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

Genetic evidence for interaction between fitA, fitB and rpoB gene products and its implication in transcription control in Escherichia coli

The fitB mutation (Fit, factor involved in transcription) inE. coli was earlier identified as an extragenic suppressor of thefitA76 mutation, which confers a temperature sensitive transcription defect. Here we show that the fitB mutation by itself confers a temperature-sensitive phenotype depending on the presence or absence of NaCl or glucose, or both, in the medium. The fitB mutation suppresses the temperature-sensitive phenotype due to thefitA24 mutation also. However, suppression off it 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

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 originallycalled fitA76 has been shown to harbour two missense mutations namelypheS5 and fit95. In order to obtain a suppressor offitA76, possibly mapping inrpoD locus, a Ts+ derivative (JV4) was isolated from afitA76 mutant. It was found that JV4 neither harbours the lesions present in the original fitA 76 nor a suppressor that maps in or nearrpoD. We show that JV4 harbours a modified form offitA76 (designatedfitA76*) together with its suppressor. The results presented here indicate that thefit95 lesion is intact in the fitA 76* mutant and the modification should be at the position of pheS5. Based on the cotransduction of the suppressor mutation and/or its wild type allelewith pps, aroD andzdj-3124::Tn10 kan we have mapped its location to 39.01 min on theE. coli chromosome. We tentatively designate the locus defined by this new extragenic suppressoras fitC and the suppressor allele asfitC4. While fitC4 could suppress the Ts phenotype of fitA76* present in JV4, it fails to suppress the Ts phenotype of the original fitA76 mutant (harbouringpheS5 and fit95). AlsofitC4 could suppress the Ts phenotype of a strain harbouringonly pheS5. Interestingly, thefitC4 Ts phenotype could also be suppressed byfit95. 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 offitA76 in both these mutants. The implications of these findings with special reference to transcription control by Fit factors in vivo are discussed

Assessing role of Cra in regulation of <i>prpB </i>and <i>yahA </i>genes of <i>Escherichia coli in vivo </i>using <i>lacZ </i>transcriptional fusions

938-944This study presents in vivo role of Cra (Catabolite repressor activator) protein in regulation of two selected genes (prpB & yahA) of Escherichia coli. Gene prpB bears Cra binding site and yahA region binds to Cra in vitro. Therefore, in pBR322 background, pprpB-lacZ and pyahA-lacZ transcriptional fusions were generated and studied expression of b-galactosidase from both fusions in cra+ and cra¯ genetic backgrounds of E. coli. Results that both the promoters could possibly be up-regulated by Cra in vivo in E. coli were discussed in relation to transcription control by accessory transcription factor(s)

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 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

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

<i>rpoB</i> alleles used in this study and their relevant characteristics.

*<p>Values given as fold increase in termination read through are from Jin <i>et al</i>, 1988.</p

Level of MMC survival in relevant strains and its implications.

<p>R (+++) – ∼100% survival.</p><p>R (++) – ∼1% survival.</p><p>S (−) – ∼0.01–0.001% survival.</p><p>S (–) – ∼ 0.0001–0.00001% survival.</p><p>S (–) – Complete loss of survival.</p

Extracellular synthesis of silver nanoparticles by the fungus <i>Emericella nidulans</i> EV4 and its application

262-265Over the last decade, nanotechnology has potentiated remarkable growth, particularly in the field of biomedical sciences including pharmacology, precisely drug delivery and surgery. It has lead to the increased demand for synthesis of nanoparticles. Biological synthesis adopting green chemistry procedures involving microorganisms, fungi and even plants took centre stage. Here, we tried out synthesis of silver nanoparticles using the fungus Emericella nidulans EV4 and studied their antibacterial activity against Pseudomonas aeruginosa NCIM 5029. Silver nanoparticles were synthesised when the cell free filtrate of the fungus E. nidulans EV4 was treated with 1.0 mM silver nitrate solution. The UV-visible spectrum of the silver nanoparticles showed a Surface Plasmon Resonance (SPR) peak at 420 nm. High resolution transmission electron microscopy analysis indicated that the nanoparticles were spherical in shape with a size range of 10-20 nm. X-ray diffraction analysis revealed the formation of face-centred cubic structure of the silver with average crystallite size of ~3.5 nm. The synthesized silver nanoparticles in solution were found to be stable for a period of 12 months without any stabilizing agents. The silver nanoparticles, synthesized as detailed above, demonstrated control over the growth of Pseudomonas aeruginosa NCIM 5029