• R Jayaraman

      Articles written in Journal of Biosciences

    • Conditional rifampicin sensitivity of arif mutant ofEscherichia coli: rifampicin induced changes in transcription specificity

      S Balachandra Dass R Jayaraman

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      Arif mutantof Escherichia coli that exhibits medium and temperature-dependent sensitivity to rifampicin is described. In the absence of rifampicin, this strain grows in minimal and rich media at 30°C and 42°C. In its presence it is viable in rich medium at both temperatures, but in minimal medium only at 30°C. In minimal-rifampicin medium at the higher temperature, RNA synthesis is decreased. The addition of certain divalent salts (MgSO4, CaCl2, BaCl2) in excess, or chelators (EDTA, EGTA, o-phenanthrolein) greatly increase viability in minimal-rifampicin medium at 42°C. Excess MgSO4 (10 mM) also increases the rate of RNA synthesis in the same medium. A model is proposed wherein therif mutation is suggested to cause a structural change in RNA polymerase that allows the binding of rifampicin and other ligands at 42°C. Rifampicin-binding is suggested to alter the conformation of RNA polymerase, impairing its ability to express genes required for growth in minimal medium. Implicit in this view is the assumption that these genes are structurally different from those expressed in rich medium in respect of certain template features recognized by RNA polymerase.

    • Modulation of gene expression by the product offitA gene inEscherichia coli

      S Balachandra Dass R Jayaraman

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      Physiological parameters such as viability, gross RNA synthesis,β-galactosidase induction, development of phages T4, T7 andλ have been studied in temperature-sensitiveEscherichia coli strains harbouring fit A76,fit A24 andfit A76fit A24 mutations in rpoB+ andrpoB240 genetic backgrounds. The efficiently of expression of these functions is influenced by thefit A alleles depending upon the medium of growth and/or temperature. Strains harbouring therpoB240 mutation and thefit A76 mutation, either alone or together with thefit A24 mutation, are rifampicin-sensitive even at the perfssive temperature. The results suggest possible interaction between thefit A gene product and RNA polymerase invivo.

    • Molecular cloning, characterization and expression of a nitrofuran reductase gene ofescherichia coli

      Ajit N Kumar R Jayaraman

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      Mini-mu derivatives carrying plasmid replicons can be used to clone genesin vivo. This method was adopted to generate phasmid clones which were later screened for their ability of restore nitrofurantoin sensitivity of a nitrofuran-resistant host by eliciting nitroreductase activity. One phasmid-derived clone (pAJ101) resulted in considerable increase in nitroreductase activity when introduced into a nitrofurantoin-resistant mutant ofEscherichia coli with reduced nitroreductase activity. Subsequently, a 1.8 kb fragment obtained from pAJ101 by partial digestion with 5au3A, was subcloned into pUC18 to yield pAJ102. The nitroreductase activity attributable to pAJ102 was capable of reducing both nitrofurantoin and nitrofurazone. The polypeptides encoded by pAJ102 were identified by the minicell method. A large, well-defined band corresponding to 37 kDa and a smaller, less-defined band corresponding to 35 kDa were detected. Tnl000 mutagenesis was used to delineate the coding segment of the 1.8 kb insert of pAJ102. A 0.8 kb stretch of DNA was shown to be part of the nitroreductase gene. The gene was mapped at 19 min on theEscherichia coli linkage map.

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

      M Hussain Munavar K Madhavi R Jayaraman

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      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.

    • Thefit genes and transcription control inEscherichia coli

      R Jayaraman

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      This article reviews the work done in the author’s laboratory on the genetics and physiology of thefit mutants ofEscherichia coli. Isolaton of thefit mutants, genetic mapping, transcription abnormalities of thefit mutants, the possible involvement of thefit gene products in transcription control and identity of the fitA gene as pheS are described.

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

      Sandhya Ramalingam M Hussain Munavar S Sudha A Ruckmani R Jayaraman

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      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.

    • Allele-specific suppression of the temperature sensitivity offitA/fitB mutants ofEscherichia coli by a new mutation (fitC4): Isolation, characterization and its implications in transcription control

      S Vidya B Praveen Kamalakar M Hussain Munavar L Sathish Kumar R Jayaraman

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      The temperature sensitive transcription defective mutant ofEscherichia coli originallycalled fitA76 has been shown to harbour two missense mutations namelypheS5 andfit95. 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 originalfitA 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 thefitA 76* mutant and the modification should be at the positionof 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 offitA76* present in JV4, it fails to suppress the Ts phenotype of theoriginal fitA76 mutant (harbouringpheS5 andfit95). 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 harbouringfitC4 and thefitA76* resembles that of theoriginal 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 factorsin vivo are discussed.

    • Bacterial persistence: some new insights into an old phenomenon

      R Jayaraman

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      Bigger discovered more than 60 years ago, at the very beginning of the antibiotic era, that populations of antibiotic-sensitive bacteria contained a very small fraction (approximately 10–6) of antibiotic-tolerant cells (persisters). Persisters are different from antibiotic-resistant mutants in that their antibiotic tolerance is non-heritable and reversible. In spite of its importance as an interesting biological phenomenon and in the treatment of infectious diseases, persistence did not attract the attention of the scientific community for more than four decades since its discovery. The main reason for this lack of interest was the difficulty in isolating sufficient numbers of persister cells for experimentation, since the proportion of persisters in a population of wild-type cells is extremely small. However, with the discovery of high-persister (hip) mutants of Escherichia coli by Moyed and his group in the early 1980s, the phenomenon attracted the attention of many groups and significant progress has occurred since then. It is now believed that persistence is the end result of a stochastic switch in the expression of some toxin–antitoxin (TA) modules (of which the hipA and hipB genes could be examples), creating an imbalance in their intracellular levels. There are also models invoking the involvement of the alarmone (p) ppGpp in the generation of persisters. However, the precise mechanisms are still unknown. Bacterial persistence is part of a wider gamut of phenomena variously called as bistability, multistability, phenotypic heterogeneity, stochastic switching processes, etc. It has attracted the attention of not only microbiologists but also a diverse band of researchers such as biofilm researchers, evolutionary biologists, sociobiologists, etc. In this article, I attempt to present a broad overview of bacterial persistence to illustrate its significance and the need for further exploration.

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