Molecular characterization of five novel plasmids from Enterococcus italicus SD1 isolated from fermented milk: An insight into understanding plasmid incompatibility.

Enterococcal plasmids have attracted considerable interest because of their indispensable role in the pathogenesis and dissemination of multidrug-resistance. In this work, five novel plasmids pSRB2, pSRB3, pSRB4, pSRB5 and pSRB7 have been identified and characterised, coexisting in Eneterococcus italicus SD1 from fermented milk. The plasmids pSRB2, pSRB3 and pSRB5 were found to replicate via theta mode of replication while pSRB4 and pSRB7 were rolling-circle plasmids. Comparative analysis of SD1-plasmids dictated that the plasmids are mosaic with novel architecture. Plasmids pSRB2 and pSRB5 are comprised of a typical iteron-based class-A theta type origin of replication, whereas pSRB3 has a Class-D theta type replication origin like pAMß1. The plasmids pSRB4 and pSRB7 shared similar ori as in pWV01. The SD1 class-A theta type plasmids shared significant homology between their replication proteins with differences in their DNA-binding domain and comprises of distinct iterons. The differences in their iterons and replication proteins restricts the "handcuff" formation for inhibition of plasmid replication, rendering to their compatibility to coexist. Similarly, for SD1 rolling circle plasmids the differences in the replication protein binding site in the origin and the replication protein supports their coexistence by inhibiting the crosstalk between the origins and replication proteins. The phylogenetic tree of their replication proteins revealed their distant kinship. The results indicate that the identified plasmids are unique to E. italicus SD1, providing further opportunities to study their utility in designing multiple gene expression systems for the simultaneous production of proteins in enterococci with the renewed concept of plasmid incompatibility.

Plasmid-Based Gene Expression Systems for Lactic Acid Bacteria: A Review.

Lactic acid bacteria (LAB) play a very vital role in food production, preservation, and as probiotic agents. Some of these species can colonize and survive longer in the gastrointestinal tract (GIT), where their presence is crucially helpful to promote human health. LAB has also been used as a safe and efficient incubator to produce proteins of interest. With the advent of genetic engineering, recombinant LAB have been effectively employed as vectors for delivering therapeutic molecules to mucosal tissues of the oral, nasal, and vaginal tracks and for shuttling therapeutics for diabetes, cancer, viral infections, and several gastrointestinal infections. The most important tool needed to develop genetically engineered LABs to produce proteins of interest is a plasmid-based gene expression system. To date, a handful of constitutive and inducible vectors for LAB have been developed, but their limited availability, host specificity, instability, and low carrying capacity have narrowed their spectrum of applications. The current review discusses the plasmid-based vectors that have been developed so far for LAB; their functionality, potency, and constraints; and further highlights the need for a new, more stable, and effective gene expression platform for LAB.

CRISPR detectives against SARS-CoV-2: a major setback against COVID-19 blowout.

The emergence of SARS-CoV-2 has brought the world to a standstill, and till date, effective treatments and diagnostics against this idiosyncratic pathogen are lacking. As compared to the standard WHO/CDC qPCR detection method, which consumes several hours for detection, CRISPR-based SHERLOCK, DETECTR, and FELUDA have emerged as rapid diagnostic tools for the detection of the RNA genome of SARS-CoV-2 within an hour with 100% accuracy, specificity, and sensitivity. These attributes of CRISPR-based detection technologies have taken themselves one step ahead of available detection systems and are emerging as an inevitable tool for quick detection of the virus. Further, the discovery of Cas13s nucleases and their orthologs has opened a new corridor for exploitation of Cas13s as an antiviral therapy against SARS-CoV-2 and other viral diseases. One such approach is Prophylactic Antiviral CRISPR in huMAN cells (PACMAN), which needs a long haul to bring into therapy. The approval of SHERLOCK as the first CRISPR-based SARS-CoV-2 test kit by the FDA, for emergency diagnosis of COVID-19 patients, has given positive hope to scientists that sooner human trials of CRISPR-based therapy will be ratified. In this review, we have extensively reviewed the present CRISPR-based approaches, challenges, and future prospects in the light of diagnostics and therapeutics against SARS-CoV-2. KEY POINTS: • The discovery of Cas12 and Cas13 siblings allowed scientists to detect the viral genes. • Cas13d's identification aided scientists in precisely cleaving the SARS-CoV-2 ssRNA. • CRISPR-Cas system acts as "molecular detector and antiviral proctor."

CRISPR/dCas system as the modulator of gene expression.

CRISPR/Cas has been a very exciting field of research because of its multifaceted applications in biological science for editing genome. This tool can be programmed to target any region of DNA of choice by designing gRNA. The potential of gRNA to recruit a CRISPR-associated protein at a specific genomic site allowed scientists to engineer genome of diverse species for research and development. The application of Cas9 has been further expanded with a recently developed catalytically inactive protein (dead Cas9). CRISPR/dCas system is widely used as a programmable vector to deliver functional cargo (transcriptional effectors) to the desired sites at the genome for targeted transcriptional repression (CRISPR interference, CRISPRi) or activation (CRISPR activation, CRISPRa). It is now possible to regulate gene expression in cells without altering the DNA sequence. These CRISPRi/a toolboxes have explored many unsolved biological issues. Further research on CRISPR system could help diagnose and treat various human diseases.

Characterization of a novel theta-type plasmid pSM409 of Enterococcus faecium RME isolated from raw milk.

Enterococcal plasmids have generated renewed interest for their indispensable role in pathogenesis and dissemination of multidrug-resistance. Recently, a novel plasmid pSM409 (4303-bp, GC% = 33.6%), devoid of antibiotic-resistance and virulence genes, has been identified in Enterococcus faecium RME, isolated from raw milk by us. pSM409 contains six open reading frames encoding a replication initiator protein (RepB) and five accessory proteins: antitoxin epsilon, bacteriocin immunity protein, HsdS, and two hypothetical proteins. Comparative sequence analysis of pSM409 reveals a mosaic pattern of similarity with different loci obtained from different theta plasmids, which dictates the plasmid to be heterogeneous or mosaic, possibly due to recombination. The pSM409 comprised of a typical theta-type origin of replication with four and a half direct repeats (iterons) of 22 nucleotides. The pSM409-RepB shared 76-82% homology with the RepB of reported theta plasmids from different genera, with dissimilarities mostly in its DNA-binding and C-terminal domain. The RepB sequence-based phylogenetic tree revealed its distinct position relative to the reported ones. The RepB grouped in the same clade has identical DNA-binding domains and their cognate iterons, possibly due to their sequence-specific interaction to initiate plasmid replication. Comparative analysis of the pSM409-iteron reveals that the repeats markedly differed from their closest homologues. This clade-specific relationship provides a new concept of classifying theta plasmids. The theta-type replicon identified in pSM409 has been found to be unique to E. faecium RME, prompting us to further investigate its utility as a vector for genetic manipulation of enterococci for health and industry.

CRISPR-Cas9 system: A new-fangled dawn in gene editing.

Till date, only three techniques namely Zinc Finger Nuclease (ZFN), Transcription-Activator Like Effector Nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9) are available for targeted genome editing. CRISPR-Cas system is very efficient, fast, easy and cheap technique for achieving knock-out gene in the cell. CRISPR-Cas9 system refurbishes the targeted genome editing approach into a more expedient and competent way, thus facilitating proficient genome editing through embattled double-strand breaks in approximately any organism and cell type. The off-target effects of CRISPR Cas system has been circumnavigated by using paired nickases. Moreover, CRISPR-Cas9 has been used effectively for numerous purposes, like knock-out of a gene, regulation of endogenous gene expression, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The execution of the CRISPR-Cas9 system has amplified the number of accessible scientific substitutes for studying gene function, thus enabling generation of CRISPR-based disease models. Even though many mechanistic questions are left behind to be answered and the system is not yet fool-proof i.e., a number of challenges are yet to be addressed, the employment of CRISPR-Cas9-based genome engineering technologies will increase our understanding to disease processes and their treatment in the near future. In this review we have discussed the history of CRISPR-Cas9, its mechanism for genome editing and its application in animal, plant and protozoan parasites. Additionally, the pros and cons of CRISPR-Cas9 and its potential in therapeutic application have also been detailed here.

Draft Genome Sequence of Cold-Tolerant Kurthia gibsonii B83, Isolated from Spinach Leaf.

Limited information is available on the whole-genome sequences of Kurthia spp. Here, we report, for the first time, the draft genome sequence of Kurthia gibsonii designated as strain B83. The strain was isolated from spinach (Spinacia oleracea L.) leaf. The genome was sequenced on the Illumina NextSeq 500 platform.

Draft Genome Sequence of Lactococcus lactis subsp. lactis W8, a Potential Nisin-Producing Starter Culture for Indian Traditional Fermented Milk (Dahi).

Dahi is a traditional Indian fermented milk consumed regularly as part of the diet because of its palatability and health benefits. Here, we report the draft genome sequence of a unique strain of Lactococcus lactis subsp. lactis, W8, a lactic acid bacterium that produces nisin while fermenting milk to dahi.