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.

A natural food preservative peptide nisin can interact with the SARS-CoV-2 spike protein receptor human ACE2.

Nisin, a food-grade antimicrobial peptide produced by lactic acid bacteria has been examined for its probable interaction with the human ACE2 (hACE2) receptor, the site where spike protein of SARS-CoV-2 binds. Among the eight nisin variants examined, nisin H, nisin Z, nisin U and nisin A showed a significant binding affinity towards hACE2, higher than that of the RBD (receptor binding domain) of the SARS-CoV-2 spike protein. The molecular interaction of nisin with hACE2 was investigated by homology modeling and docking studies. Further, binding efficiency of the most potent nisin H was evaluated through the interaction of hACE2:nisin H complex with RBD (receptor-binding domain) of SARS-CoV-2 and that of hACE2:RBD complex with nisin H. Here, nisin H acted as a potential competitor of RBD to access the hACE2 receptor. The study unravels for the first time that a globally used food preservative, nisin has the potential to bind to hACE2.

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.

A constitutive unregulated expression of ß-galactosidase in Lactobacillus fermentum M1.

A constitutively ß-galactosidase (LacL)-producing Lactobacillus fermentum M1 isolated from fermented milk was found to produce ß-galactosidase in the presence of glucose. ß-galactosidase activity produced in glucose (30 mM) medium was 2.17 U/mL as compared to 2.27 and 2.19 U/mL with galactose and lactose, respectively. When a combination of glucose (30 or 60 mM) with galactose (30 mM) was used as carbon source, ß-galactosidase activity was not repressed rather was found increased when compared to carbon sources used individually. In real-time PCR analysis of mRNA synthesized on individual and combined carbon sources, repression of the lacL gene expression was not observed. This observation suggests that the strain M1 lacked normal carbon catabolite repression. Examination of nucleotide sequence of lacL identified two catabolite responsive elements (cre): cre1 located downstream near the promoter region and cre2 within the coding sequence. Each of which differed from the 14-bp consensus by a single nucleotide. In cre1, it is C in place of highly conserved T at position 1 in the consensus. In cre 2, it is G in place of C, a residue completely conserved at position 13. Since catabolite genes in Gram-positive bacteria are regulated by carbon catabolite protein A (CcpA) through interaction with DNA at a specific cis-acting cre, it is assumed that base changes at conserved position in the cre elements disrupt CcpA binding and thereby leading to constitutive expression of lacL gene. The study noted to be the first report about the constitutive production of ß-galactosidase in L. fermentum.

Cloning, overexpression, and characterization of a novel alkali-thermostable xylanase from Geobacillus sp. WBI.

An endo-ß-1,4-xylanase gene xynA of a thermophilic Geobacillus sp. WBI from "hot" compost was isolated by PCR amplification. The gene encoding 407 residues were overexpressed in E. coli and purified by Ni-NTA chromatography. The purified enzyme (47 kDa) had a broad pH optimum of 6.0 to 9.0, and was active between 50 and 90 °C. The enzyme retained 100% of its activity when incubated at 65 °C for 1 h under alkaline condition (pH 10.0) and retained 75% activity at pH 11.0. The K(m) and V(max) of the enzyme were 0.9 mg ml(-1) and 0.8 µmol ml(-1) min(-1), respectively. In molecular dynamics simulation at 338 K (65 °C), the enzyme was found to be stable. At an elevated temperature (450 K) specific α-helix and ß-turns of the proteins were most denatured. The denaturation was less in WBI compared with its highest homolog G. stearothermophilus T-6 xylanase with difference of six residues. The results predict that these regions are responsible for the improved thermostability observed over related enzymes. The present work encourages further experimental demonstration to understand how these regions contribute thermostability to WBI xylanase. The study noted that WBI produces a xylanase with unique characteristics, specifically alkali-thermostability.

Effect of bile on nisin-mediated antibacterial activity and the expression of nisin genes of Lactococcus lactis W8.

The capability of Lactococcus lactis to produce nisin in the presence of bile in the intestinal environment remains an intriguing question. The aim of this study was to determine the effects of bile on production of nisin and the mRNA expression of nisin genes of L. lactis W8. The strain L. lactis W8 was grown on glucose in the absence and presence of bile (0.005-0.08 %) and the antibacterial activities of culture supernatants were determined. In culture with 0.035 % bile, the nisin activity was significantly reduced (400 AU/mL) within 5 h compared to that in the control without bile (2000 AU/mL), while growth of the cells was only slightly affected. In the presence of 0.07 % bile no nisin activity of the strain was manifested. Consistent with these results, mRNA expression of nisin-biosynthetic genes nisZ, nisRK, nisI, and nisF was down-regulated by 7.5-, 2.5-, 1.7-, and 6.0-fold, respectively in cells grown in the presence of bile (0.07 %) as compared to control culture without bile. The present study suggested that bile inhibited transcription of nisin genes. Nisin-production in intestine by orally administered L. lactis, thus, does not occur since complete inhibition of nisin-production by bile is observed at a concentration much lower than the physiological concentration (0.3 %) of bile present in the human intestine. The molecular mechanism underlying the bile-mediated inhibition of nisin genes remains to be elucidated. This is the first report on bile-mediated inhibition of nisin genes.

A foodgrade preparation of nisin from diluted milk fermented with Lactococcus lactis W8.

Lactococcus lactis strain W8, which contains the nisin Z gene in its genome, grew well and produced nisin in cow's milk at temperatures of 30 to 37 degrees C. Maximum production of nisin was achieved at 6 h and was 4,000 activity units (AU) per ml in skim milk and 2,400 AU/ml in 3% fat milk. The organism produced nisin even in 20 times diluted skim milk and 3% fat milk at 1,000 and 600 AU/ml, respectively. Boiling of the fermented milk (pH 4.2) made with this culture allowed the separation of the liquid part (whey) from the curd. When 20 times diluted skim milk was fermented and the whey derived from it was lyophilized, the yield of nisin was 60,000 AU/g. The antimicrobial activity of the nisin preparation was stable for at least 1 year at refrigeration temperature. L. lactis W8 may have significant applications in the food industry for a cost-effective natural nisin preparation.

Production of nisin Z by Lactococcus lactis isolated from dahi.

Lactococcus lactis CM1, an isolate from homemade "Dahi," a traditional fermented milk from India, used maltose as carbon source to produce a high level of bacteriocin. The bacterial cell mass and the bacteriocin production correlated with the initial pH of the medium and were highest when the initial pH was 11.0. The level of bacteriocin reached its peak at the late log phase with concomitant reduction of culture pH to 4.2, regardless of the initial pH of the medium. A combination of maltose and an initial medium pH of 11 resulted in the highest bacteriocin production. The antibacterial spectrum of the bacteriocin was closely similar to that of nisin and it inhibited a number of food spoilage and pathogenic bacteria. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the compound migrated close to the position of nisin (3.5 kDa). However, it had higher stability than nisin at a wide range of pH and temperature. PCR amplification using nisin gene-specific primers and sequencing of the amplified DNA revealed the structural gene for the bacteriocin to be identical to that of nisZ.

Production and characterization of nisin-like peptide produced by a strain of Lactococcus lactis isolated from fermented milk.

An isolate of Lactococcus lactis from fermented milk was found to produce a bacteriocin peptide. The isolate could grow in a medium with an initial pH of 11.0, in which it produced the bacteriocin extracellularly at the highest level. The level of the bacteriocin in the medium increased in parallel to the bacterial growth and reached its peak during the late exponential phase; thereafter it plateaued. The bacteriocin had a broad antibacterial spectrum similar to that of nisin and inhibited several related species of lactic acid bacteria and other gram-positive bacteria. The inhibitory activity of the bacteriocin was found to be stable over a wide range of pH and temperature. The molecular weight of the peptide was judged to be 2.5 kDa by SDS-polyacrylamide gel electrophoresis.