Next-generation sequencing and its potential impact on food microbial genomics

Recent efforts of researchers to elucidate the molecular mechanisms of biological systems have been revolutionized greatly with the use of high throughput and cost-effective techniques such as next generation sequencing (NGS). Application of NGS to microbial genomics is not just limited to predict the prevalence of microorganisms in food samples but also to elucidate the molecular basis of how microorganisms respond to different food-associated conditions, which in turn offers tremendous opportunities to predict and control the growth and survival of desirable or undesirable microorganisms in food. Concurrently, NGS has facilitated the development of new genome-assisted approaches for correlating genotype and phenotype. The aim of this review is to provide a snapshot of the various possibilities that these new technologies are opening up in area of food microbiology, focusing the discussion mainly on lactic acid bacteria and yeasts associated with fermented food. The contribution of NGS to a system level understanding of food microorganisms is also discussed

Predicting the functional consequences of non-synonymous single nucleotide polymorphisms in IL8 gene

Here we report an in-silico approach for identification, characterization and validation of deleterious non-synonymous SNPs (nsSNPs) in the interleukin-8 gene using three steps. In first step, sequence homology-based genetic analysis of a set of 50 coding SNPs associated with 41 rsIDs using SIFT (Sorting Intolerant from Tolerant) and PROVEAN (Protein Variation Effect Analyzer) identified 23 nsSNPs to be putatively damaging/deleterious in at least one of the two tools used. Subsequently, structure-homology based PolyPhen-2 (Polymorphism Phenotyping) analysis predicted 9 of 23 nsSNPs (K4T, E31A, E31K, S41Y, I55N, P59L, P59S, L70P and V88D) to be damaging. According to the conditional hypothesis for the study, only nsSNPs that score damaging/deleterious prediction in both sequence and structural homology-based approach will be considered as 'high-confidence' nsSNPs. In step 2, based on conservation of amino acid residues, stability analysis, structural superimposition, RSMD and docking analysis, the possible structural-functional relationship was ascertained for high-confidence nsSNPs. Finally, in a separate analysis (step 3), the IL-8 deregulation has also appeared to be an important prognostic marker for detection of patients with gastric and lung cancer. This study, for the first time, provided in-depth insights on the effects of amino acid substitutions on IL-8 protein structure, function and disease association

Adaptive response and tolerance to sugar and salt stress in the food yeast Zygosaccharomyces rouxii

The osmotolerant and halotolerant food yeast Zygosaccharomyces rouxii is known for its ability to grow and survive in the face of stress caused by high concentrations of non-ionic (sugars and polyols) and ionic (mainly Na(+) cations) solutes. This ability determines the success of fermentation on high osmolarity food matrices and leads to spoilage of high sugar and high salt foods. The knowledge about the genes, the metabolic pathways, and the regulatory circuits shaping the Z. rouxii sugar and salt-tolerance, is a prerequisite to develop effective strategies for fermentation control, optimization of food starter culture, and prevention of food spoilage. This review summarizes recent insights on the mechanisms used by Z. rouxii and other osmo and halotolerant food yeasts to endure salts and sugars stresses. Using the information gathered from S. cerevisiae as guide, we highlight how these non-conventional yeasts integrate general and osmoticum-specific adaptive responses under sugar and salts stresses, including regulation of Na(+) and K(+)-fluxes across the plasma membrane, modulation of cell wall properties, compatible osmolyte production and accumulation, and stress signalling pathways. We suggest how an integrated and system-based knowledge on these mechanisms may impact food and biotechnological industries, by improving the yeast spoilage control in food, enhancing the yeast-based bioprocess yields, and engineering the osmotolerance in other organisms

Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex.

Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5' end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding

Getting insights from genomic complexities in Zygosaccharomyces rouxii complex

Genus Zygosaccharomyces includes osmo/halotolerant yeasts that diverged from the S. cerevisiae lineage prior to its genome duplication (WGD). Recently, we delineated a novel species, Zygosaccharomyces sapae. Z. sapae has phylogenetic importance as its conspecific strain NCYC3042 has been implicated in allodiploid nature of hybrid ATCC42981. We carry out an extensive genetic survey on some Zygosaccharomyces strains (called Z. rouxii complex) to unravel intra and inter-strain DNA sequences heterogeneity; and diversity in chromosome patterns. Intragenomic copies of rDNA genes are not homogenized by concerted evolution in them, with indels and substitutions occurring mainly in internal transcribed spacer regions. This suggests a birth and death process of evolution. Variability in protein-coding sequences and ploidy level showed a marked intergenomic diversity in diploid and aneuploid cells. Functional analysis revealed a broad range tolerance to alkali metal cations, congruent with clusters obtained through genetic survey. Life cycle and breeding system are suggested to shape these diversities in this complex, making it an interesting pre-WGD model for studying evolutionary forces driving genome plasticity in low-aw environments

Complex nature of Zygosaccharomyces wild strains: a genotypic and functional characterization

Genotypic and functional characterization of Zygosaccharomyces wild strain

Pairwise comparison of D1/D2 sequences cloned from strains with intra-genomically variable D1/D2 sequences.

<p>Intra-genomic D1/D2 variants cloned from individual strains are indicated as D1/D2 copies. The number of transitional (s_i) and transversional (s_v) mutations, as well as number of indels and the involved nucleotides, were computed with MEGA6. The number of nucleotides in pairwise-aligned sequences is indicated as nt (tot), whereas the number of nucleotides in indels (N° nt) is reported in brackets. Identity (%) indicates the percentage of identical nucleotides between D1/D2 variants within individual genomes.</p

Overview of the experimental design.

<p>A) The rDNA locus in chromosome XII of the model yeast <i>Saccharomyces cerevisiae</i>, and a schematic representation of its rDNA unit. Boxes and lines represent the rDNA repeat structure and are not to scale. B) A three-step strategy is used to identify intra-genomic variants within a pool of 43 <i>Z</i>. <i>rouxii</i> related strains. Abbreviations: ETS, external transcribed spacer; ITS, internal transcribed spacer; IGS, intergenic spacer.</p

Mechanistic Basis of Antimicrobial Actions of Silver Nanoparticles

Multidrug resistance of the pathogenic microorganisms to the antimicrobial drugs has become a major impediment toward successful diagnosis and management of infectious diseases. Recent advancements in nanotechnology-based medicines have opened new horizons for combating multidrug resistance in microorganisms. In particular, the use of silver nanoparticles (AgNPs) as a potent antibacterial agent has received much attention. The most critical physico-chemical parameters that affect the antimicrobial potential of AgNPs include size, shape, surface charge, concentration and colloidal state. AgNPs exhibits their antimicrobial potential through multifaceted mechanisms. AgNPs adhesion to microbial cells, penetration inside the cells, ROS and free radical generation, and modulation of microbial signal transduction pathways have been recognized as the most prominent modes of antimicrobial action. On the other side, AgNPs exposure to human cells induces cytotoxicity, genotoxicity and inflammatory response in human cells in a cell-type dependent manner. This has raised concerns regarding use of AgNPs in therapeutics and drug delivery. We have summarized the emerging endeavors that address current challenges in relation to safe use of AgNPs in therapeutics and drug delivery platforms. Based on research done so far, we believe that AgNPs can be engineered so as to increase their efficacy, stability, specificity, biosafety and biocompatibility. In this regard, three perspectives research directions have been suggested that include 1) synthesizing AgNPs with controlled physico-chemical properties, 2) examining microbial development of resistance towards AgNPs, and 3) ascertaining the susceptibility of cytoxicity, genotoxicity, and inflammatory response to human cells upon AgNPs exposure