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Here, we report the draft genome sequence of Limosilactobacillus fermentum strain NKN-51, which was isolated from naturally processed yak cheese from the western Himalayas of India. The genome was assembled in 101 contigs with a total length of 1,879,705 bp and a GC content of 53.5%. Genome annotation predicted 1,730 protein-coding genes and 50 tRNA genes.

Plant secondary metabolites are emerging as attractive alternatives in the development of therapeutics against infectious and chronic diseases. Due to the present pandemic, therapeutics showing toxicity against bacterial pathogens and viruses are gaining interest. Plant metabolites of terpenoid and phenylpropanoid categories have known antibacterial and antiviral properties. These metabolites have also been associated with toxicity to eukaryotic cells in terms of carcinogenicity, hepatotoxicity, and neurotoxicity. Sensing methods that can report the exact antibacterial dosage, formation, and accumulation of these antibacterial compounds are needed. The whole-cell reporters for such antibacterial metabolites are cost-effective and easy to maintain. In the present study, battery of toxicity sensors containing fluorescent transcriptional bioreporters was constructed, followed by fine-tuning the response using gene-debilitated E. coli mutants. This study shows that by combining regulatory switches with chemical genetics strategy, it may be possible to detect and elucidate the mode of action of effective antibacterial plant secondary metabolites - thymol, cinnamaldehyde, eugenol, and carvacrol in both pure and complex formats. Apart from the detection of adulteration of pure compounds present in complex mixture of essential oils, this approach will be useful to detect authenticity of essential oils and thus reduce unintended harmful effects on human and animal health.

Inappropriate and disproportionate use of antibiotics is contributing immensely to the development of antibiotic resistance in bacterial species associated with food contamination. The use of natural products in combination can be a potent alternative hurdle strategy to inactivate foodborne pathogens. Here, we explored the pro-oxidant properties of essential oil linalool and vitamin C in combination with copper (LVC) in combating the foodborne pathogens Vibrio fluvialis and Salmonella enterica subsp. enterica serovar Typhi using a three-dimensional (3D) checkerboard microdilution assay. Antibacterial activity in terms of the MIC revealed that the triple combination exerted a synergistic effect compared to the effects of the individual constituents. The bactericidal effect of the triple combination was confirmed by a live/dead staining assay. Reactive oxygen species (ROS) measurements with the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay and scanning electron microscopy imaging strongly suggested that the increase in ROS production is the underlying mechanism of the enhanced antibacterial potency of the LVC combination (linalool [1.298 mM], vitamin C [8 mM], copper [16.3 μM]). In addition, the hypersensitivity of oxidative stress regulator mutants (oxyR, katG, ahpC, and sodA mutants) toward LVC corroborated the involvement of ROS in cell death. Live/dead staining and changes in cellular morphology revealed that oxidative stress did not transform the cells into the viable but nonculturable (VBNC) state; rather, killing was associated with intracellular and extracellular oxidative burst. Furthermore, the LVC combination did not display toxicity to human cells, while it effectively reduced the pathogen levels in acidic fruit juices by 3 to 4 log CFU/ml without adversely altering the organoleptic properties. This study opens a new outlook for combinatorial antimicrobial therapy. IMPORTANCE There is a need to develop effective antibacterial therapies for mitigating bacterial pathogens in food systems. We used a 3D checkerboard assay to ascertain a safe synergistic combination of food-grade components: vitamin C, copper, and the essential oil linalool. Individually, these constituents have to be added in large amounts to exert their antibacterial effect, which leads to unwanted organoleptic properties. The triple combination could exceptionally inhibit foodborne Gram-negative pathogens like Vibrio fluvialis and Salmonella enterica subsp. enterica serovar Typhi at low concentrations (linalool, 1.298 mM; vitamin C, 8 mM; copper, 16.3 μM) and displayed potent microbial inhibition in acidic beverages. We found increased susceptibility in deletion mutants of oxidative stress regulators (oxyR, katG, ahpC, and sodA mutants) due to ROS generation by Fenton’s chemistry. The results of this study show that it may be possible to use plant-based antimicrobials in synergistic combinations to control microbial contaminants.

Characterization of milk fat globule (MFG) was performed to investigate the difference in MFG membrane (MFGM) between fresh and mastitis Holstein Friesian cow milk. Lipid distribution investigated by exogenous phospholipids using microscopy showed higher phospholipid content in fresh compared to mastitic MFGM. Xanthine oxidase assay indicative of membrane impairment revealed lower activity in mastitic samples compared to fresh globules. Of note, significantly higher roughness of globule surface and zeta potential was observed in mastitis compared to fresh globules. Influence of globule membrane on the interaction with L. fermentum demonstrated preferential adhesion of bacteria to fresh compared to mastitic globules including enhanced extent of binding. Results of the present study provides an insight of the interfacial changes occurring at the globule surface as well as highlighting the importance of selective bacterial interaction with milk components for the potential development of functional food with relevance to human health.

Advances in chemical biology have led to selection of synthetic functional nucleic acids for in vivo applications. Discovery of synthetic nucleic acid regulatory elements has been a long-standing goal of chemical biologists. Availability of vast genome level genetic resources has motivated efforts for discovery and understanding of inducible synthetic genetic regulatory elements. Such elements can lead to custom-design of switches and sensors, oscillators, digital logic evaluators and cell–cell communicators. Here, we describe a simple, robust and universally applicable module for discovery of inducible gene regulatory elements. The distinguishing feature is the use of a toxic peptide as a reporter to suppress the background of unwanted bacterial recombinants. Using this strategy, we show that it is possible to isolate genetic elements of non-genomic origin which specifically get activated in the presence of DNA gyrase A inhibitors belonging to fluoroquinolone (FQ) group of chemicals. Further, using a system level genetic resource, we prove that the genetic regulation is exerted through histone-like nucleoid structuring (H-NS) repressor protein. Till date, there are no reports of in vivo selection of non-genomic origin inducible regulatory promoter like elements. Our strategy opens an uncharted route to discover inducible synthetic regulatory elements from biologically-inspired nucleic acid sequences.

In a living cell, folding of proteins is assisted by molecular chaperones and other folding helpers. In Escherichia coli (E. coli), recently an ATP independent chaperon ‘Spy’ was discovered which is highly up-regulated in the presence of protein unfolding agents like ethanol, butanol and tannic acid. Two response regulators; BaeR and CpxR have been recognized as transcriptional regulators of spy gene. However, the mechanism of genetic regulation of spy under protein denaturants like ethanol has not been studied in detail so far. Based on a combination of genetic, molecular biology and biochemical experimental data, we propose that BaeR protein is the primary regulator of spy gene in response to ethanol stress in E. coli. In addition, we expanded the experimental spectrum and validated that regulation of spy gene in the presence of zinc and copper metal stress is primarily via BaeR and CpxR regulators respectively. We also performed in-silico analysis to identify the homologs of Spy protein and their cognate regulatory elements in bacterial species belonging to enterobacteriaceae family. Based on the unique ATP-independent chaperone nature and genetic regulation of spy we also propose its importance in biosensor development and facilitated production of properly folded recombinant proteins.