8 and rpob + derivatives of nusg nusa strains namely, GJ10762 and GJ10763, gave an EOP of 1 but GJ10762 colonies could be subcultured suggesting viability while GJ10763 colonies failed to subculture on LB (Figure 3.24). Therefore, it was concluded that rpob8 rescued synthetic lethality of the rho nusa and nusg nusa double mutants. 200

11 Figure 3.24 Suppression of rho nusa and nusg nusa synthetic lethality by rpob8. The figure depicts the synthetic lethality of rho nusa and nusg nusa double mutants and the rescue of this lethality by rpob8. All strains carry the appropriate rho + or nusg + -bearing plasmids (phyd1201 and phyd751 respectively) that are IPTG dependent for replication so that the plasmids are retained on LB medium supplemented with IPTG. The strains used are, in order, rpob + and rpob8 derivatives of rho nusa, GJ10620 and GJ10621; nusg nusa, GJ10762 and GJ10763 respectively. 3.3.A.2 Construction and characterization of rho-r102s,a243e synthetic combinations with nusg and nusa 3.3.A.2.1 Construction and characterization of rho-r102s,a243e nusa synthetic lethal combination Studies performed in section 1 have identified rho-r102s,a243e mutation as an LB-sensitive, polarity-relieved suppressor obtained in the presence of H-NSΔ64 in the transposon mutagenesis screen. The rho-a243e nusa combination has also been shown to be synthetic lethal in the absence of H-NS 64 in section 3.3.A.1.1 above. Therefore, it was tested whether rho-r102s,a243e, which is a double mutant of rho with accentuated transcription termination defect that could not be suppressed by H- NS 64 expression (section 3.1.A.6), would also be lethal in combination with nusa. This was done as follows. 201

12 Using a P1 lysate prepared on the original nusa suppressor strain GJ7387, transduction was done into GJ10574 (rho-r102s,a243e ΔargG::Kan) expressing the cognate rho + gene from the IPTG dependent plasmid phyd1201. Arg + transductants were selected (Since nusa is around 92% linked to argg) and screened for synthetic lethality using Efficiency of Plating on tryptophan supplemented glucose-minimal A medium in the absence and presence of IPTG. All the eight transductants tested gave an EOP of less than 0.01following 48 hr of incubation at 37 C indicating synthetic lethality. One such lethal transductant was designated GJ10595/pHYD1201(Figure 3.25). Figure 3.25 Synthetic lethal phenotypes of rho-r102s,a243e nusa (GJ10595/pHYD1201) and rho-r102s,a243e nusg (GJ10593/pHYD751) mutants. Strains carry the rho + or nusg + - bearing plasmids (phyd1201 or phyd751) which are IPTGdependent for replication so that the plasmid is retained only in growth media supplemented with IPTG. Media used is glucoseminimal A supplemented with tryptophan. 202

13 3.3.A.2.2 rho-r102s,a243e nusa combination: Effect of H-NSΔ64 on synthetic lethality and polarity relief It had been demonstrated in section 3.3.A.1.2 that the lethality of the rho nusa synthetic combination was rescued by H-NS 64. Likewise, the synthetic combination of rho-r102s,a243e nusa was also tested for suppression of synthetic lethality by H- NS 64. GJ10595/pHYD1201 was transformed with plg-h-nsδ64 and synthetic lethality was tested using Efficiency of Plating on tryptophan supplemented glucoseminimal A Kan medium in the presence and absence of IPTG by plating appropriate dilution of overnight culture of GJ10595/pHYD1201 expressing H-NSΔ64 raised in glucose-minimal A supplemented with tryptophan, Amp, IPTG and Kan. Approximately equal number of colonies were obtained on plates with and without IPTG (EOP=1) for GJ10595/H-NSΔ64. The colonies from the IPTG plate subsequently got subcultured on the same medium suggesting viability of the strain in the absence of rho + plasmid phyd1201. Therefore, it was concluded that H-NSΔ64 rescued the rho-r102s,a243e nusa synthetic lethality. Since the rho-r102s,a243e/h-ns 64 strain had been shown to be relieved for polarity (Mel + Anth + ) both as the original suppressor GJ7368 and upon reconstruction (section 3.1.A.6, 3.1.A.7.1) and nusa-r258c has been shown to be defective for transcription termination on its own (section 3.2.A.12), therefore, the viable combination of rho-r102s,a243e nusa/h-ns 64, harbouring two independent transcription termination defective mutations, was expected to be even more defective for transcription termination so as to exhibit polarity relief at the loci tested. GJ10595/pLG-H-NSΔ64 was thus tested for polarity relief at lacz U118 and trpe9777 on melibiose and anthranilate and was found to be Mel + Anth

14 It was therefore concluded that rho-r102s,a243e is synthetic lethal in combination with nusa which is the likely consequence of its accentuated transcription termination defect due to coming together of various mutations which have been individually shown to be defective for transcription termination. This lethality however, is rescued by H-NS 64 but this combination still remains defective for transcription termination and exhibited polarity relief. 3.3.A.2.3 rho-r102s,a243e nusa synthetic lethality: Rescue by rpob8 Studies performed in section 3.3.A.1.4 identified that rpob8 suppresses rho nusa synthetic lethality. Therefore, the rho-r102s,a243e nusa combination was also tested for rescue of synthetic lethality by rpob8. This was done as follows. The rho-r102s,a243e nusa strain, GJ10595/pHYD1201 was made arge::kan, generating arginine auxotrophy, by a P1 transduction with the lysate prepared on the arge::kan strain. The Kan R Arg strain thus generated was designated GJ10626/pHYD1201. Using P1 lysate made on a rpob8 strain, transduction was then done into GJ10626/pHYD1201 selecting Arg + transductants (rpob and arge are around 50% linked) and screening Rif R (mutations in rpob have been reported to result in rifampicin resistance) (Jin and Gross, 1988). 40% of the Arg + transductants were Rif R (four out of ten tested). One of these Rif R transductants (i.e. rpob8 derivative) was designated GJ10638/pHYD1201. GJ10638/pHYD1201 and the isogenic rpob + derivative from the same cross were then grown overnight in tryptophan supplemented glucose-mimimal A medium containing Amp and IPTG and appropriate dilution was used to determine Efficiency of Plating on a pair of tryptophan supplemented glucosemimimal A Kan plates in the absence and presence of IPTG. 204

15 An EOP of 1 (see Methods section 2.19) was obtained for both GJ10638/pHYD1201 and its isogenic rpob + /phyd1201 derivative after approximately 60 hr of incubation at 37 C. These colonies could also subsequently be successfully subcultured on tryptophan supplemented glucose-minimal A medium without IPTG. It was therefore concluded that rpob8 also rescued the rho-r102s,a243e nusa synthetic lethality. 3.3.A.2.4 Construction of rho-r102s,a243e nusg double mutant and determination of synthetic lethality From previous studies it is known that nusg-g146d mutant is defective for transcription termination. Also, rho-r102s,a243e exhibits accentuated transcription termination defect. Therefore, formalism predicts that bringing together of these two profoundly transcription termination defective mutations would lead to cell death due to extremely impaired transcription termination. This was tested as follows. Transduction was done using a P1 lysate prepared on the rho-r102s,a243e mutant strain, GJ7368 into the nusg strain made Ilv i.e. GJ10552/pHYD1201 (GJ6524 ilv::kan/phyd1201) carrying the rho + gene on an IPTG dependent plasmid phyd1201. Since ilv is around 80% linked to rho, selection was made for Ilv + transductants in the presence of IPTG. The Ilv + transductants thus obtained were screened for lethality on tryptophan supplemented glucose-minimal A medium lacking IPTG. It was observed that nine out of ten Ilv + transductants exhibited slow growth phenotype on medium lacking IPTG but showed retention of the rho + plasmid phyd1201 upon subsequent subculturing on tryptophan supplemented glucoseminimal A medium containing Amp and IPTG. Retention of phyd1201 plasmid by 205

16 90% of the transductants was indicative of synthetic lethality. One such slow growing transductant was designated as GJ10593/pHYD1201. This synthetic lethality was confirmed using a more sensitive Efficiency of Plating (EOP) approach (see Methods section 2.19). Appropriate dilution of GJ10593/pHYD1201 overnight culture raised in tryptophan supplemented glucoseminimal A medium containing Amp and IPTG was used to plate on glucose-minimal A medium supplemented with tryptophan in the presence and absence of IPTG. If the combination was lethal, it was expected that the ratio of number of colonies on IPTG to +IPTG plate will be 10 2 i.e. an EOP of 0.01 should be obtained. As expected, an EOP of less than 0.01 was obtained for GJ10593/pHYD1201 after 48 hr of incubation at 37 C (Figure 3.25). The results therefore explicitly showed that rho-r102s,a243 nusg combination was synthetic lethal. 3.3.A.2.5 rho-r102s,a243e nusg synthetic lethality : Not rescued by H-NS 64 Experiments performed above have provided evidence for rescue of various synthetic lethal combinations viz. rho nusa, nusg nusa and rho-r102s,a243e nusa upon H-NS 64 expression. On similar lines rho-r102s,a243e nusg synthetic lethal combination was also tested for suppression of its synthetic lethality by H-NS 64. plg-h-nsδ64 transformants of GJ10593/pHYD1201 raised in tryptophan supplemented glucose-minimal A medium containing Amp and IPTG was plated, using an appropriate dilution, on tryptophan supplemented glucose-minimal A Kan plates in the presence and absence of IPTG. Equal number of colonies were obtained on both IPTG and +IPTG plates giving an EOP=1 following approximately 48 hr of incubation at 37 C. The colonies from IPTG plates were found to be inviable upon subsequent subculturing. 206

17 The results therefore concluded that H-NSΔ64 was unable to rescue the lethality of the rho-r102s,a243e nusg double mutant. Hence, it is concluded that rho- R102S,A243E is synthetic lethal both in combination with nusa and nusg. However, the lethality of the former combination is rescued by H-NS 64 leaving the strain still defective for transcription termination but that of the latter is not. 3.3.A.3 Construction and characterization of rho-q32r synthetic combinations with nusg and nusa 3.3.A.3.1 rho-q32r nusa combination: Test for viability and polarity relief The rho-q32r and nusa-r258c mutations were each identified as polarity relieved suppressor mutations in combination with nusg in the presence of H-NS 64. When studied in isolation, rho-q32r and nusa-r258c exhibited moderate polarity relief defect (Anth + Mel ) and lethality with pacyc184. Both these defects were however suppressed by H-NS 64 expression. nusa-r258c had been shown to be fourfold more relieved for polarity at the H19B-t R1 terminator in comparison to the wildtype strain (section 3.2.A.15). Based on the hypothesis that bringing together of two independent transcription termination defective mutations could lead to accentuation of transcription termination defect causing synthetic lethality or slow growth phenotype, rho-q32r mutation was tested in combination with nusa for viability. This was done as follows. The strain carrying rho-q32r mutation, GJ10550 was made argg using P1 transduction with an ΔargG::Kan lysate. The resultant Kan R Arg strain was designated GJ Using P1 lysate prepared on GJ7387 (orginal suppressor strain carrying nusa- R258C mutation), transduction was done into GJ10565 expressing the cognate rho + gene from an IPTG dependent plasmid phyd1201. Selection was made for Arg + 207

18 transductants (since nusa is approximately 92% linked to argg). P1 lysates were prepared on three of these Arg + transductants and a reciprocal transduction was done into GJ10535 (i.e. wild-type strain GJ6504 ΔargG::Kan), selecting for Arg + transductants which were then scored for Anth + phenotype to ascertain the inheritance of nusa-r258c mutation. One such transductant that showed the presence of nusa mutation in the reciprocal transduction was designated GJ10581/pHYD1201. Efficiency of Plating was determined by plating appropriate dilution of GJ10581/pHYD1201 overnight culture grown in LB supplemented with Amp and IPTG on LB and tryptophan supplemented glucose-minimal A medium in the absence and presence of IPTG (see Methods section 2.19). An EOP of 1 was expected if the combination was viable in the absence of rho + plasmid while if synthetic lethal, an EOP of 0.01 was expected. It was observed that equal number of colonies came up on both IPTG and +IPTG plates giving an EOP of 1 on both LB and glucose-minimal A tryptophan medium. However, the colonies on LB plate exhibited an altered morphology i.e. they were slightly smaller and looked translucent in comparison to those on the corresponding +IPTG plate (Figure 3.23). The colonies from both LB and glucoseminimal A tryptophan could be subsequently subcultured on the same medium lacking IPTG, indicating viability. Therefore the double combination of rho-q32r nusa was concluded to be viable but exhibited slow growth phenotype, as indicated by their small colony size on LB. This combination when tested for polarity relief was found to be Mel + Anth +, as against the Anth + Mel phenotype of the individual mutations (rho-q32r or nusa-r258c) suggesting accentuation of transcription termination defect in the double mutant. 208

19 3.3.A.3.2 Effect of H-NSΔ64 on polarity relief, pacyc184 lethality and slow growth phenotype of rho-q32r nusa double mutant In order to study the effect of H-NSΔ64 on polarity relief, GJ10581/pHYD1201 (rho-q32r nusa) was transformed with plg-h-nsδ64 and tested for Mel and Anth phenotypes. Efficiency of plating was also determined on LB medium in the presence and absence of IPTG to determine whether H-NSΔ64 rescued the slow growth phenotype exhibited by the double mutant. GJ10581/pLG-H-NSΔ64 became Anth + Mel, as against Anth + Mel + phenotype of GJ10581, suggesting partial suppression of polarity relief defect by H-NS 64. The EOP experiment on LB-Kan medium in the presence and absence of IPTG using appropriate dilution of overnight culture of GJ10581/pHYD1201 expressing H-NS64 from plg-h-ns 64 (raised in LB medium supplemented with Kan, Amp and IPTG), showed suppression of slow growth phenotype of the double mutant in the absence of IPTG. In correlation with the polarity relief phenotype of the double mutant in the presence of H-NSΔ64, the pacyc184 transformants of GJ10581/pLG-H-NS 64 (Tet R selection employed) exhibited delayed lethality following approximately 60 hr of incubation at 37 C. It was therefore concluded that the slow growth phenotype of the rho-q32r nusa double mutant was alleviated and its polarity relief defect was also mitigated from Mel + Anth + to Mel Anth + upon expression of H-NS 64. The combination however remained lethal with ColE1-plasmid pacyc

20 3.3.A.3.3 Construction and characterization rho-q32r nusg double mutant for synthetic lethality and polarity relief in the absence of H-NSΔ64 and pacyc184 lethality in the presence of H-NSΔ64 The rho-q32r mutation was obtained as a polarity-relieved (Mel + Anth + ) suppressor in combination with nusg mutation in the presence of H-NSΔ64. This double mutant combination was therefore both viable and polarity-relieved with H- NSΔ64. It was therefore aimed to study this double mutant for viability in the absence of H-NSΔ64. This was achieved as follows. Using a lysate made on GJ7376 (original suppressor mutant carrying rho-q32r mutation) a P1 transduction was done into the nusg strain made auxotrophic for Ilv (GJ10542) and expressing the cognate nusg + gene from the IPTG dependent plasmid, phyd751. Selection was made for Ilv + transductants (since rho and ilv are approximately 80% linked) and transductants screened for synthetic lethality on LB medium lacking IPTG (absence of IPTG inhibits replication of phyd751 thus depleting the plasmids from the growing cells). To ascertain if the transductants in the cross above have inherited the rho-q32r mutation, eight Ilv + transductants from this cross were transformed with plg-h-nsδ64 and screened for Mel and Anth phenotype on medium lacking IPTG. Six out of eight (75%) transductants had become Mel + Anth +, suggesting that these six transductants have indeed acquired the rho-q32r mutation. One such transductant was designated GJ GJ10557/pHYD751 was then used to determine Efficiency of Plating in the absence and presence of IPTG to determine whether the rho-q32r nusg double mutant was lethal in the absence of H-NSΔ64. Using appropriate dilutions of GJ10557/pHYD751 culture grown overnight in LB media supplemented with Amp and IPTG, plating was done on LB medium with and without IPTG. Equal number of 210

21 colonies were obtained on both IPTG and +IPTG plates giving an EOP=1 (Figure 3.23). The colonies from IPTG plate could be subsequently subcultured on LB without IPTG, suggesting viability of the rho-q32r nusg double combination. When tested for polarity relief on melibiose and anthranilate medium, GJ10557 was Mel + Anth + both without and with H-NSΔ64 showing defect for transcriptional polarity. In accord with its polarity-relieved phenotype (Mel + Anth + ), pacyc184 transformants of GJ10557/pLG-H-NSΔ64 exhibited delayed lethality following approximately 48 hr of incubation at 37 C. The results therefore established that rho-q32r was not lethal in combination with nusg in the absence of H-NSΔ64, was polarity-relieved (Mel + Anth + ) both in the presence and absence of H-NSΔ64 and continued to exhibit lethality with ColE1- plasmid pacyc184 in the presence of H-NSΔ A.4 Studies on rho nusg synthetic combination 3.3.A.4.1 Reproduction of rho nusg synthetic lethality Earlier studies from the lab have demonstrated rho nusg double mutant combination to be synthetic lethal in MC4100 background (Harinarayanan and Gowrishankar, 2003). However, its behaviour in the presence of H-NS 64 had not been studied. The same synthetic lethality was therefore reproduced in MG1655 strain background and the effect of H-NS 64 studied thereon. This was done as follows. The nusg strain that had been made auxotrophic for Ilv (GJ10542) expressing the cognate nusg + gene from the IPTG dependent plasmid phyd751, was transduced with a P1 lysate made on GJ3110 (MC4100 rho-a243e strain). Utilizing the property of approximately 80% linkage between ilv and rho, selection was made for Ilv + 211

22 transductants and the transductants were scored for synthetic lethality in the absence of IPTG (in the absence of IPTG phyd751 replication is inhibited resulting in depletion of the plasmid from the cells). Approximately 66% of the Ilv + transductants (six out of nine tested) were extremely slow growing on medium lacking IPTG, suggestive of synthetic lethality. One of these slow growing transductants was designated GJ10591/pHYD751 (rho nusg). Efficiency of Plating was determined for GJ10591/pHYD751on LB medium in the presence and absence of IPTG to confirm the synthetic lethal phenotype. An EOP of less than 0.01 was obtained confirming the lethality of the double mutant (Figure 3.23) in the absence of phyd751 (see Methods section 2.19). It was therefore established that rho nusg double mutant was synthetic lethal in MG1655 background also. 3.3.A.4.2 rho nusg synthetic lethal combination : Studies on viability, polarity relief and pacyc184 lethality in the presence of H-NSΔ64 In order to examine the effect of H-NS 64 on rho nusg synthetic lethality, GJ10591/pHYD751 was transformed with plg-h-ns 64 and EOP was determined on LB-Kan medium in the absence and presence of IPTG using appropriate dilution of the culture grown overnight in LB-Kan medium supplemented with Amp and IPTG. Consistent with the effect of H-NS 64 on previously studied synthetic lethal combinations like rho nusa, nusg nusa etc, an EOP of 1 was obtained for GJ10591/H- NSΔ64 following overnight incubation at 37 C (Figure 3.23). The colonies from IPTG plate could subsequently be subcultured on the same medium indicating viability. This viable combination when tested for polarity relief on melibiose and anthranilate 212

23 medium and pacyc184 lethality, GJ10591/pLG-H-NS 64 was found to be partially polarity relieved (Anth + Mel ) (Figure 3.23) and showed delayed lethality upon transformation with pacyc184 plasmid following approximately 60 hr incubation at 37 C. It was therefore concluded from these studies that H-NS 64 rescued rho nusg synthetic lethality. However, the strain remained moderately defective for transcription termination and therefore lethal with ColE1-plasmid pacyc184. Having studied all the possible synthetic double combinations in the absence and presence of H-NS 64 for their properties of synthetic lethality, polarity relief and viability with pacyc184, further studies were performed with the triple mutant combinations in the presence of H-NS 64. These are described in sections to follow. 3.3.A.5 Studies on triple mutant combinations with H-NS 64 The sections above describe in detail the five synthetic lethal double combinations (rho nusa, nusg nusa, rho nusg, rho-r102s,a243e nusa and rho-r102s,a243e nusg) and two viable synthetic double (rho-q32r nusa, rho-q32r nusg) combinations. Although the lethality in the synthetic lethal combinations, except in rho- R102S,A243E nusg, was rescued by expression of H-NS 64, the strains remained defective for transcription termination to varying extents. Since most of the double synthetic lethal combinations were made viable by H-NS 64 expression, it was aimed to study the level of transcription termination defect in triple mutant combinations in the presence of H-NS 64. The construction of two such triple mutant combinations and effect of H-NSΔ64 on their viability are described in detail in sections below. 213

24 3.3.A.5.1 Construction of nusg nusa rho-q32r triple mutant and test for viability in the presence of H-NSΔ64 Each of the three individual mutations rho-q32r, nusg-g146d and nusa-r258c has been shown to be defective for transcription termination. It has also been established that the double combination of nusg nusa is synthetic lethal. This lethality is however rescued by H-NSΔ64 expression. It was therefore asked whether addition of rho-q32r to this double mutant in the presence of H-NSΔ64 would accentuate its transcription termination defect beyond the suppression capacity of H-NSΔ64 so as to result in lethality, that is, what is the spectrum of H-NS 64 mediated suppression of transcription termination defect and what is the extreme level of defect it can suppress so as to still confer viability. This was done as follows. The nusg nusa strain GJ7419/pHYD751/pLG-H-NSΔ64 expressing nusg + from an IPTG dependent plasmid phyd751and H-NSΔ64 from the plasmid plg-h-ns 64 was made Ilv using a P1 transduction with an ilv-500::tn10 lysate constructing the strain GJ10667/pHYD751/pLG-H-NSΔ64. For the construction of the triple mutant, P1 lysate on GJ7376 (original suppressor strain carrying rho-q32r mutation) was used to transduce GJ10667/pHYD751/pLG-H-NSΔ64 and selection was made for Ilv + prototrophs (since rho and ilv are around 80% linked). To ascertain which transductants had inherited the mutant rho-q32r allele from the donor strain GJ7376, P1 lysates were made on two of these Ilv + transductants and a reciprocal transduction done into the wild-type strain GJ7364 (GJ6504 ilv::kan) employing selection for Ilv + prototrophs. The transductants were screened for polarity relief on anthranilate and pacyc184 lethality (rho-q32r single mutants are Anth + and lethal with pacyc184, section 3.1.A.8.2). All the ten transductants tested had become Anth + and lethal with pacyc184 suggesting that the donor strain indeed carried the 214

25 rho-q32r mutation. Thus the corresponding P1 donor strain carrying the three mutations rho-q32r nusg nusa was designated as GJ10669/pHYD751/pLG-H-NSΔ64. To determine the viability of the triple mutant combination in the presence of H- NSΔ64, Efficiency of Plating was determined using appropriate dilution of overnight culture of GJ10669/pHYD751/pLG-H-NS64 (grown in LB medium supplemented with mp, IPTG and Kan) and plating on LB-Kan and glucose-minimal A tryptophan Kan medium in the presence and absence of IPTG (absence of IPTG would inhibit replication of phyd751 thus depleting it from the medium). Equal number of colonies were obtained on both the IPTG and +IPTG plates giving an EOP=1 following overnight and approximately 48 hr of incubation at 37 C for LB and glucose-minimal A tryptophan plates respectively, suggesting that the triple mutant was viable in the presence of H-NSΔ64 (Figure 3.26). The colonies from the IPTG plate could subsequently be subcultured on the corresponding medium. It was therefore concluded that the triple mutant combination rho-q32r nusg nusa is viable in the presence of H-NS

26 Figure 3.26 Synthetic phenotypes of triple mutants rho-q32r nusg nusa and rho nusg nusa in the presence of H-NS 64. Cultures of phyd751- and plg-h-nsδ64- carrying derivatives of strains GJ10669 (rho-q32r nusg nusa) and GJ10668 (rho nusg nusa) were plated at a suitable dilution on LB without and with IPTG A.5.2 Construction of rho nusg nusa triple mutant and test for viability in the presence of H-NSΔ64 Similar to the studies performed in section 3.3.A.5.1, rho-a243e nusg nusa triple mutant was also constructed and tested for viability in the presence of H-NSΔ64. This was achieved as follows. The rho-a243e allele was moved into GJ10667/pHYD751/pLG-H-NSΔ64 (nusg nusa ilv-500::tn10) strain using P1 lysate on the rho-a243e mutant GJ3110 employing selection for Ilv + transductants. In order to ascertain the inheritance of the rho-a243e allele in the transductants, P1 lysates were made on two of the Ilv + transductants from the cross above and a reciprocal transduction done into the wild-type strain GJ7364 (GJ6504 ilv-500::tn10) selecting for Ilv + prototrophs and screening the transductants thus obtained for polarity relief on melibiose and anthranilate medium (rho-a243e is Mel + Anth + ). 65%-72% Ilv + transductants obtained with the two P1 lysates had become 216

27 Mel + Anth + (polarity-relieved) suggesting that the donor strain had inherited the rho- A243E allele. One of the donor P1 strains carrying the three mutations rho nusg nusa was designated GJ10668/pHYD751/pLG-H-NSΔ64. Efficiency of plating was determined by plating appropriate dilution of overnight culture of GJ10668/pHYD751/pLG-H-NSΔ64 (grown in LB supplemented with Amp, IPTG and Kan) on LB-Kan and glucose-minimal A tryptophan Kan medium in the absence and presence of IPTG. An EOP of 0.01 was obtained for GJ10668/pLG-H-NSΔ64 following 18 hr and 48 hr of incubation at 37 C for LB and glucose-minimal A tryptophan plates respectively (Figure 3.26). The result therefore indicates that the rho-a243e nusg nusa triple mutant is synthetic lethal even in the presence of H-NSΔ64. This mutant combination could therefore only be maintained in the presence both the nusg + plasmid phyd751 (or rho + plasmid phyd1201) and plg-h-ns 64 encoding H-NS A.6 Quantitative estimation of polarity relief in the viable double and triple mutants at the H19B-t R1 Rho-dependent terminator To quantitate the extent of polarity relief in many of the viable double and triple mutant combinations (with or without H-NSΔ64), β-galactosidase activities were determined from pairs of P lac -lacz fusion derivatives with or without the intervening Rho-dependent terminator t R1 of the lambdoid phage H19B. The percentage ratio of the β-galactosidase activity of the t R1 strain with respect to the +t R1 strain is a measure of the polarity relief in that particular mutant combination at the t R1 terminator. The construction of +t R1 and t R1 derivatives of various strains used for this assay and the results thus obtained are described in sections below. 217

28 3.3.A.6.1 Construction of +t R1 and t R1 derivatives of rho-q32r double and triple mutants For the construction of +t R1 and t R1 derivatives to be used for β-galactosidase assay the wild-type, nusg and nusa derivatives of +t R1 ; GJ5147, GJ10572, GJ5153 and t R1 strains RS445, GJ10573 and GJ10571 respectively, were transduced with P1 lysate prepared on ilv::kan strain to generate the Ilv derivatives of +t R1 GJ10768, GJ10769, GJ10770 and t R1 strains GJ10771, GJ10772 and GJ10773 respectively. Using P1 lysate prepared on GJ7376 (original suppressor strain carrying rho- Q32R mutation) transduction was done into GJ10768, GJ10769, GJ10770, GJ10771, GJ10772 and GJ10773 and selection made for Ilv + prototrophs. Five Ilv + transductants from each of these six transductions were checked for inheritance of rho-q32r mutation by reciprocal transduction into Anth wild-type strain GJ7366 (GJ6504 ilv::kan), selection was made for Ilv + transductants and the transductants screened for Anth + phenotype associated with rho-q32r. One Ilv + transductant from each of the six transductions using P1 on GJ7376 into GJ10768, GJ10769, GJ10770, GJ10771, GJ10772 and GJ10773 that showed Anth + phenotype in the reciprocal transduction suggesting the inheritance of rho-q32r, were designated as GJ10795, GJ10796, GJ10797, GJ10798, GJ10799 and GJ10800 respectively. Therefore, the following strain pairs (+t R1 and t R1 ) were constructed: rho-q32r, GJ10795 and GJ10798; rho-q32r nusg, GJ10796 and GJ10799; rho-q32r nusa, GJ10797 and GJ For the construction of the rho-q32r nusg nusa triple mutant in the presence of H-NSΔ64, the plg-h-nsδ64 transformants of GJ10796/pHYD751 and GJ10799/pHYD751 (phyd751 expresses the cognate nusg + gene from the IPTG dependent plasmid) were transduced with P1 on GJ7399 that harbors the nusa-r258c 218

29 mutation approximately 86% linked to trub::cm. The Cm R transductants thus obtained were checked for inheritance of the nusa-r258c mutation by a reciprocal transduction into the wild-type strain GJ6504 using P1 lysates on them. Selection was made for Cm R and the transductants scored for change of Anth phenotype of GJ6504 to Anth + upon the inheritance of nusa-r258c. One each of the transductants from cross into GJ10796/pHYD751/pLG-H-NSΔ64 and GJ10799/pHYD751/pLG-H-NSΔ64 using P1 on GJ7399 that showed change of Anth phenotype of GJ6504 to Anth + in the back transduction were designated GJ10818/pHYD751/pLG-H-NSΔ64 and GJ10817/pHYD751/pLG-H-NSΔ64 respectively. 3.3.A.6.2 Construction of +t R1 and t R1 derivatives of rho-a243e and nusg-g146d double mutant combinations expressing H-NSΔ64 The construction of rho nusa and nusg nusa strains (+t R1 and t R1 ) derivatives for quantitative estimation of polarity relief using β-galactosidase assay was done as follows. The +t R1 and t R1 derivatives of the nusa strains, GJ10572 and GJ10573, expressing the rho + gene from IPTG dependent plasmid phyd1201 and H-NSΔ64 from plg-h-nsδ64 were transduced with P1 lysate prepared on rho-a243e ilv- 500::Tn10 strain (ilv-500::tn10 is approximately 80% linked to rho). Since the rho nusa double mutant expressing H-NSΔ64 has been shown to be polarity-relieved (section 3.3.A.1.2), the Tet R transductants were selected and scored for SMG resistance (Methods section ) (rho and nusg mutants are SMG R while wild-type strain is SMG S ) and lethality following transformation with pacyc184 on medium lacking IPTG to ascertain the inheritance of rho-a243e mutation. One such SMG R transductant 219

30 each of GJ10572 and GJ10573 that was also lethal with pacyc184 was designated as GJ10813/pLG-H-NSΔ64 and GJ10814/pLG-H-NSΔ64 respectively. Similarly, to construct nusg nusa combinations with and without t R1 strain GJ10572/pHYD751/pLG-H-NSΔ64 and GJ10573/pHYD751/pLG-H-NSΔ64 (phyd751 is the IPTG dependent plasmid encoding the nusg + gene) were transduced with P1 lysate prepared on nusg-g146d arge::tn10 strain (nusg mutation approximately 50% linked to arge::tn10). Since the nusg nusa double mutant expressing H-NSΔ64 has been shown to be polarity-relieved (section 3.3.A.1.2), the Tet R transductants selected were scored for SMG resistance (Methods section ) and lethality following transformation with plasmid pacyc184 to ascertain the inheritance of nusg-g146d allele. One such SMG R transductant each from GJ10572 and GJ10573 that was also lethal with pacyc184 was designated as GJ10811/pLG-H- NSΔ64 and GJ10812/pLG-H-NSΔ64 respectively. Therefore, the following double mutant pairs (+t R1 and t R1 ) expressing H-NSΔ64 were constructed: rho nusa, GJ10813/pLG-H-NSΔ64 and GJ10814/pLG-H-NSΔ64; nusg nusa, GJ10811/pLG-H-NSΔ64 and GJ10812/pLG-H-NSΔ A.6.3 Construction of +t R1 and t R1 derivatives of rho-r102s,a243e mutant combinations The construction of +t R1 and t R1 derivatives rho-r102s,a243e (without and with H-NSΔ64) and rho-r102s,a243e nusa (with H-NSΔ64) strains for quantitative estimation of polarity relief using β-galactosidase assay was done as follows. The +t R1 and t R1 derivatives of the wild-type strain GJ5147 and RS445 without and with plg-h-nsδ64 and the nusa strain expressing H-NSΔ64, GJ10572/pLG-H- NSΔ64 and GJ10573/pLG-H-NSΔ64 were transduced with P1 lysate prepared on 220

31 GJ7368 (original suppressor strain carrying rho-r102s,a243e mutation approximately 80% linked to zif-909::tn10dtet). Since rho-r102s,a243e mutation, both in the absence and presence of H-NSΔ64 and in combination with nusa in the presence of H- NSΔ64 has been shown to be polarity relieved (section 3.1.A.7), the Tet R transductants selected on tryptophan supplemented glucose-minimal A medium, were scored for SMG resistance (Methods section ) to ascertain the inheritance of rho- R102S,A243E allele. One such SMG R transductant each from GJ5147 and RS445 was designated as GJ10801 and GJ10802, from GJ5147/pLG-H-NSΔ64 and RS445/pLG-H- NSΔ64 was designated GJ10803/pLG-H-NSΔ64 and GJ10804/pLG-H-NSΔ64, and from GJ10572/pLG-H-NSΔ64 and GJ10573/pLG-H-NSΔ64 was designated GJ10793/pLG-H-NSΔ64 and GJ10794/pLG-H-NSΔ64 respectively. 3.3.A.6.4 β-galactosidase assay for quantitative estimation of polarity relief at t R1 terminator for various viable double and triple mutant combinations The +t R1 and t R1 strains of various mutants and their synthetic viable combinations were used to determine the extent polarity relief at the Rho-dependent t R1 terminator. The percentage ratio of the β-galactosidase activity in the +t R1 strain to that of the t R1 strain is a measure of the extent of polarity relief in that particular strain. The data from these experiments, presented in Table 3.7, showed that the rho- Q32R mutation conferred polarity relief to the extent of, respectively, 30, 34 and 53% by itself and in combination with the nusg and nusa mutations in the absence of H- NSΔ64. These values were then reduced to 15, 18 and 32%, respectively, in the presence of H-NSΔ64. The rho-r102s,a243e mutation conferred 74% polarity relief in the absence of H-NSΔ64 which was reduced to 37% in the presence of H-NSΔ

32 Likewise the rho nusa, nusa nusg and rho-q32r nusa mutants with H-NSΔ64 were more polarity-relieved (40, 30 and 32% respectively than any of the corresponding single mutants with H-NSΔ64 (nusa, 5%; rho-a243e, 18%; rho-q32r, 15% and nusg, 17%) (see Table 3.4). The rho-q32r nusg nusa triple mutant (which is lethal without H-NSΔ64) exhibited 34% polarity relief with H-NSΔ64. Thus, the results obtained were consistent with the notions that combining the individual mutations leads to additive effects on polarity relief and that H-NSΔ64 expression is ameliorative in this regard. 222