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July 7, 2019

Genome editing in human pluripotent stem cells: approaches, pitfalls, and solutions.

Human pluripotent stem cells (hPSCs) with knockout or mutant alleles can be generated using custom-engineered nucleases. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nucleases are the most commonly employed technologies for editing hPSC genomes. In this Protocol Review, we provide a brief overview of custom-engineered nucleases in the context of gene editing in hPSCs with a focus on the application of TALENs and CRISPR/Cas9. We will highlight the advantages and disadvantages of each method and discuss theoretical and technical considerations for experimental design. Copyright © 2016 Elsevier Inc. All rights reserved.

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July 7, 2019

Genome sequencing and analysis of the first complete genome of Lactobacillus kunkeei strain MP2, an Apis mellifera gut isolate

Background. The honey bee (Apis mellifera) is the most important pollinator in agriculture worldwide. However, the number of honey bees has fallen significantly since 2006, becoming a huge ecological problem nowadays. The principal cause is CCD, or Colony Collapse Disorder, characterized by the seemingly spontaneous abandonment of hives by their workers. One of the characteristics of CCD in honey bees is the alteration of the bacterial communities in their gastrointestinal tract, mainly due to the decrease of Firmicutes populations, such as the Lactobacilli. At this time, the causes of these alterations remain unknown. We recently isolated a strain of Lactobacillus kunkeei (L. kunkeei strain MP2) from the gut of Chilean honey bees. L. kunkeei, is one of the most commonly isolated bacterium from the honey bee gut and is highly versatile in different ecological niches. In this study, we aimed to elucidate in detail, the L. kunkeei genetic background and perform a comparative genome analysis with other Lactobacillus species. Methods. L. kunkeei MP2 was originally isolated from the guts of Chilean A. mellifera individuals. Genome sequencing was done using Pacific Biosciences single-molecule real-time sequencing technology. De novo assembly was performed using Celera assembler. The genome was annotated using Prokka, and functional information was added using the EggNOG 3.1 database. In addition, genomic islands were predicted using IslandViewer, and pro-phage sequences using PHAST. Comparisons between L. kunkeei MP2 with other L. kunkeei, and Lactobacillus strains were done using Roary. Results. The complete genome of L. kunkeei MP2 comprises one circular chromosome of 1,614,522 nt. with a GC content of 36,9%. Pangenome analysis with 16 L. kunkeei strains, identified 113 unique genes, most of them related to phage insertions. A large and unique region of L. kunkeei MP2 genome contains several genes that encode for phage structural protein and replication components. Comparative analysis of MP2 with other Lactobacillus species, identified several unique genes of L. kunkeei MP2 related with metabolism, biofilm generation, survival under stress conditions, and mobile genetic elements (MGEs). Discussion. The presence of multiple mobile genetic elements, including phage sequences, suggest a high degree of genetic variability in L. kunkeei. Its versatility and ability to survive in different ecological niches (bee guts, flowers, fruits among others) could be given by its genetic capacity to change and adapt to different environments. L. kunkeei could be a new source of Lactobacillus with beneficial properties. Indeed, L. kunkeei MP2 could play an important role in honey bee nutrition through the synthesis of components as isoprenoids.

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July 7, 2019

Complete genome sequences of Aerococcus christensenii CCUG 28831T, Aerococcus sanguinicola CCUG 43001T, Aerococcus urinae CCUG 36881T, Aerococcus urinaeequi CCUG 28094T, Aerococcus urinaehominis CCUG 42038 BT, and Aerococcus viridans CCUG 4311T.

Strains belonging to the genus Aerococcusare causative agents of human and animal infections, including urogenital infections, bacteremia/septicemia, and infective endocarditis. This study reports the first fully closed and complete genome sequences of six type strains belonging to the genus Aerococcususing a combination of Illumina HiSeq and PacBio sequencing technologies. Copyright © 2016 Carkaci et al.

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July 7, 2019

A pigeonpea gene confers resistance to Asian soybean rust in soybean.

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

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July 7, 2019

Complete genome sequence of Salmonella enterica serovar Typhimurium strain SO3 (sequence type 302) isolated from a baby with meningitis in Mexico.

The complete genome of Salmonella entericaserovar Typhimurium strain SO3 (sequence type 302), isolated from a fatal meningitis infection in Mexico, was determined using PacBio technology. The chromosome hosts six complete prophages and is predicted to harbor 51 genomic islands, including 13 pathogenicity islands (SPIs). It carries the Salmonella virulence plasmid (pSTV). Copyright © 2016 Vinuesa et al.

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July 7, 2019

First complete genome sequence of the Dutch veterinary Coxiella burnetii strain NL3262, originating from the largest global Q fever outbreak, and draft genome sequence of its epidemiologically linked chronic human isolate NLhu3345937

The largest global Q fever outbreak occurred in The Netherlands during 2007 to 2010. Goats and sheep were identified as the major sources of disease. Here, we report the first complete genome sequence of Coxiella burnetiigoat outbreak strain NL3262 and that of an epidemiologically linked chronic human strain, both having the outbreak-related CbNL01multilocus variable-number tandem-repeat analysis (MLVA) genotype. Copyright © 2016 Kuley et al.

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July 7, 2019

Evolutionary redesign of the Atlantic cod (Gadus morhua L.) Toll-like receptor repertoire by gene losses and expansions.

Genome sequencing of the teleost Atlantic cod demonstrated loss of the Major Histocompatibility Complex (MHC) class II, an extreme gene expansion of MHC class I and gene expansions and losses in the innate pattern recognition receptor (PRR) family of Toll-like receptors (TLR). In a comparative genomic setting, using an improved version of the genome, we characterize PRRs in Atlantic cod with emphasis on TLRs demonstrating the loss of TLR1/6, TLR2 and TLR5 and expansion of TLR7, TLR8, TLR9, TLR22 and TLR25. We find that Atlantic cod TLR expansions are strongly influenced by diversifying selection likely to increase the detectable ligand repertoire through neo- and subfunctionalization. Using RNAseq we find that Atlantic cod TLRs display likely tissue or developmental stage-specific expression patterns. In a broader perspective, a comprehensive vertebrate TLR phylogeny reveals that the Atlantic cod TLR repertoire is extreme with regards to losses and expansions compared to other teleosts. In addition we identify a substantial shift in TLR repertoires following the evolutionary transition from an aquatic vertebrate (fish) to a terrestrial (tetrapod) life style. Collectively, our findings provide new insight into the function and evolution of TLRs in Atlantic cod as well as the evolutionary history of vertebrate innate immunity.

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July 7, 2019

Mechanisms involved in acquisition of blaNDM genes by IncA/C2 and IncFIIY plasmids.

blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1 and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A and ISCR1 may have been involved in acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones but different regions carrying blaNDM are found in different locations. Tn3-derived Inverted-repeat Transposable Elements (TIME) appear to have been involved in acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterisation of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements and plasmids and provide insights into the possible routes for transmission of blaNDM genes amongst species of the Enterobacteriaceae family. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Prediction of putative resistance islands in a carbapenem-resistant Acinetobacter baumannii global clone 2 clinical isolate.

We investigated the whole genome sequence (WGS) of a carbapenem-resistant Acinetobacter baumannii isolate belonging to the global clone 2 (GC2) and predicted resistance islands using a software tool.A. baumannii strain YU-R612 was isolated from the sputum of a 61-yr-old man with sepsis. The WGS of the YU-R612 strain was obtained by using the PacBio RS II Sequencing System (Pacific Biosciences Inc., USA). Antimicrobial resistance genes and resistance islands were analyzed by using ResFinder and Genomic Island Prediction software (GIPSy), respectively.The YU-R612 genome consisted of a circular chromosome (ca. 4,075 kb) and two plasmids (ca. 74 kb and 5 kb). Its sequence type (ST) under the Oxford scheme was ST191, consistent with assignment to GC2. ResFinder analysis showed that YU-R612 possessed the following resistance genes: four ß-lactamase genes bla(ADC-30), bla(OXA-66), bla(OXA-23), and bla(TEM-1); armA, aadA1, and aacA4 as aminoglycoside resistance-encoding genes; aac(6′)Ib-cr for fluoroquinolone resistance; msr(E) for macrolide, lincosamide, and streptogramin B resistance; catB8 for phenicol resistance; and sul1 for sulfonamide resistance. By GIPSy analysis, six putative resistant islands (PRIs) were determined on the YU-R612 chromosome. Among them, PRI1 possessed two copies of Tn2009 carrying bla(OXA-23), and PRI5 carried two copies of a class I integron carrying sul1 and armA genes.By prediction of resistance islands in the carbapenem-resistant A. baumannii YU-R612 GC2 strain isolated in Korea, PRIs were detected on the chromosome that possessed Tn2009 and class I integrons. The prediction of resistance islands using software tools was useful for analysis of the WGS.

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July 7, 2019

Complete genome sequence of the larvicidal bacterium Lysinibacillus sphaericus strain OT4b.25.

Lysinibacillus sphaericus OT4b.25 is a native Colombian strain isolated from coleopteran larvae in an oak forest near Bogotá D.C.; this strain has shown high levels of pathogenic activity against Culex quinquefasciatus larvae in laboratory assays compared to that of other members of the same species. Using Pacific Biosciences sequencing technology, we propose a chromosomal contig of 4,665,775 bp that, according to comparative analysis, is highly similar to that of reference strain L. sphaericus C3-41. Copyright © 2016 Rey et al.

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July 7, 2019

Haemonchus contortus: genome structure, organization and comparative genomics

One of the first genome sequencing projects for a parasitic nematode was that for Haemonchus contortus. The open access data from the Wellcome Trust Sanger Institute provided a valuable early resource for the research community, particularly for the identification of specific genes and genetic markers. Later, a second sequencing project was initiated by the University of Melbourne, and the two draft genome sequences for H. contortus were published back-to-back in 2013. There is a pressing need for long-range genomic information for genetic mapping, population genetics and functional genomic studies, so we are continuing to improve the Wellcome Trust Sanger Institute assembly to provide a finished reference genome for H. contortus. This review describes this process, compares the H. contortus genome assemblies with draft genomes from other members of the strongylid group and discusses future directions for parasite genomics using the H. contortus model. Copyright © 2016 Elsevier Ltd. All rights reserved.

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July 7, 2019

Complete nucleotide sequence of pH11, an IncHI2 plasmid conferring multi-antibiotic resistance and multi-heavy metal resistance genes in a clinical Klebsiella pneumoniae isolate.

The complete 284,628bp sequence of pH11, an IncHI2 plasmid, was determined through single-molecule, real-time (SMRT) sequencing. Harbored by a clinical Klebsiella pneumoniae strain H11, and isolated in Beijing, this plasmid contains multiple antibiotic resistance genes, including catA2, aac(6′)-Ib, strB, strA, dfrA19, blaTEM-1, blaSHV-12, sul1, qacE delta 1, ereA, arr2, and aac3. The aac(6′)-Ib is carried by a class I integron. Plasmid pH11 also carries several genes associated with resistance to heavy metals, such as tellurium, mercury, cobalt, zinc, nickel, copper, lead and cadmium. This plasmid exhibits numerous characteristics, including HipBA and RelBE toxin-antitoxin systems, two major transfer (Tra) regions closely related to those of Salmonella enterica serovar plasmid pRH-R27, a type II restriction modification system (EcoRII R-M system), several methyltransferases and methylases and genes encoding Hha and StpA. These characteristics suggest that pH11 may adapt to various hosts and environments. Multiple insertion sequence elements, transposases, recombinases, resolvases and integrases are scattered throughout pH11. The presence of these genes may indicate that horizontal gene transfer occurs frequently in pH11 and thus may facilitate the dissemination of antimicrobial resistance determinants. Our data suggest that pH11 is a chimera gradually assembled through the integration of different horizontally acquired DNA segments via transposition or homologous recombination. Copyright © 2016 Elsevier Inc. All rights reserved.

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July 7, 2019

Complete genome sequence of Vibrio parahaemolyticus FORC_023 isolated from raw fish storage water.

Vibrio parahaemolyticusis a Gram-negative halophilic bacterium that causes food-borne gastroenteritis in humans who consumeV. parahaemolyticus-contaminated seafood.The FORC_023 strain was isolated from raw fish storage water, containing live fish at a sashimi restaurant. Here, we aimed to sequence and characterize the genome of the FORC_023 strain. The genome of the FORC_023 strain showed two circular chromosomes, which contained 4227 open reading frames (ORFs), 131 tRNA genes and 37 rRNA genes. Although the genome of FORC_023 did not include major virulence genes, such as genes encoding thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), it contained genes encoding other hemolysins, secretion systems, iron uptake-related proteins and severalV. parahaemolyticusislands. The highest average nucleotide identity value was obtained between the FORC_023 strain and UCM-V493 (CP007004-6). Comparative genomic analysis of FORC_023 with UCM-V493 revealed that FORC_023 carried an additional genomic region encoding virulence factors, such as repeats-in-toxin and type II secretion factors. Furthermore,in vitrocytotoxicity testing showed that FORC_023 exhibited a high level of cytotoxicity toward INT-407 human epithelial cells. These results suggested that the FORC_023 strain may be a food-borne pathogen.© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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