Structure predictions and functional insights into Amidase_3 domain containing N-acetylmuramyl-L-alanine amidases from Deinococcus indicus DR1.

BACKGROUND: N-acetylmuramyl-L-alanine amidases are cell wall modifying enzymes that cleave the amide bond between the sugar residues and stem peptide in peptidoglycan. Amidases play a vital role in septal cell wall cleavage and help separate daughter cells during cell division. Most amidases are zinc metalloenzymes, and E. coli cells lacking amidases grow as chains with daughter cells attached to each other. In this study, we have characterized two amidase enzymes from Deinococcus indicus DR1. D. indicus DR1 is known for its high arsenic tolerance and unique cell envelope. However, details of their cell wall biogenesis remain largely unexplored. RESULTS: We have characterized two amidases Ami1Di and Ami2Di from D. indicus DR1. Both Ami1Di and Ami2Di suppress cell separation defects in E. coli amidase mutants, suggesting that these enzymes are able to cleave septal cell wall. Ami1Di and Ami2Di proteins possess the Amidase_3 catalytic domain with conserved -GHGG- motif and Zn2+ binding sites. Zn2+- binding in Ami1Di is crucial for amidase activity. AlphaFold2 structures of both Ami1Di and Ami2Di were predicted, and Ami1Di was a closer homolog to AmiA of E. coli. CONCLUSION: Our results indicate that Ami1Di and Ami2Di enzymes can cleave peptidoglycan, and structural prediction studies revealed insights into the activity and regulation of these enzymes in D. indicus DR1.

Metal-free synthesis and study of glycine betaine derivatives in water for antimicrobial and anticancer applications.

A sustainable synthesis of interesting glycine betaine derivatives from cyclic 3°-amines viz. N-methyl morpholine (NMM), N-methyl piperidine (NMP), and 1,4-diazabicyclo[2.2.2]octane (DABCO) with numerous aryl diazoacetates 1 in water and under blue LED is reported. Generally, 3°-amines and metal carbenoids (from diazoacetates with transition metal catalysts) provide C-H insertion at the α-position of the amines. Computational comparison of the metal carbenoid with the singlet carbene (metal free and generated under blue LED) realized the difference in reactivity. Next, experimental results corroborated the preliminary findings. The products were isolated either by precipitation of the solid or gel-like final products from the aqueous reaction mixture without any chromatographic purification. The reaction mechanism was realized by control experiments. These compounds exhibit selective bactericidal properties against Gram-positive S. aureus, induce lipid droplets (LDs) formation in HePG2 cells and single crystal X-ray diffraction study of their halogenated analogs reveal interesting Hal … Hal contacts.

Benzoxazine-grafted-chitosan biopolymer films with inherent disulfide linkage: Antimicrobial properties.

Synthesis and fabrication of naturally sourced biopolymers, especially chitosan, grafted with renewable small molecules have recently attracted attention as efficient antimicrobial agents and are highly desired for sustainable material development. Advantageous inherent functionalities in biobased benzoxazine extend the possibility of crosslinking with chitosan which holds immense potential. Herein, a low-temperature, greener facile methodology is adopted for the covalent confinement of benzoxazine monomers bearing aldehyde and disulfide linkages within chitosan to form benzoxazine-grafted-chitosan copolymer films. The association of benzoxazine as Schiff base, hydrogen bonding, and ring-opened structures enabled the exfoliation of chitosan galleries, and such host-guest mediated interactions demonstrated outstanding properties like hydrophobicity, good thermal, and solution stability due to the synergistic effects. Furthermore, the structures empowered excellent bactericidal properties against both E. coli and S. aureus as investigated by GSH loss, live/dead fluorescence microscopy, and morphological alteration on the cell surface by SEM. The work provides the benefits of disulfide-linked benzoxazines on chitosan, offering a promising avenue for general and eco-friendly usage in wound-healing and packaging material.

Next-generation membrane-active glycopeptide antibiotics that also inhibit bacterial cell division.

Resistance to vancomycin, a life-saving drug against Gram-positive bacterial infections necessitates developing alternative therapeutics. Herein, we report vancomycin derivatives that assimilate mechanisms beyond d-Ala-d-Ala binding. The role of hydrophobicity towards the structure and function of the membrane-active vancomycin showed that alkyl-cationic substitutions favored broad-spectrum activity. The lead molecule, VanQAmC10 delocalized the cell division protein MinD in Bacillus subtilis, implying an impact on bacterial cell division. Further examination of wild-type, GFP-FtsZ, or GFP-FtsI producing- and ΔamiAC mutants of Escherichia coli revealed filamentous phenotypes and delocalization of the FtsI protein. The findings indicate that VanQAmC10 also inhibits bacterial cell division, a property previously unknown for glycopeptide antibiotics. The conjunction of multiple mechanisms contributes to its superior efficacy against metabolically active and inactive bacteria, wherein vancomycin is ineffective. Additionally, VanQAmC10 exhibits high efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii in mouse models of infection.

Structural and functional mapping of ars gene cluster in Deinococcus indicus DR1.

Deinococcus indicus DR1 is a novel Gram-negative bacterium, isolated from the Dadri wetlands in Uttar Pradesh, India. In addition to being radiation-resistant, the rod-shaped, red-pigmented organism shows extraordinary resistance to arsenic. The proteins of the corresponding ars gene cluster involved in arsenic extrusion in D. indicus DR1 have not yet been characterized. Additionally, how these proteins regulate each other providing arsenic resistance is still unclear. Here, we present a computational model of the operonic structure and the corresponding characterization of the six proteins of the ars gene cluster in D. indicus DR1. Additionally, we show the expression of the genes in the presence of arsenic using qRT-PCR. The ars gene cluster consists of two transcriptional regulators (ArsR1, ArsR2), two arsenate reductases (ArsC2, ArsC3), one metallophosphatase family protein (MPase), and a transmembrane arsenite efflux pump (ArsB). The transcriptional regulators are trans-acting repressors, and the reductases reduce arsenate (As5+) ions to arsenite (As3+) ions for favourable extrusion. The proteins modelled using RoseTTAFold, and their conformationally stable coordinates obtained after MD simulation indicate their various functional roles with respect to arsenic. Excluding ArsB, all the proteins belong to the α + ß class of proteins. ArsB, being a membrane protein, is fully α-helical, with 12 transmembrane helices. The results show the degree of similarity or divergence of the mechanism utilized by these proteins of ars gene cluster in D. indicus DR1 to confer high levels of arsenic tolerance. This structural characterization study of the ars genes will enable new and deeper insights of arsenic tolerance.

Synthesis and Characterization of Sulfur and Sulfur-Selenium Nanoparticles Loaded on Reduced Graphene Oxide and Their Antibacterial Activity against Gram-Positive Pathogens.

Resistance to antimicrobial agents in Gram-positive bacteria has become a major concern in the last decade. Recently, nanoparticles (NP) have emerged as a potential solution to antibiotic resistance. We synthesized three reduced graphene oxide (rGO) nanoparticles, namely rGO, rGO-S, and rGO-S/Se, and characterized them using X-ray diffraction (PXRD), Raman analysis, and thermogravimetric analysis. Transmission electron microscopy confirmed spherical shape nanometer size S and S/Se NPs on the rGO surface. Antibacterial properties of all three nanomaterials were probed against Gram-positive pathogens Staphylococcus aureus and Enterococcus faecalis, using turbidometeric and CFU assays. Among the synthesized nanomaterials, rGO-S/Se exhibited relatively strong antibacterial activity against both Gram-positive microorganism tested in a concentration dependent manner (growth inhibition >90% at 200 µg/mL). Atomic force microscopy of rGO-S/Se treated cells displayed morphological aberrations. Our studies also revealed that rGO composite NPs are able to deposit on the bacterial cell surface, resulting in membrane perturbation and oxidative stress. Taken together, our results suggest a possible three-pronged approach of bacterial cytotoxicity by these graphene-based materials.

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 catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

DNA-Binding Properties of YbaB, a Putative Nucleoid-Associated Protein From Caulobacter crescentus.

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 Borrelia burgdorferi, belonging to YbaB/EbfC family of NAPs capable of binding and altering DNA conformation. YbaB, an ortholog of EbfC found in Escherichia coli and Haemophilus influenzae, 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 Caulobacter crescentus. C. crescentus has one YbaB/EbfC family gene annotated in the genome (YbaB C c ) and it shares 41% sequence identity with YbaB/EbfC family NAPs. Computational modeling revealed tweezer-like structure of YbaB C c , a characteristic of YbaB/EbfC family of NAPs. N-terminal-CFP tagged YbaB C c localized with the nucleoid and is able to compact DNA. Unlike B. burgdorferi EbfC protein, YbaB C c protein is a non-specific DNA-binding protein in C. crescentus. Moreover, YbaB C c shields DNA against enzymatic degradation. Collectively, our findings reveal that YbaB C c is a small histone-like protein and may play a role in bacterial chromosome structuring and gene regulation in C. crescentus.

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

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.

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

A fully biobased benzoxazine monomer, V-fa (using vanillin and furfurylamine) was grafted onto chitosan (CS) at different weight ratios (CXVY) 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.

SUPER STARCHY1/ONAC025 participates in rice grain filling.

NAC transcription factors (TFs) are known for their role in development and stress. This article attempts to functionally validate the role of rice SS1/ ONAC025 (LOC_Os11g31330) during seed development. The gene is seed-specific and its promoter directs reporter expression in the developing endosperm and embryo in rice transgenic plants. Furthermore, rice transgenic plants ectopically expressing SS1/ ONAC025 have a plantlet lethal phenotype with hampered vegetative growth, but increased tillers and an altered shoot apical meristem structure. The vegetative cells of these plantlets are filled with distinct starch granules. RNAseq analysis of two independent plantlets reveals the differential expression of reproductive and photosynthetic genes. A comparison with seed development transcriptome indicates differential regulation of many seed-related genes by SS1/ ONAC025. Genes involved in starch biosynthesis, especially amylopectin and those encoding seed storage proteins, and regulating seed size are also differentially expressed. In conjunction, SS1/ ONAC025 shows highest expression in japonica rice. As a TF, SS1/ ONAC025 is a transcriptional repressor localized to endoplasmic reticulum and nucleus. The article shows that SS1/ ONAC025 is a seed-specific gene promoting grain filling in rice, and negatively affecting vegetative growth.

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

Deinococcus radiodurans 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, Deinococcus indicus strain DR1 from Dadri wetland, India. This D. indicus 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 D. indicus 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 D. indicus.

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

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.

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

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.

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

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.

Biofilm formation by Exiguobacterium sp. DR11 and DR14 alter polystyrene surface properties and initiate biodegradation.

Polystyrene is a chemically inert synthetic aromatic polymer. This widely used form of plastic is recalcitrant to biodegradation. The exponential production and consumption of polystyrene in various sectors has presented a great environment risk and raised the problem of waste management. Biodegradation by bacteria has previously shown great potential against various xenobiotics but there are only a few reports concerning polyolefins. By screening wetland microbes, we found two bacterial species - Exiguobacterium sibiricum strain DR11 and Exiguobacterium undae strain DR14 which showed promising biodegradation potential against polystyrene. In this study, we report the degradation of non-irradiated solid polystyrene material after incubation with these isolates. Growth studies suggested that the Exiguobacterium strains utilize polystyrene as a carbon source. Moreover, our data suggest that polymer degradation was initiated by biofilm formation over the PS surface leading to alteration in the physical properties of the material. Surface property analysis by AFM revealed significantly enhanced roughness resulting in reduced surface hydrophobicity of polystyrene. Fourier-transfer infrared (FT-IR) spectroscopic analysis showed breakdown of polystyrene backbone by oxidation. The extent of deterioration was further determined by percent weight reduction of polystyrene after incubation with bacteria. Our data support the fact that strains of extremophile bacterium Exiguobacterium are capable of degrading polystyrene and can be further used to mitigate the environmental pollution caused by plastics.

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

Deinococcus indicus 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.

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

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., GOH and GOI) against bacterial biofilm formation. The bacterial toxicity of the deposited GO-coated surfaces was investigated for both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) 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 GOI 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, GOH is selective against S. aureus whereas GOI showed inhibition toward E. coli. 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.