Characterization and Complete Genome Analysis of a Novel Escherichia Phage, vB_EcoM-RPN242

The novel Escherichia phage vB_EcoM-RPN242 was isolated using a strain of Escherichia coli host originated from a diarrheal piglet. The phage was able to form plaques on the E. coli lawn at 15−45ºC. Moreover, it was stable over a wide pH (4−10) and temperature (4−70ºC) range. The vB_EcoM-RPN242 genome was found to be a linear, double-stranded DNA consisting of 154,840 base pairs. There were 195 protein-encoding genes and 2 tRNAs detected in the genome, however no unfavorable gene was found. According to the overall nucleotide sequence comparison, the vB_EcoM-RPN242 possibly represents a new phage species in the genus Agtrevirus.

Transcriptome Analysis Unveils the Effects of Proline on Gene Expression in the Yeast Komagataella phaffii

Komagataella phaffii yeast is one of the most important biocompounds producing microorganisms in modern biotechnology. Optimization of media recipes and cultivation strategies is key to successful synthesis of recombinant proteins. The complex effects of proline on gene expression in the yeast K. phaffii was analyzed on the transcriptome level in this work. Our analysis revealed drastic changes in gene expression when K. phaffii was grown in proline-containing media in comparison to ammonium sulphate-containing media. Around 18.9% of all protein-encoding genes were differentially expressed in the experimental conditions. Proline is catabolized by K. phaffii even in the presence of other nitrogen, carbon and energy sources. This results in the repression of genes involved in the utilization of other element sources, namely methanol. We also found that the repression of AOX1 gene promoter with proline can be partially reversed by the deletion of the KpPUT4.2 gene.

Screening And Genome Sequencing of a di-n-butyl Phthalate Degrading Bacterium J2 Isolated From Peanut Field Soil

Di-n-butyl phthalate (DBP) is commonly used plasticizers in agricultural plastic films, and is a priority pollutant due to its toxicity to human health. A newly isolated strain J2, which used DBP as its sole carbon source, was screened from peanut filed soil by continuous enrichment cultivation. Based on morphological, physiological characteristics and 16S rRNA gene sequence analysis (GenBank accession No. OK598965), it was identified as Priestia sp. J2. The research results revealed the optimal conditions for DBP degradation as 35 oC and pH 8.0. The strain could effectively degrade 97.6% DBP within 5 days. Substrate tests showed that strain J2 could utilize shorter side-chained PAEs, but could not utilize long-chained PAEs. The whole genome comprises a complete chromosome of 5,067,299 bp and four plasmids of 147,924 bp, 75,940 bp, 11,604 bp, 11,333 bp (GenBank accession No. CP086208-CP086212). This genome harbors 5,585 predicted protein-encoding genes, 130 tRNA genes, and 42 rRNA genes. Gene annotation analyses showed a DBP-degrading gene contained an open reading frame of 930 bp, and the enzyme was named Est-J2-1. The amino acid sequence of the Est-J2-1 exhibited no significant homology with those of reported DBP-degrading enzymes, suggesting the enzyme is a novel enzyme. The gene of Est-J2-1 was found to be located on the chromosome. This study provided strain resource for DBP removal from farmland and other environments.

Lentilactobacillus fungorum sp. nov., isolated from spent mushroom substrates

In Japan, during a screening of lactic acid bacteria in spent mushroom substrates, an unknown bacterium was isolated and could not be assigned to any known species. Strain YK48GT is Gram-stain-positive, rod-shaped, non-motile, non-spore-forming and catalase-negative. The isolate grew in 0–4 % (w/v) NaCl, at 15–37 °C (optimum, 30 °C) and at pH 4.0–8.0 (optimum, pH 6.0). The genomic DNA G+C content of strain YK48GT was 42.5 mol%. Based on its 16S rRNA gene sequence, strain YK48GT represented a member of the genus Lentilactobacillus and showed the highest pairwise similarity to Lentilactobacillus rapi DSM 19907T (97.86 %). Phylogenetic analyses based on amino acid sequences of 466 shared protein-encoding genes also revealed that the strain was phylogenetically positioned in the genus Lentilactobacillus but did not suggest an affiliation with previously described species. The average nucleotide identity and digital DNA–DNA hybridization values between strain YK48GT and the type strains of phylogenetically related species were 72.2–76.6% and 19.0–21.2 %, respectively, indicating that strain YK48GT represents a novel species within the genus Lentilactobacillus . Phenotypic data further confirmed the differentiation of strain YK48GT from other members of the genus Lentilactobacillus . According to the results of the polyphasic characterization presented in this study, strain YK48GT represents a novel species of the genus Lentilactobacillus , for which the name Lentilactobacillus fungorum sp. nov. is proposed. The type strain is YK48GT (=JCM 32598T=DSM 107968T).

Introduction of Modified BglBrick System in Lactococcus lactis for Straightforward Assembly of Multiple Gene Cassettes

Genetic modification of lactic acid bacteria is an evolving and highly relevant field of research that allows the engineered bacteria to be equipped with the desired functions through the controlled expression of the recombinant protein. Novel genetic engineering techniques offer the advantage of being faster, easier and more efficient in incorporating modifications to the original bacterial strain. Here, we have developed a modified BglBrick system, originally introduced in Escherichia coli and optimized it for the lactic acid bacterium Lactococcus lactis. Six different expression cassettes, encoding model proteins, were assembled in different order as parts of a modified BglBrick system in a novel plasmid pNBBX. All cassettes included nisin promoter, protein encoding gene and transcription terminator. We demonstrated successful intracellular expression of the two fluorescent proteins and display of the four protein binders on the bacterial surface. These were expressed either alone or concomitantly, in combinations of three model proteins. Thus, a modified BglBrick system developed herein enables simple and modular construction of multigene plasmids and controlled simultaneous expression of three proteins in L. lactis.

From sequences to enzymes: heterologous expression of genes from marine microbes

Heterologous expression is an easy and broadly applicable experimental approach widely used to investigate protein functions without the need to genetically manipulate the original host. The approach is used to obtain large quantities of the desired protein, which can be further analyzed from a biochemical, structural and functional perspective. The expression system consists of three main components: i) a foreign DNA sequence coding for the protein of interest; ii) a suitable expression vector; iii) a suitable host (bacterial, yeast or mammalian cells) which does not encode or express the protein of interest. Here we show how to apply an Escherichia coli-based expression system to overexpress protein encoding genes from marinemicrobes.

Comprehensive Analysis of Arabinogalactan Protein-Encoding Genes Reveals the Involvement of Three BrFLA Genes in Pollen Germination in Brassica rapa

Arabinogalactan proteins (AGPs) are a superfamily of hydroxyproline-rich glycoproteins that are massively glycosylated, widely implicated in plant growth and development. No comprehensive analysis of the AGP gene family has been performed in Chinese cabbage (Brassica rapa ssp. chinensis). Here, we identified a total of 293 putative AGP-encoding genes in B. rapa, including 25 classical AGPs, three lysine-rich AGPs, 30 AG-peptides, 36 fasciclin-like AGPs (FLAs), 59 phytocyanin-like AGPs, 33 xylogen-like AGPs, 102 other chimeric AGPs, two non-classical AGPs and three AGP/extensin hybrids. Their protein structures, phylogenetic relationships, chromosomal location and gene duplication status were comprehensively analyzed. Based on RNA sequencing data, we found that 73 AGP genes were differentially expressed in the floral buds of the sterile and fertile plants at least at one developmental stage in B. rapa, suggesting a potential role of AGPs in male reproductive development. We further characterized BrFLA2, BrFLA28 and BrFLA32, three FLA members especially expressed in anthers, pollen grains and pollen tubes. BrFLA2, BrFLA28 and BrFLA32 are indispensable for the proper timing of pollen germination under high relative humidity. Our study greatly extends the repertoire of AGPs in B. rapa and reveals a role for three members of the FLA subfamily in pollen germination.

Transcriptome-Wide Analysis of Human Liver Reveals Age-Related Differences in the Expression of Select Functional Gene Clusters and Evidence for a PPP1R10-Governed ‘Aging Cascade’

A transcriptome-wide analysis of human liver for demonstrating differences between young and old humans has not yet been performed. However, identifying major age-related alterations in hepatic gene expression may pinpoint ontogenetic shifts with important hepatic and systemic consequences, provide novel pharmacogenetic information, offer clues to efficiently counteract symptoms of old age, and improve the overarching understanding of individual decline. Next-generation sequencing (NGS) data analyzed by the Mann–Whitney nonparametric test and Ensemble Feature Selection (EFS) bioinformatics identified 44 transcripts among 60,617 total and 19,986 protein-encoding transcripts that significantly (p = 0.0003 to 0.0464) and strikingly (EFS score > 0.3:16 transcripts; EFS score > 0.2:28 transcripts) differ between young and old livers. Most of these age-related transcripts were assigned to the categories ‘regulome’, ‘inflammaging’, ‘regeneration’, and ‘pharmacogenes’. NGS results were confirmed by quantitative real-time polymerase chain reaction. Our results have important implications for the areas of ontogeny/aging and the age-dependent increase in major liver diseases. Finally, we present a broadly substantiated and testable hypothesis on a genetically governed ‘aging cascade’, wherein PPP1R10 acts as a putative ontogenetic master regulator, prominently flanked by IGFALS and DUSP1. This transcriptome-wide analysis of human liver offers potential clues towards developing safer and improved therapeutic interventions against major liver diseases and increased insights into key mechanisms underlying aging.