Ravindra Pal Singh

Synthesis and characterization of agar-based silver nanoparticles and nanocomposite film with antibacterial applications

This study describes the synthesis and characterization of silver nanoparticles and nanocomposite material using agar extracted from the red alga Gracilaria dura. Characterization of silver nanoparticles was carried out based on UV-Vis spectroscopy (421 nm), transmission electron microscopy, EDX, SAED and XRD analysis. The thermal stability of agar/silver nanocomposite film determined by TGA and DSC analysis showed distinct patterns when compared with their raw material (agar and AgNO(3)). The TEM findings revealed that the silver nanoparticles synthesized were spherical in shape, 6 nm in size with uniform dispersal. The synthesized nanoparticles had the great bactericidal activity with reduction of 99.9% of bacteria over the control value. The time required for synthesis of silver nanoparticles was found to be temperature dependent and higher the temperature less the time for nanoparticles formation. DSC and XRD showed approximately the same crystalline index (CI(DSC) 0.73).

Seaweed–microbial interactions: key functions of seaweed-associated bacteria

Seaweed-associated bacteria play a crucial role in morphogenesis and growth of seaweeds (macroalgae) in direct and/or indirect ways. Bacterial communities belonging to the phyla Proteobacteria and Firmicutes are generally the most abundant on seaweed surfaces. Associated bacterial communities produce plant growth-promoting substances, quorum sensing signalling molecules, bioactive compounds and other effective molecules that are responsible for normal morphology, development and growth of seaweeds. Also, bioactive molecules of associated bacteria determine the presence of other bacterial strains on seaweeds and protect the host from harmful entities present in the pelagic realm. The ecological functions of cross-domain signalling between seaweeds and bacteria have been reported as liberation of carpospores in the red seaweeds and settlement of zoospores in the green seaweeds. In the present review, the role of extracellular polymeric substances in growth and settlement of seaweeds spores is also highlighted. To elucidate the functional roles of associated bacteria and the molecular mechanisms underlying reported ecological phenomena in seaweeds requires a combined ecological, microbiological and biochemical approach.

Effect of quorum sensing signals produced by seaweed-associated bacteria on carpospore liberation from Gracilaria dura

Epiphytic and endophytic bacteria associated with green macroalgae Ulva (U. fasciata and U. lactuca) and red macroalgae Gracilaria (G. corticata and G. dura) have been identified from three different seasons to evaluate the effect of quorum sensing (QS) molecules on carpospores liberation from Gracilaria dura. The bacterial isolates belonging to the orders Bacillales, Pseudomonadales, Alteromonadales, and Vibrionales were present in all seasons, whereas Actinomycetales and Enterobacteriales were confined to pre-monsoon and post-monsoon seasons, respectively. Among all the Gram-negative bacteria, seven isolates were found to produce different types of N-acyl homoserine lactones (AHLs). Interestingly, Shewanella algae produced five types of AHL: C4-HSL, HC4-HSL, C6-HSL, 3-oxo-C6-HSL, and 3-oxo-C12-HSL. Subsequently, the AHLs producing bacterial isolates were screened for carpospore liberation from G. dura and these isolates were found to positively induce carpospore liberation over the control. Also, observed that carpospore liberation increased significantly in C4- and C6-HSL treated cystocarps. Sodium dodecyl sulfate and native polyacrylamide gel electrophoresis of the total protein of the C4- and C6-HSL treated cystocarps showed two specific peptide bands of different molecular weights (50 kDa and 60 kDa) as compared to the control, confirming their indirect effect on carpospore liberation.

Role of bacterial isolates in enhancing the bud induction in the industrially important red alga Gracilaria dura.

Plant growth depends on the integration of environmental cues, nitrogen fixation and phytohormone-signaling pathways. The growth and development of Gracilaria dura was significantly influenced by the association of bacterial isolates. The putative bud-inducing epiphytic Exiguobacterium homiense and endophytic Bacillus pumilus, Bacillus licheniformis were examined for their ability to fix nitrogen and produce indole-3-acetic acid (IAA). These bacterial isolates were identified to the species level by biochemical tests, fatty acid and partial 16S rRNA gene sequence analysis. The B. pumilus, B. licheniformis and E. homiense produced 445.5, 335 and 184.1 μg mL(-1) IAA and 12.51, 10.14 and 6.9 mM mL(-1) ammonium, respectively, as determined using HPLC and spectroscopy. New bud regeneration observed after the addition of total protein of the bacterial isolates suggests that IAA is conjugated with protein. The epi- and endophytic bacterial isolates were able to induce five and 10 new buds per frond, respectively, in comparison to the control, where one to two buds were observed. The combination of 25 °C and 30‰ showed the optimum condition for bud induction in G. dura when incubated with the total protein of B. pumilus. Our finding revealed for the first time that IAA coupled with nitrogen fixation induce and regenerate new buds in G. dura.

Antimicrobial compounds from seaweeds-associated bacteria and fungi

In recent decade, seaweeds-associated microbial communities have been significantly evaluated for functional and chemical analyses. Such analyses let to conclude that seaweeds-associated microbial communities are highly diverse and rich sources of bioactive compounds of exceptional molecular structure. Extracting bioactive compounds from seaweed-associated microbial communities have been recently increased due to their broad-spectrum antimicrobial activities including antibacterial, antifungal, antiviral, anti-settlement, antiprotozoan, antiparasitic, and antitumor. These allelochemicals not only provide protection to host from other surrounding pelagic microorganisms, but also ensure their association with the host. Antimicrobial compounds from marine sources are promising and priority targets of biotechnological and pharmaceutical applications. This review describes the bioactive metabolites reported from seaweed-associated bacterial and fungal communities and illustrates their bioactivities. Biotechnological application of metagenomic approach for identifying novel bioactive metabolites is also dealt, in view of their future development as a strong tool to discover novel drug targets from seaweed-associated microbial communities.

Bacterial extracellular polymeric substances and their effect on settlement of zoospore of Ulva fasciata.

The extracellular polymeric substances (EPSs) secreted by Bacillus flexus (GU592213) were estimated to have the molecular weight of approximately 1528 and 33,686 kDa with the elemental composition of Na, P, Mg, C, O, Cl and S. The (1)H NMR and FT-IR analysis of EPS confirmed the presence of different aliphatic and aromatic groups. The EPS was amorphous in nature with an average particle size of 13.969 μm (d 0.5) and roughness of 193 nm. The GC-MS analysis has revealed different monosaccharides such as fucose, ribose, xylose, galactose, mannose and glucose. Oligo and polysaccharides were detected with MALDI TOF-TOF MS. The bacterial EPS for the first time tested as a natural substratum for settle of zoospores of Ulva fasciata by incubating for various durations ranging from 2h to 48 h. The zoospore settlement on EPS coated cover slips progressively increased with incubation time in axenic cultures over controls. The EPS, thus investigated in this study was found to facilitate the primary settlement of spores that play crucial role in recruitment of macroalgal communities in coastal environment including intertidal regions.

Attenuation of quorum sensing-mediated virulence in Gram-negative pathogenic bacteria: implications for the post-antibiotic era

Quorum sensing is a cell-density dependent regulatory system, which orchestrates that quorum-sensing (QS) systems use extracellular signals to modulate the expression of a particular gene(s) in a bacterial cell, which results in virulence gene expression or biofilm formation and occasionally causes deadly plant and animal diseases. The frequent use of antibiotics to treat deadly diseases has led to the development of multiple drug-resistant bacterial strains. The increasing presence of pathogenic bacteria has thus forced us to develop alternative methods for controlling pathogen virulence. One such possible method, quorum quenching (QQ), has emerged as an interesting approach. A variety of bioactive molecules or drugs from prokaryotic or eukaryotic sources have been identified as QQ molecules, some of which are chemically synthesized, and the agonist or antagonist of their cognate receptor or metabolic intermediate was determined. Current strategies to attenuate the virulence of gene expression can be grouped into the following categories: (a) blockage of AHL–Lux-R-type binding sites, (b) inhibition of AHL–Lux-R- and Lux-I-type interactions, (c) inhibition of transporters, (d) degradation of existing AHLs by QQ enzymes and (e) inhibition of enzymes involved in the metabolic synthesis of QS molecules. This review summarises several potential QQ molecules that have been reported to attenuate QS-based virulence gene expression in serious Gram-negative pathogenic bacteria. These QQ molecules suggest possible ways of controlling the virulence effects of pathogenic bacteria in the post-antibiotic era.

Cyclodepsipeptides produced by actinomycetes inhibit cyclic-peptide-mediated quorum sensing in Gram-positive bacteria.

Cyclic peptides are commonly used as quorum-sensing autoinducers in Gram-positive Firmicutes bacteria. Well-studied examples of such molecules are thiolactone and lactone, used to regulate the expression of a series of virulence genes in the agr system of Staphylococcus aureus and the fsr system of Enterococcus faecalis, respectively. Three cyclodepsipeptides WS9326A, WS9326B and cochinmicin II/III were identified as a result of screening actinomycetes culture extracts for activity against the agr/fsr system. These molecules are already known as receptor antagonists, the first two for tachykinin and the last one for endothelin. WS9326A also inhibited the transcription of pfoA regulated by the VirSR two-component system in Clostridium perfringens. Receptor-binding assays using a fluorescence-labeled autoinducer (FITC-GBAP) showed that WS9326A and WS9326B act as receptor antagonists in this system. In addition, an ex vivo assay showed that WS9326B substantially attenuated the toxicity of S. aureus for human corneal epithelial cells. These results suggest that these three natural cyclodepsipeptides have therapeutic potential for targeting the cyclic peptide-mediated quorum sensing of Gram-positive pathogens.

Unraveling the Functions of the Macroalgal Microbiome.

Macroalgae are a diverse group of photosynthetic eukaryotic lower organisms and offer indispensable ecosystem services toward sustainable productivity of rocky coastal areas. The earlier studies have mainly focused on elucidation of the roles of the epiphytic bacterial communities in the ecophysiology of the host macroalga. However, mutualistic interactions have become topic of current interest. It is evident from recent studies that a fraction of epiphytic bacterial communities can be categorized as “core microbial species”, suggesting an obligate association. Epiphytic bacterial communities have also been reported to protect macroalgal surfaces from biofouling microorganisms through production of biologically active metabolites. Because of their intrinsic roles in the host life cycle, the host in turn may provide necessary organic nutrients in order to woo pelagic microbial communities to settle on the host surfaces. However, the precise composition of microbiomes and their functional partnership with hosts are hardly understood. In contrast, the microbial studies associated with human skin and gut and plants have significantly advanced our knowledge on microbiome and their functional interactions with the host. This has led to manipulation of the microbial flora of the human gut and of agricultural plants for improving health and performance. Therefore, it is highly imperative to investigate the functional microbiome that is closely involved in the life cycles of the host macroalgae using high-throughput techniques (metagenomics and metatranscriptomics). The findings from such investigations would help in promoting health and productivity in macroalgal species through regulation of functionally active microbiome.

Quorum Quenching Strategy Targeting Gram-Positive Pathogenic Bacteria

Quorum sensing (QS) is a cell density-dependent regulatory system that orchestrates the group behavior of unicellular organisms by synchronizing the expression of certain gene(s) within the clonal community of same species. Bacterial pathogens often employ QS system to establish efficiently an infection. A large part of low GC Gram-positive bacteria belonging to phylum Firmicutes use thiolactone/lactone peptides as communication signals so-called autoinducing peptides (AIPs) to coordinate QS circuit. In particular, QS of staphylococci, enterococci, and clostridia have been intensively studied in terms of alternative target of anti-pathogenic chemotherapy independent of bactericidal antibiotics. Thus far, a number of quorum quenching (QQ) agents that targeting the QS circuit of these Gram-positive pathogens have been developed by random screening of natural compounds or rationale design of AIP antagonists. This review summarizes those QQ agents and previews their potential as post-antibiotic drugs.