Genetic Diversity of Listeria monocytogenes Isolated From Three Commercial Tree Fruit Packinghouses and Evidence of Persistent and Transient Contamination

Whole genome analysis was performed on 501 isolates obtained from a previous survey which recovered 139 positive environmental sponge samples (i.e., up to 4 isolates per sample) from a total of 719 samples collected at 40 standardized sites in 3 commercial apple packinghouse facilities (i.e., P1, P2, and P3) over 3 successive seasons in a single production year. After excluding duplicated isolates, the data from 156 isolates revealed the clonal diversity of L. monocytogenes and allowed the detection of transient contamination, persistent contamination, and cross-area transmission events. Facility P2 with the poorest sanitary conditions had the least diversity (Shannon’s index of 0.38). P2 contained a Clonal Complex (CC) 554, serogroup IVb-v1 strain that persisted throughout the year and spread across the entire facility, a singleton Sequence Type (ST) 1003, lineage III strain that persisted through two seasons and spread across two areas of the facility, and 3 other clones from transient contaminations. P1 and P3, facilities with better sanitary conditions, had much higher diversity (i.e., 15 clones with a Shannon’s index of 2.49 and 10 clones with a Shannon’s index of 2.10, respectively) that were the result of transient contamination. Facilities P1 and P3 had the highest incidence (43.1%) of lineage III isolates, followed by lineage I (31.3%) and lineage II (25.5%) isolates. Only 1 isolate in the three facilities contained a premature stop codon in virulence gene inlA. Fourteen samples yielded 2–3 clones per sample, demonstrating the importance of choosing appropriate methodologies and selecting a sufficient number of isolates per sample for studying L. monocytogenes diversity. Only 1 isolate, belonging to CC5 and from facility P3, contained a known plasmid, and this was also the only isolate containing benzalkonium chloride tolerance genes. The persistent CC554 strain did not exhibit stronger sanitizer resistance than other isolates and did not contain any confirmed molecular determinants of L. monocytogenes stress resistance that were differentially present in other isolates, such as genes involved in sanitizer tolerance, heavy metal resistance, biofilm-forming, stress survival islet 1 (SSI-1), stress survival islet 2 (SSI-2) or Listeria genomic island (LGI2).

The emergence, spread and vanishing of a French SARS-CoV-2 variant exemplifies the fate of RNA virus epidemics and obeys the Black Queen rule

The nature and dynamics of mutations associated with the emergence, spread and vanishing of SARS-CoV-2 variants causing successive waves are complex. We determined the kinetics of the most common French variant (Marseille-4) for 10 months since its onset in July 2020. Here, we analysed and classified into subvariants and lineages 7,453 genomes obtained by next-generation sequencing. We identified two subvariants, Marseille-4A, which contains 22 different lineages of at least 50 genomes, and Marseille-4B. Their average lifetime was 4.1+/-1.4 months, during which 4.1+/-2.6 mutations accumulated. Growth rate was 0.079+/-0.045, varying from 0.010 to 0.173. All the lineages exhibited a gamma distribution. Several beneficial mutations at unpredicted sites initiated a new outbreak, while the accumulation of other mutations resulted in more viral heterogenicity, increased diversity and vanishing of the lineages. Marseille-4B emerged when the other Marseille-4 lineages vanished. Its ORF8 gene was knocked out by a stop codon, as reported in several mink lineages and in the alpha variant. This subvariant was associated with increased hospitalization and death rates, suggesting that ORF8 is a nonvirulence gene. We speculate that the observed heterogenicity of a lineage may predict the end of the outbreak.

Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast

Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in E. coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remains limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNACUA support the incorporation of ncAAs into proteins produced in S. cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were translationally active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the translational activity of previously reported Methanosarcina mazei, Methanosarcina barkeri, and chimeric M. mazei and M. barkeri PylRSs. Using the yeast strain RJY100, and pairing these aaRSs with the M. barkeri tRNACUA, we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS to the orthogonal translation machinery toolkit in yeast potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery.

Genetic profiles and invasion ability of Listeria monocytogenes isolated from bovine carcasses in Southern Brazil

The goals of this study were to evaluate the persistence and the virulence potential of Listeria monocytogenes isolated from beef carcasses obtained in processing facilities in the Southern region of Rio Grande do Sul, Brazil, based on pulsed field gel electrophoresis (PFGE), invasion ability in human colorectal carcinoma cells (HCT-116), InlA expression by western blot (WB) and identification of mutation points in the inlA . PFGE profiles demonstrated that L. monocytogenes isolates were grouped based on their previously identified lineages and serogroups (lineage I: serogroups IIb, n = 2, and IVb, n = 5; lineage II, serogroup IIc, n = 5), isolates with indistinguishable genetic profiles by this method were obtained from different slaughterhouses and sampling steps, with up to 3-year interval. Seven isolates showed high invasion ability (2.4 to 7.4%, lineage I, n = 6, lineage II, n = 1) in HCT and expressed InlA. Five isolates showed low cell invasion ability (0.6 to 1.4%, lineage I, n = 1, lineage II, n = 4) and did not express InlA, and two of them (lineage II, serogroup IIc) presented mutations in inlA leading to a premature stop codon (PMSC) type 19, at position 326 (GAA → TAA). The results demonstrated that most of L. monocytogenes isolates from Lineage I expressed InlA and were the most invasive in HCT indicating their high virulence potential, while most isolates from Lineage II showed attenuated invasion due to non-expression of InlA and the presence of PMSC type 19 in inlA . The obtained results demonstrated that L. monocytogenes with indistinguishable PFGE profiles can be persisting or being reintroduced in beef processing facilities in the studied region and differences on their virulence potential based on their lineages and serogroups.

Comparative Analysis of the Complete Mitochondrial Genomes of Five Species of Ricaniidae (Hemiptera: Fulgoromorpha) and Phylogenetic Implications

Ricaniidae is a relatively small planthopper family with about 69 genera and 442 species worldwide. Members of this family occur throughout the warm temperate and tropical regions. Some species cause devastating damage to major agricultural and economic plants. However, the relationship between Ricaniidae and other families of Fulgoroidea needs to be further explored. The morphological definitions of the two biggest genera, Pochazia Amyot & Serville, 1843 and Ricania Germar, 1818 (the type genus of Ricaniidae) remain controversial. In this study, mitogenomes of five representatives in these two genera were decoded using the next-generation sequence method and genome assembly. Results showed that their complete mitogenomes are circular DNA molecules with 15,457 to 16,411 bp. All protein-coding genes (PCGs) begin with the start codon ATN, GTG or TTG and end with TAA, TAG, an incomplete stop codon single T or an incomplete stop codon single A. A lost DHU arm was discovered in the trnS gene of the five mitogenomes and the trnV gene within Pochaziaconfusa, Pochazia guttifera and Ricania simulans. The remnant tRNAs folded into clover-leaf structures. The sliding window, genetic distance, and Ka/Ks analyses indicated that the cox1 gene is the slowest evolving and is relatively conserved. The phylogenetic tree topologies support (Delphacidae + (((Issidae + (Lophopidae + Caliscelidae)) + (Flatidae + Ricaniidae)) + (Achilidae + (Dictyopharidae + Fulgoridae)))) as the best topology, as recognized by both PhyloBayes, RAxML and MrBayes based on four data sets (PCG, PCGRNA, PCG12, PCG12RNA). The monophyly of Ricaniidae and the sister group status of two families Flatidae and Ricaniidae are supported, but all analyses failed to support the monophyly of Pochazia and Ricania. The diagnoses between these two genera cannot be resolved until more evidence is acquired.

Caenorhabditis elegans ETR-1/CELF has broad effects on the muscle cell transcriptome, including genes that regulate translation and neuroblast migration

Migration of neuroblasts and neurons from their birthplace is central to the formation of neural circuits and networks. ETR-1 is the Caenorhabditis elegans homolog of the CELF1 (CUGBP, ELAV-like family 1) RNA-processing factor involved in neuromuscular disorders. etr-1 regulates body wall muscle differentiation. Our previous work showed that etr-1 in muscle has a non-autonomous role in neuronal migration, suggesting that ETR-1 is involved in the production of a signal emanating from body wall muscle that controls neuroblast migration and that interacts with Wnt signaling. etr-1 is extensively alternatively-spliced, and we identified the viable etr-1(lq61) mutant, caused by a stop codon in alternatively-spliced exon 8 and only affecting etr-1 isoforms containing exon 8. We took advantage of viable etr-1(lq61) to identify potential RNA targets of ETR-1 in body wall muscle using a combination of fluorescence activated cell sorting (FACS) of body wall muscles from wild-type and etr-1(lq61) and subsequent RNA-seq. This analysis revealed genes whose splicing and transcript levels were controlled by ETR-1 exon 8 isoforms, and represented a broad spectrum of genes involved in muscle differentiation, myofilament lattice structure, and physiology. Genes with transcripts underrepresented in etr-1(lq61) included those involved in ribosome function and translation, similar to potential CELF1 targets identified in chick cardiomyocytes. This suggests that at least some targets of ETR-1 might be conserved in vertebrates, and that ETR-1 might generally stimulate translation in muscles. As proof-of-principle, a functional analysis of a subset of ETR-1 targets revealed genes involved in AQR and PQR neuronal migration. One such gene, lev-11/tropomyosin, requires ETR-1 for alternative splicing, and another, unc-52/perlecan, requires ETR-1 for the production of long isoforms containing 3′ exons. In sum, these studies identified gene targets of ETR-1/CELF1 in muscles, which included genes involved in muscle development and physiology, and genes with novel roles in neuronal migration.

Mitogenomes of Nine Asian Skipper Genera and Their Phylogenetic Position (Lepidoptera: Hesperiidae: Pyrginae)

In this study, complete mitochondrial genomes of nine species representing three tribes in the subfamily Pyrginae sensu lato were newly sequenced. The mitogenomes are closed double-stranded circular molecules, with the length ranging from 15,232 bp to 15,559 bp, which all encode 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a control region. The orientation and gene order of these nine mitogenomes are identical to the inferred ancestral arrangement of insects. All PCGs exhibit the typical start codon ATN except for cox1 (using CGA) and cox2 (using TTG) in Mooreana trichoneura. Most of the PCGs terminate with a TAA stop codon, while cox1, cox2, nad4, and nad5 end with the incomplete codon single T. For the different datasets, we found that the one comprising all 37 genes of the mitogenome produced the highest nodal support, indicating that the inclusion of RNAs improves the phylogenetic signal. This study re-confirmed the status of Capila, Pseudocoladenia, and Sarangesa; namely, Capila belongs to the tribe Tagiadini, and Pseudocoladenia and Sarangesa to the tribe Celaenorrhini. Diagnostic characters distinguishing the two tribes, the length of the forewing cell and labial palpi, are no longer significant. Two populations of Pseudocoladenia dan fabia from China and Myanmar and P. dan dhyana from Thailand are confirmed as conspecific.

Validation that human microbiome phages use alternative genetic coding with TAG stop read as Q

Metagenomic findings suggesting that bacteriophages (phages) can use genetic codes different from those of their host bacteria reveal a new dimension of phage-host interaction dynamics. Whereas reassignment of stop codons to code for amino acids has been predicted, there has been no proteomic validation of alternative coding in phages. In fact, one code where the stop codon TAG is reassigned to glutamine (code 15) has never been experimentally validated in any biological system. Here, we characterized stop codon reassignment in two crAss-like phages found in the human gut microbiome using LC-MS/MS-based metaproteomics. The proteome data from several phage structural proteins clearly demonstrates reassignment of the TAG stop codon to glutamine, establishing for the first time the expression of genetic code 15.

High homozygosity of inversions in sunflower species largely averts accumulation of deleterious mutations

Recombination is critical both for accelerating adaptation and for the purging of deleterious mutations. Chromosomal inversions can act as recombination modifiers that suppress local recombination and, thus, are predicted to accumulate such mutations. In this study, we investigated patterns of recombination, transposable element abundance and coding sequence evolution across the genomes of 1,445 individuals from three sunflower species, as well as within nine inversions segregating within species. We also analyzed the effects of inversion genotypes on 87 phenotypic traits to test for overdominance. We found significant negative correlations of long terminal repeat retrotransposon abundance and deleterious mutations with recombination rates across the genome in all three species. However, we failed to detect an increase in these features in the inversions, except for a modest increase in the proportion of stop codon mutations in several very large or rare inversions. Moreover, there was little evidence of phenotypic overdominance in inversion heterozygotes, consistent with observations of minimal deleterious load. On the other hand, significantly greater load was observed for inversions in populations polymorphic for a given inversion compared to populations monomorphic for one of the arrangements, suggesting that the local state of inversion polymorphism affects deleterious load. These seemingly contradictory results can be explained by the geographic structuring and consequent excess homozygosity of inversions in wild sunflowers. Inversions contributing to local adaptation often exhibit geographic structure; such inversions represent ideal recombination modifiers, acting to facilitate adaptive divergence with gene flow, while largely averting the accumulation of deleterious mutations due to recombination suppression.

Characterization, Comparison of Four New Mitogenomes of Centrotinae (Hemiptera: Membracidae) and Phylogenetic Implications Supports New Synonymy

To explore the phylogenetic relationships of the subfamily Centrotinae from the mitochondrial genome data, four complete mitogenomes (Anchon lineatus, Anchon yunnanensis, Gargara genistae and Tricentrus longivalvulatus) were sequenced and analyzed. All the newly sequenced mitogenomes contain 37 genes. Among the 13 protein-coding genes (PCGs) of the Centrotinae mitogenomes, a sliding window analysis and the ratio of Ka/Ks suggest that atp8 is a relatively fast evolving gene, while cox1 is the slowest. All PCGs start with ATN, except for nad5 (start with TTG), and stop with TAA or the incomplete stop codon T, except for nad2 and cytb (terminate with TAG). All tRNAs can fold into the typical cloverleaf secondary structure, except for trnS1, which lacks the dihydrouridine (DHU) arm. The BI and ML phylogenetic analyses of concatenated alignments of 13 mitochondrial PCGs among the major lineages produce a well-resolved framework. Phylogenetic analyses show that Membracoidea, Smiliinae and Centrotinae, together with tribes Centrotypini and Leptobelini are recovered as well-supported monophyletic groups. The tribe Gargarini (sensu Wallace et al.) and its monophyly are supported.