Involvement of Antibiotic Efflux Machinery in Glutathione-Mediated Decreased Ciprofloxacin Activity in Escherichia coli.

We have analyzed the contribution of different efflux components to glutathione-mediated abrogation of ciprofloxacin's activity in Escherichia coli and the underlying potential mechanism(s) behind this phenomenon. The results indicated that glutathione increased the total active efflux, thereby partially contributing to glutathione-mediated neutralization of ciprofloxacin's antibacterial action in E. coli However, the role of glutathione-mediated increased efflux becomes evident in the absence of a functional TolC-AcrAB efflux pump.

Cyclic AMP receptor protein (CRP) regulates the expression of cspA, cspB, cspG and cspI, members of cspA family, in Escherichia coli.

Escherichia coli K-12 contains nine paralogs of CspA, CspA-CspI, collectively known as CspA family of cold-shock proteins (CSPs). In spite of the high degree of similarity among themselves, only five (cspA, B, E, G and I) are induced during cold-stress. In the present study, we show that cspB, cspG and cspI, the members of cspA family, known to be induced in response to cold shock, are regulated by cyclic AMP receptor protein (CRP) , a global regulator involved in sugar metabolism, during growth at 37 °C as well as at 15 °C, as seen by green fluorescent protein (gfp) promoter fusions assays. Interestingly, cspA is selectively regulated by CRP during growth at 15 °C but not at 37 °C. The regulation of cspA, cspB, cspG and cspI by CRP was found to be through an indirect mechanism as determined by electrophoretic mobility shift assay (EMSA). These results substantiate our earlier study demonstrating a role for CRP during growth at low temperature.

Cyclic AMP receptor protein regulates cspD, a bacterial toxin gene, in Escherichia coli.

cspD, a member of cspA family of cold shock genes in Escherichia coli, is not induced during cold shock. Its expression is induced during stationary phase. CspD inhibits DNA replication, and a high level of the protein is toxic to cells. Recently, CspD was proposed to be associated with persister cell formation in E. coli. Here, we show that cyclic AMP receptor protein (CRP) upregulates cspD transcription. Sequence analysis of the cspD upstream region revealed two tandem CRP target sites, CRP site-I (the proximal site centered at -83.5 with respect to the transcription start) and CRP site-II (the distal site centered at -112.5). The results from electrophoretic mobility shift assays showed that CRP indeed binds to these two target sites in PcspD. The promoter-proximal CRP target site was found to play a major role in PcspD activation by CRP, as studied by transcriptional fusions carrying mutations in the target sites. The results from in vitro transcription assays demonstrated that CRP activates PcspD transcription in the absence of additional factors other than RNA polymerase. The requirement for activating region 1 of CRP in PcspD activation, along with the involvement of the 287, 265, and 261 determinants of the α-CTD, suggest that CRP activates by a class I-type mechanism. However, only moderate activation in vitro was observed compared to high activation in vivo, suggesting there might be additional activators of PcspD. Overall, our findings show that CRP, a global metabolic regulator in E. coli, activates a gene potentially related to persistence.

Increased ultraviolet radiation sensitivity of Escherichia coli grown at low temperature.

The repair of DNA damage caused by ultraviolet radiation (UVR) is well understood in both lower and higher organisms. Genetic studies carried out at optimum temperature for growth, 37 °C in Escherichia coli, have revealed the major pathways of DNA repair. We show that E. coli cells grown at 20 °C are more sensitive to UVR than cells grown at 37 °C. The analysis of knockout mutants demonstrates that cells impaired in recombinational DNA repair pathways show increased UV sensitivity at 20 °C. Cells with mutations in the nucleotide excision repair (NER) pathway genes are highly sensitive to UVR when grown at 37 °C and retain that sensitivity when grown at 20 °C, whereas wild-type cells are not sensitive when grown at 37 °C but become more sensitive to UVR when grown at low temperatures. Our results taken along with reports from the literature suggest that the UVR sensitivity of E. coli cells at low temperature could be due to impaired NER function.

Involvement of pnp in survival of UV radiation in Escherichia coli K-12.

Polynucleotide phosphorylase (PNPase), a multifunctional protein, is a 3'→5' exoribonuclease or exoDNase in the presence of inorganic phosphate (P(i)), and extends a 3'-OH of RNA or ssDNA in the presence of ADP or dADP. In Escherichia coli, PNPase is known to protect against H(2)O(2)- and mitomycin C-induced damage. Recent reports show that Bacillus subtilis PNPase is required for repair of H(2)O(2)-induced double-strand breaks. Here we show that absence of PNPase makes E. coli cells sensitive to UV, indicating that PNPase has a role in survival of UV radiation damage. Analyses of various DNA repair pathways show that in the absence of nucleotide excision repair, survival of UV radiation depends critically on PNPase function. Consequently, uvrA pnp, uvrB pnp and uvrC pnp strains show hypersensitivity to UV radiation. Whereas the pnp mutation is non-epistatic to recJ, recQ and recG mutations with respect to the UV-sensitivity phenotype, it is epistatic to uvrD, recB and ruvA mutations, implicating it in the recombinational repair process.

Molecular and Biochemical Analysis of Duplicated Cytosolic CuZn Superoxide Dismutases of Rice and in silico Analysis in Plants.

Superoxide dismutases (SODs, EC 1.15.1.1) are ubiquitous antioxidant metalloenzymes important for oxidative stress tolerance and cellular redox environment. Multiple factors have contributed toward the origin and diversity of SOD isoforms among different organisms. In plants, the genome duplication events, responsible for the generation of multiple gene copies/gene families, have also contributed toward the SOD diversity. However, the importance of such molecular events on the characteristics of SODs has not been studied well. This study investigated the effects of divergence on important characteristics of two block-duplicated rice cytosolic CuZn SODs (OsCSD1, OsCSD4), along with in silico assessment of similar events in other plants. The analysis revealed heterogeneity in gene length, regulatory regions, untranslated regions (UTRs), and coding regions of two OsCSDs. An inconsistency in the database-predicted OsCSD1 gene structure was also identified and validated experimentally. Transcript analysis showed differences in the basal levels and stress responsiveness of OsCSD1 and OsCSD4, and indicated the presence of two transcription start sites in the OsCSD1. At the amino acid level, the two OsCSDs showed differences at 18 sites; however, both exist as a homodimer, displaying typical CuZn SOD characteristics, and enhancing the oxidative stress tolerance of Escherichia coli cells. However, OsCSD4 showed higher specific activity as well as stability. The comparison of the two OsCSDs with reported thermostable CSDs from other plants identified regions likely to be associated with stability, while the homology modeling and superposition highlighted structural differences. The two OsCSDs displayed heteromeric interaction capability and forms an enzymatically active heterodimer (OsCSD1:OsCSD4) on co-expression, which may have significance as both are cytosolic. In silico analysis of 74 plant genomes revealed the prevalence of block duplications for multiple CSD copies (mostly cytosolic). The divergence and clustering analysis of CSDs suggested the possibility of an ancestral duplication event in monocots. Conserved SOD features indicating retention of SOD function among CSD duplicates were evident in few monocots and dicots. In most other species, the CSD copies lacked critical features and may not harbor SOD function; however, other feature-associated functions or novel functions might be present. These aspects of divergent CSD copies encoding co-localized CSDs may have implications in plant SOD functions in the cytosol and other organelles.

Cyclic AMP receptor protein regulates cspE, an early cold-inducible gene, in Escherichia coli.

cspE, a member of the cspA family of cold shock proteins in Escherichia coli, is an early cold-inducible protein. The nucleic acid melting ability and transcription antiterminator activity of CspE have been reported to be critical for growth at low temperature. Here, we show that the cyclic AMP receptor protein (CRP), a global regulator involved in sugar metabolism, upregulates cspE in E. coli. Sequence analysis of the cspE upstream region revealed a putative CRP target site centered at -61.5 relative to the transcription start. The binding of CRP to this target site was demonstrated using electrophoretic mobility shift assays. The presence of this site was shown to be essential for P(cspE) activation by CRP. Mutational analysis of the binding site indicated that the presence of an intact second core motif is more important than the first core motif for CRP-P(cspE) interaction. Based on the promoter architecture, we classified P(cspE) as a class I CRP-dependent promoter. This was further substantiated by our data demonstrating the involvement of the AR1 domain of CRP in P(cspE) transcription. Furthermore, the substitutions in the key residues of the RNA polymerase α-subunit C-terminal domain (α-CTD), which are important for class I CRP-dependent transcription, showed the involvement of 265 and 287 determinants in P(cspE) transcription. In addition, the deletion of crp led to a growth defect at low temperature, suggesting that CRP plays an important role in cold adaptation.

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.

Intra-individual and intra-species heterogeneity in nuclear rDNA ITS region of Vigna species from subgenus Ceratotropis.

The extent of intra-individual and intra-species heterogeneity in the nuclear rDNA internal transcribed spacer (ITS) was investigated among the 'Asiatic Vigna' species (subgenus Ceratotropis). High intra- and inter-individual ITS polymorphism was observed among Vigna radiata accessions, where multiple ITS length variants ranging from approximately 700 to approximately 770 bp were detected on PCR amplification. Subsequent analysis revealed that the variants are 'heteroduplex ITS fragments' generated during the PCR process. Analysis of ITS from wild and cultivated forms of ten Vigna species from subgenus Ceratotropis revealed substantial intra-species divergence in four species: Vigna umbellata, Vigna trilobata, V. radiata and Vigna minima. However, no other species analysed showed intra-individual ITS heterogeneity as observed in V. radiata. The results demonstrate differential evolution of ITS sequence among wild and cultivated forms of V. radiata. Evidence indicates that intra-species hybridization and a slow 'molecular drive' are responsible for this phenomenon. Sequence analysis of 5.8S, ITS1 and ITS2 and secondary-structure analysis of ITS regions indicate that the ITS variants do not belong to pseudogenic rDNA repeat units. Further, reverse transcriptase-PCR (RT-PCR) analysis showed that rDNA repeat units harbouring certain intra-individual ITS variants were transcriptionally inactive, indicating the regulation of these loci by epigenetic gene silencing. The V. radiata ITS variants, when analysed together, did not cause any phylogenetic errors at the species level.

Posttranscriptional regulation of cspE in Escherichia coli: involvement of the short 5'-untranslated region.

Escherichia coli K-12 contains nine paralogs of CspA, namely CspA-CspI. In spite of considerable sequence similarity among these genes, the individual members of this family show significant differences in their expression regulation. Among these nine members, cspA, B, G and I have been reported to be cold-induced. The unusually long 5'-untranslated region (5'-UTR) of these four and other cold-induced genes has often been associated with their inducibility. Sequence analysis of the cspE upstream region revealed two promoter-like motifs having high scores. We identified the promoter site and established that cspE has a much shorter 5'-UTR compared to other cold-induced genes. Our results showed that cspE is induced to about threefold at both the transcript and the protein level in response to cold-shock. Its transcript half-life increases significantly upon cold-shock. Furthermore, we demonstrated that RNase E, a key endonuclease responsible for mRNA degradation in E. coli, regulates cspE transcript stability, possibly through the assembly of a degradosome. In silico analysis of the cspE 5'-UTR revealed alternative secondary structures at 37 and 15 degrees C. A point mutation that was predicted to relax the secondary structure of the 5'-UTR at 15 degrees C showed considerable reduction in transcript stability, indicating that alternative transcript secondary structures might be the cause of the differential stability.

The cyclic AMP receptor protein (CRP) regulates mqsRA, coding for the bacterial toxin-antitoxin gene pair, in Escherichia coli.

Bacterial persisters represent a small number of slow-growing antibiotic-tolerant cells among populations of rapidly growing cells, and are the main cause of frequent recurrent infections. MqsR-MqsA, the toxin-antitoxin (TA) pair, is the most frequently induced TA system associated with antibiotic persistence in Escherichia coli. In this study, we show that the cyclic AMP receptor protein (CRP) indirectly upregulates mqsRA transcription. We also show that CRP plays an important role in antibiotic persistence, which seems to be partially mediated through MqsRA. Overall, this study highlights the role of CRP as an important regulator of antibiotic persistence in E. coli.

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.

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.

Antioxidant supplementation enhances bacterial peritonitis in mice by inhibiting phagocytosis.

Antioxidants are known to exhibit numerous health benefits including anti-ageing, anti-apoptotic and immuno-stimulatory effects. However, we present the data showing counterproductive effects of therapeutically relevant antioxidants on bacterial clearance by the immune system in a murine peritonitic model. The antioxidants ascorbic acid, glutathione and N-acetylcysteine augmented morbidity and mortality in mice carrying Eshcerichia coli-induced acute bacterial peritonitis. Treatment of peritonitic mice with antioxidants significantly increased their bacterial load in the range of 0.3-2 logs. Antioxidant administration to peritonitic mice resulted in decreased numbers of macrophages, B-cells and dendritic cells at the primary site of infection and increased neutrophil infiltration. Serum TNF-α levels were also decreased in antioxidant-treated peritonitic mice. In vitro experiments showed that antioxidants reduced the phagocytic efficacy of peritoneal macrophages by ~60-75% and also decreased E. coli-induced oxidative burst in macrophages cells. Taken together, our data indicate that the antioxidants increased the severity of peritonitis by decreasing the phagocytic efficiency, oxidative burst, and TNF-α production, and increasing neutrophil infiltration. Based on these results, we propose that antioxidant supplementation during the course of bacterial infection is not recommended as it could be detrimental for the host. In addition, the present study underlines the importance of timing and context of antioxidant administration rather than indiscriminate usage to gain the best possible therapeutic advantage of these redox compounds.

Assessment of hybridization among wild and cultivated Vigna unguiculata subspecies revealed by arbitrarily primed polymerase chain reaction analysis.

BACKGROUND AND AIMS: Intra-species hybridization and incompletely homogenized ribosomal RNA repeat units have earlier been reported in 21 accessions of Vigna unguiculata from six subspecies using internal transcribed spacer (ITS) and 5S intergenic spacer (IGS) analyses. However, the relationships among these accessions were not clear from these analyses. We therefore assessed intra-species hybridization in the same set of accessions. METHODOLOGY: Arbitrarily primed polymerase chain reaction (AP-PCR) analysis was carried out using 12 primers. The PCR products were resolved on agarose gels and the DNA fragments were scored manually. Genetic relationships were inferred by TREECON software using unweighted paired group method with arithmetic averages (UPGMA) cluster analysis evaluated by bootstrapping and compared with previous analyses based on ITS and 5S IGS. PRINCIPAL RESULTS: A total of 202 (86 %) fragments were found to be polymorphic and used for generating a genetic distance matrix. Twenty-one V. unguiculata accessions were grouped into three main clusters. The cultivated subspecies (var. unguiculata) and most of its wild progenitors (var. spontanea) were placed in cluster I along with ssp. pubescens and ssp. stenophylla. Whereas var. spontanea were grouped with ssp. alba and ssp. tenuis accessions in cluster II, ssp. alba and ssp. baoulensis were included in cluster III. Close affinities of ssp. unguiculata, ssp. alba and ssp. tenuis suggested inter-subspecies hybridization. CONCLUSIONS: Multi-locus AP-PCR analysis reveals that intra-species hybridization is prevalent among V. unguiculata subspecies and suggests that grouping of accessions from two different subspecies is not solely due to the similarity in the ITS and 5S IGS regions but also due to other regions of the genome.

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.

A novel mutation spatially remote from the G-domain in IF2 affects the cold stress adaptation of Escherichia coli.

In this paper, we describe a new mutation, gicD1, that gives a cold-sensitive phenotype in bacterial cell growth. Complementation analysis showed gicD1 to be allelic to infB. We identify gicD1 to be a valine to isoleucine substitution in initiation factor-2 (IF2) of a residue that seems to be well conserved in eubacterial IF2 proteins. This mutation lies in a region distant from the G-domain to which all earlier reported cold-sensitive mutations cluster. We describe a novel phenotype of the mutant that is suppression of rpsL31-mediated streptomycin resistance in cold. We provide evidence that mutant IF2 specifically interacts with rpsL31 in cold, leading to a bacteriostatic effect on host cells.