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July 7, 2019

A vast genomic deletion in the C56BL/6 genome affects different genes within the Ifi200 cluster on chromosome 1 and mediates obesity and insulin resistance.

Obesity, the excessive accumulation of body fat, is a highly heritable and genetically heterogeneous disorder. The complex, polygenic basis for the disease consisting of a network of different gene variants is still not completely known.In the current study we generated a BAC library of the obese-prone NZO strain to clarify the genomic alteration within the gene cluster Ifi200 on chr.1 including Ifi202b, an obesity gene that is in contrast to NZO not expressed in the lean B6 mouse. With the PacBio sequencing data of NZO BAC clones we identified a deletion spanning approximately 261.8 kb in the B6 reference genome. The deletion affects different members of the Ifi200 gene family which also includes the original first exon and 5′-regulatory parts of the Ifi202b gene and suggests to be the relevant cause of its expression deficiency in B6. In addition, the generation and characterization of congenic mice carrying the critical fragment on the B6 background demonstrate its crucial role for obesity and insulin resistance.Our data reveal the reconstruction of a complex genomic region on mouse chr.1 resulting from deletions and duplications of Ifi200 genes and suggest to be relevant for the development of obesity. The results further demonstrate the complexity of the disease and highlight the importance for studying rare genetic variants as they can be causal for large effects.

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July 7, 2019

Complete genome sequence of Thermus brockianus GE-1 reveals key enzymes of xylan/xylose metabolism.

Thermus brockianus strain GE-1 is a thermophilic, Gram-negative, rod-shaped and non-motile bacterium that was isolated from the Geysir geothermal area, Iceland. Like other thermophiles, Thermus species are often used as model organisms to understand the mechanism of action of extremozymes, especially focusing on their heat-activity and thermostability. Genome-specific features of T. brockianus GE-1 and their properties further help to explain processes of the adaption of extremophiles at elevated temperatures. Here we analyze the first whole genome sequence of T. brockianus strain GE-1. Insights of the genome sequence and the methodologies that were applied during de novo assembly and annotation are given in detail. The finished genome shows a phred quality value of QV50. The complete genome size is 2.38 Mb, comprising the chromosome (2,035,182 bp), the megaplasmid pTB1 (342,792 bp) and the smaller plasmid pTB2 (10,299 bp). Gene prediction revealed 2,511 genes in total, including 2,458 protein-encoding genes, 53 RNA and 66 pseudo genes. A unique genomic region on megaplasmid pTB1 was identified encoding key enzymes for xylan depolymerization and xylose metabolism. This is in agreement with the growth experiments in which xylan is utilized as sole source of carbon. Accordingly, we identified sequences encoding the xylanase Xyn10, an endoglucanase, the membrane ABC sugar transporter XylH, the xylose-binding protein XylF, the xylose isomerase XylA catalyzing the first step of xylose metabolism and the xylulokinase XylB, responsible for the second step of xylose metabolism. Our data indicate that an ancestor of T. brockianus obtained the ability to use xylose as alternative carbon source by horizontal gene transfer.

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July 7, 2019

Identification of a Pseudomonas aeruginosa PAO1 DNA methyltransferase, its targets, and physiological roles.

DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this study, we used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We identified a conserved sequence motif targeted by an adenine methyltransferase of a type I R-M system and quantified the presence of N(6)-methyladenine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in the PAO1 methylation status were dependent on growth conditions and affected P. aeruginosa pathogenicity in a Galleria mellonella infection model. Furthermore, we found that methylated motifs in promoter regions led to shifts in sense and antisense gene expression, emphasizing the role of enzymatic DNA methylation as an epigenetic control of phenotypic traits in P. aeruginosa Since the DNA methylation enzymes are not encoded in the core genome, our findings illustrate how the acquisition of accessory genes can shape the global P. aeruginosa transcriptome and thus may facilitate adaptation to new and challenging habitats.IMPORTANCE With the introduction of advanced technologies, epigenetic regulation by DNA methyltransferases in bacteria has become a subject of intense studies. Here we identified an adenosine DNA methyltransferase in the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is responsible for DNA methylation of a conserved sequence motif. The methylation level of all target sequences throughout the PAO1 genome was approximated to be in the range of 65 to 85% and was dependent on growth conditions. Inactivation of the methyltransferase revealed an attenuated-virulence phenotype in the Galleria mellonella infection model. Furthermore, differential expression of more than 90 genes was detected, including the small regulatory RNA prrF1, which contributes to a global iron-sparing response via the repression of a set of gene targets. Our finding of a methylation-dependent repression of the antisense transcript of the prrF1 small regulatory RNA significantly expands our understanding of the regulatory mechanisms underlying active DNA methylation in bacteria. Copyright © 2017 Doberenz et al.

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July 7, 2019

Complete genome sequence of a Staphylococcus epidermidis strain with exceptional antimicrobial activity.

Staphylococcus epidermidis is a Gram-positive bacterium that is prevalent on human skin. The species is associated with skin health, as well as with opportunistic infections. Here, we report the complete genome sequence of S. epidermidis 14.1.R1, isolated from human skin. In bacterial interference assays, the strain showed exceptional antimicrobial activity. Copyright © 2017 Lassen et al.

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July 7, 2019

Hybrid sequencing and map finding (HySeMaFi): optional strategies for extensively deciphering gene splicing and expression in organisms without reference genome.

Using second-generation sequencing (SGS) RNA-Seq strategies, extensive alterative splicing prediction is impractical and high variability of isoforms expression quantification is inevitable in organisms without true reference dataset. we report the development of a novel analysis method, termed hybrid sequencing and map finding (HySeMaFi) which combines the specific strengths of third-generation sequencing (TGS) (PacBio SMRT sequencing) and SGS (Illumina Hi-Seq/MiSeq sequencing) to effectively decipher gene splicing and to reliably estimate the isoforms abundance. Error-corrected long reads from TGS are capable of capturing full length transcripts or as large partial transcript fragments. Both true and false isoforms, from a particular gene, as well as that containing all possible exons, could be generated by employing different assembly methods in SGS. We first develop an effective method which can establish the mapping relationship between the error-corrected long reads and the longest assembled contig in every corresponding gene. According to the mapping data, the true splicing pattern of the genes was reliably detected, and quantification of the isoforms was also effectively determined. HySeMaFi is also the optimal strategy by which to decipher the full exon expression of a specific gene when the longest mapped contigs were chosen as the reference set.

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July 7, 2019

Genome sequence of the thermotolerant foodborne pathogen Salmonella enterica serovar Senftenberg ATCC 43845 and phylogenetic analysis of loci encoding increased protein quality control mechanisms.

Salmonella enterica subsp. enterica bacteria are important foodborne pathogens with major economic impact. Some isolates exhibit increased heat tolerance, a concern for food safety. Analysis of a finished-quality genome sequence of an isolate commonly used in heat resistance studies, S. enterica subsp. enterica serovar Senftenberg 775W (ATCC 43845), demonstrated an interesting observation that this strain contains not just one, but two horizontally acquired thermotolerance locus homologs. These two loci reside on a large 341.3-kbp plasmid that is similar to the well-studied IncHI2 R478 plasmid but lacks any antibiotic resistance genes found on R478 or other IncHI2 plasmids. As this historical Salmonella isolate has been in use since 1941, comparative analysis of the plasmid and of the thermotolerance loci contained on the plasmid will provide insight into the evolution of heat resistance loci as well as acquisition of resistance determinants in IncHI2 plasmids. IMPORTANCE Thermal interventions are commonly used in the food industry as a means of mitigating pathogen contamination in food products. Concern over heat-resistant food contaminants has recently increased, with the identification of a conserved locus shown to confer heat resistance in disparate lineages of Gram-negative bacteria. Complete sequence analysis of a historical isolate of Salmonella enterica serovar Senftenberg, used in numerous studies because of its novel heat resistance, revealed that this important strain possesses two distinct copies of this conserved thermotolerance locus, residing on a multireplicon IncHI2/IncHI2A plasmid. Phylogenetic analysis of these loci in comparison with homologs identified in various bacterial genera provides an opportunity to examine the evolution and distribution of loci conferring resistance to environmental stressors, such as heat and desiccation.

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July 7, 2019

Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts.

Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species.

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July 7, 2019

The histidine decarboxylase gene cluster of Lactobacillus parabuchneri was gained by horizontal gene transfer and is mobile within the species.

Histamine in food can cause intolerance reactions in consumers. Lactobacillus parabuchneri (L. parabuchneri) is one of the major causes of elevated histamine levels in cheese. Despite its significant economic impact and negative influence on human health, no genomic study has been published so far. We sequenced and analyzed 18 L. parabuchneri strains of which 12 were histamine positive and 6 were histamine negative. We determined the complete genome of the histamine positive strain FAM21731 with PacBio as well as Illumina and the genomes of the remaining 17 strains using the Illumina technology. We developed the synteny aware ortholog finding algorithm SynOrf to compare the genomes and we show that the histidine decarboxylase (HDC) gene cluster is located in a genomic island. It is very likely that the HDC gene cluster was transferred from other lactobacilli, as it is highly conserved within several lactobacilli species. Furthermore, we have evidence that the HDC gene cluster was transferred within the L. parabuchneri species.

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July 7, 2019

Acquisition of virulence factors in livestock-associated MRSA: Lysogenic conversion of CC398 strains by virulence gene-containing phages.

Staphylococcus aureus MRSA strains belonging to the clonal complex 398 (CC398) are highly prevalent in livestock and companion animals but may also cause serious infections in humans. CC398 strains in livestock usually do not possess well-known virulence factors that can be frequently found in other MRSA sequence types (ST). Since many staphylococcal virulence genes are residing on the genomes of temperate phages, the question arises why livestock-associated (LA-) CC398 strains are only rarely infected by those phages. We isolated and characterized four temperate phages (P240, P282, P630 and P1105) containing genes of the immune evasion cluster (IEC) and/or for the Panton-Valentine leucocidin (PVL). Sequence analysis of the phage genomes showed that they are closely related to known phages and that the DNA region encoding lysis proteins, virulence factors and the integrase exhibits numerous DNA repeats which may facilitate genomic rearrangements. All phages lysed and lysogenized LA-CC398 strains. Integration of IEC phage P282 was detected at ten sites of the hosts’ chromosome. The prophages were stably inherited in LA-CC398 and enterotoxin A, staphylokinase and PVL toxin were produced. The data demonstrate that lysogenic conversion of LA-CC398 strains by virulence-associated phages may occur and that new pathotypes may emerge by this mechanism.

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July 7, 2019

Tripartite species interaction: eukaryotic hosts suffer more from phage susceptible than from phage resistant bacteria.

Evolutionary shifts in bacterial virulence are often associated with a third biological player, for instance temperate phages, that can act as hyperparasites. By integrating as prophages into the bacterial genome they can contribute accessory genes, which can enhance the fitness of their prokaryotic carrier (lysogenic conversion). Hyperparasitic influence in tripartite biotic interactions has so far been largely neglected in empirical host-parasite studies due to their inherent complexity. Here we experimentally address whether bacterial resistance to phages and bacterial harm to eukaryotic hosts is linked using a natural tri-partite system with bacteria of the genus Vibrio, temperate vibriophages and the pipefish Syngnathus typhle. We induced prophages from all bacterial isolates and constructed a three-fold replicated, fully reciprocal 75 × 75 phage-bacteria infection matrix.According to their resistance to phages, bacteria could be grouped into three distinct categories: highly susceptible (HS-bacteria), intermediate susceptible (IS-bacteria), and resistant (R-bacteria). We experimentally challenged pipefish with three selected bacterial isolates from each of the three categories and determined the amount of viable Vibrio counts from infected pipefish and the expression of pipefish immune genes. While the amount of viable Vibrio counts did not differ between bacterial groups, we observed a significant difference in relative gene expression between pipefish infected with phage susceptible and phage resistant bacteria.These findings suggest that bacteria with a phage-susceptible phenotype are more harmful against a eukaryotic host, and support the importance of hyperparasitism and the need for an integrative view across more than two levels when studying host-parasite evolution.

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