M. Hussain Munavar

Aberrant transcriptionin fit mutants ofEscherichia coli and its alleviation by suppressor mutations

Earlier work from this laboratory had identified, mapped and characterised an intragenic suppressor(fitA24) as well as an extragenic suppressor(fitB) for the temperature-sensitive transcription defective mutationfitA76 inEscherichia coli In this communication we report the results of experiments on RNA synthesis and decay of pulse labelled RNA in strains harbouringfit A76,fitB, fitA24, fitA76-fitA24, fitA76-fitB mutation(s) as well as in the isogenicfitA+ fitB+ strain. Taken together with earlier results, this indicates that thefitA andfitB gene products could be involved in the expression of some classes of genes including genes coding for ribosomal proteins. The implications of these results for thein vivo control of transcription inEscherichia coli are discussed.

Extragenic suppression of the temperature-sensitivity of afitA mutation by afitB mutation inEscherichia coli: Possible interaction between FitA and FitB gene products in transcription control

Temperature-sensitivity due to thefit A76 mutation is suppressed by a mutation in a locus mapping in between the Pps and FitA loci, at approximately 37.3 min on theE. coli circular genetic map. Genetic evidence suggests that the suppressor mutation might define a distinct genetic locus which has been named FitB. Suppression by thefit B mutation is independent of the medium of growth unlike the previously described intragenic suppression which is manifested only in rich media. Infit B/fit B’ merodiploids there is a drastic reduction of suppression efficiency in minimal medium. The possible mode of action of the FitA and FitB gene products in transcription control is discussed.

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.

Genetic evidence for interaction betweenfitA, fitB andrpoB gene products and its implication in transcription control inEscherichia coli

ThefitB mutation (Fit, factor involved in transcription) inE. coli was earlier identified as an extragenic suppressor of thefitA76 mutation, which confers a temperaturesensitive transcription defect. Here we show that thefitB mutation by itself confers a temperature-sensitive phenotype depending on the presence or absence of NaCl or glucose, or both, in the medium. ThefitB mutation suppresses the temperature-sensitive phenotype due to thefitA24 mutation also. However, suppression offit A24 byfit B is restricted to rich medium, unlike suppression in thefitA76 fitB combination where it is independent of the medium. The strain harbouringfitA76, fitA24 andfitB mutations shows the extragenically suppressed (as infit A76 fit B) phenotype. Severalrif (rpoB) alleles isolated in afitB genetic background affect growth of thefit B mutant, depending on the medium of growth, temperature, and presence or absence of rifampicin. We propose a model for interaction betweenfitA andfitB gene products and involvement of thefit genes in transcription controlin vivo.

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 NalR 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 NalR 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.

Elucidation of the lesions present in the transcription defectivefitA76 mutant ofEscherichia coli: Implication of phenylalanyl tRNA synthetase subunits as transcription factors

Earlier reports from our laboratory dealt with the identification, mapping and characterization of a temperature sensitive mutant (fitA76) with a primary transcription defect at 42‡C and two of its suppressors (fitA24 andfitB). We report here the cloning and molecular characterization of a 2-1 kb DNA fragment which complemented the Ts phenotype of thefitA76 andfitA24 mutants but not that due to thefitB mutant. Cloning of this fragment in the T7 expression vector pT7.5 revealed the synthesis of a 33 kDa protein. The fragment hybridized with the Kohara phages 322 and 323 whose overlapping regions includepheS,pheT andrplT genes. Nucleotide sequencing showed that the fragment contains the entirepheS gene and the N-terminal portion ofpheT. Although these results implied that thefitA andpheS genes could be one and the same, earlier data had ruled out such a possibility. In order to know whether thefitA76 mutation defines a novel allele ofpheS, thepheS region of thefitA76 mutant was also sequenced, revealing a G → A nucleotide transition at position 293 of the coding region. This lesion is the same as that reported for thepheS5 mutant. However, it is shown that thefitA76 mutant is primarily transcription-defective while thepheS5 mutant is primarily translation-defective. These results suggested that thefitA76 mutant might harbour another mutation, in addition topheS5. In this report, we present genetic evidence for a second mutation (namedfit95) in thefitA76 mutant. Thefit95 by itself confers a Ts phenotype on rich media devoid of sodium chloride. It is proposed that the subunits of phenylalanyl tRNA synthetase could act as transcription factors (Fit) also.

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 → C673transversion) 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.

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.

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.