trans-translation system is important for maintaining genome integrity during DNA damage in bacteria.

DNA integrity in bacteria is regulated by various factors that act on the DNA. trans-translation has previously been shown to be important for the survival of Escherichia coli cells exposed to certain DNA-damaging agents. However, the mechanisms underlying this sensitivity are poorly understood. In this study, we explored the involvement of the trans-translation system in the maintenance of genome integrity using various DNA-damaging agents and mutant backgrounds. Relative viability assays showed that SsrA-defective cells were sensitive to DNA-damaging agents, such as nalidixic acid (NA), ultraviolet radiation (UV), and methyl methanesulfonate (MMS). The viability of SsrA-defective cells was rescued by deleting sulA, although the expression of SulA was not more pronounced in SsrA-defective cells than in wild-type cells. Live cell imaging using a Gam-GFP fluorescent reporter showed increased double-strand breaks (DSBs) in SsrA-defective cells during DNA damage. We also showed that the ribosome rescue function of SsrA was sufficient for DNA damage tolerance. DNA damage sensitivity can be alleviated by partial uncoupling of transcription and translation by using sub-lethal concentrations of ribosome inhibiting antibiotic (tetracycline) or by mutating the gene coding for RNase H (rnhA). Taken together, our results highlight the importance of trans-translation system in maintaining genome integrity and bacterial survival during DNA damage.

Horizontal transfer of domains in ssrA gene among Enterobacteriaceae.

The tmRNA (transfer messenger RNA), encoded by ssrA gene, is involved in rescuing of stalled ribosomes by a process called trans-translation. Additionally, regions of the ssrA gene (coding for tmRNA) were reported to serve as integration sites for various bacteriophages. Though variations in ssrA genes were reported, their functional relevance is less studied. In this study, we investigated the horizontal gene transfer (HGT) of ssrA among the members of Enterobacteriaceae. This was done by predicting recombination signals in ssrA gene (belonging to Enterobacteriaceae) using RDP5 (Recombination Detection Program 5). Our results revealed 7 recombination signals in ssrA gene belonging to different species. We further showed that the recombination signals were more in the domains present in the 3' end than 5' end of tmRNA. Of note, the mRNA region was reported in many recombination signals. Further, members belonging to genera Yersinia, Erwinia, Dickeya and Enterobacter were highly represented in the recombination signals. Sequence analysis revealed the presence of integration sites for different class of bacteriophages in ssrA gene. The locations of phage recognition sites are comparable with recombination signals. Taken together, our results revealed a diverse nature of HGT and recombination which possibly due to transduction mediated by phages.

Two new mutations in dnaJ suppress DNA damage hypersensitivity and capsule overproduction phenotypes of Δlon mutant of Escherichia coli by modulating the expression of clpYQ (hslUV) and rcsA genes.

Lon is a major ATP-dependent protease of E. coli involved in degradation of abnormal misfolded proteins and specific regulatory proteins. Absence of Lon in E. coli results in sensitivity to DNA damaging agents and over-production of capsular polysaccharide due to accumulation of Lon substrates, SulA (cell division inhibitor induced upon DNA damage) and RcsA (activator of cps genes), respectively. In a previous study, we identified that a G232D mutation, termed faa (for function affecting alternative-lon-protease), in the E. coli co-chaperone DnaJ, results in suppression of lon mutant phenotypes. Additionally, inactivation of the trans-translation system was found to have an additive effect on faa activity. In the present work, we employed random mutagenesis approach to isolate novel mutations in dnaJ which could phenotypically compensate the absence of Lon. Using a lacZ-based Lon reporter strain, we were able to isolate two new mutations in dnaJ as lon suppressors. These mutations, namely, flm-1 (H33Y) and flm-2 (P34S), affected the highly conserved HPD motif of DnaJ. Both mutations suppressed lon phenotypes to variable extent and the suppression was also differentially modulated by mutations in ssrA that affect trans-translation. We show that ClpYQ protease up-regulated in both mutants should degrade SulA, since inactivation of clpQ abolished the resistance to DNA damaging agents. On the other hand, we found suppression of capsule overproduction phenotype was independent of ClpYQ in both mutants but resulted due to down-regulation of rcsA in flm-1. Thus, our findings highlight the intricate redundancy of cellular proteolysis networks in bacteria which can compensate the absence of Lon via distinct mechanisms.

Unveiling the molecular basis for pleiotropy in selected rif mutants of Escherichia coli: Possible role for Tyrosine in the Rif binding pocket and fast movement of RNA polymerase.

Rifampicin (RIF) is still a first line of antibiotic in the treatment of bacterial diseases, in particular the Mycobacterial infections. The antimicrobial activity of RIF is attributed to its ability to inhibit transcription by binding to the ß subunit of bacterial RNA polymerase (encoded by rpoB). Continued use of this drug resulted in the emergence of RIF resistant rpoB mutations in a high frequency that compels the use of RIF almost exclusively in drug combinations. As of date, a broad array of rif mutations have been isolated and characterized by different research groups. Studies on rpoB mutations strengthen the view that the ß subunit of RNA polymerase (RNAP) is very crucial in modulating transcription thereby leading to differential gene expression. Very recently we have reported the transcriptome profile of rpoB12 mutant that provides molecular evidence that presence of rpoB12 mutation modulates the transcription of about 450 genes. Here we present a maiden report that rpoB mutations that substitute Tyr at the Rif binding pocket (RBP) of ß subunit of RNA polymerase are able to suppress the over-production of colanic acid capsular polysaccharide (Ces phenotype) in Δlon mutant of Escherichia coli. Further analyses of the rif mutants involving their growth pattern on LB at higher temperature (42 °C), LB media without NaCl, survival in LB media with acidic pH (pH - 3) and motility revealed that only rpoB12 (His526Tyr) and rpoB137 (Ser522Tyr) affected all the above mentioned physiological parameters in addition to the elicitation of Ces phenotype. These two rif mutations confer fast movement to RNAP and they bear Tyr as the substituted amino acid in the RBP. This is perhaps the first study that brings out the possible role of Tyr in the RBP and its participation in the global gene expression. This study also envisages the point that amino acid residues that share the properties of Tyr in the RBP can be employed as a tool to bring out differential gene expression which would certainly have basic and applied values for the mankind.

Suppression of Δlon phenotypes in Escherichia coli by N-terminal DnaK peptides.

Δlon mutant of Escherichia coli becomes hypersensitive to DNA damaging agents and over-produce capsule due to stabilization of the Lon substrates, namely, SulA and RcsA, respectively. These phenotypes were earlier found to be suppressed in Δlon ssrA::cat/pUC4 K and Δlon faa (DnaJ, G232D) strains, called as "Alp" strains. We observed that a plasmid carrying an E. coli chromosomal fragment harboring few genes, a heat shock gene htpY and a portion of dnaK capable of encoding truncated N-terminal ATPase domain (244 aa) could suppress lon mutant phenotypes. Deletion of htpY did not affect the efficiency of suppression. Clones expressing DnaK' (244 aa) peptide alone could suppress both Δlon phenotypes in copy number dependent manner. Inactivation of clpQ did not affect the MMSR phenotype of Δlon strain carrying dnaK' clones indicating that ClpYQ protease does not degrade SulA. We hypothesize that the high levels of defective DnaK'-DnaJ chaperone complex formed in these strains might lead to aggregation of SulA and RcsA and, thereby the suppression of Δlon phenotypes. Systematic deletion analysis of dnaK' revealed that, ∼220 aa N-terminal DnaK peptide is required for suppression of cps-lac over-expression and ∼169 aa peptide is enough for the suppression of MMSS phenotype of Δlon mutant.

Microarray based transcriptome profile data of ∆lon and ∆lon rpoB12 strains of Escherichia coli.

The data presented in this article shows the microarray based transcriptome profiles of ∆lon and ∆lon rpoB12 strains of Escherichia coli. The rif mutation namely, rpoB12 was isolated spontaneously in the background of ∆lon strain (over-produces colanic acid capsular polysaccharide) as a suppressor for over-production of colanic acid capsular polysaccharide (Meenakshi and Munavar, 2015) [1]. The E. coli strains were grown in LB medium at 30 °C overnight in duplicates. Total RNA from each samples were isolated and microarray based transcriptome profiles were studied and compared. The detailed methodology and data are given in this article. The interpretation of these data are discussed in the research article, "Evidence for Up and Down Regulation of 450 genes by rpoB12 (rif) Mutation and their Implications in Complexity of Transcription Modulation in Escherichia coli" (Meenakshi and Munavar, 2018) [2].

A putative curved DNA region upstream of rcsA in Escherichia coli plays a key role in transcriptional regulation by H-NS.

It is well established that in Escherichia coli, the histone-like nucleoid structuring (H-NS) protein also functions as negative regulator of rcsA transcription. However, the exact mode of regulation of rcsA transcription by H-NS has not been studied extensively. Here, we report the multicopy effect of dominant-negative hns alleles on the transcription of rcsA based on expression of cps-lac transcriptional fusion in ∆lon, ∆lon rpoB12, ∆lon rpoB77 and lon+ strains. Our results indicate that H-NS defective in recognizing curved DNA fails to repress rcsA transcription significantly, while nonoligomeric H-NS molecules still retain the repressor activity to an appreciable extent. Together with bioinformatics analysis, our study envisages a critical role for the putative curved DNA region present upstream of rcsA promoter in the transcriptional regulation of rcsA by H-NS.

Evidence for up and down regulation of 450 genes by rpoB12 (rif) mutation and their implications in complexity of transcription modulation in Escherichia coli.

Analyses of mutations in rpoB subunit of Escherichia coli that lead to resistance to rifampicin have been invaluable in providing insight into events during transcription continue to be discovered. Earlier we reported that rpoB12 suppresses over-expression of cps genes in Δlon mutant of E. coli, by interfering with the transcription of rcsA. Here we report Microarray based Transcriptome profile of Δlon and Δlon rpoB12 strains. The data analyses clearly reveal that rpoB12 mutation results in the differential expression of ∼450 genes. The transcription profiles of some of the genes namely, rcsA, gadE, csgD, bolA, ypdI, dnaJ, clpP, csrA and hdeA are significantly altered, particularly the genes implicated in virulence. Some of the phenotypic traits namely, biofilm formation, motility, curli synthesis and ability to withstand acidic stress in a lon+rpoB12 strain were assessed. The results clearly indicate that rpoB12 up-regulates biofilm formation and curli synthesis while it makes the cells sensitive for growth in acidic medium and inhibits motility almost completely. Furthermore, rpoB12 modulates the expression profile of a significant number of genes involved in stress responses, genes encoding small RNAs. Thus, this study reveals the versatile role of the rpoB12 mutation, especially its impact on the regulation of genes related to virulence and highlights its medical importance.

Glu571 of PheT plays a pivotal role in the thermal stability of Escherichia coli PheRS enzyme.

As of date the two temperature sensitive mutations isolated in pheST operon include pheS5 (G293 →A293 ) and pheT354. Recently, we reported that G673 of pheS defines a hot spot for intragenic suppressors of pheS5. In this investigation, in 13 independent experiments, a collection of temperature sensitive mutants were isolated by localized mutagenesis. Complementation using clones bearing pheS+ , pheT+ , and pheS+ T+ indicated that 34 mutants could harbor lesion(s) in pheS and four could be in pheT and one mutant might be a double mutant. Surprisingly, all the 34 pheS mutants harbored the very same (G293 →A293 ) transition mutation as present in the classical pheS5 mutant. Most unexpectedly, the four pheT mutants isolated harbored the same G1711 →A1711 transition, a mutation which is hitherto unreported. Since all the four pheT mutants were defined by the same G1711 →A1711 base change, we believe that getting other mutations could be hard hitting and therefore it is proposed that G1711 itself could be a "hot spot" for emergence of Ts mutations in pheT and similarly G293 itself could be a "hot spot" for Ts lesions in pheS. These results clearly imply a vital role for Glutamic acid571 (Glu571 ) of PheT and reinforce criticality of Glycine98 (Gly98 ) of PheS in the thermal stability of PheRS enzyme.

Ascribing a novel role for tmRNA of Escherichia coli in resistance to mitomycin C.

AIM: The ssrA mutants were found to be more sensitive to mitomycin C (MMC) and our aim was to study this phenomenon in detail. MATERIALS & METHODS: Strains were constructed by P1 transduction. pssrA+ plasmid was constructed by PCR-based cloning and transformation was done by CaCl2 method. Relative viability analyses were done to assess the extent of viability of strains in relevant conditions. Gram staining was used for microscopic analysis. RESULTS: ssrA mutants become sensitive specifically to MMC, that too in a strain-specific manner. Precise tagging function of SsrA is necessary for conferring resistance to MMC. sulA::kan restored the viability of ssrA::cat mutants in a strain-specific manner. CONCLUSION: This study for the first time implicates SsrA in progression of efficient cell division and resistance to MMC.

Suppression of capsule expression in Δlon strains of Escherichia coli by two novel rpoB mutations in concert with HNS: possible role for DNA bending at rcsA promoter.

Analyses of mutations in genes coding for subunits of RNA polymerase always throw more light on the intricate events that regulate the expression of gene(s). Lon protease of Escherichia coli is implicated in the turnover of RcsA (positive regulator of genes involved in capsular polysaccharide synthesis) and SulA (cell division inhibitor induced upon DNA damage). Failure to degrade RcsA and SulA makes lon mutant cells to overproduce capsular polysaccharides and to become sensitive to DNA damaging agents. Earlier reports on suppressors for these characteristic lon phenotypes related the role of cochaperon DnaJ and tmRNA. Here, we report the isolation and characterization of two novel mutations in rpoB gene capable of modulating the expression of cps genes in Δlon strains of E. coli in concert with HNS. clpA, clpB, clpY, and clpQ mutations do not affect this capsule expression suppressor (Ces) phenotype. These mutant RNA polymerases affect rcsA transcription, but per se are not defective either at rcsA or at cps promoters. The results combined with bioinformatics analyses indicate that the weaker interaction between the enzyme and DNA::RNA hybrid during transcription might play a vital role in the lower level expression of rcsA. These results might have relevance to pathogenesis in related bacteria.

G673 could be a novel mutational hot spot for intragenic suppressors of pheS5 lesion in Escherichia coli.

The pheS5 Ts mutant of Escherichia coli defined by a G293 → A293 transition, which is responsible for thermosensitive Phenylalanyl-tRNA synthetase has been well studied at both biochemical and molecular level but genetic analyses pertaining to suppressors of pheS5 were hard to come by. Here we have systematically analyzed a spectrum of Temperature-insensitive derivatives isolated from pheS5 Ts mutant and identified two intragenic suppressors affecting the same base pair coordinate G673 (pheS19 defines G673 → T673 ; Gly225 → Cys225 and pheS28 defines G673 → C673 ; Gly225 → Arg225). In fact in the third derivative, the intragenic suppressor originally named pheS43 (G673 → C673 transversion) is virtually same as pheS28. In the fourth case, the very pheS5 lesion itself has got changed from A293 → T293 (named pheS40). Cloning of pheS(+), pheS5, pheS5-pheS19, pheS5-pheS28 alleles into pBR322 and introduction of these clones into pheS5 mutant revealed that excess of double mutant protein is not at all good for the survival of cells at 42°C. These results clearly indicate a pivotal role for Gly225 in the structural/functional integrity of alpha subunit of E. coli PheRS enzyme and it is proposed that G673 might define a hot spot for intragenic suppressors of pheS5.

Selective alleviation of Mitomycin C sensitivity in lexA3 strains of Escherichia coli demands allele specificity of rif-nal mutations: a pivotal role for rpoB87-gyrA87 mutations.

Very recently, we have reported about an unconventional mode of elicitation of Mitomycin C (MMC) specific resistance in lexA3 (SOS repair deficient) mutants due to a combination of Rif-Nal mutations (rpoB87-gyrA87). We have clearly shown that UvrB is mandatory for this unconventional MMC resistance in rpoB87-gyrA87-lexA3 strains and uvrB is expressed more even without DNA damage induction from its LexA dependent promoter despite the uncleavable LexA3 repressor. The rpoB87 allele is same as the rpoB3595 which is known to give rise to a fast moving RNA Polymerase and gyrA87 is a hitherto unreported Nal(R) allele. Thus, it is proposed that the RNA Polymerase with higher elongation rate with the mutant DNA Gyrase is able to overcome the repressional hurdle posed by LexA3 to express uvrB. In this study we have systematically analysed the effect of three other rpoB (rif) mutations-two known to give rise to fast moving RNAP (rpoB2 and rpoB111) and one to a slow moving RNAP (rpoB8) and four different alleles of gyrA Nal(R) mutations (gyrA199, gyrA247, gyrA250, gyrA259) isolated spontaneously, on elicitation of MMC resistance in lexA3 strains. Our results indicate that in order to acquire resistance to 0.5 µg/ml MMC cells require both rpoB87 and gyrA87 but resistance to 0.25 µg/ml of MMC can be brought about by either rpoB87, gyrA87, fast moving rpoB mutations or other nal mutations also. We have also depicted increased constitutive uvrB expression in strains carrying fast moving RNAP (rpoB2 and rpoB111) with gyrA87 and another nal mutation with rpoB87 and expression level in these strains is lesser than rpoB87-gyrA87 strain. These results evidently suggest an allele specific role for the rif-nal mutations to acquire MMC resistance in lexA3 strains via increased constitutive uvrB expression and a pivotal role for rpoB87-gyrA87 combination to elicit higher levels of resistance.

Evidence for involvement of UvrB in elicitation of 'SIR' phenotype by rpoB87-gyrA87 mutations in lexA3 mutant of Escherichia coli.

An unconventional DNA repair termed SIR (SOS Independent Repair), specific to mitomycin C (MMC) damage elicited by a combination of specific Rif(R) (rpoB87) and Nal(R) (gyrA87) mutations in SOS un-inducible strains of Escherichia coli was reported by Kumaresan and Jayaraman (1988). We report here that the rpoB87 mutation defines a C(1565)→T(1565) transition changing S(522)→F(522) and gyrA87 defines a G(244)→A(244) transition changing D(82)→N(82). The reconstructed lexA3 rpoB87 gyrA87 strain (DM49RN) exhibited resistance to MMC but not to UV as expected. When mutations in several genes implicated in SOS/NER were introduced into DM49RN strain, uvrB mutation alone decreased the MMC resistance and suppressed SIR phenotype. This was alleviated about two fold by a plasmid clone bearing the uvrB(+) allele. Neither SulA activity as measured based on filamentation and sulA::gfp fluorescence analyses nor the transcript levels of sulA as seen based on RT-PCR analyses indicate a change in sulA expression in DM49RN strain. However, uvrB transcript levels are increased with or without MMC treatment in the same strain. While the presence of lexA3 allele in a plasmid clone was found to markedly decrease the MMC resistance of the DM49RN strain, the additional presence of uvrB(+) allele in the same clone alleviated the suppression of MMC resistance by lexA3 allele to a considerable extent. These results indicate the increased expression of uvrB in the DM49RN strain is probably from the LexA dependent promoter of uvrB. The sequence analyses of various uvrB mutants including those isolated in this study using localized mutagenesis indicate the involvement of the nucleotide phosphate binding domain (ATPase domain) and the ATP binding domain and/or the DNA binding domain of the UvrB protein in the MMC repair in DM49RN. The possible involvement of UvrB protein in the MMC damage repair in DM49RN strain in relation to DNA repair is discussed.

Evidence that the supE44 mutation of Escherichia coli is an amber suppressor allele of glnX and that it also suppresses ochre and opal nonsense mutations.

Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.

Allele-specific suppression of the temperature sensitivity of fitA/fitB mutants of Escherichia coli by a new mutation (fitC4): isolation, characterization and its implications in transcription control.

The temperature sensitive transcription defective mutant of Escherichia coli originally called fitA76 has been shown to harbour two missense mutations namely pheS5 and fit95. In order to obtain a suppressor of fitA76, possibly mapping in rpoD locus, a Ts+ derivative (JV4) was isolated from a fitA76 mutant. It was found that JV4 neither harbours the lesions present in the original fitA76 nor a suppressor that maps in or near rpoD. We show that JV4 harbours a modified form of fitA76 (designated fitA76*) together with its suppressor. The results presented here indicate that the fit95 lesion is intact in the fitA76* mutant and the modification should be at the position of pheS5. Based on the cotransduction of the suppressor mutation and/or its wild type allele with pps, aroD and zdj-3124::Tn10 kan we have mapped its location to 39.01 min on the E. coli chromosome. We tentatively designate the locus defined by this new extragenic suppressor as fitC and the suppressor allele as fitC4. While fitC4 could suppress the Ts phenotype of fitA76* present in JV4, it fails to suppress the Ts phenotype of the original fitA76 mutant (harbouring pheS5 and fit95). Also fitC4 could suppress the Ts phenotype of a strain harbouring only pheS5. Interestingly, the fitC4 Ts phenotype could also be suppressed by fit95. The pattern of decay of pulse labelled RNA in the strains harbouring fitC4 and the fitA76* resembles that of the original fitA76 mutant implying a transcription defect similar to that of fitA76 in both these mutants. The implications of these findings with special reference to transcription control by Fit factors in vivo are discussed.