isolation-propagation-identification-bacterial-species-with

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

Shiv Nadar University

Microbial load of nurses' white coats in a hospital was determined on polyester cotton blend and polyester fabric after the first and second work shift. Contamination on the blend fabric was significantly higher (P < .05) than on polyester. After the second shift, bacterial colonies on polyester and blend fabrics increased by ~98% and ~70%, respectively. Among the isolates detected on polyester after the second shift, Escherichia coli was the most abundant (47.8%), followed by staphylococci (19.1%).

Bacterial contamination of nurses' white coats after first and second shift.

 strain DR1, a red-pigmented, arsenic- and radiation-resistant bacterium, was isolated from a water sample of the Dadri wetland, Uttar Pradesh, India. Here, we report a draft genome sequence of this strain, which may provide useful information regarding the genes and pathways involved in heavy-metal bioremediation.

Draft Genome Sequence of  DR1, a Novel Strain Isolated from a Freshwater Wetland.

Bacterial cell division is a complex process brought about by the coordinated action of multiple proteins. Separation of daughter cells during the final stages of division involves cleavage of new cell wall laid down at the division septum. In E. coli, this process is governed by the action of N-acetylmuramoyl-L-alanine amidases AmiA/B/C, which are regulated by their LytM activators EnvC and NlpD. While much is known about the regulation of septum cleavage in E. coli, the mechanism of daughter cell separation is not clear in Caulobacter crescentus, a dimorphic crescent-shaped bacterium. In this work, we characterized the role of AmiC, the only annotated amidase in C. crescentus. AmiC from C. crescentus is functional in E. coli and restores cell separation defects seen in E. coli amidase mutants, suggesting that AmiC has septum splitting activity. The medial localization of AmiC was independent of DipM, an LytM domain-containing endopeptidase. Our results indicate that enzymatic activity is essential for medial recruitment of AmiC. Overexpression of AmiC causes cell separation defects and formation of chains. Finally, overexpression of AmiC in cells inhibited for cell division leads to lysis. Collectively, our findings reveal that regulation of daughter cell separation in C. crescentus differs from that of E. coli and can serve as a model system to study bacterial cytokinesis.

Amidase activity is essential for medial localization of AmiC in Caulobacter crescentus.

Graphene oxide (GO) is a promising and remarkable nanomaterial that exhibits antimicrobial activity due to its specific surface-interface interactions. In the present work, for the first time, we have reported the antibacterial activity of GO-coated surfaces prepared by two different methods (Hummers' and improved, i.e., GO and GO) against bacterial biofilm formation. The bacterial toxicity of the deposited GO-coated surfaces was investigated for both Gram-negative () and Gram-positive () models of bacteria. The mechanism of inhibition is different on the coated surface than that in suspension, as determined by measurement of the percentage inhibition of biofilm formation, Ellman's assay, and colony forming unit (CFU) studies. The difference in the nature, degree of oxidative functionalities, and size of the synthesized GO nanoparticles mitigates biofilm formation. To better understand the antimicrobial mechanism of GO when coated on surfaces, we were able to demonstrate that beside reactive oxygen species-mediated oxidative stress, the physical properties of the GO-coated substrate effectively inactivate bacterial cell proliferation, which forms biofilms. Light and atomic force microscopy (AFM) images display a higher inhibition in the proliferation of planktonic cells in Gram-negative bacteria as compared to that in Gram-positive bacteria. The existence of a smooth surface with fewer porous domains in GO inhibits biofilm formation, as demonstrated by optical microscopy and AFM images. The oxidative stress was found to be lower in the coated surface as compared to that in the suspensions as the latter enables exposure of both a large fraction of the active edges and functionalities of the GO sheets. In suspension, GO is selective against  whereas GO showed inhibition toward . This study provides new insights to better understand the bactericidal activity of GO-coated surfaces and contributes to the design of graphene-based antimicrobial surface coatings, which will be valuable in biomedical applications.

Graphene Oxide-Coated Surface: Inhibition of Bacterial Biofilm Formation due to Specific Surface-Interface Interactions.

Arsenic prevalence in the environment impelled many organisms to develop resistance over the course of evolution. Tolerance to arsenic, either as the pentavalent [As(V)] form or the trivalent form [As(III)], by bacteria has been well studied in prokaryotes, and the mechanism of action is well defined. However, in the rod-shaped arsenic tolerant Deinococcus indicus DR1, the key enzyme, arsenate reductase (ArsC) has not been well studied. ArsC of D. indicus belongs to the Grx-linked prokaryotic arsenate reductase family. While it shares homology with the well-studied ArsC of Escherichia coli having a catalytic cysteine (Cys 12) and arginine triad (Arg 60, 94, and 107), the active site of D.indicus ArsC contains four residues Glu 9, Asp 53, Arg 86, and Glu 100, and with complete absence of structurally equivalent residue for crucial Cys 12. Here, we report that the mechanism of action of ArsC of D. indicus is different as a result of convergent evolution and most likely able to detoxify As(V) using a mix of positively- and negatively-charged residues in its active site, unlike the residues of E. coli. This suggests toward the possibility of an alternative mechanism of As (V) degradation in bacteria.

Structure and function prediction of arsenate reductase from Deinococcus indicus DR1.

Antimicrobial drug resistance is one of the most critical problems that plagued the human race in modern times. Discovery of novel antibiotics is important to counter this threat. Accordingly, herein we have reported the discovery of substituted benzimidazole class of molecules with antimicrobial property (specifically against Staphylococcus aureus). They were initially identified through a random screening and a novel catalytic synthetic strategy was utilized to access them. in vitro screening and phenotypic profiling revealed the antimicrobial nature. De novo transcriptome and gene analyses predicted the putative targets. This work provides a solid foundation for developing the benzimidazoles as a target specific antimicrobial preclinical candidate.

Transcriptome analysis predicts mode of action of benzimidazole molecules against Staphylococcus aureus UAMS-1.

 exhibits growth medium-dependent morphological variation in cell shape, but there is no evidence whether this phenomenon is observed in other members of the Deinococcaceae family. In this study, we isolated a red-pigmented, aerobic,  strain DR1 from Dadri wetland, India. This  strain exhibited cell-morphology transition from rod-shaped cells to multi-cell chains in a growth-medium-dependent fashion. In response to addition of 1% casamino acids in the minimal growth medium, rod-shaped cells formed multi-cell chains. Addition of all 20 amino acids to the minimal medium was able to recapitulate the phenotype. Specifically, a combination of L-methionine, L-lysine, L-aspartate, and L-threonine caused morphological alterations. The transition from rod shape to multi-cell chains is due to delay in daughter cell separation after cell division. Minimal medium supplemented with L-ornithine alone was able to cause cell morphology changes. Furthermore, a comparative UPLC analysis of PG fragments isolated from  cells propagated in different growth media revealed alterations in the PG composition. An increase in the overall cross-linkage of PG was observed in muropeptides from nutrient-rich TSB and NB media versus PYE medium. Overall our study highlights that environmental conditions influence PG composition and cell morphology in .

Amino Acid-Dependent Alterations in Cell Wall and Cell Morphology of  DR1.

A fully biobased benzoxazine monomer, V-fa (using vanillin and furfurylamine) was grafted onto chitosan (CS) at different weight ratios (CV) using "grafting to" benign Schiff base chemistry. Incorporation of V-fa onto CS increased the tensile strength and improved chemical resistance of the CS-graft-V-fa films. Reversible labile linkages, expansion of CS galleries and leaching out of phenolic species from biobased polymer films led to an improved antibacterial activity against Staphylococcus aureus, which is ∼125 times higher than the bare CS film, V-fa and oligomeric V-fa. The leached out species from films were analyzed extensively by NMR, FTIR, GPC, ABTS and HRMS analysis. Oxidative-stress seems to be responsible for antibacterial activity. Current work illustrates an attractive synthetic approach and the improved antibacterial performance of biobased CS-graft-poly(V-fa) films which may hold as a potential alternative for wound-healing and implant applications in future.

Antibacterial performance of fully biobased chitosan-grafted-polybenzoxazine films: Elaboration and properties of released material.

Violacein is a naturally found pigment that is used by some gram negative bacteria to defend themselves from various gram positive bacteria. As a result, this molecule has caught attention for its potential biomedical applications and has already shown promising outcomes as an antiviral, an antibacterial, and an anti-tumor agent. Understanding the interaction of this molecule with a cellular membrane is an essential step to extend its use in the pharmaceutical paradigm. Here, the interaction of violacein with a lipid monolayer formed at the air-water interface is found to depend on electrostatic nature of lipids. In presence of violacein, the two dimensional (2D) pressure-area isotherms of lipids have exhibited changes in their phase transition pressure and in-plane elasticity. To gain insights into the out-of-plane structural organization of lipids in a membrane, X-ray reflectivity (XRR) study on a solid supported lipid monolayer on a hydrophilic substrate has been performed. It has revealed that the increase in membrane thickness is more pronounced in the zwitterionic and positively charged lipids compared to the negatively charged one. Further, the lipid molecules are observed to decrease their tilt angle made with the normal of lipid membrane along with an alteration in their in-plane ordering. This has been quantified by grazing incidence X-ray diffraction (GIXD) experiments on the multilayer membrane formed in an environment with controlled humidity. The structural reorganization of lipid molecules in presence of violacein can be utilized to provide a detailed mechanism of the interaction of this molecule with cellular membrane.

Discerning perturbed assembly of lipids in a model membrane in presence of violacein.

Nucleoid-associated proteins (NAPs) or histone-like proteins (HLPs) are DNA-binding proteins present in bacteria that play an important role in nucleoid architecture and gene regulation. NAPs affect bacterial nucleoid organization via DNA bending, bridging, or forming aggregates. EbfC is a nucleoid-associated protein identified first in , belonging to YbaB/EbfC family of NAPs capable of binding and altering DNA conformation. YbaB, an ortholog of EbfC found in  and , also acts as a transcriptional regulator. YbaB has a novel tweezer-like structure and binds DNA as homodimers. The homologs of YbaB are found in almost all bacterial species, suggesting a conserved function, yet the physiological role of YbaB protein in many bacteria is not well understood. In this study, we characterized the YbaB/EbfC family DNA-binding protein in  has one YbaB/EbfC family gene annotated in the genome (YbaB   ) and it shares 41% sequence identity with YbaB/EbfC family NAPs. Computational modeling revealed tweezer-like structure of YbaB   , a characteristic of YbaB/EbfC family of NAPs. N-terminal-CFP tagged YbaB   localized with the nucleoid and is able to compact DNA. Unlike  EbfC protein, YbaB   protein is a non-specific DNA-binding protein in . Moreover, YbaB   shields DNA against enzymatic degradation. Collectively, our findings reveal that YbaB   is a small histone-like protein and may play a role in bacterial chromosome structuring and gene regulation in 

Article	Year
Bacterial contamination of nurses' white coats after first and second shift. American journal of infection control\| PubMed ID: 27671361	2017
Draft Genome Sequence of DR1, a Novel Strain Isolated from a Freshwater Wetland. Genome announcements\| PubMed ID: 28774987	2017
Amidase activity is essential for medial localization of AmiC in Caulobacter crescentus. Current genetics\| PubMed ID: 29167986	2018
Graphene Oxide-Coated Surface: Inhibition of Bacterial Biofilm Formation due to Specific Surface-Interface Interactions. ACS omega\| PubMed ID: 30023683	2017
Structure and function prediction of arsenate reductase from Deinococcus indicus DR1. Journal of molecular modeling\| PubMed ID: 30610463	2019
Transcriptome analysis predicts mode of action of benzimidazole molecules against Staphylococcus aureus UAMS-1. Drug development research\| PubMed ID: 30793347	2019
Amino Acid-Dependent Alterations in Cell Wall and Cell Morphology of DR1. Frontiers in microbiology\| PubMed ID: 31333600	2019
Antibacterial performance of fully biobased chitosan-grafted-polybenzoxazine films: Elaboration and properties of released material. Carbohydrate polymers\| PubMed ID: 33357864	2021
Discerning perturbed assembly of lipids in a model membrane in presence of violacein. Biochimica et biophysica acta. Biomembranes\| PubMed ID: 33989532	2021
DNA-Binding Properties of YbaB, a Putative Nucleoid-Associated Protein From . Frontiers in microbiology\| PubMed ID: 34777284	2021

Richa Priyadarshini

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