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September 22, 2019  |  

The DNA methylome of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N6-methyl-adenine (m6A), N4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we began by investigating the DNA methylome of Sulfolobus acidocaldarius. The strategy behind this initial study entailed the use of combined digestion assays, dot blots, and genome resequencing, which utilizes specific restriction enzymes, antibodies specifically raised against m6A and m5C and single-molecule real-time (SMRT) sequencing, respectively, to identify DNA methylations occurring in exponentially growing cells. The previously identified restriction-modification system, specific of S. acidocaldarius, was confirmed by digestion assay and SMRT sequencing while, the presence of m6A was revealed by dot blot and identified on the characteristic Dam motif by SMRT sequencing. No m5C was detected by dot blot under the conditions tested. Furthermore, by comparing the distribution of both detected methylations along the genome and, by analyzing DNA methylation profiles in synchronized cells, we investigated in which cellular pathways, in particular the cell cycle, this m6A methylation could be a key player. The analysis of sequencing data rejected a role for m6A methylation in another defense system and also raised new questions about a potential involvement of this modification in the regulation of other biological functions in S. acidocaldarius.

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September 22, 2019  |  

Conserved genomic and amino acid traits of cold adaptation in subzero-growing Arctic permafrost bacteria.

Permafrost accounts for 27% of all soil ecosystems and harbors diverse microbial communities. Our understanding of microorganisms in permafrost, their activities and adaptations, remains limited. Using five subzero-growing (cryophilic) permafrost bacteria, we examined features of cold adaptation through comparative genomic analyses with mesophilic relatives. The cryophiles possess genes associated with cold adaptation, including cold shock proteins, RNA helicases, and oxidative stress and carotenoid synthesis enzymes. Higher abundances of genes associated with compatible solutes were observed, important for osmoregulation in permafrost brine veins. Most cryophiles in our study have higher transposase copy numbers than mesophiles. We investigated amino acid (AA) modifications in the cryophiles favoring increased protein flexibility at cold temperatures. Although overall there were few differences with the mesophiles, we found evidence of cold adaptation, with significant differences in proline, serine, glycine and aromaticity, in several cryophiles. The use of cold/hot AA ratios of >1, used in previous studies to indicate cold adaptation, was found to be inadequate on its own. Comparing the average of all cryophiles to all mesophiles, we found that overall cryophiles had a higher ratio of cold adapted proteins for serine (more serine), and to a lesser extent, proline and acidic residues (fewer prolines/acidic residues).

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September 22, 2019  |  

Inferring the minimal genome of Mesoplasma florum by comparative genomics and transposon mutagenesis.

The creation and comparison of minimal genomes will help better define the most fundamental mechanisms supporting life. Mesoplasma florum is a near-minimal, fast-growing, nonpathogenic bacterium potentially amenable to genome reduction efforts. In a comparative genomic study of 13 M. florum strains, including 11 newly sequenced genomes, we have identified the core genome and open pangenome of this species. Our results show that all of the strains have approximately 80% of their gene content in common. Of the remaining 20%, 17% of the genes were found in multiple strains and 3% were unique to any given strain. On the basis of random transposon mutagenesis, we also estimated that ~290 out of 720 genes are essential for M. florum L1 in rich medium. We next evaluated different genome reduction scenarios for M. florum L1 by using gene conservation and essentiality data, as well as comparisons with the first working approximation of a minimal organism, Mycoplasma mycoides JCVI-syn3.0. Our results suggest that 409 of the 473 M. mycoides JCVI-syn3.0 genes have orthologs in M. florum L1. Conversely, 57 putatively essential M. florum L1 genes have no homolog in M. mycoides JCVI-syn3.0. This suggests differences in minimal genome compositions, even for these evolutionarily closely related bacteria. IMPORTANCE The last years have witnessed the development of whole-genome cloning and transplantation methods and the complete synthesis of entire chromosomes. Recently, the first minimal cell, Mycoplasma mycoides JCVI-syn3.0, was created. Despite these milestone achievements, several questions remain to be answered. For example, is the composition of minimal genomes virtually identical in phylogenetically related species? On the basis of comparative genomics and transposon mutagenesis, we investigated this question by using an alternative model, Mesoplasma florum, that is also amenable to genome reduction efforts. Our results suggest that the creation of additional minimal genomes could help reveal different gene compositions and strategies that can support life, even within closely related species.

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September 22, 2019  |  

The genome of the marine medaka Oryzias melastigma.

Marine medaka (Oryzias melastigma) is considered to be a useful fish model for marine and estuarine ecotoxicology studies and has good potential for field-based population genomics because of its geographical distribution in Asian estuarine and coastal areas. In this study, we present the first whole-genome draft of O. melastigma. The genome assembly consists of 8,602 scaffolds (N50 = 23.737 Mb) and a total genome length of 779.4 Mb. A total of 23,528 genes were predicted, and 12,670 gene families shared with three teleost species (Japanese medaka, mangrove killifish and zebrafish) were identified. Genome analyses revealed that the O. melastigma genome is highly heterozygous and contains a large number of repeat sequences. This assembly represents a useful genomic resource for fish scientists.© 2018 John Wiley & Sons Ltd.

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September 22, 2019  |  

Coordinated regulation of core and accessory genes in the multipartite genome of Sinorhizobium fredii.

Prokaryotes benefit from having accessory genes, but it is unclear how accessory genes can be linked with the core regulatory network when developing adaptations to new niches. Here we determined hierarchical core/accessory subsets in the multipartite pangenome (composed of genes from the chromosome, chromid and plasmids) of the soybean microsymbiont Sinorhizobium fredii by comparing twelve Sinorhizobium genomes. Transcriptomes of two S. fredii strains at mid-log and stationary growth phases and in symbiotic conditions were obtained. The average level of gene expression, variation of expression between different conditions, and gene connectivity within the co-expression network were positively correlated with the gene conservation level from strain-specific accessory genes to genus core. Condition-dependent transcriptomes exhibited adaptive transcriptional changes in pangenome subsets shared by the two strains, while strain-dependent transcriptomes were enriched with accessory genes on the chromid. Proportionally more chromid genes than plasmid genes were co-expressed with chromosomal genes, while plasmid genes had a higher within-replicon connectivity in expression than chromid ones. However, key nitrogen fixation genes on the symbiosis plasmid were characterized by high connectivity in both within- and between-replicon analyses. Among those genes with host-specific upregulation patterns, chromosomal znu and mdt operons, encoding a conserved high-affinity zinc transporter and an accessory multi-drug efflux system, respectively, were experimentally demonstrated to be involved in host-specific symbiotic adaptation. These findings highlight the importance of integrative regulation of hierarchical core/accessory components in the multipartite genome of bacteria during niche adaptation and in shaping the prokaryotic pangenome in the long run.

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September 22, 2019  |  

Genome-based evolutionary history of Pseudomonas spp.

Pseudomonas is a large and diverse genus of Gammaproteobacteria. To provide a framework for discovery of evolutionary and taxonomic relationships of these bacteria, we compared the genomes of type strains of 163 species and 3 additional subspecies of Pseudomonas, including 118 genomes sequenced herein. A maximum likelihood phylogeny of the 166 type strains based on protein sequences of 100 single-copy orthologous genes revealed thirteen groups of Pseudomonas, composed of two to sixty three species each. Pairwise average nucleotide identities and alignment fractions were calculated for the data set of the 166 type strains and 1224 genomes of Pseudomonas available in public databases. Results revealed that 394 of the 1224 genomes were distinct from any type strain, suggesting that the type strains represent only a fraction of the genomic diversity of the genus. The core genome of Pseudomonas was determined to contain 794 genes conferring primarily housekeeping functions. The results of this study provide a phylogenetic framework for future studies aiming to resolve the classification and phylogenetic relationships, identify new gene functions and phenotypes, and explore the ecological and metabolic potential of the Pseudomonas spp.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019  |  

Insect symbionts as valuable grist for the biotechnological mill: an alkaliphilic silkworm gut bacterium for efficient lactic acid production.

Insects constitute the most abundant and diverse animal class and act as hosts to an extraordinary variety of symbiotic microorganisms. These microbes living inside the insects play critical roles in host biology and are also valuable bioresources. Enterococcus mundtii EMB156, isolated from the larval gut (gut pH >10) of the model organism Bombyx mori (Lepidoptera: Bombycidae), efficiently produces lactic acid, an important metabolite for industrial production of bioplastic materials. E. mundtii EMB156 grows well under alkaline conditions and stably converts various carbon sources into lactic acid, offering advantages in downstream fermentative processes. High-yield lactic acid production can be achieved by the strain EMB156 from renewable biomass substrates under alkaline pretreatments. Single-molecule real-time (SMRT) sequencing technology revealed its 3.01 Mbp whole genome sequence. A total of 2956 protein-coding sequences, 65 tRNA genes, and 6 rRNA operons were predicted in the EMB156 chromosome. Remarkable genomic features responsible for lactic acid fermentation included key enzymes involved in the pentose phosphate (PP)/glycolytic pathway, and an alpha amylase and xylose isomerase were characterized in EMB156. This genomic information coincides with the phenotype of E. mundtii EMB156, reflecting its metabolic flexibility in efficient lactate fermentation, and established a foundation for future biotechnological application. Interestingly, enzyme activities of amylase were quite stable in high-pH broths, indicating a possible mechanism for strong EMB156 growth in an alkaline environment, thereby facilitating lactic acid production. Together, these findings implied that valuable lactic acid-producing bacteria can be discovered efficiently by screening under the extremely alkaline conditions, as exemplified by gut microbial symbionts of Lepidoptera insects.

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September 22, 2019  |  

Unexpected invasion of miniature inverted-repeat transposable elements in viral genomes

Transposable elements (TEs) are common and often present with high copy numbers in cellular genomes. Unlike in cellular organisms, TEs were previously thought to be either rare or absent in viruses. Almost all reported TEs display only one or two copies per viral genome. In addition, the discovery of pandoraviruses with genomes up to 2.5-Mb emphasizes the need for biologists to rethink the fundamental nature of the relationship between viruses and cellular life.

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September 22, 2019  |  

Horizontal transfer and proliferation of Tsu4 in Saccharomyces paradoxus.

Recent evidence suggests that horizontal transfer plays a significant role in the evolution of of transposable elements (TEs) in eukaryotes. Many cases of horizontal TE transfer (HTT) been reported in animals and plants, however surprisingly few examples of HTT have been reported in fungi.Here I report evidence for a novel HTT event in fungi involving Tsu4 in Saccharomyces paradoxus based on (i) unexpectedly high similarity between Tsu4 elements in S. paradoxus and S. uvarum, (ii) a patchy distribution of Tsu4 in S. paradoxus and general absence from its sister species S. cerevisiae, and (iii) discordance between the phylogenetic history of Tsu4 sequences and species in the Saccharomyces sensu stricto group. Available data suggests the HTT event likely occurred somewhere in the Nearctic, Neotropic or Indo-Australian part of the S. paradoxus species range, and that a lineage related to S. uvarum or S. eubayanus was the likely donor species. The HTT event has led to massive proliferation of Tsu4 in the South American lineage of S. paradoxus, which exhibits partial reproductive isolation with other strains of this species because of multiple reciprocal translocations. Full-length Tsu4 elements are associated with both breakpoints of one of these reciprocal translocations.This work shows that comprehensive analysis of TE sequences in essentially-complete genome assemblies derived from long-read sequencing provides new opportunities to detect HTT events in fungi and other organisms. This work also provides support for the hypothesis that HTT and subsequent TE proliferation can induce genome rearrangements that contribute to post-zygotic isolation in yeast.

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September 22, 2019  |  

Heterogeneous and flexible transmission of mcr-1 in hospital-associated Escherichia coli.

The recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route of mcr-1 among Enterobacteriaceae species in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis of Escherichia coli isolates collected in a hospital in Hangzhou, China. We found that mcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread of mcr-1 The mcr-1 gene was found on either chromosomes or plasmids, but in most of the E. coli isolates, mcr-1 was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission of mcr-1 and the coexistence of mcr-1 with other genes encoding ß-lactamases and fluoroquinolone resistance in the E. coli isolates. These findings indicate that mcr-1 is heterogeneously disseminated in both commensal and pathogenic strains of E. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology of mcr-1 among hospital-associated E. coli strains. IMPORTANCE Colistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergent mcr-1 colistin resistance gene threatens the clinical utility of colistin and has gained global attention. How mcr-1 spreads in hospital settings remains unknown and was investigated by whole-genome sequencing of mcr-1-carrying Escherichia coli in this study. The findings revealed extraordinary flexibility of mcr-1 in its spread among genetically diverse E. coli hosts and plasmids, nosocomial transmission of mcr-1-carrying E. coli, and the continuous emergence of novel Inc types of plasmids carrying mcr-1 and new mcr-1 variants. Additionally, mcr-1 was found to be frequently associated with other genes encoding ß-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology of mcr-1 and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat. Copyright © 2018 Shen et al.

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September 22, 2019  |  

Complete genome sequencing and comparative genomic analysis of Helicobacter apodemus isolated from the wild Korean striped field mouse (Apodemus agrarius) for potential pathogenicity

The Helicobacter bacterial genus comprises of spiral-shaped gram-negative bacteria with flagella that colonize the gastro-intestinal (GI) tract of humans and various mammals (Solnick and Schauer, 2001). In particular, Helicobacter pylori was classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) in 1994, and has been shown to occur with a high prevalence in humans, although this varies between geographical regions, ethnic groups, and various populations (Kusters et al., 2006; Goh et al., 2011). To date, more than 37 Helicobacter species have been identified in addition to H. pylori (Péré-Védrenne et al., 2017). Furthermore, non-H. pylori Helicobacters (NHPH) have been shown to infect both humans and animals, and NHPH infections are associated with intestinal carcinoma, and mucinous adenocarcinoma (Swennes et al., 2016). Despite the demonstrated association between NHPH and disease, most studies to date have investigated H. pylori in humans; thus, it is necessary to characterize NHPH and elucidate its role in the GI tract of wild rodents which are potential Helicobacter carriers (Taylor et al., 2007; Mladenova-Hristova et al., 2017).

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September 22, 2019  |  

Novel enterobacter lineage as leading cause of nosocomial outbreak involving carbapenemase-producing strains.

We investigated unusual carbapenemase-producing Enterobacter cloacae complex isolates (n = 8) in the novel sequence type (ST) 873, which caused nosocomial infections in 2 hospitals in France. Whole-genome sequence typing showed the 1-year persistence of the epidemic strain, which harbored a blaVIM-4 ST1-IncHI2 plasmid, in 1 health institution and 2 closely related strains harboring blaCTX-M-15 in the other. These isolates formed a new subgroup in the E. hormaechei metacluster, according to their hsp60 sequences and phylogenomic analysis. The average nucleotide identities, specific biochemical properties, and pangenomic and functional investigations of isolates suggested isolates of a novel species that had acquired genes associated with adhesion and mobility. The emergence of this novel Enterobacter phylogenetic lineage within hospitals should be closely monitored because of its ability to persist and spread.

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September 22, 2019  |  

Wheat microbiome bacteria can reduce virulence of a plant pathogenic fungus by altering histone acetylation.

Interactions between bacteria and fungi have great environmental, medical, and agricultural importance, but the molecular mechanisms are largely unknown. Here, we study the interactions between the bacterium Pseudomonas piscium, from the wheat head microbiome, and the plant pathogenic fungus Fusarium graminearum. We show that a compound secreted by the bacteria (phenazine-1-carboxamide) directly affects the activity of fungal protein FgGcn5, a histone acetyltransferase of the SAGA complex. This leads to deregulation of histone acetylation at H2BK11, H3K14, H3K18, and H3K27 in F. graminearum, as well as suppression of fungal growth, virulence, and mycotoxin biosynthesis. Therefore, an antagonistic bacterium can inhibit growth and virulence of a plant pathogenic fungus by manipulating fungal histone modification.

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September 22, 2019  |  

Whole-genome sequencing and comparative analysis of two plant-associated strains of Rhodopseudomonas palustris (PS3 and YSC3).

Rhodopseudomonas palustris strains PS3 and YSC3 are purple non-sulfur phototrophic bacteria isolated from Taiwanese paddy soils. PS3 has beneficial effects on plant growth and enhances the uptake efficiency of applied fertilizer nutrients. In contrast, YSC3 has no significant effect on plant growth. The genomic structures of PS3 and YSC3 are similar; each contains one circular chromosome that is 5,269,926 or 5,371,816?bp in size, with 4,799 or 4,907 protein-coding genes, respectively. In this study, a large class of genes involved in chemotaxis and motility was identified in both strains, and genes associated with plant growth promotion, such as nitrogen fixation-, IAA synthesis- and ACC deamination-associated genes, were also identified. We noticed that the growth rate, the amount of biofilm formation, and the relative expression levels of several chemotaxis-associated genes were significantly higher for PS3 than for YSC3 upon treatment with root exudates. These results indicate that PS3 responds better to the presence of plant hosts, which may contribute to the successful interactions of PS3 with plant hosts. Moreover, these findings indicate that the existence of gene clusters associated with plant growth promotion is required but not sufficient for a bacterium to exhibit phenotypes associated with plant growth promotion.

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September 22, 2019  |  

Comparative genomics of Salmonella enterica serovar Montevideo reveals lineage-specific gene differences that may influence ecological niche association.

Salmonella enterica serovar Montevideo has been linked to recent foodborne illness outbreaks resulting from contamination of products such as fruits, vegetables, seeds and spices. Studies have shown that Montevideo also is frequently associated with healthy cattle and can be isolated from ground beef, yet human salmonellosis outbreaks of Montevideo associated with ground beef contamination are rare. This disparity fuelled our interest in characterizing the genomic differences between Montevideo strains isolated from healthy cattle and beef products, and those isolated from human patients and outbreak sources. To that end, we sequenced 13 Montevideo strains to completion, producing high-quality genome assemblies of isolates from human patients (n=8) or from healthy cattle at slaughter (n=5). Comparative analysis of sequence data from this study and publicly available sequences (n=72) shows that Montevideo falls into four previously established clades, differentially occupied by cattle and human strains. The results of these analyses reveal differences in metabolic islands, environmental adhesion determinants and virulence factors within each clade, and suggest explanations for the infrequent association between bovine isolates and human illnesses.

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