Narendra Jawali

Involvement of Reactive Oxygen Species in the Action of Ciprofloxacin against Escherichia coli

ABSTRACT Ciprofloxacin is an important and commonly used member of the fluoroquinolone group of antibiotics. Ciprofloxacin inhibits DNA topoisomerase II and DNA topoisomerase IV activities, eventually leading to bacterial cell death. In addition, an increase of reactive oxygen species in the bacterial cells in response to ciprofloxacin has been shown. We investigated the role of reactive oxygen species in the antibacterial action of ciprofloxacin by studying the effects of different antioxidant compounds on ciprofloxacin susceptibility of Escherichia coli. Among the antioxidants checked, glutathione and ascorbic acid provided substantial protection against ciprofloxacin. The involvement of superoxide anion (O2−) and hydrogen peroxide (H2O2) in the antibacterial action of ciprofloxacin was analyzed using superoxide dismutase, catalase, and alkyl hydroperoxide reductase knockout strains of E. coli. The effects of multicopy sod genes on ciprofloxacin susceptibility of E. coli were also analyzed. On the basis of our results, we conclude that O2− and H2O2 may be involved in antibacterial action of ciprofloxacin. Our findings that glutathione gave protection against other fluoroquinolones and not against nonfluoroquinolone antibiotics imply that reactive oxygen species may have a similar role in the antibacterial action of all these fluoroquinolones and that glutathione-mediated protection is not a general phenomenon but specific to fluoroquinolones. These observations are of significance, as fluoroquinolones are important antibiotics with immense therapeutic value, and the effectiveness of treatment by these drugs may be affected by dietary intake and cellular levels of these antioxidants.

Antioxidant enzyme activities and protein profiling under salt stress in indica rice genotypes differing in salt tolerance

The effect of NaCl stress (0–300 mM) was investigated in terms of antioxidant enzymes activity and their isozymatic pattern and SDS-PAGE proteins banding pattern in three rice cultivars (cvs), Panvel-3 (tolerant), Kalarata (moderately tolerant) and Karjat-3 (sensitive). Interestingly, superoxide dismutase activity in roots and glutathione reductase activity in both shoots and roots were decreased significantly under high salinity levels in Karjat-3 and Kalarata, while, a sharp increase was observed in Panvel-3. Catalase and peroxidase activities were increased with salt stress of all cvs, with highest magnitude in Panvel-3. There was an induction of two new POX isoforms in Panvel-3 and Kalarata in stressed plants. Four SOD isoforms were observed in all the genotypes, irrespective of non/saline conditions. Total 33 proteins bands ranging from 17–154.5 kDa were either expressed de novo or up/down-accumulated due to NaCl stress. In Panvel-3, three new bands, one of 32 kDa in shoots while two of 37 and 116.7 kDa appeared in roots under salt stress. A new band of 37 kDa was also observed in roots of Kalarata under salinity stress. Thus, salinity tolerance nature of Panvel-3 may be correlated with higher enzyme activities and expression of some new polypeptides under salt stress.

Effects of glutathione and ascorbic acid on streptomycin sensitivity of Escherichia coli.

ABSTRACT We examined the effects of antioxidants and the role of reactive oxygen species (ROS) on the antibacterial action of aminoglycosides in Escherichia coli. We concluded that reduced streptomycin sensitivity in the presence of glutathione and ascorbic acid is not due to the antioxidant-mediated scavenging of ROS.

Comparative quantitative analysis of headspace volatiles and their association with BADH2 marker in non-basmati scented, basmati and non-scented rice (Oryza sativa L.) cultivars of India

For the first time, 91 Indian rice (Oryza sativa L.) cultivars, belonging to non-basmati scented (77), basmati (9) and non-scented (5) categories, were quantitatively analysed for 23 headspace volatiles. In addition, the BADH2 marker related to 2-acetyl-1-pyrroline (2AP) synthesis is validated among the cultivars representing these categories. The non-basmati scented cultivars (15) excelled in 2-acetyl-1-pyrroline (2AP) content compared to basmati cultivars. The major aroma volatile (2AP) correlated positively with 1-tetradecene and indole, and negatively with benzyl alcohol. On the basis of nonanal, octanal, decanal and 1-octen-3-ol contents, basmati cultivars were different from non-scented cultivars. Benzyl alcohol, 2-phenylethanol, 2-amino acetophenone, indole, 1-hexanol and nonanoic acid exhibited significant variation among rice categories under study. This study reports 16 non-basmati scented cultivars with variations in the BADH2 locus rendering a marker based on 8bp deletion in BADH2 as unsuitable for MAS in rice cultivars under study.

N-Acetylcysteine-Mediated Modulation of Bacterial Antibiotic Susceptibility

N-Acetylcysteine (NAC) acts as a precursor for glutathione biosynthesis (1, 2), in addition to performing several other biological functions in mammals and bacteria (1, 5). As per our earlier studies (3, 4), glutathione is an important modulator of antibiotic activity in bacteria; consequently, it is of interest to study the effect of NAC on bacterial antibiotic susceptibility. It becomes even more important since NAC is supplied as a mucolytic agent in combination with antibiotics during treatment of lower respiratory tract infection (1). Here we report the effect of NAC on various antibiotics against different bacterial strains, including opportunistic respiratory pathogens like Klebsiella and Pseudomonas.

Resveratrol induced inhibition of Escherichia coli proceeds via membrane oxidation and independent of diffusible reactive oxygen species generation.

Resveratrol (5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol), a redox active phytoalexin with a large number of beneficial activities is also known for antibacterial property. However the mechanism of action of resveratrol against bacteria remains unknown. Due to its extensive redox property it was envisaged if reactive oxygen species (ROS) generation by resveratrol could be a reason behind its antibacterial activity. Employing Escherichia coli as a model organism we have evaluated the role of diffusible reactive oxygen species in the events leading to inhibition of this organism by resveratrol. Evidence for the role of ROS in E. coli treated with resveratrol was investigated by direct quantification of ROS by flow cytometry, supplementation with ROS scavengers, depletion of intracellular glutathione, employing mutants devoid of enzymatic antioxidant defences, induction of adaptive response prior to resveratrol challenge and monitoring oxidative stress response elements oxyR, soxS and soxR upon resveratrol treatment. Resveratrol treatment did not result in scavengable ROS generation in E. coli cells. However, evidence towards membrane damage was obtained by potassium leakage (atomic absorption spectrometry) and propidium iodide uptake (flow cytometry and microscopy) as an early event. Based on the comprehensive evidences this study concludes for the first time the antibacterial property of resveratrol against E. coli does not progress via the diffusible ROS but is mediated by site-specific oxidative damage to the cell membrane as the primary event.

Characterisation of aromatic rice (Oryza sativa L.) germplasm and correlation between their agronomic and quality traits

Eighty-eight aromatic cultivars collected from Maharashtra state were assessed for determinants of kernel quality (kernel size-shape, test weight and aroma) and grain morphology such as awning, lemma and palea characters, pubescence, colour of sterile lemma and apiculus colour. We, report seven cultivars—‘Girga’, ‘Kothmirsal’, ‘Kala bhat’, ‘Chimansal’, ‘Jiri’, ‘Kalsal’ ‘Velchi’ and ‘Kali kumud’ as indigenous to southern India. Of the 69 cultivars characterized for agronomic traits 36 cultivars were exquisite genotypes and possessed one or more superior traits such as early flowering, dwarf stature, higher number of productive tiller per plant; long panicles; higher number of filled grains per panicle and strong aroma. Variability in aromatic cultivars was assessed on the basis of nine traits placed aromatic rice cultivars in five clusters. Number of cultivars in each cluster ranged from 1 to 33. 27 significant correlations were obtained in the physical, agronomic and grain morphology traits. Aroma was found to be negatively associated with days to 50% flowering as well as with filled grains per panicle. However, correlation between panicle length and effective tillers with aroma was not observed. Therefore, to increase the yield, improvement in length of panicle and increasing number of productive tillers in medium or mild scented cultivars would be the best strategy.

Shikimate pathway modulates the elicitor-stimulated accumulation of fragrant 2-hydroxy-4-methoxybenzaldehyde in Hemidesmus indicus roots

Enzymatic route to fragrant 2-hydroxy-4-methoxybenzaldehyde (MBALD) formation in Hemidesmus indicus roots is not known. Earlier studies with H. indicus excised roots suggested a possible origin of MBALD via central phenylpropanoid pathway. Different elicitors (e.g., chitosan, methyl jasmonate, yeast extract) were tested for their relative efficiency in uplifting MBALD accumulation in roots, amongst which, treatment with yeast extract for 18 h showed maximum accumulation in excised roots. As benzoate pathways originate either directly from shikimate or via phenylpropanoid pathway, this study aimed at finding the roles of shikimate pathway in uplifting/enhancing MBALD accumulation in H. indicus roots upon elicitation. In fact, a sharp increase in shikimate dehydrogenase (SKDH; E.C. 1.1.1.25) along with phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.24) activities was noted on a time-course basis in yeast extract-treated roots as compared to the untreated ones. PAL as well as phenylpropanoid C₂ side-chain cleavage activities (leading to p-hydroxybenzaldehyde, the first benzoate product formed in the MBALD pathway) were compared in elicited roots, non-elicited roots and glyphosate-treated elicited roots at different concentrations of glyphosate. It was observed that glyphosate treatment, in addition to 25% suppressions of phenylalanine ammonia-lyase and C₂ chain-cleavage enzyme activities as compared to elicited one, also resulted in around 40% suppression of MBALD accumulation, when used in conjunction with yeast extract treatment; in contrast, shikimic acid content was increased as compared to glyphosate untreated ones. These findings suggest that shikimate pathway plays an important role in modulating MBALD biosynthesis in H. indicus roots.

Phylogenetic Analysis of Subgenus Vigna Species Using Nuclear Ribosomal RNA ITS: Evidence of Hybridization among Vigna unguiculata Subspecies

Molecular phylogeny among species belonging to subgenus Vigna (genus Vigna) was inferred based on internal transcribed spacer (ITS) sequences of 18S-5.8S-26S ribosomal RNA gene unit. Analysis showed a total of 356 polymorphic sites of which approximately 80% were parsimony informative. Phylogenetic reconstruction by neighbor joining and maximum parsimony methods placed the 57 Vigna accessions (belonging to 15 species) into 5 major clades. Five species viz. Vigna heterophylla, Vigna pubigera, Vigna parkeri, Vigna laurentii, and Vigna gracilis whose position in the subgenus was previously not known were placed in the section Vigna. A single accession (Vigna unguiculata ssp. tenuis, NI 1637) harbored 2 intragenomic ITS variants, indicative of 2 different types of ribosomal DNA (rDNA) repeat units. ITS variant type-I was close to ITS from V. unguiculata ssp. pubescens, whereas type-II was close to V. unguiculata ssp. tenuis. Transcript analysis clearly demonstrates that in accession NI 1637, rDNA repeat units with only type-II ITS variants are transcriptionally active. Evidence from sequence analysis (of 5.8S, ITS1, and ITS2) and secondary structure analysis (of ITS1 and ITS2) indicates that the type-I ITS variant probably does not belong to the pseudogenic rDNA repeat units. The results from phylogenetic and transcript analysis suggest that the rDNA units with the type-I ITS may have introgressed as a result of hybridization (between ssp. tenuis and ssp. pubescens); however, it has been epigenetically silenced. The results also demonstrate differential evolution of ITS sequence among wild and cultivated forms of V. unguiculata.

Differential response of indica rice genotypes to NaCl stress in relation to physiological and biochemical parameters

Abstract The effect of NaCl stress was studied in indica rice cultivars, Panvel-3 (tolerant), Kalarata (moderately tolerant) and Karjat-3 (sensitive) under various levels (50 – 300 mM) of salinity besides control (0 mM). Salinity stress decreased germination percentage, plant growth, biomass production and chlorophyll pigments, and increased malondialdehyde level (lipid peroxidation) and free proline accumulation. Salinity-induced decrease in germination percentage, biomass production, chlorophyll and total protein contents were significantly higher in Karjat-3 than Panvel-3. The free proline content at all the levels as well as the magnitude of increase in accumulation with increasing salinity was also highest in Panvel-3, as at 300 mM NaCl concentration, it was 8 times more than control, while it was about 5 times more in Karjat-3 and around 4 times in Kalarata. The level of lipid peroxidation was lowest at all levels of salinity in Panvel-3 as compared with Karjat-3, however, Kalarata showed intermediate results. Results showed salinity tolerance of Panvel-3 was manifested by lower decrease in germination, plant growth and chlorophyll pigments and associated with higher proline accumulation and lower lipid peroxidation under high salinity stress.