Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3′→5′ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.

The LORF5 Gene Is Non-essential for Replication but Important for Duck Plague Virus Cell-to-Cell Spread Efficiently in Host Cells

Duck plague virus (DPV) can cause high morbidity and mortality in many waterfowl species within the order Anseriformes. The DPV genome contains 78 open reading frames (ORFs), among which the LORF2, LORF3, LORF4, LORF5, and SORF3 genes are unique genes of avian herpesvirus. In this study, to investigate the role of this unique LORF5 gene in DPV proliferation, we generated a recombinant virus that lacks the LORF5 gene by a two-step red recombination system, which cloned the DPV Chinese virulent strain (DPV CHv) genome into a bacterial artificial chromosome (DPV CHv-BAC); the proliferation law of LORF5-deleted mutant virus on DEF cells and the effect of LORF5 gene on the life cycle stages of DPV compared with the parent strain were tested. Our data revealed that the LORF5 gene contributes to the cell-to-cell transmission of DPV but is not relevant to virus invasion, replication, assembly, and release formation. Taken together, this study sheds light on the role of the avian herpesvirus-specific gene LORF5 in the DPV proliferation life cycle. These findings lay the foundation for in-depth functional studies of the LORF5 gene in DPV or other avian herpesviruses.

A Novel Cre/lox-Based Genetic Tool for Repeated, Targeted and Markerless Gene Integration in Yarrowia lipolytica

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by the limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of the protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed that the genetic tool was effective for the integration of other genes, loci, and strains. Thus, this study significantly promotes the application of the Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

Optimization of a Lambda-RED Recombination Method for Rapid Gene Deletion in Human Cytomegalovirus

Human cytomegalovirus (HCMV) continues to be a major cause of morbidity in transplant patients and newborns. However, the functions of many of the more than 282 genes encoded in the HCMV genome remain unknown. The development of bacterial artificial chromosome (BAC) technology contributes to the genetic manipulation of several organisms including HCMV. The maintenance of the HCMV BAC in E. coli cells permits the rapid generation of recombinant viral genomes that can be used to produce viral progeny in cell cultures for the study of gene function. We optimized the Lambda-Red Recombination system to construct HCMV gene deletion mutants rapidly in the complete set of tested genes. This method constitutes a useful tool that allows for the quick generation of a high number of gene deletion mutants, allowing for the analysis of the whole genome to improve our understanding of HCMV gene function. This may also facilitate the development of novel vaccines and therapeutics.

Genome-wide estimation of recombination, mutation and positive selection enlightens diversification drivers of Mycobacterium bovis

Genome sequencing has reinvigorated the infectious disease research field, shedding light on disease epidemiology, pathogenesis, host–pathogen interactions and also evolutionary processes exerted upon pathogens. Mycobacterium tuberculosis complex (MTBC), enclosing M. bovis as one of its animal-adapted members causing tuberculosis (TB) in terrestrial mammals, is a paradigmatic model of bacterial evolution. As other MTBC members, M. bovis is postulated as a strictly clonal, slowly evolving pathogen, with apparently no signs of recombination or horizontal gene transfer. In this work, we applied comparative genomics to a whole genome sequence (WGS) dataset composed by 70 M. bovis from different lineages (European and African) to gain insights into the evolutionary forces that shape genetic diversification in M. bovis. Three distinct approaches were used to estimate signs of recombination. Globally, a small number of recombinant events was identified and confirmed by two independent methods with solid support. Still, recombination reveals a weaker effect on M. bovis diversity compared with mutation (overall r/m = 0.037). The differential r/m average values obtained across the clonal complexes of M. bovis in our dataset are consistent with the general notion that the extent of recombination may vary widely among lineages assigned to the same taxonomical species. Based on this work, recombination in M. bovis cannot be excluded and should thus be a topic of further effort in future comparative genomics studies for which WGS of large datasets from different epidemiological scenarios across the world is crucial. A smaller M. bovis dataset (n = 42) from a multi-host TB endemic scenario was then subjected to additional analyses, with the identification of more than 1,800 sites wherein at least one strain showed a single nucleotide polymorphism (SNP). The majority (87.1%) was located in coding regions, with the global ratio of non-synonymous upon synonymous alterations (dN/dS) exceeding 1.5, suggesting that positive selection is an important evolutionary force exerted upon M. bovis. A higher percentage of SNPs was detected in genes enriched into “lipid metabolism”, “cell wall and cell processes” and “intermediary metabolism and respiration” functional categories, revealing their underlying importance in M. bovis biology and evolution. A closer look on genes prone to horizontal gene transfer in the MTBC ancestor and included in the 3R (DNA repair, replication and recombination) system revealed a global average negative value for Taijima’s D neutrality test, suggesting that past selective sweeps and population expansion after a recent bottleneck remain as major evolutionary drivers of the obligatory pathogen M. bovis in its struggle with the host.

Marker-free genome engineering in Amycolatopsis using the pSAM2 site-specific recombination system

ABSTRACTActinobacteria belonging to the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as biodegradation or bioconversion. Despite their industrial importance, tools and methods for the genetic manipulation of Amycolatopsis are less developed than in other actinobacteria such as Streptomyces. Moreover, most of the existing methods do not support convenient marker-free genome engineering. Here, we report the use of the pSAM2 site-specific recombination system for the efficient deletion of marker genes or large DNA regions in Amycolatopsis. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the Xis and Int proteins from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the recombination sites attL and attR on each side of a region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei DSM 40773 by obtaining with high efficiency (>95%) a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster.IMPORTANCEThe genus Amycolatopsis is regarded as an important source of diverse specialized metabolites. Members of this genus are used in industry for the production of valuable antibiotics such as rifamycins or vancomycin. Amycolatopsis spp. also present a great interest for biotechnological applications such as biodegradation or bioconversion. Despite their importance, their genetic manipulation was somehow hampered by the lack of efficient tools. Here we report the successful use of the pSAM2 site-specific recombination system to construct unmarked deletion mutants, allowing marker recycling, or to create large deletions in A. mediterranei DSM 40773. The high efficiency of this site-specific recombination system and it possible application to other Amycolatopsis species open new opportunities for marker-free genome engineering in this genus.

Annotated expression and activity data for murine recombinase alleles and transgenes: the CrePortal resource

Recombinase alleles and transgenes can be used to facilitate spatio-temporal specificity of gene disruption or transgene expression. However, the versatility of this in vivo recombination system relies on having detailed and accurate characterization of recombinase expression and activity to enable selection of the appropriate allele or transgene. The CrePortal (http://www.informatics.jax.org/home/recombinase) leverages the informatics infrastructure of Mouse Genome Informatics to integrate data from the scientific literature, direct data submissions from the scientific community at-large, and from major projects developing new recombinase lines and characterizing recombinase expression and specificity patterns. Searching the CrePortal by recombinase activity or specific recombinase gene driver provides users with a recombinase alleles and transgenes activity tissue summary and matrix comparison of gene expression and recombinase activity with links to generation details, a recombinase activity grid, and associated phenotype annotations. Future improvements will add cell type-based activity annotations. The CrePortal provides a comprehensive presentation of recombinase allele and transgene data to assist researchers in selection of the recombinase allele or transgene based on where and when recombination is desired.

Guardian of the Genome: An Alternative RAG/Transib Co-Evolution Hypothesis for the Origin of V(D)J Recombination

The appearance of adaptive immunity in jawed vertebrates is termed the immunological ‘Big Bang’ because of the short evolutionary time over which it developed. Underlying it is the recombination activating gene (RAG)-based V(D)J recombination system, which initiates the sequence diversification of the immunoglobulins and lymphocyte antigen receptors. It was convincingly argued that the RAG1 and RAG2 genes originated from a single transposon. The current dogma postulates that the V(D)J recombination system was established by the split of a primordial vertebrate immune receptor gene into V and J segments by a RAG1/2 transposon, in parallel with the domestication of the same transposable element in a separate genomic locus as the RAG recombinase. Here, based on a new interpretation of previously published data, we propose an alternative evolutionary hypothesis suggesting that two different elements, a RAG1/2 transposase and a Transib transposon invader with RSS-like terminal inverted repeats, co-evolved to work together, resulting in a functional recombination process. This hypothesis offers an alternative understanding of the acquisition of recombinase function by RAGs and the origin of the V(D)J system.

Quorum Sensing-1 Signaling of N-hexanoyl-L-Homoserine Lactone Contributes to Virulence in Avian Pathogenic Escherichia Coli

Avian pathogenic E. coli (APEC) caused avian colibacillosis is mostly common in poultry industry worldwide. APEC virulence factors lead to pathogenesis and the quorum sensing (QS) system is actively involved in the regulation of these virulence factors. Signaling molecules in QS are known as autoinducers (AIs). In QS-1, E. coli encodes a single LuxR homolog, i.e SdiA, but does not express the LuxI homolog, an acyl-homoserine lactone (AHL) synthase of producing AI-1. Avian pathogenic E. coli (APEC) regulates its virulence genes expression in response to exogenous AHLs, but regulatory mechanisms of AHL and QS-1 are still unknown. This study targeted the APEC CE129 isolate as the reference strain, and the Yersinia enterocolitica yenI gene was expressed into APEC CE129. CE129/pyenI was conferred the ability to produce AHL signal. The CE129 SdiA mutant strain with an in-frame sdiA (AHL receptor) gene deletion was constructed by a λRed recombination system, which lost the ability to sense AHL. AimsThe goal of this study was to explore the function of QS-1 upon virulence and elucidate the regulatory effect of QS-1/AHL signals in the APEC strain.ResultsAdherence and invasion assays revealed that QS-1 affected APEC adherence and survival ability. APEC biofilm formation was also suppressed under C6HSL. Interestingly, APEC exhibited different phenotypes of acid tolerance and flagella expression when compared to enterotoxigenic E. coli or enterohemorrhagic E. coli (ETEC and EHEC, respectively). These findings enhance our understanding of the QS mechanism.ConclusionsQS-1 affected APEC biofilm formation, adhesion, and survival ability, but did not affect bacterial acid resistance (AR) or flagella expression, which were observed in ETEC and EHEC. The findings of this study have laid the foundation for further clarifying the complex mechanism of QS in future investigations on the virulence of APEC.