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July 7, 2019

Novel recA-independent horizontal gene transfer in Escherichia coli K-12.

In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F’-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10-12 CFU/recipient per hour.

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July 7, 2019

Common cell shape evolution of two nasopharyngeal pathogens.

Respiratory infectious diseases are the third cause of worldwide death. The nasopharynx is the portal of entry and the ecological niche of many microorganisms, of which some are pathogenic to humans, such as Neisseria meningitidis and Moraxella catarrhalis. These microbes possess several surface structures that interact with the actors of the innate immune system. In our attempt to understand the past evolution of these bacteria and their adaption to the nasopharynx, we first studied differences in cell wall structure, one of the strongest immune-modulators. We were able to show that a modification of peptidoglycan (PG) composition (increased proportion of pentapeptides) and a cell shape change from rod to cocci had been selected for along the past evolution of N. meningitidis. Using genomic comparison across species, we correlated the emergence of the new cell shape (cocci) with the deletion, from the genome of N. meningitidis ancestor, of only one gene: yacF. Moreover, the reconstruction of this genetic deletion in a bacterium harboring the ancestral version of the locus together with the analysis of the PG structure, suggest that this gene is coordinating the transition from cell elongation to cell division. Accompanying the loss of yacF, the elongation machinery was also lost by several of the descendants leading to the change in the PG structure observed in N. meningitidis. Finally, the same evolution was observed for the ancestor of M. catarrhalis. This suggests a strong selection of these genetic events during the colonization of the nasopharynx. This selection may have been forced by the requirement of evolving permissive interaction with the immune system, the need to reduce the cellular surface exposed to immune attacks without reducing the intracellular storage capacity, or the necessity to better compete for adhesion to target cells.

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July 7, 2019

Tandem repeats in rodents genome and their mapping.

Tandemly-repeated sequences represent a unique class of eukaryotic DNA. Their content in the genome of higher eukaryotes mounts to tens of percents. However, the evolution of this class of sequences is poorly-studied. In our paper, 62 families of Mus musculus tandem repeats are analyzed by bioinformatic methods, and 7 of them are analyzed by fluorescence in situ hybridization. It is shown that the same tandem repeat sets co-occure only in closely related species of mice. But even in such species we observe differences in localization on the chromosomes and the number of individual tandem repeats. With increasing evolutionary distance only some of the tandem repeat families remain common for different species. It is shown, that the use of a combination of bioinformatics and molecular biology techniques is very perspective for further studies of the evolution of tandem repeats.

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July 7, 2019

First complete genome sequences of Staphylococcus aureus subsp. aureus Rosenbach 1884 (DSM 20231T), determined by PacBio Single-Molecule Real-Time Technology.

The first complete genome sequences of Staphylococcus aureus subsp. aureus Rosenbach 1884 strain DSM 20231(T), the type strain of the bacterium causing staphylococcal disease, were determined using PacBio RS II. The sequences represent the chromosome (2,755,072 bp long; G+C content, 32.86%) and a plasmid (27,490 bp long; G+C content, 30.69%). Copyright © 2015 Shiroma et al.

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July 7, 2019

Complete genome sequence of Piscirickettsia salmonis LF-89 (ATCC VR-1361) a major pathogen of farmed salmonid fish.

Piscirickettsia salmonis, the causative agent of salmonid rickettsial septicemia (SRS), is a significant threat to the healthy and sustainable production of salmonid farming industry. This Gram-negative bacterium, originally isolated from a coho salmon in Southern Chile, produces a systemic infection characterized by colonization of several fish organs. P. salmonis is able to infect, survive, and replicate inside salmonid macrophages however little is known about its mechanisms of pathogenesis. Here, we present the whole genome sequence and annotation of the P. salmonis reference strain LF-89 (ATCC VR-1361). The genome contains one circular chromosome of 3,184,851bp and three plasmids, pPSLF89-1 (180,124bp), pPSLF89-2 (33,516bp) and pPSLF89-3 (51,573bp). A total of 2850 protein-coding genes, 56 tRNAs and six copies of 5S-16S-23S rRNA. Copyright © 2015 Elsevier B.V. All rights reserved.

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July 7, 2019

The assembly and characterisation of two structurally distinct cattle MHC class I haplotypes point to the mechanisms driving diversity.

In cattle, there are six classical MHC class I genes that are variably present between different haplotypes. Almost all known haplotypes contain between one and three genes, with an allele of Gene 2 present on the vast majority. However, very little is known about the sequence and therefore structure and evolutionary history of this genomic region. To address this, we have refined the MHC class I region in the Hereford cattle genome assembly and sequenced a complete A14 haplotype from a homozygous Holstein. Comparison of the two haplotypes revealed extensive variation within the MHC class Ia region, but not within the flanking regions, with each gene contained within a conserved 63- to 68-kb sequence block. This variable region appears to have undergone block gene duplication and likely deletion at regular breakpoints, suggestive of a site-specific mechanism. Phylogenetic analysis using complete gene sequences provided evidence of allelic diversification via gene conversion, with breakpoints between each of the extracellular domains that were associated with high guanine-cytosine (GC) content. Advancing our knowledge of cattle MHC class I evolution will help inform investigations of cattle genetic diversity and disease resistance.

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July 7, 2019

Isolation and characterization of an interactive culture of two Paenibacillus species with moderately thermophilic desulfurization ability.

To isolate and characterize novel thermophilic bacteria capable of biodesulfurization of petroleum.A culture containing two Paenibacillus spp. (denoted “32O-W” and “32O-Y”) was isolated by repeated passage of a soil sample at up to 55 °C in medium containing dibenzothiophene (DBT) as sulfur source. Only 32O-Y metabolized DBT, apparently via the 4S pathway; maximum activity occurred from 40 to 45 °C, with some activity up to at least 50 °C. 32O-W enhanced DBT metabolism by 32O-Y (by 22-74 % at 40-50 °C). With sulfate as sulfur source, 32O-Y and 32O-W grew well up to 58 and 63 °C, respectively. Selection of a mixed culture of 32O-Y and 32O-W at 54 °C increased DBT metabolism 36-42 % from 40 to 45 °C. Genome sequencing identified desulfurization gene homologs in the strains consistent with their desulfurization properties.The 32O-Y/32O-W culture may be a useful starting point for development of an improved thermophilic petroleum biodesulfurization process.

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July 7, 2019

Sequencing of plasmids pAMBL1 and pAMBL2 from Pseudomonas aeruginosa reveals a blaVIM-1 amplification causing high-level carbapenem resistance.

Carbapenemases are a major concern for the treatment of infectious diseases caused by Gram-negative bacteria. Although plasmids are responsible for the spread of resistance genes among these pathogens, there is limited information on the nature of the mobile genetic elements carrying carbapenemases in Pseudomonas aeruginosa.We combined data from two different next-generation sequencing platforms, Illumina HiSeq2000 and PacBio RSII, to obtain the complete nucleotide sequences of two blaVIM-1-carrying plasmids (pAMBL1 and pAMBL2) isolated from P. aeruginosa clinical isolates.Plasmid pAMBL1 has 26?440 bp and carries a RepA_C family replication protein. pAMBL1 is similar to plasmids pNOR-2000 and pKLC102 from P. aeruginosa and pAX22 from Achromobacter xylosoxidans, which also carry VIM-type carbapenemases. pAMBL2 is a 24?133 bp plasmid with a replication protein that belongs to the Rep_3 family. It shows a high degree of homology with a fragment of the blaVIM-1-bearing plasmid pPC9 from Pseudomonas putida. Plasmid pAMBL2 carries three copies of the blaVIM-1 cassette in an In70 class 1 integron conferring, unlike pAMBL1, high-level resistance to carbapenems.We present two new plasmids coding for VIM-1 carbapenemase from P. aeruginosa and report that the presence of three copies of blaVIM-1 in pAMBL2 produces high-level resistance to carbapenems.© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Genome sequence of the haloarchaeon Haloterrigena jeotgali type strain A29(T) isolated from salt-fermented food.

Haloterrigena jeotgali is a halophilic archaeon within the family Natrialbaceae that was isolated from shrimp jeotgal, a traditional Korean salt-fermented food. A29(T) is the type strain of H. jeotgali, and is a Gram-negative staining, non-motile, rod-shaped archaeon that grows in 10 %-30 % (w/v) NaCl. We present the annotated H. jeotgali A29(T) genome sequence along with a summary of its features. The 4,131,621 bp genome with a GC content of 64.9 % comprises 4,215 protein-coding genes and 127 RNA genes. The sequence can provide useful information on genetic mechanisms that enable haloarchaea to endure a hypersaline environment.

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July 7, 2019

Draft genome sequence of the extremely halophilic archaeon Haladaptatus cibarius type strain D43T isolated from fermented seafood

An extremely halophilic archaeon, Haladaptatus cibarius D43 T , was isolated from traditional Korean salt-rich fermented seafood. Strain D43 T shows the highest 16S rRNA gene sequence similarity (98.7 %) with Haladaptatus litoreus RO1-28 T , is Gram-negative staining, motile, and extremely halophilic. Despite potential industrial applications of extremely halophilic archaea, their genome characteristics remain obscure. Here, we describe the whole genome sequence and annotated features of strain D43 T . The 3,926,724 bp genome includes 4,092 protein-coding and 57 RNA genes (including 6 rRNA and 49 tRNA genes) with an average G?+?C content of 57.76 %.

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July 7, 2019

Complete chloroplast genome sequence of MD-2 pineapple and its comparative analysis among nine other plants from the subclass Commelinidae.

Pineapple (Ananas comosus var. comosus) is known as the king of fruits for its crown and is the third most important tropical fruit after banana and citrus. The plant, which is indigenous to South America, is the most important species in the Bromeliaceae family and is largely traded for fresh fruit consumption. Here, we report the complete chloroplast sequence of the MD-2 pineapple that was sequenced using the PacBio sequencing technology.In this study, the high error rate of PacBio long sequence reads of A. comosus’s total genomic DNA were improved by leveraging on the high accuracy but short Illumina reads for error-correction via the latest error correction module from Novocraft. Error corrected long PacBio reads were assembled by using a single tool to produce a contig representing the pineapple chloroplast genome. The genome of 159,636 bp in length is featured with the conserved quadripartite structure of chloroplast containing a large single copy region (LSC) with a size of 87,482 bp, a small single copy region (SSC) with a size of 18,622 bp and two inverted repeat regions (IRA and IRB) each with the size of 26,766 bp. Overall, the genome contained 117 unique coding regions and 30 were repeated in the IR region with its genes contents, structure and arrangement similar to its sister taxon, Typha latifolia. A total of 35 repeats structure were detected in both the coding and non-coding regions with a majority being tandem repeats. In addition, 205 SSRs were detected in the genome with six protein-coding genes contained more than two SSRs. Comparative chloroplast genomes from the subclass Commelinidae revealed a conservative protein coding gene albeit located in a highly divergence region. Analysis of selection pressure on protein-coding genes using Ka/Ks ratio showed significant positive selection exerted on the rps7 gene of the pineapple chloroplast with P less than 0.05. Phylogenetic analysis confirmed the recent taxonomical relation among the member of commelinids which support the monophyly relationship between Arecales and Dasypogonaceae and between Zingiberales to the Poales, which includes the A. comosus.The complete sequence of the chloroplast of pineapple provides insights to the divergence of genic chloroplast sequences from the members of the subclass Commelinidae. The complete pineapple chloroplast will serve as a reference for in-depth taxonomical studies in the Bromeliaceae family when more species under the family are sequenced in the future. The genetic sequence information will also make feasible other molecular applications of the pineapple chloroplast for plant genetic improvement.

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July 7, 2019

The genome of the Saprophytic fungus Verticillium tricorpus reveals a complex effector repertoire resembling that of its pathogenic relatives.

Vascular wilts caused by Verticillium spp. are destructive plant diseases affecting hundreds of hosts. Only a few Verticillium spp. are causal agents of vascular wilt diseases, of which V. dahliae is the most notorious pathogen, and several V. dahliae genomes are available. In contrast, V. tricorpus is mainly known as a saprophyte and causal agent of opportunistic infections. Based on a hybrid approach that combines second and third generation sequencing, a near-gapless V. tricorpus genome assembly was obtained. With comparative genomics, we sought to identify genomic features in V. dahliae that confer the ability to cause vascular wilt disease. Unexpectedly, both species encode similar effector repertoires and share a genomic structure with genes encoding secreted proteins clustered in genomic islands. Intriguingly, V. tricorpus contains significantly fewer repetitive elements and an extended spectrum of secreted carbohydrate- active enzymes when compared with V. dahliae. In conclusion, we highlight the technical advances of a hybrid sequencing and assembly approach and show that the saprophyte V. tricorpus shares many hallmark features with the pathogen V. dahliae.

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July 7, 2019

Keeping an eye on P. aeruginosa.

This month’s Genome Watch looks at how whole-genome sequencing (WGS) can be used to track the source of Pseudomonas aeruginosa infection and to investigate the transition and adaptation of this opportunistic pathogen from the environment to the human host.

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July 7, 2019

Retrohoming of a mobile group II intron in human cells suggests how eukaryotes limit group II intron proliferation.

Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a “ribozyme”) and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed “retrohoming”. Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.

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