Beneficial effects of bioinspired silver nanoparticles on zebrafish embryos including a gene expression study.

Background and purpose: Many sectors use nanoparticles and dispose of them in the aquatic environment without deciding the fate of these particles. Experimental approach: To identify a benign species of nanoparticles which can cause minimum harm to the aquatic environment, a comparative study was done with chemically synthesized silver nanoparticles (AgNPs) and green tea mediated synthesis (GT/AgNP) in both in vitro using human alveolar cancer cell line (A549) and normal cell line (L132), and in in vivo with zebrafish embryos. Key results: The in vitro studies revealed that GT/AgNPs were less toxic to normal cells than cancer cells. The GT/AgNPs showed high biocompatibility for zebrafish embryos monitored microscopically for their developmental stages and by cumulative hatchability studies. The reduced hatchability found in the AgNPs-treated group was correlated by differential gene expression of zebrafish hatching enzymes (ZHE) (ZHE1 and ZHE2). Conclusion: The results indicated that nanoparticles can affect the hatching of zebrafish embryos and elicit toxicity at the gene level.

The role of probiotics as wound healers: an overall view.

A wound is an injury to the skin or damage to the body tissue. The healing process differs between various kinds of wounds. Treatment of hard-to-heal (chronic) wounds becomes challenging for healthcare practitioners, especially if patients have underlying health complications such as diabetes. Infection of wounds is another factor that interferes with the healing process and extends its duration. Active research is being conducted into the development of advanced wound dressing technologies. These wound dressings are intended to manage the exudate, reduce bacterial infection and speed up the healing process. Probiotics have been receiving much attention because of their potential application in the clinical field, especially in diagnostics and treatment strategies of various infectious and non-infectious diseases. The host immune-modulatory response and antimicrobial activity of probiotics are expanding their role in the development of improved wound dressing technology.

Rise of Bacterial Small Proteins and Peptides in Therapeutic Applications.

BACKGROUND: Polypeptides that comprise less than 100 amino acids (50 amino acids in some cases) are referred to as small proteins (SPs), however, as of date, there is no strict definition. In contrast to the small polypeptides that arise due to proteolytic activity or abrupt protein synthesis, SPs are coded by small open reading frames (sORFs) and are conventionally synthesized by ribosomes. PURPOSE OF THE REVIEW: Although proteins that contain more than 100 amino acids have been studied exquisitely, studies on small proteins have been largely ignored, basically due to the unsuccessful detection of these SPs by traditional methodologies/techniques. Serendipitous observation of several small proteins and elucidation of their vital functions in cellular processes opened the floodgate of a new area of research on the new family of proteins called "Small proteins". Having known the significance of such SPs, several advanced techniques are being developed to precisely identify and characterize them. CONCLUSION: Bacterial small proteins (BSPs) are being intensely investigated in recent days and that has brought the versatile role of BSPs into the limelight. In particular, identification of the fact that BSPs exhibit antimicrobial activity has further expanded its scope in the area of therapeutics. Since the microbiome plays an inevitable role in determining the outcome of personalized medicine, studies on the secretory small proteins of the microbiome are gaining momentum. This review discusses the importance of bacterial small proteins and peptides in terms of their therapeutic applications.

Unveiling the molecular basis for pleiotropy in selected rif mutants of Escherichia coli: Possible role for Tyrosine in the Rif binding pocket and fast movement of RNA polymerase.

Rifampicin (RIF) is still a first line of antibiotic in the treatment of bacterial diseases, in particular the Mycobacterial infections. The antimicrobial activity of RIF is attributed to its ability to inhibit transcription by binding to the ß subunit of bacterial RNA polymerase (encoded by rpoB). Continued use of this drug resulted in the emergence of RIF resistant rpoB mutations in a high frequency that compels the use of RIF almost exclusively in drug combinations. As of date, a broad array of rif mutations have been isolated and characterized by different research groups. Studies on rpoB mutations strengthen the view that the ß subunit of RNA polymerase (RNAP) is very crucial in modulating transcription thereby leading to differential gene expression. Very recently we have reported the transcriptome profile of rpoB12 mutant that provides molecular evidence that presence of rpoB12 mutation modulates the transcription of about 450 genes. Here we present a maiden report that rpoB mutations that substitute Tyr at the Rif binding pocket (RBP) of ß subunit of RNA polymerase are able to suppress the over-production of colanic acid capsular polysaccharide (Ces phenotype) in Δlon mutant of Escherichia coli. Further analyses of the rif mutants involving their growth pattern on LB at higher temperature (42 °C), LB media without NaCl, survival in LB media with acidic pH (pH - 3) and motility revealed that only rpoB12 (His526Tyr) and rpoB137 (Ser522Tyr) affected all the above mentioned physiological parameters in addition to the elicitation of Ces phenotype. These two rif mutations confer fast movement to RNAP and they bear Tyr as the substituted amino acid in the RBP. This is perhaps the first study that brings out the possible role of Tyr in the RBP and its participation in the global gene expression. This study also envisages the point that amino acid residues that share the properties of Tyr in the RBP can be employed as a tool to bring out differential gene expression which would certainly have basic and applied values for the mankind.

Microarray based transcriptome profile data of ∆lon and ∆lon rpoB12 strains of Escherichia coli.

The data presented in this article shows the microarray based transcriptome profiles of ∆lon and ∆lon rpoB12 strains of Escherichia coli. The rif mutation namely, rpoB12 was isolated spontaneously in the background of ∆lon strain (over-produces colanic acid capsular polysaccharide) as a suppressor for over-production of colanic acid capsular polysaccharide (Meenakshi and Munavar, 2015) [1]. The E. coli strains were grown in LB medium at 30 °C overnight in duplicates. Total RNA from each samples were isolated and microarray based transcriptome profiles were studied and compared. The detailed methodology and data are given in this article. The interpretation of these data are discussed in the research article, "Evidence for Up and Down Regulation of 450 genes by rpoB12 (rif) Mutation and their Implications in Complexity of Transcription Modulation in Escherichia coli" (Meenakshi and Munavar, 2018) [2].

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.

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.

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.

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.